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在亞洲人群包括中國人群中，顱內動脈粥樣硬化（ICAS）發病率很高，是缺血性卒中和短暫性腦缺血發作（TIA）的首要病因。然而，目前ICAS 並未被深入研究。因此我們在一系列研究中通過運用傳統及創新的神經影像學方法，來研究ICAS 的臨床及血流動力學特徵，以期促進其全面評價及危險分層。, 既往研究發現亞洲人群和西方人群在頭頸部動脈粥樣硬化的發生和發展上存在種族差異。爲了進一步驗證這些種族差異，我們開展了一項社區研究，以探索無癥狀性顱內外動脈粥樣硬化在中國社區成年居民中的發病情況，以及二者之間的相互關係。在該研究中，我們分別採用經顱多普勒（TCD）和頸部血管超聲（CD）來評價顱內和顱外動脈的粥樣硬化。在研究納入的537 例研究對象中，我們發現顱內動脈粥樣硬化的發展優先於頸動脈粥樣硬化，而且不同階段的頸動脈粥樣硬化與顱內動脈粥樣硬化並無獨立相關關係。該結果提示，在中國人群的全身系統性粥樣硬化的過程中，顱內動脈粥樣硬化可能是一個比較早期而且相對獨立的過程，這與西方人群的情況不同。本研究結果進一步支持東西方人群在顱內外動脈粥樣硬化進程上存在的種族差異。, 根據既往研究結果，病因為癥狀性ICAS的缺血性卒中或TIA患者卒中復發的風險很高。目前，癥狀性ICAS患者的危險分層大多基於其動脈管腔的狹窄程度。然而，管腔的解剖學狹窄程度並不一定與其血流動力學的嚴重程度成比例，而後者也可能影響相關患者的卒中復發風險。因此，我們進行了以下的一系列研究， 以評價癥狀性ICAS的血流動力學特徵，同時初步探索其在相關患者危險分層中的應用價值。, 我們首先進行了三項研究，採用一種基於磁共振血管成像（MRA）的創新方法來評價癥狀性ICAS的血流動力學嚴重程度。基於時間飛躍法（TOF）MRA的信號對比機制，我們提出了一項名為信號強度比值（SIR）的參數來定量地評價癥狀性ICAS 的血流動力學效應；該參數代表TOF MRA 影像上經過背景信號強度校正后的ICAS 病變后和病變前的信號強度比值。在一項初步研究中，我們確定了該參數的評價和計算方法。在26例癥狀性ICAS病變中，我們發現該參數的計算操作簡便，在臨床上可行，且在同一評價者的兩次評價中具有很高的可重複性。在隨後的一項研究中，我們在102例癥狀性ICAS病變中發現該參數在兩位評價者之間具有顯著的可重複性。在第三項研究中，我們在36例具有癥狀性ICAS的缺血性卒中患者中發現SIR與患者的急性梗死灶體積顯著相關，但我們並未發現該參數與患者1年的卒中復發風險相關，可能由於該研究的樣本量過小。以上三項研究證實，SIR作為一種基於TOF MRA的評價癥狀性ICAS血流動力學嚴重程度的方法，具有可重複性及臨床可行性；而其對於相關患者危險分層的價值需要在前瞻性的較大型研究中進一步驗證。, 在如下的另外兩項研究中，我們採用計算機流體動力學（CFD）技術對癥狀性ICAS患者的計算機斷層掃描血管成像（CTA）影像進行重建，從而評價其ICAS 病變的血流動力學特徵。首先，在一項初步研究中，我們探索了採用臨床常規CTA影像進行CFD模型重建的可行性。在10例癥狀性ICAS病變中，9例病變的CTA原始圖像成功重建為CFD模型。重建的CFD模型可以定量地反映ICAS病變的各種血流動力學特徵。該初步研究證實了基於臨床常規CTA進行CFD建模從而評價ICAS血流動力學特徵的可行性。在隨後的一項研究中，我們探索了CFD 模型反映的癥狀性ICAS 的血流動力學特徵對於相關患者卒中復發的預測價值。在32例具有70-99%管腔狹窄的癥狀性ICAS病例中，我們發現病變前後血流動力學參數的變化（包括壓力，剪切應變率及血流速度）可能預測患者的卒中復發風險。以上兩項研究證實，基於臨床常規CTA進行CFD模型重建從而定量評價癥狀性ICAS的血流動力學特徵具可行性，同時，這些血流動力學特徵可能對相關患者的卒中復發風險具有預測價值。, 綜上所述，通過以上研究，我們進一步證實了亞洲人群和西方人群在顱內外動脈粥樣硬化的進程上存在的種族差異。更重要的是，我們的研究證實評價癥狀性ICAS病變的血流動力學特徵具有臨床意義。對於相關患者，採用以上研究中的兩種方法評價癥狀性ICAS的血流動力學特徵，可能對患者的危險分層具有潛在的指導意義。在未來的前瞻性大樣本研究中，上述方法對癥狀性ICAS患者卒中復發風險的預測價值需要進一步證實，以期促進相關的臨床決策，從而在長遠目標上降低相關患者的卒中復發風險。, Intracranial atherosclerosis (ICAS) is of high prevalence in Asia, which is the leading cause for ischemic stroke and transient ischemic attack (TIA) in Asians, including the Chinese. However, it has not been fully appreciated or adequately investigated in relevant studies. In this thesis, we tried to delineate the hemodynamics and clinical implications of ICAS, by using several traditional and novel neuroimaging methods., Previous studies had suggested differences in atherogenesis of intra- and extracranial arteries between Asians and Caucasians. To find further evidence, we performed a study to investigate asymptomatic ICAS and carotid atherosclerosis and their correlations in community-dwelling Chinese adults, by using transcranial Doppler and carotid duplex ultrasonography, respectively. For the 537 subjects studied, we found more advanced asymptomatic ICAS than carotid atherosclerosis, and there were no independent correlations between different stages of carotid atherosclerosis and presence of ICAS. The results suggested that atherogenesis of intracranial arteries might be a relatively independent course in systemic atherosclerosis in the Chinese population, which is unlike the case in Caucasians. By combing with previous findings, results of this study further supported the existence of racial differences in cervicocerebral atherogenesis between Asians and Caucasians., According to previous studies, stroke patients with symptomatic ICAS are at high risk of recurrence. Currently, risk stratification of symptomatic ICAS are usually based on the percentage of luminal stenosis. However, the anatomic severity does not always proportionate to its hemodynamic significance, which may also impact on the risk of stroke recurrence in symptomatic ICAS. Therefore, we performed a series of studies as follows to evaluate the hemodynamics of symptomatic ICAS, and to assess its value in risk stratification of those with such lesions., We first performed three studies based on time-of-flight (TOF) magnetic resonance angiography (MRA), to gauge the hemodynamic significance of symptomatic ICAS. Based on the signal contrast mechanism of TOF MRA, we developed a novel index, signal intensity ratio (SIR), representing changes of signal intensities (SI) across an ICAS on maximum intensity projections of TOF MRA, to quantify its hemodynamic significance: SIR = (mean post-stenotic SI - mean background SI) / (mean pre-stenotic SI - mean background SI). In a pilot study to establish the methodology of this index, we found it easy to perform, and highly reproducible between repetitive measurements by a same observer in 26 symptomatic ICASs. In a subsequent study, we also found this index to be substantially reproducible between measurements from two observers in 102 symptomatic ICAS lesions. In a third study, we tried to investigate the relationships between SIR of ICAS, other imaging features, and 1 year outcomes of patients with symptomatic ICAS. In the 36 patients enrolled, SIR was found to be significantly, linearly and negatively correlated to acute infarct volume on diffusion-weighted MR images. However, we did not establish a definite correlation between SIR and recurrent ischemic stroke, probably due to the small sample size. These studies suggested that SIR as evaluated on MRA was a feasible and reproducible method to gauge the hemodynamic and functional significance of ICAS. The role of this index in predicting further recurrent risks in those with symptomatic ICAS needs to be verified in future studies., In another two studies, we applied the computational fluid dynamics (CFD) modeling technique in processing computed tomography angiography (CTA) images, to evaluate the hemodynamic characteristics of ICAS. In a pilot study, we tested the feasibility of CFD modeling of ICAS based on CTA images. Among 10 cases of symptomatic ICAS identified on CTA, the CTA source images of 9 were successfully processed to CFD models, which were able to quantitatively delineate the hemodynamic environment across the lesions. This pilot study demonstrated the feasibility of constructing CFD models of ICAS out of routinely obtained CTA source images. Then in a second study, we preliminarily explored the values of hemodynamics of ICAS revealed by such CFD models, in predicting recurrent risks in patients with symptomatic ICAS of 70-99% luminal stenosis. In the 32 cases evaluated, we found that changes of hemodynamic features across an ICAS lesion, including the changes of pressure, shear strain rate, and blood flow velocity, may be able to predict the recurrent risk in this patient subset. Therefore, it was feasible to model hemodynamics of symptomatic ICAS based on CTA images, and future prospective studies with larger sample sizes are warranted to further validate the role of CFD modeling in risk stratification of affected patients., In conclusion, in this thesis we found further evidence to support the existence of racial differences in atherogenesis of cervicocerebral arteries between Caucasians and Asians. More importantly, we demonstrated that hemodynamics of symptomatic ICAS could be of clinical significance in characterization of such lesions. In patients with symptomatic ICAS, the two methods to evaluate hemodynamic features of ICAS as used in this thesis, may yield potential values in predicting the recurrent risk of these patients. In the near future, prospective studies enrolling more patients are warranted to further validate findings in this thesis, to embrace more reasonable and comprehensive evaluation of symptomatic ICAS, so as to facilitate decision making in clinical scenarios and patient selection in clinical studies, which in the long run may help reduce the risk of stroke recurrence in affected patients., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Leng, Xinyi., Thesis (Ph.D.) Chinese University of Hong Kong, 2014., Includes bibliographical references (leaves 131-146)., s also in Chinese., http://library.cuhk.edu.hk/record=b6115571, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

糖調節蛋白78 (Glucose-regulated protein 78),也稱之Bip,是70kDa熱休克蛋白家族成员之一。已有的研究表明，Bip 是一個具有多功能的蛋白，參與眾多的生物調控過程，包括蛋白折疊，調節鈣平衡，以及作為內質網緊張（ER stress) 的感應器。有研究表明，Bip可以在細胞膜上定位，作為Nodal信號通路的一個輔助受體發揮作用。大量的研究表明，Bip在疾病和代謝方面也發揮重要作用。它參與胰島素的生物合成，並可以提高長期高血糖下β細胞的功能。同時具有抗細胞凋亡的作用。然而Bip在胚胎髮育中的生物功能卻知之甚少。, 高等脊椎動物腎臟發育中經歷形成3種腎臟形式：前腎，中腎和後腎。腎單位是這3種形式的基本結構和功能單位。在兩棲類，前腎在胚胎時期發揮作用，在胚胎的兩側各只有一個腎單位。這使得爪蟾成為前腎研究的一個非常好的模型。, 在此項研究中，我們採用非洲爪蛙作為動物模型來研究Bip在胚胎髮育過程中，尤其是在前腎發育中的生物功能。Bip是一個母性因子，在尾芽期，Bip 表達在粘液腺，前腎，肝以及耳囊。 Bip在前腎清晰明確的表達，表明Bip可能在前腎的發育中發揮作用。我們利用BipMO來進行敲低功能實驗，免疫印記顯示BipMO能阻斷帶Flag標記Bip的翻譯。通過原位雜交技術檢測前腎的不同標記基因的表達發現，敲低Bip抑制前腎的形成，表明Bip的正常表達是前腎發育所必須的。, 為了研究Bip調節前腎的發育的分子機制， 我們使用Affmetrix基因芯片分析在Bip敲低情況下的不同時期胚胎中基因的表達譜，發現在Bip敲低表達的胚胎中，視黃酸信號通路的一些重要的組分的表達受到抑制。爪蛙胚胎原腸胚的動物帽細胞具有多能性， 使用激活素和視黃酸一同處理動物帽細胞可以誘導其分化成為原腎組織。在此體外分化體系中敲低Bip表達，前腎標記基因表達降低，顯示在這一體外系統中前腎的分化受到抑制。該實驗結果與體內實驗結果一致。在體外培養的HEK293T細胞中敲低Bip，抑制視黃酸處理後視黃酸信號通路螢光素報告的活性。 lhx1是前腎發育早期表達標記之一，對於前腎原基的初始化具有重要的作用，同是它也是視黃酸信號通路的靶基因。共同註射BipMO和lhx1表明，前腎的異常可以明顯降低，顯示lhx1可以部份拯救由於Bip缺失所造成的腎臟發育缺陷。該實驗表明Bip通過調節視黃酸信號通路，來調控lhx1的表達前腎的形成。我們進一不發現，敲低Bip後，前腎異常形成的區域內，細胞凋亡增加，增殖減少。該結果在細胞水平上解釋了Bip敲低表達時前腎形成異常的一個原因。, 综述所述，Bip正確表達对胚胎前肾的发育極為重要。它胚胎发育过程中通过視黃酸信号通路調控lhx1的表達，從而对前肾的形成发挥重要作用。, Glucose-regulated protein 78 (Grp78), also known as Bip, belongs to heat shock protein 70kDa family. It has been implicated in various biological processes including protein folding, regulation of calcium homeostasis, and serving as a sensor of ER (Endoplasmic Reticulum) stress. Moreover, it can localize in cell membrane, acting as co-receptor of nodal signaling. It is essential for insulin biosynthesis. In addition, Bip plays important roles in a number of diseases. For example, BIP can improve β-cell function in the prolonged hyperglycemia. Knockdown of BIP in β-cell can induce apoptosis. However, little is known about its function during embryonic development., In high vertebrate, three sets of nephric forms develop successively during embryonic kidney development. They are pronephros, mesonephros, and metanephros. Nephron is the basic structural and functional unit of all these three forms. In amphibian, the pronephros performs function at the embryonic stages, which has only one nephron on either side of the body. It makes Xenopus a very good model for pronephros study., In this study, we took advantage of Xenopus leavis as an animal model to investigate Bip function during embryonic development, especially its role in pronephros development. We first examined the expression of Bip in developing embryos. Whole mount in situ hybridization showed that Bip was expressed in the cement gland, pronephros, liver and ear vesicle during tailbud stages. It was expressed in the pronephros strongly and clearly which suggested that Bip might play roles in pronephros development. We performed loss-of-function experiment by using morpholino oligonucleotide (MO) knock down translation of endogenous Bip expression. Depletion of Bip impaired formation of pronephros revealed by reduction expression of different pronephros maker genes. The pluripotent animal caps can differentiate into pronephros tissue when treated with activin and all-trans retinoic acid (atRA) in vitro kidney induction assay. In line with our in vivo observation, knockdown of Bip inhibited pronephros differentiation that can normally achieved by combined effects of activin and atRA in animal cap assay., In order to investigate the molecular mechanisms as how Bip regulated pronephros development, we performed Affymetrix DNA microarray assay to generate gene expression profile in Bip morphants. We found that some components of RA signaling were inhibited when Bip was knockdown. Moreover, knockdown of Bip caused reduction of RA target genes expression after treatment with RA. Consistent with above observations, luciferase activities of RA signaling reporter was reduced in HEK293T cells when BIP expression was depleted by RNAi. lhx1 is one of RA target genes and has been implicated playing essential roles in pronephros development. The inhibition of pronephros formation induced by Bip depletion can be partially rescued by co-overpression, suggesting 1) lhx1 is downstream of Bip in the regulatory network of pronephros formation; and 2) Bip regulates pronephros formation through RA signaling via lhx1. We also found increased apoptosis and decreased cell proliferation at pronephros-forming region in Bip morphants. That could explain the reason of pronephros malformation when Bip is downregulated., Taken together, Bip is essential for pronephros development. It functions through RA signaling during the complex developmental processes., Detailed summary in vernacular field only., Shi, Weili., Thesis (Ph.D.) Chinese University of Hong Kong, 2014., Includes bibliographical references (leaves 125-143)., s also in Chinese., http://library.cuhk.edu.hk/record=b6115577, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

研究背景及實驗目的: 非酒精性脂肪性肝炎(NASH)使得肥胖和2 型糖尿病變得複雜，肝臟炎症的持續產生是其主要的發病機理。CXCL10 是一種促進炎症的細胞因數，其在肥胖和2 型糖尿病中的表達顯著升高。CXCL10 以及其受體CXCR3 是否在NASH 的發生發展中起作用尚不清楚。在本研究中，我們探索了CXCL10 以及其受體CXCR3 在脂肪性肝炎中的功能， 並評估了CXCL10 在NASH 中的臨床價值。, 實驗方法：CXCL10 基因敲除鼠，CXCR3 敲除鼠以及野生型C57BL/6 小鼠給予蛋氨酸膽鹼缺乏食(MCD)4 周或者8 周。CXCL10 的信號通路以及下游靶點通過細胞因數分析，cDNA array, 蛋白DNA 結合實驗，自噬溶酶體系統分析進行檢測。為了闡明CXCL10 抑制對NASH 的預防治療作用，我們給MCD 餵養的小鼠注射抗CXCL10 抗體。用不同濃度的CXCL10 抗體以及CXCR3 抑制劑NIBR2130 幹預MCD 培養的肝細胞株AML-12。臨床研究中，我們收集了147個非酒精性脂肪肝患者以及73 個健康對照的血清，用酶聯免疫吸附試驗檢測血清中CXCL10 的水準。, 結果：野生型小鼠給予MCD 餵養後，CXCL10 以及CXCR3 的表達升高，並出現脂肪性肝炎的表現。然而，MCD 飼養的CXCL10 以及CXCR3 基因敲除鼠中，脂肪性肝炎明顯減輕。CXCL10 通過促炎細胞因數的產生以及NK-κB 信號通路促進MCD 飼養的小鼠NASH 的發生。CXCL10 通過促進脂質合成的基因SREBP-1c, ChREBP 和 SCD-1 引起脂肪變性，並通過CYP2E1 以及 C/EBPβ 的上調引起氧化應激。值得注意的是，自噬的損傷在CXCL10 以及CXCR3 導致的脂肪性肝炎的進展中起重要作用。 MCD 飼養的野生型小鼠中p62 以及LC3-II 表達明顯高於CXCL10 以及CXCR3 基因敲除鼠。通過抗CXCL10 抗體中和CXCL10 可以減輕MCD 食引起的小鼠脂肪性肝炎以及MCD 培養液引起的AML-12 細胞損傷。高選擇性的CXCR3 抑制劑NIBR2130 也可以抑制MCD 引起的肝細胞損傷。我們進一步研究了CXCL10 的臨床應用價值，發現NASH 患者血清以及肝臟中CXCL10 的水準明顯升高。更重要的是，血液中CXCL10 的水準與肝小葉炎症程度有關，是NASH 的獨立危險因素。, 結論：我們的研究首次發現CXCL10 以及其受體CXCR3 通過促進炎症，脂質聚集，氧化應激以及自噬缺乏在NASH 的發病中起重要作用。抑制CXCL10 或者CXCR3 為NASH 患者的治療提供了新的方法。CXCL10 可作為NASH 患者非侵入性診斷的標誌物。, Background and aims: Non-alcoholic steatoheaptitis (NASH) complicates obesity and type 2 diabetes, while recruitment and perpetuation of liver inflammation is central to its pathogenesis. Expression of C-X-C motif chemokine 10 (CXCL10), a proinflammatory cytokine, correlates positively with obesity and type 2 diabetes. Whether CXCL10 and its receptor CXCR3 play a role in NASH is unknown. In this study, we investigated the functional significance of CXCL10 and its receptor CXCR3 in steatoheaptitis. Moreover, the clinical impact of CXCL10 in NASH was examined., Methods: Gene-deleted CXCL10 (CXCL10-/-), CXCL10 receptor CXCR3 (CXCR3-/-) and C57BL/6 wildtype (WT) mice were fed methionine and choline-deficient (MCD) diet for 4 or 8 weeks. Cytokine profiling assay, cDNA array, protein-DNA binding activity assay and autophagosome-lysosome system analysis of CXCL10 signaling and downstream targets were performed. In other experiments, we injected neutralizing anti-CXCL10 monoclonal antibodies (mAb) into MCD diet-fed WT mice, while AML-12 cells were cultured in MCD medium in the presence of anti-CXCL10 mAb or CXCR3 inhibitor (NIBR2130) for 24 hours. Human serum was obtained from 147 patients with biopsy-proven non-alcoholic fatty liver disease and 73 controls. Circulating CXCL10 levels were determined by enzyme-linked immunosorbent assay., Results: MCD-fed WT mice developed steatohepatitis with higher hepatic CXCL10 and CXCR3 expression. CXCL10-/- and CXCR3-/- mice were refractory to MCDinduced steatohepatitis. In WT mice with steatohepatitis, but not in CXCL10-/- mice, CXCL10 was associated with the induction of pro-inflammatory chemokines and cytokines, as well as activation of nuclear factor-κB (NF-κB) signaling. CXCL10 expression was linked to steatosis through lipogenic factors, including liver X receptors and its downstream targets (SREBP-1c, ChREBP and SCD-1), and also to oxidative stress (up-regulation of CYP2E1 and C/EBPβ). In particular, autophagy deficiency was involved in CXCL10- and CXCR3-induced steatohepatitis as indicated by p62 and LC3-I/II protein accumulation in MCD-fed WT mice than in CXCL10-/- and CXCR3-/- mice. Moreover, the impaired autophagic function was related to the reduction of lysosomal function in CXCL10- or CXCR3-induced NASH. Blockade of CXCL10 by anti-CXCL10 mAb protected against MCD-induced steatohepatitis in vivo and against MCD-mediated injury to AML-12 cells in vitro. The highly selective CXCR3 antagonist NIBR2130 also inhibited MCD-induced injury in AML-12 hepatocytes. We further investigated the clinical impact of CXCL10 and found circulating and hepatic CXCL10 levels were significantly higher in human NASH. Importantly, circulating CXCL10 level was correlated with the degree of lobular inflammation and was an independent risk factor for NASH patients., Conclusions: We demonstrate for the first time that CXCL10 and its receptor CXCR3 plays a pivotal role in the pathogenesis of NASH by promoting inflammation, fatty acid accumulation, oxidative stress and autophagy deficiency. Blockade of CXCL10 or CXCR3 is a potential novel approach for NASH intervention. CXCL10 is a noninvasive biomarker for NASH patients., Detailed summary in vernacular field only., Zhang, Xiang., Thesis (Ph.D.) Chinese University of Hong Kong, 2014., Includes bibliographical references (leaves 145-167)., s also in Chinese., http://library.cuhk.edu.hk/record=b6115759, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Xiong, Lei., Thesis (M.Phil.)--Chinese University of Hong Kong, 2013., Includes bibliographical references (leaves 79-97)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., s also in Chinese., http://library.cuhk.edu.hk/record=b5884411, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Wang, Shuxin., Thesis (Ph.D.)--Chinese University of Hong Kong, 2013., Includes bibliographical references (leaves 191-212)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., s also in Chinese; appendixes includes Chinese., http://library.cuhk.edu.hk/record=b5884521, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

養分與睡眠的關聯是一個在睡眠科學上極具爭議性的課題。減低攝取蛋白質和碳水化合物會導致失眠，增加攝取總脂肪和油份會導致失眠。維生素和礦物質也被認為與失眠有關。此論文嘗試通過研究一般香港中國人的食習慣和失眠情況，進一步了解營養物質與失眠之間的關聯。此論文將會深入探討營養成分對失眠的影響。, 背景和目標： 失眠是常見的睡眠障礙和公共衛生問題。失眠可分為三個亞型：難以啟動睡眠(DIM)、難以維持睡眠(DMS) 和過早覺醒類型(EMA)。然而，有關的研究多着重於外國人口。針對研究香港中國人口的失眠情況與營養成分關聯的資料相對比較少。此研究目的是找出在香港中國人口失眠與營養成分之間的關聯，有助研究失眠與營養成分之間的機制。據推測，失眠與營養成分之間於香港中國人口有關聯。香港中國人口失眠症患者的食特點跟其他地區人口會有所不同。失眠的三個亞型和營養成分之間的關聯會有所不同。, 研究方法： 十三間學校被邀請進行了橫斷面研究。一百三十八位青少年(六十一男、七十七女) 以及一百七十三位成年人（八十四男、 八十九女）應邀參加這項研究。有關日常營養攝取量的資料，以自行申報的三天膳食記錄表取得。有關失眠症狀的評估，以自行申報的標準睡眠問卷（ISI）獲得。有關焦慮和抑鬱的評估，以自行申報的醫院焦慮抑鬱量表（HADS）取得。, 研究結果與結論：分析顯示，失眠與減低攝取維生素A有關聯(成年人組別p = 0.02、青少年組別p = 0.01)，與減低攝取維生素D有關聯(成年人組別p = 0.02、青少年組別p = 0.01）和與減低攝取維生素E有關聯(成年人組別p = 0.02、青少年組別p = 0.01)。失眠綜合症與難以啟動睡眠(DIM)、難以維持睡眠(DMS) 和過早覺醒類型(EMA) 與減低攝取飽和脂肪、碳水化合物、維生素A 、維生素D、和維生素E有關聯。此研究證實了香港中國人口的失眠與營養成分之間有關聯。證實了香港中國人口失眠症患者的食特點跟其他地區人口有不同。證實了失眠的三個亞型和營養成分之間的關聯有不同。我們於這項研究成功找到與失眠有關的營養成分，有助研發以天然營養物質來解決香港中國人的失眠問題。, The association of nutrients and sleep is a debatable question in sleep science. Some literatures suggest that sleep is enhanced by certain nutrients, while some other literatures suggest that sleep is deprived by certain nutrients causing insomnia. This dissertation attempts to address the association between nutrients and insomnia of Hong Kong Chinese Population., Background and Objective: Insomnia is a common sleep disorder and a major public health issue. Insomnia could be classified into three subtypes: Difficulty in Initiating Sleep (DIS), Difficulty in Maintaining Sleep (DMS), and Early Morning Awakening (EMA). Vitamins and minerals are thought to be associated with insomnia. From literature reviews, studies in western population and in Asian population found that protein and carbohydrates, fat and oil are associated with insomnia. Insomnia could be affected by the availability of nutritional substances in individual’s diet. However, limited studies are done in Hong Kong Chinese population on the association between insomnia and nutrient components. The aim of this study is to find out the association between insomnia and nutrient components in-take in Hong Kong Chinese population., Hypothesis: It is hypothesized insomnia and nutrient components would also have association in Hong Kong Chinese population. It is hypothesized the dietary characteristic of insomniac in Hong Kong Chinese population would be different from that of non-Hong Kong Chinese population, and it is hypothesized each insomnia subtype and nutrient components would have different association., Method: A community-based cross-sectional study is conducted in 13 schools. There are 138 adolescents (61 male and 77 female) and 173 adults (84 male and 89 female) participated in this study. Information of daily nutrient intake is obtained by a self-administrated 3-day food diary, the assessment of insomnia symptom is obtained by a standard sleep questionnaire Insomnia Severity Index (ISI), and the assessment of anxiety and depression is obtained by Hospital Anxiety and Depression Scale (HADS)., Results and Conclusion: Agree with the hypothesis, insomnia and nutrient component have association in Hong Kong Chinese population. The dietary characteristic of insomniac in Hong Kong Chinese population is different from that of non-Hong Kong Chinese population. Each insomnia subtype and nutrient component has different association. Multivariance analysis shows insomnia subtype Difficult Initiating Sleep (DIS), Difficult Maintaining Sleep (DMS), Early Morning Awakening (EMA), and overall insomnia syndrome associate with decreased in-take of vitamin A, vitamin D and vitamin E in both adults and adolescents. Decreased intake of saturated fat associates with insomnia subtype DMS and decreased intake of carbohydrate associates with insomnia subtype EMA in this study. Information from this study shines lights on the relationship of insomnia and nutrients in-take in the general population of Hong Kong Chinese., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Lau, Yin Wah Vivien., Thesis (M.Phil.)--Chinese University of Hong Kong, 2013., Includes bibliographical references (leaves 82-90)., s also in Chinese., --- p.i-iv, Acknowledgements: --- p.v, Table of contents: --- p.vi-viii, List of Lists: --- p.ix, List of Tables: --- p.ix, List of Figures: --- p.ix, Objective --- p.1, Chapter Chapter 1: --- Introduction, Chapter 1.1 --- Sleep Research --- p.2, Chapter 1.1.1 --- Background and History of Sleep Research --- p.2-3, Chapter 1.1.2 --- Sleep Function and Consequence --- p.3-4, Chapter 1.1.3 --- Neurotransmitters and Neuromodulators --- p.4-5, Chapter 1.2 --- Insomnia --- p.5, Chapter 1.2.1 --- The Definition of Insomnia --- p.6, Chapter 1.2.1.1 --- Many Different Definitions of Insomnia Diagnostic Criteria --- p.6, Chapter 1.2.1.2 --- Diagnostic Criteria used for Insomnia in This Study --- p.6-8, Chapter 1.2.1.3 --- Symptoms and Syndrome of Insomnia --- p.9-10, Chapter 1.2.2 --- The Cost of Insomnia --- p.10-11, Chapter 1.2.3 --- The Common Causes of Insomnia --- p.11, Chapter 1.2.4 --- Cognitive-Behavioral Model of Insomnia --- p.12, Chapter 1.2.5 --- Treatments of Insomnia --- p.14, Chapter 1.2.6 --- Confounding Factors of Insomnia --- p.14, Chapter Chapter 2: --- Age, Education and Body Mass Effect on Sleep Pattern, Chapter 2.1 --- Age --- p.16, Chapter 2.2 --- Education --- p.17, Chapter 2.3 --- Body Mass --- p.17, Chapter Chapter 3: --- Mood, Pain, Sleep Hygiene, Drug, Caffeine and Alcohol Effect on Sleep Pattern, Chapter 3.1 --- Mood --- p.18, Chapter 3.2 --- Pain --- p.18, Chapter 3.3 --- Sleep Hygiene --- p.18, Chapter 3.4 --- Drug --- p.20, Chapter 3.5 --- Caffeine --- p.20, Chapter 3.6 --- Alcohol --- p.20, Chapter Chapter 4: --- Nutrient Components, Chapter 4.1 --- Macro-nutrient --- p.21, Chapter 4.1.1 --- Carbohydrate --- p.21-22, Chapter 4.1.2 --- Fatty Acid --- p.22-23, Chapter 4.1.3 --- Protein --- p.23-24, Chapter 4.2 --- Micro-nutrient --- p.24, Chapter 4.2.1 --- Vitamin B₁ (Thiamine) --- p.24, Chapter 4.2.2 --- Vitamin B₂ (Riboflavin) and Vitamin B₃ (Niacin) --- p.24-25, Chapter 4.2.3 --- Vitamin B₆ (Pyridoxine) --- p.25, Chapter 4.2.4 --- Vitamin B₁₂ (Cobalamin, Folate) --- p.25-26, Chapter 4.2.5 --- Vitamin A and Vitamin D --- p.26-27, Chapter 4.2.6 --- Tryptophan, Tyrosine, Choline and Phosphatidylcholine (Lecithin) --- p.27-28, Chapter 4.2.7 --- Vitamin E and Vitamin C --- p.30, Chapter 4.2.8 --- Iron --- p.30, Chapter Chapter 5: --- Nutrient Components and Insomnia, Chapter 5.1 --- Introduction --- p.32, Chapter 5.2 --- Social Perspective of Insomnia and Nutrients --- p.33, Chapter 5.3 --- Biochemical Perspective of Insomnia and Nutrients --- p.33-34, Chapter Chapter 6: --- Material and Method, Chapter 6.1 --- Sampling Method --- p.35, Chapter 6.1.1 --- Background --- p.35, Chapter 6.1.2 --- Method --- p.35, Chapter 6.1.3 --- Population --- p.35, Chapter 6.1.4 --- Questionnaire --- p.36, Chapter 6.1.5 --- Food Diary --- p.36, Chapter 6.2 --- Participant Recruitment Criteria --- p.38, Chapter 6.2.1 --- Major Inclusion Criteria for This Study --- p.38, Chapter 6.2.2 --- Major Exclusion Criteria for This Study --- p.38, Chapter 6.2.3 --- Ethical Considerations --- p.38, Chapter 6.3 --- Statistic Analysis --- p.39, Chapter 6.4 --- Quality Assessment and Data Extraction --- p.39, Chapter 6.5 --- Hypothesis --- p.40, Chapter Chapter 7: --- Results, Chapter 7.1 --- Demographic Data --- p.41, Chapter 7.2 --- Overall Insomnia --- p.43, Chapter 7.2.1 --- Difficult Initiating Sleep (DIS) --- p.52, Chapter 7.2.2 --- Difficulty Maintaining Sleep (DMS) --- p.52, Chapter 7.2.3 --- Early Morning Awakening (EMA) --- p.61, Chapter 7.2.4 --- Insomnia Syndrome --- p.61, Chapter Chapter 8: --- Discussion and Limitation, Chapter 8.1 --- Age and Insomnia --- p.71, Chapter 8.2 --- Alcohol and Insomnia --- p.72, Chapter 8.3 --- Caffeine and Insomnia --- p.72, Chapter 8.4 --- Carbohydrate and Insomnia --- p.72-73, Chapter 8.5 --- Vitamin E and Insomnia --- p.73, Chapter 8.6 --- Vitamin A and Insomnia --- p.74, Chapter 8.7 --- Vitamin D and Insomnia --- p.74, Chapter 8.8 --- Saturated Fat and Insomnia --- p.75, Chapter 8.9 --- Summary --- p.76, Chapter Chapter 9: --- Limitation and Implications, Chapter 9.1 --- Limitation of This Study --- p.77, Chapter 9.2 --- Implication to Further Study --- p.77-78, Chapter 9.3 --- Implication to Clinical Intervention --- p.78-79, Chapter Chapter 10: --- Executive Summary --- p.80-81, Bibliography --- p.82-90, http://library.cuhk.edu.hk/record=b5549250, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

大腸癌是全世界最常見的癌症之一，亦是一個癌症死亡率的首要原因。大腸癌患者約50%在病程中會出現轉移病灶。近十年來，雖然多種被批准用於臨床治療的新化療藥顯著提高了大腸癌的治療效果，但是轉移性大腸癌病人的預後仍然很差。隨著各種分子生物技術的進步，新的治療標靶可能在大腸癌細胞株中被發現，並得以在病人標本中驗證。, 在本研究中，我們採用即時定量多聚酶鏈反應(qPCR)陣列分析，比較大腸癌細胞株和正常大腸細胞株基因表達譜，試圖識別潛在的新的治療標靶。結果提示，與正常大腸細胞株 CCD-18Co 比較，Jagged 2 (JAG2) 和 Frizzled-3 (FZD3)基因 在大腸癌細胞株 SW480 和 SW620 中表達升高。病人大腸癌組織的免疫組織化學染色 (IS) 檢查進一步證實了上述結果，大腸癌組織較其癌旁正常組織表達3.1倍JAG2和6.6倍FZD3蛋白。因此， 我們假設JAG2和FZD3在大腸癌的發生中起重要作用。, 為了檢驗該假設的真偽，我們運用RNA 干擾的方法進行功能缺失研究。通過該方法，大腸癌細胞株中JAG2 信使RNA和蛋白均能夠被下調，但是FZD3蛋白卻沒有顯示降低。為了弄清JAG2基因的功能，我們進行了單層細胞劃痕傷口癒合試驗和Matrigel 侵襲試驗。結果提示，JAG2 基因下調顯著抑制大腸癌細胞遷移和侵襲的能力。, 為了調查參與上述功能的機制，我們利用腫瘤轉移相關基因的qPCR陣列分析，試圖檢測出JAG2基因敲除後上調或下調表達的轉移相關基因。結果顯示組織蛋白酶K (CTSK)，一種溶酶體半胱氨酸蛋白酶，在JAG2基因沉默的大腸癌細胞株中表達下調。為了闡明CTSK 活性在大腸癌細胞株侵襲能力中起到的作用，我們採用CTSK抑制劑處理大腸癌細胞株HCT116和DLD-1，發現這兩種細胞株的侵襲能力分別下降了36%和59%。總之, 這些發現表明CTSK可能是JAG2的下游靶基因，活性CTSK可能參與了JAG2介導的大腸癌細胞株侵襲能力。, 以前的研究表明p38 MAPK通路參與癌細胞遷和侵襲能力的調控。通過Western blot方法，磷酸化的p38和磷酸化的STAT3被發現在JAG2基因沉默的大腸癌細胞中表達降低。p38抑制劑處理的 HCT116和DLD-1細胞降低了侵襲能力下降，同時遷移能力也由於p38抑制劑的處理而降低，支持p38可調控癌細胞遷移和侵襲能力的事實。, 總之，我們的結果顯示JAG2高表達通過啟動CTSK和p38 MAPK通路，可能促進大腸癌轉移。因此，JAG2可能成為轉移性大腸癌治療的潛在標靶。, Colorectal cancer (CRC) is one of the most frequent cancers worldwide and is a leading cause of cancer mortality. Around 50% of patients with CRC will experience metastases. Although significant progress has been made in CRC treatment within the last decade with the approval of multiple new chemotherapeutic agents, the prognosis for patients with metastatic CRC remains poor. With the advancement of molecular techniques, novel therapeutic targets are able to be discovered in CRC cell lines and validated in patient samples., Therefore in this project, I aim to identify potential novel therapeutic targets by comparing the gene expression profile of colon cancer cell lines and a normal colon cell line using quantitative polymerase chain reaction (qPCR) arrays. Results showed that Jagged 2 (JAG2) and Frizzled-3 (FZD3) were up-regulated in the CRC cell lines SW480 and SW620 as compared to the normal colon cell line CCD-18Co. Those results were further validated by immunohistochemical staining (IS), which detected up-regulated JAG2 (3.1-fold) and FZD3 (6.6-fold) proteins expression in CRC tissues as compared to adjacent normal tissues. Thus I hypothesized that JAG2 and FZD3 may play an important role in CRC carcinogenesis., In order to study the roles of FZD3 and JAG2 in CRC, loss-of-function studies by RNA interference (RNAi) were carried out. While the expression of FZD3 protein failed to be down-regulated by RNAi, JAG2 expression was successfully knocked down in CRC cell lines at both the mRNA and protein levels. Functional analyses using the monolayer scratch wound-healing assay and Matrigel invasion assay showed that JAG2 knockdown significantly inhibited migration and invasion in CRC cell lines., To investigate the mechanisms involved, a tumour metastasis qPCR array was used to examine the changes in the expression level of metastasis-related genes after JAG2 gene knockdown. Results showed that the expression of Cathepsin K (CTSK), a lysosomal cystein protease, was found to be down-regulated in CRC cell lines following JAG2 silencing. To demonstrate the importance of CTSK activity in CRC cell invasion, HCT116 and DLD-1 CRC cell lines were treated with a CTSK inhibitor and its effect were assessed by the Matrigel invasion assay. Results showed that CTSK inhibition led to a 36% and 59% reduction in number of invaded cells in HCT116 and DLD-1 cell lines, respectively. Taken together, these findings show that CTSK may be a downstream target of JAG2 and that active CTSK may involve in JAG2 mediated invasion in CRC cell lines., Previous works by others have shown that the p38 MAPK pathway is involved in the regulation of migration and invasive activity of cancer cell lines. Using Western blot analysis, the expression of phosphorylated p38 MAPK and phosphorylated STAT3 were found to be down-regulated following JAG2 depletion in CRC cell lines. In support of a role for p38 MAPK in the regulation of cancer cell migration and invasive capability, treatment with a p38 MAPK inhibitor was found to reduce the percentage of invasive cells and distance moved by migratory cells in HCT116 and DLD-1 cell lines., In conclusion, my results show that JAG2 over-expression in CRC may promote cancer cell migration and invasion through activation of CTSK and the p38 MAPK pathway. Therefore, JAG2 may be a potential therapeutic target for treatment of metastatic CRC., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., He, Wan., Thesis (Ph.D.)--Chinese University of Hong Kong, 2013., Includes bibliographical references (leaves 164-207)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., s also in Chinese., in English --- p.i, in Chinese --- p.iv, Acknowledgements --- p.vi, Chapter Chapter 1 --- Introduction --- p.1, Chapter 1.1 --- Colorectal Cancer (CRC) --- p.1, Chapter 1.1.1 --- Epidemiology and Incidence --- p.1, Chapter 1.1.2 --- Histology --- p.2, Chapter 1.1.3 --- Gender and Age --- p.4, Chapter 1.1.4 --- Etiology of CRC --- p.4, Chapter 1.1.4.1 --- Environment --- p.4, Chapter 1.1.4.2 --- Hereditary Factors --- p.5, Chapter 1.1.4.3 --- Dietary Factors --- p.6, Chapter 1.1.4.4 --- Obesity --- p.6, Chapter 1.1.4.5 --- Tobacco and alcoho --- p.7, Chapter 1.1.4.6 --- Inflammatory bowel disease (IBC) --- p.7, Chapter 1.1.5 --- Genetic Changes in CRC --- p.8, Chapter 1.1.5.1 --- Chromosomal Aberration --- p.8, Chapter 1.1.5.2 --- Tumor Suppressor Genes --- p.10, Chapter 1.1.5.2.1 --- APC gene --- p.10, Chapter 1.1.5.2.2 --- P53 gene --- p.11, Chapter 1.1.5.2.3 --- SMAD4 gene --- p.11, Chapter 1.1.5.3 --- Oncogenes --- p.12, Chapter 1.1.5.3.1 --- Epidermal Growth Factor Receptor (EGFR) gene --- p.12, Chapter 1.1.5.3.2 --- RAS gene and BRAF gene --- p.13, Chapter 1.1.5.4 --- Proposed Two-hit Model for the Multistep Pathogenesis of CRC --- p.15, Chapter 1.1.6 --- Clinical Presentation and Diagnosis --- p.16, Chapter 1.1.7 --- Theatment --- p.16, Chapter 1.1.7.1 --- Surgery --- p.16, Chapter 1.1.7.2 --- Radiotherapy (RT) --- p.17, Chapter 1.1.7.3 --- Concurrent Chemotherapy --- p.17, Chapter 1.1.7.4 --- Target Therapy --- p.18, Chapter 1.1.7.5 --- Colorectal Cancer Treatment by Stage --- p.19, Chapter 1.1.7.6 --- Novel Strategies --- p.20, Chapter 1.1.7.6.1 --- Epigenetic therapy --- p.20, Chapter 1.1.7.6.2 --- Immunotherapy --- p.21, Chapter 1.2 --- Pathways Involved in CRC Carcinogenesisand Progression --- p.22, Chapter 1.2.1 --- Wnt Signaling Pathway --- p.22, Chapter 1.2.2 --- Notch Signaling --- p.23, Chapter 1.2.3 --- Nuclear Factor-kappa B (NF-κB) Signaling Pathway --- p.23, Chapter 1.2.4 --- Phosphatidylinositol 3-kinase (PI3K) Signaling Pathway --- p.24, Chapter 1.2.5 --- Crosstalk Among WNT, NOTCH, NF-κB and PI3K Signaling Pathway in CRC --- p.24, Chapter 1.3 --- Hypothesis and Objectives of this Study --- p.28, Chapter Chapter 2 --- Identification of Differentially Expressed Genes between Colorectal Cancer Cell Lines and A Normal Colon Cell Line --- p.29, Chapter 2.1 --- Background --- p.29, Chapter 2.2 --- Materials and Methods --- p.33, Chapter 2.2.1 --- Cell Lines --- p.33, Chapter 2.2.2 --- Identification of Differetially Expressed Genes by qPCR Arrays --- p.33, Chapter 2.2.2.1 --- Total RNA Extraction --- p.33, Chapter 2.2.2.2 --- RNA Quality Contol --- p.34, Chapter 2.2.2.3 --- Reverse Transcription (RT) --- p.34, Chapter 2.2.2.4 --- PCR Arrays --- p.34, Chapter 2.3 --- Results --- p.36, Chapter 2.3.1 --- Differentially Expressed Genes in WNT Signaling Pathway --- p.36, Chapter 2.3.2 --- Differentially Expressed Genes in Notch Signaling Pathway --- p.40, Chapter 2.3.3 --- Differentially Expressed Genes in NF-κB Signaling Pathway --- p.43, Chapter 2.3.4 --- Differentially Expressed Genes in PI3K-AKT Signaling Pathway --- p.46, Chapter 2.3.5 --- Choice of over-expressed genes for further validation and characterization --- p.49, Chapter 2.4 --- Discussions --- p.53, Chapter 2.4.1 --- WNT Signaling Pathway --- p.53, Chapter 2.4.2 --- NOTCH Signaling Pathway --- p.54, Chapter 2.4.3 --- NF-κB Signaling Pathway --- p.55, Chapter 2.4.4 --- PI3K-AKT Signaling Pathway --- p.56, Chapter 2.4.5 --- Choice of over-expressed genes for further validation and characterization --- p.56, Chapter Chapter 3 --- JAG2, FZD3 and NOTCH4 Expression in Colorectal Cancer Cell Lines and Colorectal Cancer Tissues --- p.59, Chapter 3.1 --- Background --- p.59, Chapter 3.1.1 --- JAG2 Ligand --- p.59, Chapter 3.1.2 --- FZD3 Receptor --- p.61, Chapter 3.1.3 --- NOTCH4 Receptor --- p.62, Chapter 3.2 --- Materials and Methods --- p.64, Chapter 3.2.1 --- CRC Cell Lines --- p.65, Chapter 3.2.2 --- CRC Tissues --- p.65, Chapter 3.2.3 --- Quantitative RT-PCR --- p.66, Chapter 3.2.4 --- Detection of JAG2, FZD3 and NOTCH4 Protein Expression in CRC Tissues by Immunohistochemical Staining (IS) --- p.67, Chapter 3.2.5 --- Western Blot Assays --- p.68, Chapter 3.2.5.1 --- Protein extraction --- p.68, Chapter 3.2.5.2 --- SDS-PAGE gel electrophroresis --- p.68, Chapter 3.2.5.3 --- Protein blotting --- p.68, Chapter 3.2.6 --- Detection of JAG2 and FZD3 Protein Expression in CRC and Normal Colon Cell Lines by Western Blotting --- p.69, Chapter 3.2.7 --- Statistical Analysis --- p.70, Chapter 3.3 --- Results --- p.71, Chapter 3.3.1 --- JAG2 and FZD3 but not NOTCH4 mRNA were Over -expressed in CRC Cell Lines --- p.71, Chapter 3.3.2 --- Over-expression of JAG2 and FZD3 Proteins in CRC Tissues --- p.72, Chapter 3.3.3 --- FZD3 Over-expression Correlated with Tumour-Node Metastasis (TNM) stages --- p.76, Chapter 3.3.4 --- JAG2 and FZD3 Protein Expression in Colorectal Cancer and Normal Cell Lines --- p.77, Chapter 3.4 --- Discussions --- p.78, Chapter Chapter 4 --- Functional Analyses of JAG2 and FZD3 in CRC Cell Lines by RNA Interference --- p.81, Chapter 4.1 --- Background --- p.81, Chapter 4.2 --- Materials and Methods --- p.84, Chapter 4.2.1 --- Transfection of siRNA into CRC Cell Lines --- p.84, Chapter 4.2.2 --- Cell Proliferation Assay --- p.85, Chapter 4.2.3 --- Monolayer Scratch Wound Healing Assay --- p.85, Chapter 4.2.4 --- Matrigel Invasion Assay --- p.86, Chapter 4.2.5 --- Statistical Analysis --- p.87, Chapter 4.3 --- Results --- p.88, Chapter 4.3.1 --- Knockdown of JAG2 and FZD3 Expression by RNA Interference --- p.88, Chapter 4.3.2 --- Effect of JAG2 Knockdown on Migration of CRC Cell Lines --- p.91, Chapter 4.3.3 --- JAG2 Knockdown by siRNA 2 Transfection Reduced Migratory Capability of HCT116, DLD-1and HT29 cell lines --- p.94, Chapter 4.3.4 --- JAG2 Knockdown Impaired the Invasiveness of HCT116 and DLD-1 Cell Lines --- p.97, Chapter 4.3.5 --- Decreased Migratory and Invasive Capabilities Induced by JAG2 Knockdown was not Due to Reduced Cell Proliferation --- p.100, Chapter 4.4 --- Discussions --- p.102, Chapter Chapter 5 --- NOTCH Pathway Inactivation by JAG2 Silencing Reduces Oncogenic Properties of HT29 but not HCT116 andDLD-1 CRC Cell Lines --- p.106, Chapter 5.1 --- Background --- p.106, Chapter 5.2 --- Materials and Methods --- p.109, Chapter 5.2.1 --- CRC Cell lines --- p.109, Chapter 5.2.2 --- Pharmacological Inhibition of NOTCH signaling by DAPT --- p.109, Chapter 5.2.3 --- Combination of DAPT Treatment and JAG2 Silencing by siRNA --- p.109, Chapter 5.2.4 --- Western Blotting --- p.109, Chapter 5.2.5 --- Cell Proliferation Assay (MTS Assay) --- p.110, Chapter 5.2.6 --- Monolayer Scratch Wound Healing Assay --- p.110, Chapter 5.2.7 --- Matrigel Invasion Assay --- p.111, Chapter 5.2.8 --- Statistical Analysis --- p.111, Chapter 5.3 --- Results --- p.112, Chapter 5.3.1 --- JAG2 Silencing Down-regulates Notch Pathway Signaling in CRC Cell Lines --- p.112, Chapter 5.3.2 --- Inhibition of NOTCH Signaling by DAPT Treatment in CRC Cell Lines --- p.112, Chapter 5.3.3 --- NOTCH Inhibition Does not Significantly Affect Cell Proliferation in CRC Cell Lines --- p.114, Chapter 5.3.4 --- Suppression of NOTCH Signaling by DAPT Inhibits Migration in HT29 but not in HCT116 and DLD-1 CRC Cell Lines --- p.115, Chapter 5.3.5 --- Suppression of NOTCH Signaling by DAPT does not Significantly Affect Invasiveness of HCT116 and DLD-1 CRC Cell Lines --- p.117, Chapter 5.4 --- Discussions --- p.118, Chapter Chapter 6 --- JAG2 Knockdown Inhibits Invasion in CRC Cell Lines through Inactivation of Cathepsin K --- p.121, Chapter 6.1 --- Background --- p.121, Chapter 6.2 --- Materials and Methods --- p.123, Chapter 6.2.1 --- Human Tumour Metastasis RT2 Profiler[superscript TM] PCR Array --- p.123, Chapter 6.2.2 --- Measurement of CTSK Gene expression level by Quantitative Real-Time PCR --- p.123, Chapter 6.2.3 --- Immunohistochemical Staining (IS) of CTSK in CRC Tissues --- p.124, Chapter 6.2.4 --- Pharmacological Inhibitior of CTSK in CRC Cell Lines --- p.124, Chapter 6.2.5 --- Inhibition of CTSK in CRC Cell Lines for Migration Study --- p.124, Chapter 6.2.6 --- Inhibition of CTSK in CRC Cell Lines for Invasion Study --- p.125, Chapter 6.2.7 --- Western Blotting --- p.125, Chapter 6.2.8 --- Statistical Analysis --- p.125, Chapter 6.3 --- Results --- p.126, Chapter 6.3.1 --- Identification of Metastasis Related Genes Which were Down-regulated by JAG2 Knockdown in HCT116 Cells --- p.126, Chapter 6.3.2 --- Validation of Down-regulation of CTSK Gene by JAG2 Knockdown in HCT116 Cell Line by qRT-PCR --- p.126, Chapter 6.3.3 --- JAG2 Knockdown Reduced Expression of Active CTSK Protein in CRC Cell Lines --- p.128, Chapter 6.3.4 --- CTSK Protein Expression in CRC Tissue Samples --- p.130, Chapter 6.3.5 --- Pharmacological Inhibition of CTSK Suppressed Invasiveness of CRC Cell Lines --- p.131, Chapter 6.3.6 --- Pharmacological Inhibition of CTSK did not Affect Migration of CRC Cell Lines --- p.132, Chapter 6.4 --- Discussions --- p.133, Chapter Chapter 7 --- Depletion of JAG2 Inhibits Migration and Invasion in CRC Cell Lines through Inactivation of p38 MAPK/HSP27 Pathway --- p.137, Chapter 7.1 --- Background --- p.137, Chapter 7.2 --- Materials and Methods --- p.140, Chapter 7.2.1 --- Pharmocological Inhibition of p38 MAPK Phosphorylation CRC Cell Lines --- p.140, Chapter 7.2.2 --- Inhibition of p38 MAPK Phosphorylation for Migration Study in CRC Cell Lines --- p.140, Chapter 7.2.3 --- Inhibition of p38 MAPK Phosphorylation for Invasion Study in CRC Cell Lines --- p.140, Chapter 7.2.4 --- Knockdown of STAT3 by RNA interference --- p.141, Chapter 7.2.5 --- Knockdown of STAT3 for Migration Study in CRC Cell Lines --- p.141, Chapter 7.2.6 --- Knockdown of STAT3 for Invasion Study in CRC Cell Lines --- p.141, Chapter 7.2.7 --- Western Blotting --- p.141, Chapter 7.2.8 --- Statistical Analysis --- p.142, Chapter 7.3 --- Results --- p.143, Chapter 7.3.1 --- JAG2 Knockdown Inhibits p38 MAPK / HSP27 Pathway in CRC Cell Lines --- p.143, Chapter 7.3.2 --- Inhibition of p38 MAPK / HSP27 Signaling Pathway Down-regulated Invasive Capability of CRC Cell Line --- p.145, Chapter 7.3.3 --- Inhibition of p38 MAPK / HSP27 Signaling Pathway Down-regulated Migration of CRC Cell lines --- p.147, Chapter 7.3.4 --- JAG2 Knockdown Inactivated p38 MAPK / HSP27 Pathway Independently of NOTCH Pathway in CRC Cell Lines --- p.149, Chapter 7.3.5 --- JAG2 Knockdown Inhibits STAT3 Activation in CRC Cell Lines --- p.151, Chapter 7.3.6 --- STAT3 Silencing Reduced Invasive Capability in CRC Cell Lines --- p.152, Chapter 7.3.7 --- STAT3 Silencing Reduced Migratory Capability in CRC Cell Lines --- p.154, Chapter 7.3.8 --- Inhibition of p38 MAPK Activity Suppressed STAT3 Activation in HCT116 Cells --- p.156, Chapter 7.4 --- Discussions --- p.157, Chapter Chapter 8 --- Conclusions and Future Works --- p.161, Chapter 8.1 --- Conclusions --- p.161, Chapter 8.2 --- Future work --- p.163, References --- p.164, Chapter Appendix 1 --- List of Figures and Tables --- p.208, Chapter Appendix 2 --- Abbrevations used in this thesis --- p.212, http://library.cuhk.edu.hk/record=b5549838, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Tang, Yongjiang., Thesis (Ph.D.)--Chinese University of Hong Kong, 2013., Includes bibliographical references (leaves 159-202)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., http://library.cuhk.edu.hk/record=b5884488, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

背景: 香港和廣州市是華南兩大發達城市，都面臨著人口老化的嚴峻形勢。由於醫療水準的提高，癡呆成為一個非常嚴重的公共健康問題。由於缺乏有效的治療手段，早期發現和干預成為減少認知功能損害及癡呆發病的最有效的措施。研究怎樣保護長者的認知功能對於公眾健康具有越來越重要的意義。越來越多的證據表明休閒活動有益於認知功能。研究表明體育鍛煉，腦力活動以及社會活動有益於認知健康，可以減少癡呆發病的風險。然而由於概念上的差異和研究方法的不同，使得目前的研究結果很難進行比較。由於社會背景會顯著影響休閒活動的參與，研究社會背景怎樣影響休閒活動對於長者認知功能的作用具有重要的意義。香港和廣州為華南兩大城市，其人口種族，基因，健康狀況及人口學特徵相似。然而在過去的一百多年中，由於歷史發展的不同，兩城市有著不同的社會系統。以上這些因素對於研究不同設計背景下的認知功能的對照研究提供了方便。研究兩地認識休閒活動與認知功能的關係，有助於評價不同社會環境對於休閒活動影響認知功能的以及社會因素對認知功能的影響。, 研究目的: 本研究對於兩地長者認知功能的特點以及休閒活動的參與情況進行了比較。研究的主要目的是評估休閒活動與認知功能的關係，以及兩個城市中休閒活動與認知功能的關係。, 方法: 這是一個橫斷面研究。557名非癡呆住在社區的長者參與了研究，其中香港260名，廣州297名。兩組長者在年齡，性別以及教育程度上沒有差異。休閒活動分為體育活動，智力活動，社會活動以及消遣活動。休閒活動的參與通過三種方式進行評估：種類，次數以及每週參與的小時數。一組評估量表包括：簡短精神狀態量表，詞語記憶，延遲回憶，詞語流暢性測驗，連線測驗，數位劃消測驗及Stroop測驗，用於評估兩地長者的認知功能。, 兩地長者的人口學特徵，認知功能以及休閒活動的參與進行了比較，多元線性回歸用於分析每一類休閒活動與認知功能的關係，同時控制可能的混雜因素包括年齡，性別，教育程度，職業，婚姻狀況，居住情況，吸煙、酒情況，慢性疾病以及精神狀態。分層回歸用於分析每類休閒活動與認知功能的關係，同時控制其他三類休閒活動以及與認知功能顯著相關的混雜因素。, 結果: 多元回歸分析表明與家人居住在一起者休閒活動的總類較多 (p=0.01)，休閒活動的時間較長 (p=0.02)。协方差分析檢驗顯示除了每週看電視的時間 （p=0.07），香港長者參與的休閒活動在種類，次數以及每週參與的小時數都明顯多於廣州長者，差異具有顯著性。兩地長者的認知功能測驗的分數未見顯著差異。體育活動（腦-身體鍛煉及有氧鍛煉）的種類與延遲回憶及詞語分類測驗顯著相關。智力活動與所有的認知功能測驗顯著相關。社會活動與語詞回憶和詞語流暢性測驗顯著相關。休閒活動與語詞回憶及連線測驗顯著相關。, 多元線性回歸分析了休閒活動與認知功能的關係的同時，控制了其他三類休閒活動以及與認知功能顯著相關的協變數。分析結果顯示智力活動的種類與簡易精神狀態量表，語詞回憶，延遲回憶，詞語流暢性測驗以及數位劃消測驗顯著相關 （p<0.001）。體育活動和社會活動與認知功能未見明顯相關。消遣活動的時間與連線測驗顯著相關 （p=0.01）。休閒活動與認知功能的相關性兩地未見明顯顯著差異。, 結論: 香港的長者參與了較多的休閒活動，但是認知功能測驗的分數與廣州長者卻沒有顯著差別。結果可能與之前的研究結果相矛盾，即參與較多的休閒活動與良好的認知功能相關，這可能與兩地的社會人口學的差異相關。以前的研究證明晚年的婚姻狀態與癡呆或者認知功能的下降相關。未婚或者喪偶的長者罹患癡呆症或者認知功能下降的風險性較高。香港未婚或喪偶長者較廣州多，或許這可以解釋為什麼香港長者參與較多的休閒活動，但是認知功能測驗卻未明顯優於廣州長者。同時也表明，除了休閒活動，社會因素（婚姻及居住狀況）對認知功能也有影響。, 我們的研究表明參與智力活動尤其是參與各種各樣的智力活動與長者良好的認知功能相關。智力活動與認知功能的相關性在兩地沒有顯著差別，表明智力活動可在不同的社會環境中用於保護長者的認知功能。其他三類的活動與認知功能未發現有顯著相關性，這可能與智力活動的混雜效應有關，也可能與各活動之間其構成成分的重疊有關。儘管這樣，休閒活動對於認知功能的保護作用扔值得進一步研究。, Background: The two most developed cities in southern China, Hong Kong (HK) and Guangzhou (GZ), are facing rapid population aging. As a result of improvements in medical care, dementia has emerged as a crucial public health problem. With limited treatment options available, early detection and intervention are likely to be the most effective strategies to reduce subsequent impairments and morbidity. Research into the prevention of cognitive decline among older persons is crucial for public health. There is increasing evidence that participation in leisure activities has a favorable effect on cognitive function. Studies have reported that physical exercise, cognitive activity and social engagement are beneficial for cognitive health and may reduce the risk of dementia. However, interpretation of the available evidence is hampered by conceptual discrepancies and methodological variations. As the social context may significantly influence leisure activity participation, it is interesting to explore how social contexts play a role in modulating the effects of leisure activity on cognitive function in older adults. HK and GZ are two major cities in southern China, and they share very similar ethnic, genetic, health and demographic characteristics. However, owing to differences in historical development, the two cities have been run with different social systems over the past few decades. This provided a natural case-control experiment for studying the effect of the social context on cognition. Hence, this study examined the association between leisure activity participation and cognition in the two cities to evaluate the cognitive modulating effects of leisure activities in different social environments., Objectives of the studies: The main study objectives were to compare the cognitive characteristics and leisure activity participation of the two groups; to examine the association between leisure activity participation and cognitive function and the specific associations in HK and GZ; and to explore the modulating effect of social factors on cognitive function., Methodology: This was a cross-sectional study. Convenience sampling was used to recruit 557 participants aged 60 years and over without dementia. Of these, 260 were recruited in HK and 297 in GZ. The two groups were recruited with similar demographic characteristics (age, gender and education). Leisure activities were classified as physical, intellectual, social and recreational activities. Leisure activity participation was measured in terms of the total number and total hours of participation per week for each category of activities. A battery of cognitive tests including the Cantonese version of the Mini Mental State Exam (CMMSE), word list learning test, delayed recall test, Category Verbal Fluency Test (CVFT), trail making test, digit cancellation test and Stroop test were used to measure participants’ cognitive function., Differences in the participants’ demographic characteristics, cognitive performances and leisure activity participation were computed. A multiple linear regression of cognitive performance on leisure activity was performed, while controlling for other categories of activities and potential confounders that were significantly associated with cognitive function., Results: The multiple linear regression revealed that living arrangement had a significant positive association with the total number of leisure activities (p=0.01) and total hours of leisure activity participation (p=0.02). Analysis of covariance showed that participants in HK participated in more leisure activities than those in GZ, as measured by the total number of subtypes and hours per week, except total hours of recreational activities per week (p=0.07). No significant differences were found between the cognitive performances of the older persons in the two cities. Pearson’s correlation and x² tests were performed to identify the leisure activities and potential confounders that were significantly correlated with cognitive performance. The total number and total hours of intellectual activity were significantly correlated with CMMSE scores (p<0.001 and p<0.001).The total number of subtypes and total hours per week of intellectual activity (p<0.001 and p<0.001), social activity (p<0.001 and p<0.01) and recreational activity (p<0.001 and p<0.01) were significantly correlated with the word list learning test. The total number of physical activities (p<0.01), total number of intellectual activities (p<0.001) and total hours of intellectual activity (p<0.01) were significantly correlated with the delayed recall test. The total number of physical and intellectual activities (p<0.01 and p<0.001), and total hours of intellectual and social activity (p<0.01 and p<0.001) were correlated with the CVFT. The total number of intellectual activities (p<0.01)and total hours of recreational activity (p<0.01) were significantly correlated with the trail making test (p<0.001). The total number and total hours of intellectual activity were significantly correlated with the digit cancellation test (p<0.001 and p<0.001). The total number and total hours of intellectual activity were significantly correlation with the Stroop test (p<0.01 and p<0.001)., Multiple linear regression using the enter method was conducted to measure the association between leisure activities and cognitive performance. The results showed that the total number of intellectual activities was significantly associated with better performance on cognitive tests, including the CMMSE (p<0.001), word list learning test (p<0.001), delayed recall test (p<0.001), CVFT (p<0.001) and digit cancellation test (p=0.01). Total hours of recreational activity was significantly associated with the trail making test (p=0.01). Multiple linear regression using the enter method also revealed that marital status was significantly associated with the CMMSE (p=0.002), word list learning test (p=0.003), delayed recall test (p=0.002), trail making test (p<0.001) and digit cancellation test (p=0.01)., Conclusions: HK participants participated in more leisure activities than GZ participants. However, HK participants did not show better cognitive performance than GZ participants. This finding appears to be inconsistent with previous studies that found that participation in more leisure activities predicted better cognitive functioning. This inconsistency might be explained by socio-demographic differences between the two cities. Some previous studies have found an association between late-life marital status and the risk of cognitive impairment. Older persons who were unmarried or widowed were at higher risk of dementia or cognitive decline. There were more unmarried or widowed participants among HK participants. The results indicate that in addition to leisure activities, social factors (marital or living status) might also contribute to the preservation of cognitive function among the elderly., Our results underscore the significance of intellectual activity, especially participation in a variety of intellectual activities, in maintaining better cognitive functioning in older persons. Furthermore, a similar significant association between intellectual activity and cognitive function was found for participants in both HK and GZ, suggesting that the protective effect of intellectual activity could be generalized to different social environments. We failed to find significant associations between physical, social and recreational activities and cognitive function. However, the protective effect of leisure activity participation is recommended for further investigation in future studies., Detailed summary in vernacular field only., Su, Xiufang., Thesis (Ph.D.) Chinese University of Hong Kong, 2013., Includes bibliographical references (leaves 88-94)., s also in Chinese; appendixes in Chinese., http://library.cuhk.edu.hk/record=b6116334, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Lau, Hoi Lam., Thesis (Ph.D.)--Chinese University of Hong Kong, 2013., Includes bibliographical references (leaves 271-289)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., http://library.cuhk.edu.hk/record=b5884288, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

本研究的目的是評估長期服用氯胺酮對青少年認知功能和精神健康狀況的影響。 自2009年12月至2011年12月，共300名受試者入組。受試者分為3組：氯胺酮組，氯胺酮及多種藥物組和健康對照組，每組有100名受試者入組。精神狀況評估包括問卷篩查和麵談。所有受試者均完成一套詳細的認知測試。該測試涵蓋一般智慧、語詞記憶、視覺記憶、執行功能、動作速度和語言。, 氯胺酮組受試者主要濫用氯胺酮，而氯胺酮及多種藥物組受試者除氯胺酮外主要濫用可卡因和冰毒。兩組氯胺酮濫用者最常見的共患精神障礙是抑鬱障礙。在單因素分析中,兩組氯胺酮濫用者在幾乎所有的測試中得分低於健康對照。多因素分析控制混雜因素如年齡、性別、教育程度和Beck 抑鬱量表總分後，兩組氯胺酮濫用組與健康對照組在語詞記憶和視覺記憶仍存在顯著差異。本研究進一步將氯胺酮組及氯胺酮多種藥物組分別分為現用藥者和戒斷者。在氯胺酮組中現用藥者在詞記憶、視覺記憶、動作速度和部分執行功能測試上得分低於戒斷者和健康對照，並且現用者Beck 抑鬱量表總分高於戒斷者和健康對照，而戒斷者和健康對照在認知測試和Beck 抑鬱量表總分沒有顯著差別。但在氯胺酮及多種藥物組，現用藥者和戒斷者均在記憶測試中得分低於及Beck 抑鬱量表總分高於健康對照。另外, 女性氯胺酮濫用組視覺記憶得分低於男性，但女性在語詞記憶得分普遍高於男性。, 本研究認為氯胺酮或氯胺酮合用多種藥物均能導致記憶和執行功能的損害。這種損害主要與近期濫用氯胺酮有關，並且氯胺酮組現用藥者語詞記憶損害較氯胺酮合用多種藥物現用藥者嚴重。單純氯胺酮導致的記憶和執行功能損害在戒斷1月後明顯好轉但氯胺酮合用多種藥物者戒斷一月後未能見到記憶功能好轉。超過半數的氯胺酮濫用者共患抑鬱障礙。本研究的結果為治療氯胺酮濫用有用資訊，亦有助於戒毒者鞏固其戒斷行為。但氯胺酮所致認知損害的可逆性還需要前瞻性或縱向研究進一步證實，並且這種可逆性損害的機制還不明確。未來的研究還需要進一步明確氯胺酮對人體的損害作用是否具有性別差異性。, The objective of this study was to evaluate the long-term effect of ketamine use on both the cognition and psychological well-being of youths in Hong Kong., Three hundred participants were recruited for the study, which lasted from December 2009 to December 2011. Participants were divided into three groups of 100 each: primarily ketamine (Primarily K) users, poly-drug ketamine (Poly K) users and healthy controls (HCs). Psychiatric assessments included screening with self-rating questionnaires and face-to-face interviews. All participants completed a detailed cognitive battery covering general intelligence, verbal memory, visual memory, executive function, motor speed and language., The participants in the Primarily K group predominantly used ketamine, whereas those in the Poly K group used ketamine in addition to secondary drugs, of which cocaine and methamphetamine were the most frequent. Depressive disorder was the most common psychiatric disorder in both ketamine groups. Univariate analysis also showed the two ketamine groups to score poorly on most of the cognitive tests relative to the HC group. After adjusting for age, sex, education and Beck Depression Inventory (BDI) score, verbal and visual memory remained impaired in both ketamine groups in comparison with the HC group. Ketamine use in the past month was independently related to memory impairment in the Primarily K group. In subgroup analyses of Primarily K users, verbal and visual memory, motor speed, and some of the executive function indexes were significantly impaired in current users but not in ex-users. These findings suggest that the cognitive influence of ketamine is reversible. Moreover, the current ketamine users had a higher BDI score than the ex-users or HCs. However, the ex- and current poly-drug ketamine users exhibited a similar degree of memory impairment compared with the HCs. The female Primarily K users showed more visual memory impairment than their male counterparts, although females generally performed better than males in verbal memory., In conclusion, the use of ketamine alone and in conjunction with other psychotropic drugs is associated with deficits in memory and executive function. The observed memory impairment was related primarily to recent ketamine use, with current Primarily K users presenting with a more severe memory deficit than current Poly K users. However, the Primarily K group realised improvement in cognitive impairment after abstaining from ketamine, whereas the Poly K group did not. In addition to cognitive functioning difficulties, more than half of the ketamine users suffered from depressive disorder. Moreover, the findings suggest that women may be more sensitive than men to visual memory impairment following chronic ketamine use. The findings of this study will be helpful in treating ketamine abuse, and reinforce the efficacy of abstinence from drugs. Further longitudinal research is needed to determine the reversibility of ketamine’s effects and the mechanism by which that reversibility takes place. Further study is also needed to clarify the drug’s sex-specific effects., Detailed summary in vernacular field only., Liang, Huajun., Thesis (Ph.D.)--Chinese University of Hong Kong, 2013., Includes bibliographical references (leaves 102-119)., also in Chinese., DECLARATION OF ORIGINALITY --- p.I, ACKNOWLEDGEMENTS --- p.II, PUBLICATIONS AND PRESENTATIONS --- p.1, LIST OF ABBREVIATIONS --- p.XI, LIST OF TABLES AND FIGURES --- p.XIII, p.XV, 摘要 --- p.XVIII, CONTENTS --- p.XX, Chapter CHAPTER 1 --- BACKGROUND --- p.1, Chapter 1.1 --- Introduction to ketamine and ketamine misuse --- p.1, Chapter 1.2 --- Effects profile of ketamine in the brain --- p.4, Chapter 1.2.1 --- Glutamate system dysfunction underlying ketamine actions --- p.4, Chapter 1.2.2 --- Different effect patterns between acute and repeated administration --- p.7, Chapter 1.2.3 --- Acute and chronic effects of ketamine on human cognitive functions --- p.9, Chapter 1.2.4 --- Sex differences in addiction --- p.11, Chapter 1.3 --- Introduction to cognition and intelligence and their assessments --- p.13, Chapter 1.3.1 --- Memory and memory assessments --- p.13, Chapter 1.3.2 --- Executive function and assessments of executive function --- p.16, Chapter 1.3.3 --- Intelligence and intelligence tests --- p.19, Chapter 1.4 --- Study hypotheses --- p.31, Chapter CHAPTER 2 --- METHODS --- p.29, Chapter 2.1 --- Study design --- p.29, Chapter 2.2 --- Study subjects --- p.31, Chapter 2.2.1 --- Subject recruitment sites --- p.31, Chapter 2.2.2 --- Inclusion criteria --- p.32, Chapter 2.3 --- Data collection --- p.33, Chapter 2.3.1 --- Demographic information --- p.33, Chapter 2.3.2 --- Drug use pattern and severity --- p.34, Chapter 2.3.3 --- Psychiatric comorbidities --- p.34, Chapter 2.3.4 --- Cognitive function evaluation --- p.40, Chapter 2.4 --- Statistical methods --- p.44, Chapter CHAPTER 3 --- RESULTS --- p.45, Chapter 3.1 --- Demographics and basic information --- p.45, Chapter 3.2 --- Drug use patterns --- p.47, Chapter 3.3 --- Comorbid psychiatric problems --- p.51, Chapter 3.4 --- Cognitive functions --- p.59, Chapter 3.4.1 --- Cognitive functions among primarily ketamine, poly-drug ketamine and control groups --- p.59, Chapter 3.4.2 --- Cognitive functions in current and ex-primarily ketamine users --- p.60, Chapter 3.4.3 --- Cognitive functions in current and ex-poly-drug ketamine users --- p.70, Chapter 3.4.4 --- Cognitive functions in current primarily ketamine and current poly-drug ketamine users --- p.79, Chapter 3.4.5 --- Cognitive functions in female and male primarily ketamine users --- p.80, Chapter CHAPTER 4 --- DISCUSSION --- p.86, Chapter 4.1 --- Demographics and drug use patterns --- p.86, Chapter 4.2 --- Effects of ketamine on psychological health --- p.87, Chapter 4.3 --- Effects of ketamine on cognitive functions --- p.88, Chapter 4.3.1 --- Effects of primarily ketamine use on memory --- p.90, Chapter 4.3.2 --- Effects of primarily ketamine use on executive functions --- p.92, Chapter 4.3.3 --- Effects of poly-drug ketamine use on cognitive functions --- p.96, Chapter 4.3.4 --- Sex-specific effects of ketamine use on cognitive functions --- p.98, Chapter CHAPTER 5 --- LIMITATIONS AND CONCLUSIONS --- p.98, Chapter 5.1 --- Limitations --- p.98, Chapter 5.2 --- Conclusions --- p.99, REFERENCES --- p.102, http://library.cuhk.edu.hk/record=b5549725, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Liu, Xin., Thesis (Ph.D.)--Chinese University of Hong Kong, 2013., Includes bibliographical references (leaves 157-164)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., http://library.cuhk.edu.hk/record=b5884509, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Xiao, Yajie., Thesis (Ph.D.)--Chinese University of Hong Kong, 2013., Includes bibliographical references (leaves 156-190)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., http://library.cuhk.edu.hk/record=b5884361, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Lau, Wai Hung., Thesis (M.Phil.)--Chinese University of Hong Kong, 2013., Includes bibliographical references (leaves 141-153)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., s also in Chinese., http://library.cuhk.edu.hk/record=b5884358, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Wei, Lihua., Thesis (Ph.D.)--Chinese University of Hong Kong, 2013., Includes bibliographical references (leaves 166-196)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., http://library.cuhk.edu.hk/record=b5884487, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Yang, Yaling., Thesis (Ph.D.)--Chinese University of Hong Kong, 2013., Includes bibliographical references (leaves 187-207)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., s also in Chinese., http://library.cuhk.edu.hk/record=b5884289, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Zhu, Yin., Thesis (Ph.D.)--Chinese University of Hong Kong, 2013., Includes bibliographical references (leaves 162-203)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese; appendixes includes Chinese., http://library.cuhk.edu.hk/record=b5884315, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Wang, Jia., Thesis (Ph.D.)--Chinese University of Hong Kong, 2013., Includes bibliographical references (leaves 99-115)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., http://library.cuhk.edu.hk/record=b5884434, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Shen, Jiayun., Thesis (Ph.D.)--Chinese University of Hong Kong, 2013., Includes bibliographical references (leaves 166-199)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., http://library.cuhk.edu.hk/record=b5884459, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Gao, Zhibo., Thesis (Ph.D.)--Chinese University of Hong Kong, 2013., Includes bibliographical references (leaves 147-157)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., http://library.cuhk.edu.hk/record=b5884454, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

He, Jun., Thesis (Ph.D.)--Chinese University of Hong Kong, 2013., Includes bibliographical references (leaves 136-156)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., http://library.cuhk.edu.hk/record=b5884462, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Zhou, Yu., Thesis (M.Phil.)--Chinese University of Hong Kong, 2013., Includes bibliographical references (leaves 89-107)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., s also in Chinese., http://library.cuhk.edu.hk/record=b5884428, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Jiang, Guozhi., Thesis (Ph.D.)--Chinese University of Hong Kong, 2013., Includes bibliographical references (leaves 147-157)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., http://library.cuhk.edu.hk/record=b5884338, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Tang, Xiaolin., Thesis (Ph.D.)--Chinese University of Hong Kong, 2013., Includes bibliographical references (leaves 132-144)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., http://library.cuhk.edu.hk/record=b5884431, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Zhang, Yang., Thesis (Ph.D.)--Chinese University of Hong Kong, 2013., Includes bibliographical references (leaves 179-201)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., http://library.cuhk.edu.hk/record=b5884369, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

研究背景和目的, 高血壓和糖尿病是人群中常見的疾病，兩者常共同存在，其共存的病理生理機制非常複雜，其中腎素血管景張素系統功能紊亂起重要作用。多個研究表明血管緊張素轉化晦抑制劑和血管緊張素II 1 型受體阻滯劑通過調節不同基因的表達，發揮其保護心血管和腎臟功能的效用。然而，目前仍缺乏遠兩類藥物影響全基因表達譜的全面調查。因此，本研究應用全基因表達譜晶片技術，檢測分析了高血壓和糖尿病並發的病人在服用安慰劑、雷米普利(ramipril)和替米沙坦(telmisartan)後的全基因表達譜的變化，從而全面評估了血管緊張素轉化臨抑制劑和血管繁張素II 1 型受體阻滯劑對相關基因的轉錄調控作用。, 方法, 11 名患有高血壓和糖尿病的病人(男性5 名)在服用安慰劑最少2 星期后，以隨機吹序接受為期各6 星期的雷米普利和替米沙坦治療，並分別在安慰劑期和2 個藥物治療期結束后提取心A 進行全基因表達譜分析。, 結果, 與服用安慰劑時的全基因表達譜相比，雷米普利治療后有267 個基因的表達降低， 99 個基因的表達增強。表達差異幅度為-2.0 至1.3 (P < 0.05) 。表達下降的基因主要與血管平滑肌收縮、炎症反應和氧化壓力相關。表達增強的基因主要與心血管炎症反應負調節相關。基因共表達網絡分析表明， 2 個共表達基因組與雷米普利的降血壓作用相闕， 3 個共表達基因組與肥胖相關。, 與服用安慰劑時的全基因表達譜相比， 替米拉)、坦治療后有55 個基因表達降低， 158 個基因的表達增強。表達差異幅度為-1. 9 至1.3 (P < 0.05) 。表達增強的基因主要與脂質代謝、糖代謝和抗炎症因子作用相關。基因共表達網絡分析表明， 2 個共表達基因組與替米沙坦對24 小時舒張壓負荷量的作用相關， 2 個共表達基因組則與總膽固醇， 低密度脂蛋白膽固醇和C 反應蛋白相關。, 結論, 本論文描述了高血壓和2 型糖尿病病患全基因組表達的總體模式及經藥物治療後表達譜的相應改變， 為今後進一步研究腎素血管緊張素系統抑制劑和高血壓、糖尿病發展進程的相互作用提供了方向。, Background and aim: Pathophysiological mechanisms underpinning the coexistence of hypertension and type 2 diabetes are complex systemic responses involving dysregulation of the renin-angiotensin system (RAS). We conducted this study to investigate the genome wide gene expression changes in patients with both hypertension and diabetes at three treatment stages, including placebo, ramipril and telmisartan. This study aimed to obtain a panoramic view of interactions between gene transcription and antihypertensive therapy by RAS inhibition., Methods: 11 diabetic patients (S men) with hypertension were treated with placebo for at least 2 weeks followed by 6 weeks randomised crossover treatment with ramipril Smg daily and telmisartan 40mg daily, respectively. Total RNA were extracted from leukocytes at the end of placebo and each treatment period, and were hybridized to the whole transcript microarray. The limma package for R was used to identify differentially expressed genes between placebo and the 2 active treatments. The weighted gene coexpression network analysis (WGCNA) was applied to identify groups of genes (modules) highly correlated to a common biological function in pathogenesis and progression of hypertension and diabetes., Results: There were 267 genes down-regulated and 99 genes up-regulated with ramipril. Fold changes of gene expression were ranged from -2.0 to 1.3 (P < 0.05). The down-regulated genes were involved in vascular signalling pathways responsible for vascular smooth muscle contraction, inflammation and oxidative stress. The up-regulated genes were associated with negative regulation of cardiovascular inflammation. The WGCNA identified 17 coexpression gene modules related to ramipril. The midnight blue (57 genes, r < -0.44, P < 0.05) and magenta (190 genes, r < -0.44, P < 0.05) modules were significantly correlated to blood pressure differences between placebo and ramipril., There were 55 genes down-regulated and 158 genes up-regulated with telmisartan. Fold changes of gene expression were ranged from -1.9 to 1.3 (P < 0.05). The down-regulated genes were mainly associated with cardiovascular inflammation and oxidative stress. The up-regulated genes were associated with lipid and glucose metabolism and anti-inflammatory actions. The WGCNA identified 8 coexpression gene modules related to telmisartan. The black (56 genes, r = 0.46, P = 0.03) and turquoise (1340 genes, r = -0.48, P = 0.02) modules were correlated with diastolic blood pressure load. The blue (1027 genes) module was enriched with genes correlated with total cholesterol (r = - 0.52, P = 0.01), LDL-C (r = - 0.58, P = 0.004), and hsCRP (r = -0.57, P = 0.006). The green module (272 genes) was significantly correlated with LDL-C (r = - 0.44, P = 0.04) and hsCRP (r = - 0.59, P = 0.004)., Conclusion: Genome wide gene expression profiling in this study describes the general pattern and treatment responses in patients with hypertension and type 2 diabetes, which suggests future directions for further investigations on the interaction between actions of the RAS blockers and disease progression., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Deng, Hanbing., "December 2011.", Thesis (Ph.D.)--Chinese University of Hong Kong, 2012., Includes bibliographical references (leaves 198-256)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., Declaration --- p.i, Publications --- p.ii, p.iv, 論文摘要 --- p.vi, Acknowledgements --- p.viii, Table of Contents --- p.x, List of tables --- p.xiv, List of figures --- p.xv, List of appendices --- p.xvii, List of abbreviations --- p.xviii, Chapter Chapter 1 --- Introduction --- p.1, Chapter 1.1 --- Overview --- p.1, Chapter 1.2 --- Epidemiology --- p.6, Chapter 1.2.1 --- Epidemiology of hypertension --- p.9, Chapter 1.2.2 --- Epidemiology of type 2 diabetes --- p.10, Chapter 1.3 --- Aetiology --- p.13, Chapter 1.3.1 --- Ageing --- p.13, Chapter 1.3.1.1 --- Age-induced artery stiffness --- p.14, Chapter 1.3.1.2 --- Age-related endothelial dysfunction --- p.14, Chapter 1.3.2 --- The renin-angiotensin system (RAS) --- p.16, Chapter 1.3.2.1 --- The local RAS --- p.20, Chapter 1.3.2.2 --- The RAS and insulin resistance --- p.22, Chapter 1.3.2.3 --- The RAS and inflammation --- p.26, Chapter 1.3.2.4 --- The RAS and oxidative stress --- p.28, Chapter 1.3.3 --- Obesity --- p.31, Chapter 1.3.3.1 --- Obesity and renin-angiotensin system (RAS) --- p.33, Chapter 1.3.3.2 --- Obesity and insulin resistance --- p.36, Chapter 1.3.3.3 --- Obesity and oxidative stress --- p.38, Chapter 1.3.3.4 --- Obesity and sympathetic nervous system (SNS) --- p.38, Chapter 1.4 --- Pharmacogenomics of antihypertensive therapy --- p.39, Chapter 1.4.1 --- Angiotensin-converting enzyme inhibitors (ACEIs) --- p.41, Chapter 1.4.2 --- Angiotensin II type 1 receptor blockers (ARBs) --- p.43, Chapter Chapter 2 --- Aim --- p.59, Chapter Chapter 3 --- Methods --- p.60, Chapter 3.1 --- Subjects --- p.60, Chapter 3.1.1 --- Subject recruitment protocol --- p.60, Chapter 3.1.2 --- Definition of type 2 diabetes --- p.62, Chapter 3.1.3 --- Definition of obesity --- p.62, Chapter 3.1.4 --- Definition of dyslipidaemia --- p.63, Chapter 3.2 --- Study design and procedure --- p.64, Chapter 3.2.1 --- Blood pressure assessments --- p.65, Chapter 3.2.2 --- Anthropometric measurements --- p.68, Chapter 3.2.3 --- Medical history, life style and side effect evaluation --- p.68, Chapter 3.2.4 --- RNA isolation --- p.68, Chapter 3.2.5 --- RNA quality assessment --- p.70, Chapter 3.2.6 --- Oligonucleotide microarrays --- p.71, Chapter 3.2.7 --- DNA extraction --- p.75, Chapter 3.2.8 --- Biomedical measurements --- p.76, Chapter 3.2.8.1 --- Glycosylated haemoglobin Alc (HbA₁c) --- p.77, Chapter 3.2.8.2 --- Fasting plasma glucose (FP G) --- p.77, Chapter 3.2.8.3 --- Fasting insulin --- p.77, Chapter 3.2.8.4 --- Plasma urate --- p.77, Chapter 3.2.8.5 --- High sensitive C-reactive protein (hsCRP) --- p.78, Chapter 3.2.8.6 --- Fasting plasma triglycerides (TG) --- p.78, Chapter 3.2.8.7 --- Fasting plasma cholesterols --- p.78, Chapter 3.2.8.8 --- Renal and liver functions --- p.78, Chapter 3.2.8.9 --- Urinary parameters --- p.79, Chapter 3.3 --- Statistical Analysis --- p.79, Chapter 3.3.1 --- Statistical analysis of clinical and biomedical data --- p.79, Chapter 3.3.2 --- Analysis of microarray data --- p.80, Chapter 3.3.2.1 --- Raw data assessment --- p.80, Chapter 3.3.2.2 --- Data normalisation --- p.92, Chapter 3.3.2.3 --- Data filtering --- p.96, Chapter 3.3.2.4 --- Linear models for assessment of differential expression --- p.96, Chapter 3.3.2.5 --- Weighted gene coexpression network analysis --- p.101, Chapter 3.3.2.6 --- Network visualisation and gene ontology analysis --- p.102, Chapter 3.3.3 --- Sample size calculation --- p.103, Chapter Chapter 4 --- Results --- p.104, Chapter 4.1 --- Demographic and biomedical characteristics at baseline --- p.104, Chapter 4.1.1 --- Hypertension and diabetes status at baseline --- p.108, Chapter 4.1.2 --- Prevalence of dyslipidaemia --- p.108, Chapter 4.1.3 --- Prevalence of obesity --- p.109, Chapter 4.1.4 --- Prevalence of metabolic syndrome --- p.109, Chapter 4.1.5 --- Inflammation markers --- p.110, Chapter 4.2 --- Blood pressure response to the RAS blockers --- p.110, Chapter 4.2.1 --- Clinic blood pressure --- p.110, Chapter 4.2.2 --- 24-hour ambulatory blood pressure --- p.112, Chapter 4.3 --- Biomedical characteristics --- p.118, Chapter 4.4 --- Compliance, side effects and adverse events --- p.120, Chapter 4.5 --- Gene expression differences between treatments --- p.121, Chapter 4.5.1 --- Gene expression differences between placebo and ramipril --- p.121, Chapter 4.5.1.1 --- Expression changes in genes related to regulation of transcription with ramipril --- p.122, Chapter 4.5.1.2 --- Expression changes with ramipril in genes related to molecular mechanism of cardiovascular changes in hypertension --- p.125, Chapter 4.5.1.3 --- Expression changes in genes related to blood pressure with ramipril --- p.128, Chapter 4.5.1.4 --- Expression changes in genes related to fatty acid metabolism with ramipril --- p.130, Chapter 4.5.1.5 --- Expression changes in genes related to inflammation with ramipril --- p.130, Chapter 4.5.1.6 --- Expression changes in genes related to oxidative stress with ramipril --- p.133, Chapter 4.5.1.7 --- Power estimation --- p.133, Chapter 4.5.2 --- Gene expression differences between placebo and telmisartan --- p.135, Chapter 4.5.2.1 --- Changes in regulation oftranscription with telmisartan --- p.137, Chapter 4.5.2.2 --- Expression changes in genes related to glucose metabolism with telmisartan --- p.141, Chapter 4.5.2.3 --- Expression changes in genes related to lipid metabolism with telmisartan --- p.143, Chapter 4.5.2.4 --- Expression changes in genes related to inflammation with telmisartan --- p.143, Chapter 4.5.2.5 --- Power estimation --- p.145, Chapter 4.5.3 --- WGCNA for comparison between placebo and ramipriI --- p.147, Chapter 4.5.3.1 --- Midnight blue module and clinical responses to ramipril --- p.152, Chapter 4.5.3.2 --- Magenta module and blood pressure responses to ramipril --- p.154, Chapter 4.5.3.3 --- Yellow module and clinical responses to ramipril --- p.158, Chapter 4.5.3.4 --- Red module and clinical responses to ramipril --- p.161, Chapter 4.5.3.5 --- Salmon module and clinical responses to ramipril --- p.163, Chapter 4.5.4 --- WGCNA for comparison between placebo and telmisaItan --- p.168, Chapter 4.5.4.1 --- Diastolic blood pressure load and gene coexpression modules --- p.168, Chapter 4.5.4.2 --- Lipids, hsCRP and gene coexpression modules --- p.172, Chapter Chapter 5 --- Discussion --- p.176, Chapter 5.1 --- Gene expression changes related to ramipril --- p.177, Chapter 5.1.1 --- Gene expression changes and blood pressure reduction by ramipri1 --- p.177, Chapter 5.1.2 --- Gene expression changes and vascular protection by ramipri1 --- p.181, Chapter 5.1.3 --- Obesity and gene expression changes by ramipril --- p.183, Chapter 5.2 --- Gene expression changes related to telmisartan --- p.185, Chapter 5.2.1 --- Blood pressure and coexpressed gene modules with telmisartan --- p.185, Chapter 5.2.2 --- Lipid metabolism and gene expression changes by telmisartan --- p.187, Chapter 5.2.3 --- Glucose metabolism and gene expression changes by telmisartan --- p.189, Chapter 5.2.4 --- hsCRP and gene expression changes by telmisartan --- p.190, Chapter 5.3 --- Limitations of this study and future directions of research --- p.191, Chapter Chapter 6 --- Conclusion --- p.194, References --- p.198, Appendices --- p.257, http://library.cuhk.edu.hk/record=b5549577, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

由於慢性疾病的流行程度有著全球性上升的趨勢，它經已成為一個公共衛生的問題，為醫療系統帶來沉重的負擔。慢性疾病的發病率以老年人為最高，慢性疾病對老年人的生理、心理、社交及經濟等，構成尤其嚴重的後果。由於香港的人口持續老化，所以預計患有慢性疾病的人口數目在將來幾十年會不斷增加。但是，現時對患有慢性疾病的老年人所提供的照顧不足，再加上本地老年人擁有的多種特徵，例如社會經濟地位較低，健康讀寫能力較弱，及同時患有多重疾病，都有可能對醫護人員提供的護理造成障礙。提升慢性病患者掌管健康的能力，例如提供自我管理的支援，增強他們的信心，及協助他們作出有關健康的判斷及決策，有機會能解決慢性疾病所引起的問題。雖然過往的研究已經發現自我管理教育課程能夠改善慢性病患者的生理、心理及社交健康，及提升患者的健康行為，可是這類課程對老年人的成效，依然缺乏足夠科研證據的支持。, 作者在這論文中進行了兩項研究，去探討自我管理教育課程對患有慢性疾病的長者的健康行為、生理、心理、社交、生活質素及醫療服務的使用的影響。甲項研究是一個半實驗性研究，探討¬「慢性疾病自我管理課程」對患有不同種類慢性疾病的長者的效果。乙項研究是一個隨機控制實驗，研究「糖尿病自我管理課程」對患有非胰島素依賴的長者的效果。, 甲項研究招募了患有一種或以上慢性疾病，及居住在社區的長者進行研究。三百零二名治療組的參加者接受了一個為期六星期的「慢性疾病自我管理課程」，當中包括六課以小組模式進行的課堂，每堂為兩小時三十分。課程由專業人員或非專業的長者義工組長帶領。二百九十八名對照組的參加者則繼續接受六個月慣常的護理。每位參加者都會在基線及六個月後接受測試，測試包括自我管理行為、自我效能感、健康狀況及醫療服務的使用。, 利用單向共變數分析法，結果顯示治療組的所有的自我管理行為和自我效能感測試都有顯著改善 (p < .05)。在十項健康狀況測試中，有五項有明顯改善 (p < .05)。另外，醫療服務的使用則沒有明顯改變。, 乙項研究是利用隨機方法，分別把九十位及八十七位患有非胰島素依賴的長者分配到治療組及對照組。治療組的參加者參與了為期八堂，每星期一堂，每堂兩小時的「糖尿病自我管理課程」。對照組參加者則在八星期內繼續接受慣常的護理。所有參加者都會在基線及八星期後接受測試，測試包括身高體重指數、腰臀比例、血糖及血壓水平、糖尿病相關的認識、糖尿病指定及總稱的生活質素、及營養攝取。, 利用單向共變數分析法，結果顯示治療組的糖尿病相關的認識 (p < .0005)，糖尿病指定生活質素的滿意分類 (p = .045)，及總稱生活質素的精神健康分類 (p = .003)皆有明顯的改善。治療組的總能量 (p = .018)及飽和脂肪攝取 (p = .03)都有明顯減少。在各生理及人體測量指標及其他生活質素測試，則沒有明顯改變。, 此研究增加對疾病指定及非疾病指定的自我管理教育課程於社區上患有慢性疾病的長者的成效的認識。研究結果發現針對長者而設計的課程有機會改善長者的行為、心理及社交狀況，長者亦可以通過課程學習自我管理技巧及改變健康行為，從而改善健康。由長者義工組長帶領的課程有可能跟由專業人員帶領的課程的效果相近。研究結果象徵著自我管理課程需要融入醫療系統的慣常服務當中，以達致最大的成效。本論文亦為如何於各個護理層面及本地環境推行自我管理課程作出詳細討論。對於將來的研究發展，本論文建議加長跟進測試的時間及利用更大的實驗樣本探討自我管理課程於長者身上的成效，疾病指定及非疾病指定課程的效果亦需要作出比較，個別自我管理課程的特徵對課程成效的影響亦需要詳盡地探討。, The global epidemic of chronic disease has become a public health issue and created a huge burden on health care systems and societies. Older population is highly susceptible to chronic disease. The high prevalence of chronic disease among older adults results in a series of physical, psychosocial and financial consequences in this patient group. In Hong Kong, as the population continues to age, the number of people having chronic disease is expected to increase rapidly in next few decades. The care for older adults with chronic disease is yet suboptimal. Local older people are predisposed to a number of characteristics, such as low socioeconomic status, poor health literacy and multiple morbidities, which may hinder professionals to provide effective care. Empowering patients through supporting self-management, increasing confidence and assisting decision-making of people with chronic disease has been found to be a solution to the problem. Although literature has suggested that self-management education programmes may improve physical and psychosocial outcomes, and promote health-related behaviours among people with chronic disease, the evidence of the effects of such programmes in older adults is still lacking., Two studies have been conducted to examine the effects of self-management education programmes in improving health behaviours, physical and psychological status, quality of life and health care utilization in older people with chronic disease. Study One is a quasi-experimental trial exploring the effects of the Chronic Disease Self-Management Programme (CDSMP) in older adults with a wide range of chronic diseases. Study Two is a randomized controlled trial evaluating the effects of the Diabetes Mellitus Self-Management Programme (DMSMP) in older adults with non-insulin-dependent diabetes mellitus., In Study One, community-dwelling older people with one or more chronic disease were recruited. The intervention group (n = 302) received the 6-week CDSMP, which consisted of 6 group sessions with each session lasting for 2.5 hours. The programme was facilitated either by professional and older lay leaders. The control group (n = 298) continued their usual care for 6 months. Self-management behaviours, self-efficacy, health status, and health care utilization of participants were assessed at baseline and 6 months., The one-way analysis of covariance showed that the intervention group has significant improvements in all self-management behaviours and self-efficacy outcomes, and 5 out of 10 health status measures (all p < .05). No significant change was detected in the use of health care services., In Study Two, older people with non-insulin-dependent diabetes mellitus were randomly assigned to either the intervention (n = 90) or control (n = 87) group. The intervention group attended the DMSMP comprising 8 weekly 2-hour sessions. The control group received usual care for 8 weeks. Body mass index, waist-to-hip ratio, blood glucose and blood pressure levels, diabetes-related knowledge, disease-specific and generic quality of life, and nutritional intakes were measured at baseline and 8 weeks., Using the one-way analysis of covariance, the intervention group found significant improvements in diabetes-related knowledge (p < .0005), the satisfaction subscale score in the diabetes-specific quality of life measure (p = .045), and the mental health score in the generic quality of life measure (p = .003). Significant reductions of total energy (p = .018) and saturated fat intakes (p = .03) were also demonstrated in the intervention group. No significant change was detected in the physiological outcomes, anthropometric indices and other quality of life and nutritional measures., The present studies enrich the knowledge of the effects of disease-specific and generic self-management education programmes for older adults with chronic disease living in the community. It demonstrated that the programmes specifically tailored for older adults may improve a wide range of behavioural and psychosocial outcomes. Older adults may be able to learn new skills for self-management and change behaviours to improve their health. The effects of using older lay persons to lead such programmes may be similar with those using professional staff. The findings imply that self-management programmes need to be integrated into the routine service of health care systems and community care in order to have maximal effects. The implementation of self-management support at different levels of care and under the local context was discussed. Further studies should be conducted to explore the effects of self-management programmes on older people using prolonged follow-ups and larger sample size. The comparative effects of disease-specific and generic self-management programme should be evaluated. The individual influences of various essential features of self-management interventions need to be determined explicitly., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Chan, Lap Sun., Thesis (Ph.D.)--Chinese University of Hong Kong, 2012., Includes bibliographical references (leaves 265-302)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese; some appendixes also in Chinese., Chapter CHAPTER ONE --- INTRODUCTION, The epidemiology of chronic disease --- p.1, Causes of the epidemiology of chronic disease --- p.3, Risk factors of chronic disease --- p.4, Self-management approach in managing chronic disease --- p.5, The research problem --- p.8, Chapter CHAPTER TWO --- LITERATURE REVIEW, Challenges in managing chronic disease --- p.10, Specific concerns for older people --- p.12, Chronic disease management --- p.16, Introduction --- p.16, Patient-centred care --- p.18, Frameworks for improving care of chronic disease --- p.20, Chronic Care Model --- p.20, Innovative Care for Chronic Conditions --- p.22, Service delivery model of chronic disease management --- p.24, Global strategies in chronic disease management --- p.26, Empirical evidence of the Chronic Care Model --- p.29, Self-management --- p.34, Definitions --- p.34, Conceptualizing self-management --- p.37, Patient-professional relationship --- p.37, The goal of self-management --- p.40, Self-management tasks and skills --- p.41, Perspectives and barriers of self-management in older adults with chronic disease --- p.44, Self-management education and support --- p.50, Introduction --- p.50, Comparison with traditional patient education --- p.51, Characteristics of self-management education --- p.53, Theoretical basis in self-management education --- p.53, Self-efficacy theory --- p.56, Teaching problem-solving skills and making action plans --- p.58, Individualizing self-management education --- p.59, Continuity of self-management support --- p.60, Framework of delivering self-management support services --- p.62, Global implementation of self-management education --- p.65, Empirical evidence of the effects of self-management interventions --- p.70, Effects of self-management interventions in general --- p.71, Effects of self-management interventions for older adults with chronic disease --- p.77, Effects of self-management interventions for patients with chronic disease in Hong Kong --- p.85, Methodological issues in self-management studies --- p.88, Establishing self-management interventions for older adults --- p.90, Establishing self-management interventions under the local context --- p.93, Summary of Literature Review --- p.94, Chapter CHAPTER THREE --- METHODS (STUDY ONE), Introduction --- p.97, Methodology --- p.98, Research objectives --- p.98, Null hypotheses --- p.99, Study design --- p.100, Participants --- p.101, Recruitment and procedure --- p.101, Intervention --- p.103, Adaptations of programme delivery for local older participants --- p.106, Sample size calculation --- p.107, Outcome measures --- p.108, The questionnaire --- p.108, The Abbreviated Mental Test, Hong Kong version (AMT) --- p.109, Frailty Index (FI) --- p.109, Statistical analysis --- p.112, Primary analysis --- p.112, Secondary analysis --- p.112, Focus group --- p.114, Chapter CHAPTER FOUR --- RESULTS (STUDY ONE), Participants --- p.116, Baseline --- p.118, Comparing baseline and 6 months outcomes of intervention group --- p.123, Comparing baseline and 6 months outcomes of control group --- p.125, Comparing outcomes between intervention and control groups at 6 months --- p.127, Subgroup analysis --- p.130, Comparison among age subgroups --- p.132, Comparison among education level subgroups --- p.133, Comparison among frailty level subgroups --- p.133, Comparing professional staff-led and older lay-led programmes at 6 months --- p.140, Focus group --- p.142, Chapter CHAPTER FIVE --- DISCUSSION (STUDY ONE), Introduction --- p.145, Demographics characteristics --- p.145, Baseline outcomes --- p.147, Effects of the CDSMP on older adults with chronic disease --- p.148, Self-management behaviours and self-efficacy --- p.148, Health status --- p.148, Health care utilization --- p.149, Comparing with existing literature --- p.150, Effects of age, education level and frailty level on the outcomes --- p.154, Age --- p.154, Education level --- p.154, Frailty level --- p.155, Effects of leaders on the outcomes --- p.156, Qualitative findings --- p.157, Feasibility of training older people to be lay leaders --- p.160, Summary of the discussion --- p.162, Chapter CHAPTER SIX --- METHODS (STUDY TWO), Introduction --- p.164, Methodology --- p.166, Research objectives --- p.166, Null hypothesis --- p.166, Study design --- p.167, Pilot study --- p.168, Participants --- p.169, Recruitment and procedure --- p.170, Intervention --- p.172, Educational talks --- p.174, Exercise practice --- p.174, Goal setting and problem-solving --- p.177, Issues of designing self-management programme for local older adults --- p.177, Sample size calculation --- p.178, Outcome measures --- p.179, Diabetes Knowledge scale (DKN) --- p.179, 24-hour food recall --- p.180, Anthropometric measurements --- p.181, Clinical health indicators --- p.182, Quality of life --- p.183, Statistical analysis --- p.185, Primary analysis --- p.185, Secondary analysis --- p.186, Focus group --- p.186, Chapter CHAPTER SEVEN --- RESULTS (STUDY TWO), Participants --- p.188, Baseline --- p.190, Comparing baseline and 8 weeks outcomes of intervention group --- p.193, Comparing baseline and 8 weeks outcomes of control group --- p.195, Comparing outcomes between intervention and control group at 8 weeks --- p.197, Nutritional intakes --- p.200, Comparing baseline, 8 weeks and 6 months outcomes of intervention group --- p.203, Focus group --- p.206, Chapter CHAPTER EIGHT --- DISCUSSION (STUDY TWO), Introduction --- p.208, Demographics characteristics --- p.209, Baseline outcomes --- p.210, Effects of the DMSMP on older adults with type 2 DM --- p.213, Knowledge and nutritional intakes --- p.213, Anthropometric measures and clinical health indicators --- p.214, Quality of life --- p.217, Long-term effects of the DMSMP on intervention group participants --- p.219, Comparing with existing literature --- p.220, Comparing with a local study --- p.226, Qualitative findings --- p.227, Summary of the discussion --- p.231, Chapter CHAPTER NINE --- CONCLUSION, Overall effects of self-management interventions for older adults with chronic disease --- p.232, Strengths of the study --- p.238, Using a more stringent study design --- p.238, Incorporated essential features of self-management interventions into current programmes --- p.239, Demonstrated a collaborative model between health and social sectors --- p.240, Limitations of the study --- p.241, The integrity of study sample --- p.241, Issues in the representativeness of study sample --- p.241, High attrition rate in the longitudinal follow-up (The DMSMP) --- p.243, Unknown uptake rate --- p.244, The study design --- p.244, Non-randomized allocation of participants (The CDSMP) --- p.244, The lack of control for attention effect --- p.246, The implementation of study intervention --- p.247, Using multiple components --- p.247, The absence of blinding (The DMSMP) --- p.248, The evaluation and statistical analysis --- p.248, Short duration of follow-up --- p.248, Limitations of post-hoc analyses --- p.249, Diffusion of self-management interventions for older adults --- p.250, Considerations to the adoption of current self-management interventions --- p.251, Relative advantage --- p.251, Compatibility --- p.251, Complexity --- p.252, Trialability --- p.252, Observability --- p.253, Experience of implementing self-management interventions in the UK and the US --- p.254, Considerations to the implementation of current self-management interventions --- p.256, Strategies applied in promoting the adoption and implementation of current self-management interventions --- p.257, Recommendations for local implementation of self-management interventions --- p.259, Recommendations for future research --- p.261, Conclusion --- p.264, http://library.cuhk.edu.hk/record=b5549430, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

背景及目的:偶爾發惡夢通常被認為是普遍以及自限性的現象，然而頻繁的惡夢困擾也許代表睡眠問題的一種臨床症狀，或者是具有預測自殺傾向的潛在精神病理學病症。在這項研究中，我們的目標是：1) 瞭解頻繁惡夢在普通人群中的流行性以及與之相關的因素; 2) 調查頻繁惡夢在臨床人口中的流行性以及長期病程; 3) 探討在抑鬱症病人中與頻繁惡夢相關的各種社會心理學的和臨床的因素以及有關的潛在生物學標誌。, 方法:第一部分研究: 這是一項在社區內分兩個階段進行的研究。在第一階段, 我們對8558 個成人進行睡眠問卷的調查; 在第二階段, 我們對其中252 位研究對象進行詳細的精神病理學臨床評估和人格個性的剖析。, 第二部分研究: 這是一項在社區內進行的橫斷面研究。受訪者包括6359 個孩童 (年齡9.2 ± 1.8 歲, 女孩49.9%) 和他們的親生父母 (n=9855), 研究中收集的數據包括社會人口統計學的資料、與睡眠、行為以及家庭環境相關的資訊。, 第三部分研究: 這一項研究訪問了在香港一間大學的附屬公共精神科門診的1231 位病人(年齡42.5±11.3 歲，女性68.2%)。在基線時, 受訪者完成一份詳細的睡眠問卷調查。我們全面追溯了受訪者過往的臨床病歷。此外, 我們在問卷調查完成1 年後對這些病例進行跟進。, 第四部分研究: 對參與過第三部分研究並且被診斷為憂鬱症的病人,我們進行為期 4 年的前瞻性觀察研究。 研究內容包括結構式精神病學訪談以及全面的問卷調查(包括睡眠問卷, 醫院焦慮憂鬱量表,人格量表,SF-12 健康調查量表) 。, 第五部分研究: 這是一項病例-對照研究。研究對象包括35 例受到頻繁惡夢困擾的憂鬱病患者、以及與他們年齡、性別匹配的35 例無惡夢困擾的憂鬱症病患者和35 例健康對照者。研究評估包括臨床診斷、憂鬱症狀嚴重程度測量、心理社會因素量表、多導睡眠圖和腕動計的客觀睡眠檢查、24 小時尿液兒茶酚胺和皮質醇濃度、以及唾液皮質醇濃度的測量。, 結果:以一星期一次或者以上為標準, 頻繁惡夢患病率在一般人群的成人和兒童當中分別為5.1%和5.2%。在成人中, 女性、低家庭月收入、失眠症狀、睡眠呼吸障礙的症狀、 及睡眠相關的日間症狀與發惡夢的頻率有顯著的關聯 。患有頻繁惡夢的成人比起其他沒有惡夢困擾的成人患上精神疾病的風險要高出5.74 倍(95% CI 2.03-16.26),尤其是情緒病(odds ratio [OR] = 15.57,95% CI 3.77-64.37)。排除併發的精神障礙, 患有頻繁惡夢的成人在人格量表中神經質得分顯著高于其他沒有惡夢的成人 (p<0.05)。 在兒童中, 頻繁惡夢與低家庭月收入、 父母親發惡夢的頻率、 失眠症狀、類睡症症狀和日間症狀有明顯的關聯。同時, 在控制了相關的危險因素後, 頻繁惡夢與兒童的過度活躍 (OR = 1.68, 95% CI 1.16-2.44)、 頻繁情緒失控 (OR = 1.76, 95% CI1.27-2.44)、和成績不好 (OR = 1.62, 95% CI 1.11-2.36)有關。失眠和頻繁惡夢的共病性在成人和小孩當中都非常普遍。在臨床人口中, 患有頻繁惡夢的終生及1 年患病率分別為 22.5% 和21.7%。頻繁惡夢在憂鬱症和焦慮症患者中更為高發。在4 年的前瞻性跟進研究中, 憂鬱症患者基線和隨訪的惡夢患病率分別為32%和19%。大約有三分之一的憂鬱症患者持續地患有頻繁惡夢;而在憂鬱病患者中, 頻繁惡夢的新發病率為10%。基線時失眠和頻繁惡夢的共病是隨訪時病情未緩解的危險因數(OR = 3.25. 95% CI 1.56-6.77)。 持續地患有頻繁惡夢和新發病的頻繁惡夢的憂鬱病患者在隨訪時憂鬱和焦慮症狀上要更為嚴重,並且更可能在過往的4 年裡有自殺和住院的紀錄。同時, 在恢複期的憂鬱病患者當中, 患有頻繁惡夢與生活質量的各方面受損以及自殺意念 (OR= 8.40, 95% C.I. 1.79-39.33)有關。此外, 我們的病例-對照研究顯示頻繁惡夢與抑鬱型憂鬱、比較嚴重的憂鬱病症、以及高度的自殺傾向有關。同時, 患有頻繁惡夢的憂鬱病患者在多導睡眠圖檢查中的快速眼動期顯示更高的快速眼動頻率, 和在縱向的腕動計睡眠檢查中顯示更大的每晚之間的變異性 。, 結論:頻繁惡夢在一般人群中並非罕見, 並和不同的因素有關， 其中包括遺傳易感性 、社會人口特點、社會心理因素、睡眠問題的共病性和精神病理學症狀。同時, 頻繁惡夢在臨床人口中, 特別是憂鬱病患者代表著一類常見的、持續的、和令人困擾的睡眠問題。我們研究提示應該加強對患有頻繁惡夢的憂鬱病患者的臨床關注,同時需要更多的研究以瞭解在憂鬱病患者中進行針對睡眠問題的臨床治療的有效性。隨著認知神經科學研究的進一步發展, 未來的研究應該探討與惡夢困擾和精神病理學有關的認知神經科學機理。, Objectives: Frequent nightmares may represent a sleep disorder or/and in association with psychopathology. We aimed to examine the epidemiology of frequent nightmares in both general and clinical populations., Methods: Part I & Part II: A community-based study with questionnaire assessment of 6359 children and 8558 adults in phase 1 was followed by clinical evaluation of psychopathology and personality of 252 adult subjects in phase 2., Part III: A study with sleep questionnaire assessment in the psychiatric outpatients (n=1231)., Part IV: A 4-year, prospective study in a cohort of depressive patients depression (n= 371, response rate = 88.5%) with a standardized interview and psychometric inventories., Part V: A case-control study was conducted with clinical, psychosocial, sleep andbiological measurements., Major Results: Prevalence of frequent nightmares was 5.1% and 5.2% in the adults and children, respectively, of the local general population. Female, low family income, sleep-related symptoms, psychopathology, and neuroticism trait were associated with nightmares in the general adult population. In children, frequent nightmares were associated with socioeconomic status, parental predisposition, sleep-related symptoms and childhood psychopathology. The prevalence rates of recurrent nightmares in depressed subjects at baseline and 4-year follow-up were 32% and 19%, respectively. The comorbidity of nightmares and insomnia disturbances reported at baseline was a significant risk factor of non-remission at follow-up (OR = 3.25. 95% C.I. 1.56-6.77). Subjects with persistence or incidence of frequent nightmares had more severe depression. Meanwhile, among those remitted depressed subjects, residual nightmares were associated with various aspects of impaired quality of life and suicidal ideation (OR= 8.40, 95% C.I. 1.79-39.33)., ConclusionsFrequent nightmares were associated with a constellation of personal, sleep and psychopathological factors in general population, and represent a common complaint with detrimental effects in the psychiatric populations. There is a need of enhanced clinical attention in patients with frequent nightmare complaints., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Li, Xin., Thesis (Ph.D.)--Chinese University of Hong Kong, 2012., Includes bibliographical references (leaves 173-199)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese; some appendixes also in Chinese., p.I, ACKNOWLEDGE --- p.V, THESIS/ASSESSMENT COMMITTEE --- p.VII, TABLE OF CONTENTS --- p.VIII, TABLE LIST --- p.XV, FIGURE LIST --- p.XVII, Chapter CHAPTER 1 --- GENERAL INTRODUCTION, Chapter 1.1 --- Dream Research: A Historical Perspective --- p.1, Chapter 1.2 --- Nightmares: Definitions and Differentials --- p.2, Chapter 1.2.1 --- Clinical and Research Definitions of Nightmares --- p.2, Chapter 1.2.2 --- Differentials of Nightmares --- p.4, Chapter 1.3 --- Epidemiological Studies of Nightmares in Adults --- p.6, Chapter 1.4 --- Epidemiological Studies of Nightmares in Children --- p.6, Chapter 1.5 --- Associated Factors of Frequent Nightmares in General Populations --- p.7, Chapter 1.5.1 --- Psychological Correlates of Frequent Nightmares - Anxiety and Personality Dimensions --- p.8, Chapter 1.5.2 --- Stress as a Precipitating Factor of Frequent Nightmares --- p.8, Chapter 1.6 --- Nightmare Disturbances and Major Psychopathology --- p.9, Chapter 1.6.1 --- Posttraumatic Nightmares and PTSD Prevalence, Phenomenology and Associations --- p.10, Chapter 1.6.2 --- Nightmare Disturbances and Other Psychiatric Disorders --- p.12, Chapter 1.7 --- Prognostic Implications of Nightmare Disturbances in Predicting Suicidality Clinical and Community-Based Evidence --- p.13, Chapter 1.8 --- Summary and Research Directions --- p.15, Chapter CHAPTER 2 --- Part I Study - Epidemiology of Frequent Nightmares in Hong Kong Chinese Adults: A Community-based 2-Phase Study, Chapter 2.1 --- INTRODUCTION --- p.26, Chapter 2.1.1 --- Objectives and Hypotheses of the Study --- p.26, Chapter 2.2 --- METHODS, Chapter 2.2.1 --- Overview of the Project --- p.27, Chapter 2.2.2 --- Subjects and Measurements, Chapter 2.2.2.1 --- Study Subjects in Phase 1 --- p.28, Chapter 2.2.2.2 --- Study Instruments in Phase 1 --- p.29, Chapter 2.2.2.3 --- Study Subjects in Phase 2 --- p.30, Chapter 2.2.2.4 --- Study Instruments in Phase 2 --- p.32, Chapter 2.2.3 --- Statistical Analysis --- p.33, Chapter 2.3 --- RESULTS, Chapter 2.3.1 --- Phase 1 Results: Socio-demographic Characteristics and Other Sleep Problems in Relation to Frequent Nightmares --- p.34, Chapter 2.3.2. --- Phase 2 Results: Psychopathology and Psychosocial Characteristics in Relation to Frequent Nightmares --- p.38, Chapter 2.4 --- DISCUSSIONS, Chapter 2.4.1 --- Frequent nightmares in Hong Kong Chinese Adults - Prevalence and Gender Difference --- p.40, Chapter 2.4.2 --- Sleep Correlates of Frequent Nightmares --- p.40, Chapter 2.4.3 --- Sociodemographic Features and Psychopathology in Relation to Frequent Nightmares --- p.41, Chapter 2.4.4 --- Personality Dimensions Independently Associated with Frequent Nightmares --- p.42, Chapter 2.4.5 --- Strengths and Limitations of the Study --- p.43, Chapter CHAPTER 3 --- Part II Study - Epidemiology and Familial Aggregation of Frequent Nightmares in Hong Kong Chinese Children, Chapter 3.1 --- INTRODUCTION --- p.44, Chapter 3.1.1 --- Objectives and Hypotheses of the Study --- p.45, Chapter 3.2 --- METHODS, Chapter 3.2.1 --- Overview of the Project --- p.45, Chapter 3.2.2 --- Subjects and Measurements, Chapter 3.2.2.1 --- Study Subjects --- p.46, Chapter 3.2.2.2 --- Study Instruments --- p.47, Chapter 3.2.3 --- Statistical Analysis --- p.48, Chapter 3.3 --- RESULTS, Chapter 3.3.1 --- Prevalence and Associated Factors of Frequent Nightmares in Hong Kong Chinese Children --- p.49, Chapter 3.3.2 --- Association of Frequent Nightmares with Parent-Reported Mood, Behaviors, and Academic Performance --- p.54, Chapter 3.4 --- DISCUSSIONS, Chapter 3.4.1 --- Prevalence and Sleep Correlates of Frequent Nightmares in Children --- p.56, Chapter 3.4.2 --- Stronger Paternal Effect in the Familial Aggregation of Frequent Nightmares --- p.56, Chapter 3.4.3 --- Frequent Nightmares in Children Associations with Adverse Neurobehavioral Outcomes and Academic Performance --- p.57, Chapter 3.4.4 --- Strengths and Limitations of the Study --- p.58, Chapter 3.4.5 --- Summary --- p.59, Chapter CHPATER 4 --- Part III Study - A Clinical Epidemiologic Study of Frequent Nightmares among Psychiatric Patients, Chapter 4.1 --- INTRODUCTION --- p.60, Chapter 4.1.1 --- Objectives and Hypotheses of the Study --- p.61, Chapter 4.2 --- METHODS, Chapter 4.2.1 --- Procedure and Study Subjects --- p.61, Chapter 4.2.2. --- Study Instrument --- p.62, Chapter 4.2.3 --- Statistical Analysis --- p.63, Chapter 4.3 --- RESULTS --- p.64, Chapter 4.4 --- DISCUSSIONS --- p.70, Chapter 4.4.1 --- Comorbidity of Nocturnal Sleep Disturbances in Psychiatric Patients --- p.70, Chapter 4.4.2 --- Nocturnal Sleep Disturbances in Association with Suicidal Risk --- p.71, Chapter 4.4.3 --- Nightmare Disturbances and Psychopharmacologic Treatments --- p.72, Chapter 4.4.4. --- Limitations of the study --- p.73, Chapter 4.4.5 --- Summary --- p.73, Chapter CHAPTER 5 --- Part IV Study - A Prospective Study of Nightmare Disturbances in a Cohort of Psychiatric Outpatients with Major Depressive Disorder (MDD), Chapter 5.1 --- INTRODUCTION --- p.74, Chapter 5.1.1 --- Major Depressive Disorder --- p.74, Chapter 5.1.2 --- Disturbed Dreaming and Nightmares in Depression - Phenomenology and Correlates --- p.75, Chapter 5.1.2.1 --- Phenomenological Characteristics of Disturbed Dreaming in Depression --- p.75, Chapter 5.1.2.2 --- Disturbed dreaming in Relation to the Treatments and Clinical Outcome of Depression --- p.76, Chapter 5.1.2.3 --- Recent Findings on Frequent Nightmares in Depression --- p.77, Chapter 5.1.3 --- Insomnia and Depression - Associations with Frequent Nightmares --- p.78, Chapter 5.1.4 --- Nightmare Disturbances in the Context of Residual Symptomatology of Depression --- p.79, Chapter 5.1.5 --- Objectives and Hypotheses of the Study --- p.80, Chapter 5.2 --- METHODS, Chapter 5.2.1 --- Overview of the Study - Procedure and Study Subjects --- p.88, Chapter 5.2.2 --- Measurements, Chapter 5.2.2.1 --- Sleep Questionnaire (Baseline and Follow-up) --- p.89, Chapter 5.2.2.2 --- Psychometric Instruments (Follow-up) --- p.89, Chapter 5.2.2.3 --- Psychiatric Diagnosis, Suicidal ideation and Related Clinical History --- p.90, Chapter 5.2.3 --- Statistical Analysis --- p.91, Chapter 5.2.3.1 --- Analysis on the Frequent Nightmares as a Predictor of Remission Outcome in Depression --- p.92, Chapter 5.2.3.2 --- Analysis on the Progress of Frequent Nightmares in the Depressed Patients --- p.93, Chapter 5.2.3.3 --- Analysis on the Frequent Nightmares as a Residual Symptom in the Remitted Depressed Patients --- p.93, Chapter 5.3 --- RESULTS, Chapter 5.3.1 --- Study Recruitment and Overall Sample Characteristics --- p.93, Chapter 5.3.2 --- Factors Associated With the Remission Outcome in Depression --- p.97, Chapter 5.3.2.1 --- Socio-Demographic & Clinical Characteristics --- p.97, Chapter 5.3.2.2 --- Baseline Nightmare and Insomnia Disturbances --- p.98, Chapter 5.3.3 --- Longitudinal Course of Frequent Nightmares in Depression --- p.101, Chapter 5.3.4 --- Factors Associated with the Progress of Frequent Nightmares in the Depressed Patients --- p.102, Chapter 5.3.4.1 --- Factors Associated with Persistence of Frequent Nightmares --- p.102, Chapter 5.3.4.2 --- Factors Associated with Incidence of Frequent Nightmares --- p.103, Chapter 5.3.4.3 --- Residual Nightmare Disturbances in Remitted Depressed Patients --- p.108, Chapter 5.4 --- DISCUSSIONS, Chapter 5.4.1 --- Prevalence of Frequent Nightmares and Its Association with the Remission Outcome in Depression --- p.114, Chapter 5.4.2 --- Persistence and Incidence of Frequent Nightmares in Depression --- p.115, Chapter 5.4.3 --- Frequent nightmares as a Residual Symptom in Depression --- p.116, Chapter 5.4.3.1 --- Prevalence & Correlates of Residual Nightmares in Remitted Depressed Patients --- p.116, Chapter 5.4.3.2 --- Impaired Functional Outcomes and Suicidal Ideation Associated with Residual Nightmare Disturbances --- p.118, Chapter 5.4.4. --- Study Strengths and Limitations --- p.119, Chapter CHAPTER 6 --- Part V study - An In-depth Clinical, Polysomnographic and Neurobiological Investigation of Depressive Patients with Recurrent Nightmares: A Case-Control Study, Chapter 6.1 --- INTRODUCTION --- p.120, Chapter 6.1.1 --- Objective Sleep Abnormalities in Depression --- p.120, Chapter 6.1.2 --- Objective Studies of Nightmares --- p.121, Chapter 6.1.3 --- Hormonal Responses in Relation to Recurrent Nightmare Disturbances. --- p.122, Chapter 6.1.4 --- Objectives and Hypotheses of the Study --- p.124, Chapter 6.2 --- METHODS, Chapter 6.2.1 --- Procedure and Study Subjects --- p.124, Chapter 6.2.2 --- Measurements, Chapter 6.2.2.1 --- In-Home Assessments --- p.127, Chapter 6.2.2.2 --- Laboratory Assessments --- p.128, Chapter 6.2.2.3 --- Diagnostic Assessment and Psychometric Inventories --- p.130, Chapter 6.2.3 --- Statistical Analysis --- p.133, Chapter 6.3 --- RESULTS, Chapter 6.3.1. --- Study Recruitment --- p.135, Chapter 6.3.2 --- Nightmare-Related History and Characteristics of Depressed Subjects with Frequent Nightmares --- p.137, Chapter 6.3.3 --- Clinical Characteristics of Depressed Subjects With and Without Frequent Nightmares --- p.138, Chapter 6.3.4 --- Comparison on the Demographic Features and Self-reported Sleep Measures --- p.139, Chapter 6.3.5 --- Comparison on the Psychometric Measurements --- p.141, Chapter 6.3.6 --- Comparison on the Actigraphic Measurements --- p.145, Chapter 6.3.7 --- Comparison on the Polysomnographic Data --- p.148, Chapter 6.3.8 --- Comparison on the Mood States and Self-reported Sleep Measures in Association with the Presence of Nightmares --- p.150, Chapter 6.3.9 --- Comparison on the Endocrine Data --- p.152, Chapter 6.4 --- DISCUSSIONS, Chapter 6.4.1 --- Nightmare-Related History in Depression --- p.154, Chapter 6.4.2 --- Treatment Seeking in Depressed Patients for Nightmare Disturbances --- p.155, Chapter 6.4.3 --- Frequent Nightmares in Depression - Clinical Correlates and Psychosocial Characteristics --- p.156, Chapter 6.4.4 --- Subjective and Objective Sleep Features in Relation to Frequent Nightmares in Depression --- p.157, Chapter 6.4.5 --- Neuroendocrine Measurements in Relation to Frequent Nightmares in Depression --- p.160, Chapter 6.4.6 --- Study Strengths and Limitations --- p.161, Chapter CHAPTER 7 --- GENERAL DISCUSSIONS, Chapter 7.1 --- Recapitulation and Elaboration of Our Major Findings, Chapter 7.1.1 --- Epidemiology of Frequent nightmares --- p.162, Chapter 7.1.2 --- Etiology of Frequent Nightmares --- p.163, Chapter 7.1.2.1 --- Predisposing Factors of Frequent nightmares --- p.163, Chapter 7.1.2.2 --- Precipitating Factors of Frequent Nightmares --- p.165, Chapter 7.1.2.3 --- Perpetuating Factors - The Interplay of Nightmares, Insomnia and Psychopathology --- p.165, Chapter 7.1.4 --- Nightmares in the Context of Psychopathology Implications for Clinical Management --- p.168, Chapter 7.2 --- Future Research Directions --- p.169, Chapter 7.2.1 --- Genetic Studies and Interventional Studies on Nightmares --- p.169, Chapter 7.2.2 --- Linking Nightmares and Mood Disturbances to Memory Consolidation: A Hypothesis from A Neurocognitive Perspective --- p.171, Chapter 7.2.2.1 --- Sleep and Memory Consolidation --- p.171, Chapter 7.2.2.2 --- Dreaming as Off-line Processing of Memory in the Psychopathological Context --- p.171, Chapter 7.3 --- Summary --- p.172, REFERENCES --- p.173, APPENDICES, Chapter A --- Study Instruments of Part I & Part II Studies --- p.200, Chapter B --- Study Instruments of Part III Study --- p.217, Chapter C --- Study Instruments of Part IV Study --- p.220, Chapter D --- Study Instruments of Part V Study --- p.231, Chapter E --- Documentation of Permission to Republish the Copyrighted Materials in the Dissertation, Chapter F --- Li SX, Zhang B, Li AM, Wing YK. Prevalence and correlates of frequent nightmares: a community-based 2-phase study. Sleep. 2010;33:774-80., Chapter G --- Li SX, Yu MW, Lam SP, Zhang J, Li AM, Lai KY, Wing YK. Frequent nightmares inchildren: familial aggregation and associations with parentreported behavioral and mood problems. Sleep. 2011;34:487-93., Chapter H --- Li SX, Lam SP, Yu MWM, Zhang J, Wing YK. Nocturnal sleep disturbances as a predictor of suicide attempts among psychiatric outpatients: a clinical, epidemiologic, prospective study. J Clin Psychiatry 2010;71:1440-6., Chapter I --- Li SX, Lam SP, Chan JWY, Yu MWM, Wing YK. Residual Sleep Disturbances in Patients Remitted From Major Depressive Disorder: A 4- Year Naturalistic Follow-up Study [In press], Chapter J --- Curriculum Vitae, http://library.cuhk.edu.hk/record=b5549562, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

全球人類健康的頭號殺手--腫瘤目前仍是難以攻克的醫學難題。腫瘤的發生是一個復雜的過程，主要由促癌基因的異常增多或激活及抑癌基因（TSG）的缺失或功能喪失的累積效果導致。近年來基於非基因序列改變所致基因表達水平變化的表觀遺傳學的研究進展表明，啟動子區CpG島甲基化所致的表觀遺傳沉默是抑癌基因轉錄失活的重要機制。Ras和Wnt信號轉導通路在癌病的發生和發展過程中均起到重要的作用，因此針對該兩種信號通路的拮抗因子的表觀遺傳調控及功能學研究將為我們提供有研究及應用前景的候選抑癌基因。, 作為一種重要的原癌基因，Ras家族基因具有致癌活性的點突變及其導致的過度激活的Ras信號通路被發現廣泛存在於大約30%的人類腫瘤中。然而在一些缺乏Ras基因突變的腫瘤類型中，持續激活的Ras信號通路仍然普遍存在並具有重要作用，昭示著除了Ras基因點突變以外的信號轉導異常激活的機制。與GTP的結合可激活Ras，而RasGAP家族蛋白可通過水解GTP達到使Ras失活的作用。通過采用微陣列比較基因組雜交(aCGH)的實驗手段我們發現6p21.3染色體區具有半接合子缺失， 並於此區域發現了候選抑癌基因RASA5。在以往的研究報道中，RASA5被命名為SynGAP且其功能研究僅限於神經系統。我們的研究發現不同於RasGAP家族的其它基因RASA2-4，RASA5廣泛表達於人類正常器官組織中，並特異性地在腫瘤細胞，特別是鼻咽癌(NPC)，食管鱗狀上皮細胞癌(ESCC)和乳腺癌這些具有野生型Ras基因但Ras信號通路仍被過度激活的細胞中被表觀遺傳沉默。RASA5的異位表達可有效促進腫瘤細胞的雕亡，抑制腫瘤細胞的生長、遷移及“幹性（stemness）“。同時，使用siRNA敲除內源性RASA5可以激發細胞的克隆形成及上皮-間質(EMT)轉化。RASA5的抑癌功能是通過調低Ras-GTP水平並進而抑制其下遊信號通路的活性實現的。過量表達具有致癌活性的點突變的Ras或RasGAP結構域缺失均可部分逆轉這種抑癌作用。此項研究首次證明了RASA5的抑癌功能。, Wnt/Dvl/β-catenin信號轉導通路在人類腫瘤中存在廣泛的異常激活。我們發現DACT (Dpr/Frodo)家族成員TUSC-T2的表觀遺傳沉默是一種普遍存在於人類腫瘤中的現象。TUSC-T2編碼一種胞質蛋白，外源性表達TUSC-T2可促進腫瘤細胞雕亡並導致腫瘤細胞的克隆形成能力下降。TUSC-T2可與Dvl蛋白結合並下調其活化水平，從而保護GSK-3β蛋白不被Dvl蛋白抑制。GSK-3β可與Axin及APC蛋白形成蛋白質復合物，該復合物可捕捉並降解細胞內信號分子β-catenin。TUSC-T2的過量表達可以抑制β-catenin的激活及其向細胞核內的富集，並進一步阻止β-catenin在細胞核內與Lef/Tcf轉錄因子家族的作用及下遊特定原癌基因，例如c-Myc, CCND1及Fibronectin的表達。因此TUSC-T2具有抑制腫瘤細胞增殖、遷移及上皮-間質(EMT)轉化的作用。, 綜上所述，我們的研究結果表明RASA5及TUSC-T2是具有抑癌功能的Ras或Wnt/Dvl/β-catenin信號轉導通路抑制因子，其表觀遺傳沉默導致的轉錄失活對於腫瘤的發生發展具有重要意義。同時，針對這兩種抑癌基因的進一步研究將為我們提供富有應用前景的腫瘤標記物。值得註意的是，RASA5課題的研究開創性地闡明了Ras信號通路的拮抗因子的表觀遺傳沉默是一種Ras信號轉導通路於腫瘤細胞中異常激活的新機制。, Cancer is the top killer of the world, as well as the medical problem difficult to overcome. The conversion of a normal cell to a cancer cell is usually caused by upregulation of oncogenes and downregulation of tumor suppressor genes (TSGs). Epigenetic silencing has been proved to be important in TSGs inactivation, often through methylation of CpG-rich promoter regions. Ras and Wnt signaling pathways are both important for the tumorigenesis, epigenetic and functional studies of antagonists to Ras and Wnt signaling would provide us with candidate TSGs., Ras is a well-known oncogene. Aberrant mutations of Ras genes occur in approximately 30% of human tumors, causing constitutively activated Ras signaling. However, in certain types of tumors with wild type Ras genes, abnormally activated Ras signaling is still a common and critical event, suggesting alternative mechanisms for Ras signaling hyperactivation. Ras is active when it is bound to GTP, while the hydrolysis of bound GTP and inactivation of Ras is catalyzed by Ras GTPase activating proteins (RasGAPs). Using 1-Mb array CGH (aCGH), we refined a small hemizygous deletion at the 6p21.3 chromosome region that contains a RasGAP family member gene RASA5, which used to be named as SynGAP and studied only in the neuron systems. We demonstrated that RASA5, rather than other RasGAP family members RASA2-4, is broadly expressed in human normal tissues while frequently epigenetically silenced in multiple tumors, especially in certain tumor types such as nasopharyngeal (NPC), esophageal (ESCC) and breast carcinomas (BrCa) with wild-type Ras while Ras cascade is still constitutively active. Ectopic expression of RASA5 led to apoptosis, growth and migration inhibition, as well as ‘stemness’ repression of tumor cells. Meanwhile, knockdown of RASA5 by siRNA promoted the tumor cell colony formation as well as epithelial-mesenchymal transition (EMT). The tumor-suppressive function of RASA5 was exerted through downregulating Ras-GTP level and further inactivating Ras signaling. Such an inhibitory effect could be partially abrogated in the presence of mutated, activated Ras or by deletion of the RasGAP domain. For the first time, our study refined the role of RASA5 as a tumor suppressor., Wnt/DVL/β-catenin signaling pathway is aberrantly activated in a wide range of human cancers. We identified a DACT (Dpr/Frodo) family member TUSC-T2 as an epigenetically downregulated gene in human tumors. TUSC-T2 encodes a punctate cytoplasmic protein. Ectopic expression of TUSC-T2 dramatically inhibited tumor cell colony formation in silenced tumor cell lines, mainly through inducing apoptosis. TUSC-T2 interacts and downregulates Dishevelled (Dvl) protein, thus protecting glycogen synthase kinase 3β (GSK-3β) from inactivation by Wnt/Dvl and allowing GSK-3β to form a complex with Axin and APC to promote the phosphorylation and proteasomal degradation of β-catenin. Overexpression of TUSC-T2 disrupted β-catenin activation and accumulation in nuclei, thus preventing its binding to transcription factors of the Lef/Tcf family. This caused the downregulation of β-catenin target oncogenes such as c-Myc, CCND1 and Fibronectin as well as the inhibition of tumor cell proliferation and migration. We also observed that TUSC-T2 could inhibit tumor cell EMT., Taken together, our data demonstrate that RASA5 and TUSC-T2 are functional tumor suppressors epigenetically silenced in multiple tumors through acting as negative regulators of the Ras or Wnt/Dvl/β-catenin cancer pathways, and could be developed as promising biomarkers for human tumors. Of note, our study reveals that epigenetic silencing of the Ras antagonist represents a new mechanism responsible for Ras aberrant activation in cancers with wild-type Ras., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Fan, Yichao., Thesis (Ph.D.)--Chinese University of Hong Kong, 2012., Includes bibliographical references (leaves 184-216)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., Acknowledgements --- p.i, List of abbreviations --- p.ii-iii, List of tables --- p.iv, List of Figures --- p.v-vii, List of Publications --- p.viii-ix, in English --- p.x-xii, in Chinese --- p.xiii-xiv, Table of Contents --- p.xv, Chapter Chapter 1 --- Introduction and Literature Review --- p.1, Chapter 1.1 --- Cancer epigenetics --- p.4, Chapter 1.1.1 --- Epigenetic modifications --- p.5, Chapter 1.1.1.1 --- DNA Methylation --- p.5, Chapter 1.1.1.2 --- Histone modifications --- p.10, Chapter 1.1.1.3 --- RNA interference --- p.14, Chapter 1.1.1.4 --- Nucleosome positioning --- p.15, Chapter 1.1.2 --- Epigenetic alteration induced Tumor suppressor genes (TSGs) silencing during carcinogenesis --- p.17, Chapter 1.2 --- Epigenetic alterations in cancer pathways --- p.23, Chapter 1.2.1 --- Brief introduction of cancer pathways --- p.23, Chapter 1.2.2 --- Ras pathway --- p.25, Chapter 1.2.2.1 --- Ras pathway and carcinogenesis --- p.25, Chapter 1.2.2.2 --- Epigenetic regulation of RasGAP proteins in carcinogenesis --- p.28, Chapter 1.2.2.3 --- Epigenetic silencing of other negative regulators of Ras signaling --- p.30, RAS association domain family (RASSF) proteins --- p.30, PTEN --- p.32, Sprouty (SPRY) proteins --- p.33, Chapter 1.2.2.4 --- Hypomethylation induced Ras oncogenes activation --- p.35, Chapter 1.2.2.5 --- Ras mediates epigenetic regulation through feedback loop --- p.36, Chapter 1.2.3 --- Wnt pathway --- p.43, Chapter 1.2.3.1 --- Wnt signaling pathway and carcinogenesis --- p.43, Chapter 1.2.3.2 --- Epigenetic silencing of negative regulators of Wnt signaling --- p.45, Chapter 1.2.3.3 --- DACT family proteins and carcinogenesis --- p.48, Chapter 1.3 --- Application of tumor specific epigenetic alterations as tumor biomarkers and therapeutic targets --- p.49, Chapter 1.3.1 --- The potential and advantage of tumor specific epigenetic alterations used as tumor biomarkers and therapeutic targets --- p.49, Chapter 1.3.2 --- Epigenetic-disrupted regulators of Ras signaling as tumor biomarkers and therapeutic targets --- p.50, Chapter 1.3.3 --- Epigenetic-disrupted regulators of Wnt signaling as tumor biomarkers and therapeutic targets --- p.52, Chapter Chapter 2 --- Aims of this study --- p.54, Chapter 2.1 --- To identify epigenetically silenced candidate TSGs as antagonists to Ras or Wnt signaling --- p.55, Chapter 2.2 --- To elucidate the functional of candidate TSGs --- p.56, Chapter Chapter 3 --- Materials and Methods --- p.57, Chapter 3.1 --- Cell lines, tumor samples and routine cell line maintenance --- p.57, Chapter 3.2 --- Drug and stress treatments --- p.59, Chapter 3.3 --- DNA and RNA extraction --- p.59, Chapter 3.4 --- Semi-quantitative RT-PCR and Real time PCR --- p.60, Chapter 3.5 --- Direct sequencing of PCR products --- p.67, Chapter 3.6 --- CpG island analysis --- p.67, Chapter 3.7 --- Bisulfite treatment --- p.67, Chapter 3.8 --- Methylation-specific PCR (MSP) and bisulfite genomic sequencing --- p.68, Chapter 3.9 --- Plasmid extraction --- p.69, Chapter 3.9.1 --- Bacteria culture --- p.69, Chapter 3.9.2 --- Mini-scale preparation of plasmid DNA --- p.70, Chapter 3.9.3 --- Large-scale endotoxin-free plasmids extraction --- p.71, Chapter 3.10 --- Construction of expression plasmids --- p.71, Chapter 3.10.1 --- Gene cloning and plasmids construction of RASA5 --- p.71, Chapter 3.10.2 --- Gene cloning and plasmids construction of TUSC-T2 --- p.74, Chapter 3.11 --- Immunofluorescence Staining --- p.74, Chapter 3.12 --- Colony formation assay --- p.76, Chapter 3.13 --- Apoptosis assay --- p.77, Chapter 3.14 --- Luciferase reporter assay --- p.78, Chapter 3.15 --- Protein preparation and Western blot --- p.79, Chapter 3.16 --- Ras Activity Assay --- p.80, Chapter 3.17 --- Wound healing assay --- p.81, Chapter 3.18 --- Matrigel invasion assay --- p.81, Chapter 3.19 --- RNA Interference --- p.81, Chapter 3.20 --- Statistical analysis --- p.82, Chapter Chapter 4: --- Epigenetic disruption of Ras signaling through silencing of a Ras GTPase-activating protein RASA5 in human cancers --- p.83, Chapter 4.1 --- Identification of RASA5 as a downregulated gene residing in the 6p21.3 deletion region --- p.86, Chapter 4.2 --- RASA5 is widely expressed in human normal tissues but downregulated in tumor cell lines --- p.91, Chapter 4.3 --- The tumor-specific downregulation pattern of RASA5 is unique in the RASA family genes --- p.95, Chapter 4.4 --- RASA5 promoter CpG methylation resulted in its transcription inactivation --- p.96, Chapter 4.5 --- Frequent methylation of RASA5 promoter in multiple primary tumors --- p.101, Chapter 4.6 --- Cloning and characterization of human RASA5 --- p.104, Chapter 4.7 --- RASA5 inhibits tumor cell clonogenicity through inducing apoptosis --- p.108, Chapter 4.8 --- RasGAP domain is required for the tumor suppressive function of RASA5 --- p.111, Chapter 4.9 --- Certain cancer types harbor wild type Ras but active Ras signaling, with RASA5 epigenetically silenced --- p.114, Chapter 4.10 --- RASA5 antagonizes Ras signaling pathway --- p.117, Chapter 4.10.1 --- RASA5 represses Ras signaling through downregulating Ras-GTP level --- p.117, Chapter 4.10.2 --- Oncogenic mutant form of Ras abrogated colony formation inhibitory effect of RASA5 on tumor cells --- p.120, Chapter 4.10.3 --- Knockdown of RASA5 promoted the tumor cell colony formation and Ras signaling activation --- p.122, Chapter 4.10.4 --- RASA5 inhibits ERK1/2 nuclei translocation and activation --- p.123, Chapter 4.10.5 --- RASA5 negatively regulates Ras target gene expression --- p.125, Chapter 4.11 --- RASA5 inhibits tumor cell migration and invasion through the Ras/Rac/cofilin signaling --- p.127, Chapter 4.12 --- RASA5 suppresses tumor cell epithelial-mesenchymal transition (EMT) and stemness --- p.133, Chapter 4.13 --- RASA5 appears in the cellcell interaction region nanotubes --- p.139, Chapter 4.14 --- Discussion --- p.141, Chapter Chapter 5: --- The Wnt/Dvl signaling antagonist TUSC-T2 is a pro-apoptotic tumor suppressor epigenetically silenced in tumors and inhibits tumor cell proliferation and migration --- p.150, Chapter 5.1 --- Expression of TUSC-T2 is downregulated in human tumors --- p.150, Chapter 5.2 --- TUSC-T2 promoter methylation results in its transcriptional inactivation --- p.151, Chapter 5.3 --- Cloning and characterization of TUSC-T2 --- p.155, Chapter 5.4 --- TUSC-T2 inhibits tumor cell clonogenicity through inducing apoptosis --- p.157, Chapter 5.5 --- TUSC-T2 inhibits Wnt/Dvl/β-catenin pathway --- p.161, Chapter 5.6 --- TUSC-T2 suppresses cell migration and EMT through upregulating E-cadherin --- p.165, Chapter 5.7 --- Discussion --- p.171, Chapter Chapter 6: --- Conclusions --- p.176, Chapter 6.1. --- RasGAP family member RASA5 is epigenetically silenced in human cancers, acting as a tumor suppressor through negatively regulating Ras signaling --- p.177, Chapter 6.2. --- DACT family member TUSC-T2 functions as a candidate TSG silenced by promoter methylation and inhibits Wnt/Dvl/β-catenin pathway --- p.178, Chapter Chapter 7: --- Future Studies --- p.181, Chapter 7.1. --- Further functional study of RASA5 and TUSC-T2 --- p.181, Chapter 7.2. --- Clinical application of epigenetic silenced candidate TSGs --- p.182, Chapter 7.3. --- Further screening of candidate TSGs as antagonists to cancer pathways --- p.183, Reference list --- p.184, http://library.cuhk.edu.hk/record=b5549467, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

大动脉闭塞性疾病是脑卒中常见病因，包括颅内外狭窄性血管病变。本研究旨在分析卒中患者颅内外大动脉狭窄斑块的血管造影特征以及相关治疗方案。进一步了解颅内斑块的形态学变化，以及颅外大动脉狭窄的血管造影特征和侧枝循环状态，对于研究其发病机制及临床治疗有指导意义。, 研究目的, 研究1：通过前瞻性纵向研究，利用三维旋转血管造影（3D-RA），探讨颅内斑块形态学变化。, 研究2：通过病例对照研究分析症状性放射性闭塞性血管病变(ORV)的血管造影特征及侧枝循环状态。, 研究3：通过病例对照研究分析放射性血管病变（RIV）患者进行颈动脉支架治疗（CAS）后血管造影特征及临床预后。, 研究方法, 研究1：24例颅内重度狭窄(>70%)的急性缺血性卒中患者，严格控制其危险因素，应用3D-RA研究其发病时及12个月后颅内斑块的形态学变化。, 研究2：分析96例ORV以及115例非放疗所致严重颈动脉狭窄（>70%）的缺血性卒中患者血管造影特点，比较其病变分布，形态学改变及侧枝循环状态。, 研究3：比较63例ORV以及87例动脉粥样硬化性颈动脉狭窄的卒中患者的血管造影及预后。主要终点事件包括短暂性脑缺血发作，卒中和死亡。次要终点事件为24个月时支架内再狭窄。, 结果, 研究1：颅内动脉粥样硬化性斑块的厚长比不能预测其稳定性。12个月的血管造影提示：13例（50%)斑块逆转；10例（38.5%)斑块无明显变化；3例（11.5%)斑块进展。, 研究2：ORV更多累及颈总动脉，多见双侧颈动脉受累（54% vs 22%)或出现闭塞（30% vs 9%），常见椎动脉受累(28% vs 14%)（均P<0.05）。ORV常见代偿性软脑膜动脉、前后交通动脉开放，及逆向眼动脉血流。, 研究3：两组间围手术期并发症，长期生存率和卒中复发率无统计学差异。, 结论, 研究1：3D-RA可评价颅内斑块形态学变化；颅内光滑斑块亦可为易损斑块。严格控制危险因素可能逆转斑块。, 研究2： ORV患者更多见颈动脉及椎基底动脉狭窄-闭塞性病变，并伴随侧枝循环开放。侧枝循环代偿功能减退可能诱导ORV患者发生卒中。, 研究3：RIV患者与对照组相比，CAS的耐受性和临床预后无明显差异。, Large artery occlusive disease, encompassing stenosis in intracranial and extracranial vasculature, is the most common stroke subtype worldwide. In this thesis, we aimed to investigate angiographic plaque morphology and treatments in stroke patients attributed to intracranial and/or extracranial stenosis. A better understanding of intracranial plaque morphology, angiographic characteristics and collateral circulations of extracranial occlusive vasculopathy may help clarify pathogenesis and formulate treatment., Objectives, Study 1: In this prospective longitudinal study, we investigated the intracranial plaque morphology of acute stroke patients by three-dimensional rotational angiography (3D-RA)., Study 2: We aimed to delineate the angiographic attributes and collateral circulations in symptomatic occlusive radiation vasculopathy (ORV) patients by a case-controlled study., Study 3: We investigated the angiographic and clinical outcome of carotid artery stenting (CAS) in stroke patients attributed to ORV., Methods, Study 1: Twenty-four patients with acute strokes attributed to a >70% intracranial stenosis were recruited to undergo 3D-RA at baseline and in 12 months after an intensive control of atherosclerotic risks. We described the degree of stenosis and morphology that might be associated with plaque vulnerability., Study 2: We performed digital subtraction angiograms (DSA) in 96 patients who had first-ever ischemic strokes attributed to ORV, and 115 referent patients who had no radiotherapy (RT) but symptomatic high-grade (>70%) atherosclerotic carotid stenoses. We compared the lesions’ distribution, morphology, and the resultant alteration of collateral flows in both patient groups., Study 3: We compared the angiographic and clinical outcome of CAS in 63 symtomatic ORV patients and 87 patients with spontaneous atheromatous carotid stenoses. Primary end-points were transient ischemic attack, stroke and death of all causes. Secondary end-point was instent restenosis in 24 months., Results, Study 1: Inracranial atherosclerotic plaque is a dynamic lesion.Thickness-to-length ratio may not indicate plaque vulnerability. In 12-month angiogram, 13 patients (50%) had plaque regression, 10 (38.5%) had static plaque, and 3 (11.5%) had plaque progression., Study 2: Compared with spontaneous atheromatous carotid disease, ORV lesions diffusely involved common carotid artery, and were more frequently bilateral (54% vs 22%), associated with complete occlusion in one or both carotid arteries (30% vs 9%), vertebral artery steno-occlusions (27% vs 14%) (all p<0.05). ORV patients showed more established collateral circulations through leptomeningeal arteries, anterior communicating artery, posterior communicating artery and retrograde flow in ophthalmic artery., Study 3: We found no significant differences in the frequency of periprocedural complications, the rates of patient survival and stroke recurrence between ORV and control groups., Conclusions, Study 1: Evaluation of intracranial plaque morphology is feasible with 3D-RA. Smooth plaques might also be vulnerable in intracranial vasculature. Intensive risk factor control may halt progression of intracranial plaques., Study 2: ORV patients had more steno-occlusions over carotid and vertebral arteries amid mature collateral circulations at initial stroke presentation. Decompensation of collateral flows may precipitate stroke in ORV., Study 3: The durability and clinical outcome of CAS in ORV patients were comparable to those in patients with spontaneous atherosclerotic carotid stenosis., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Zou, Xinying., Thesis (Ph.D.)--Chinese University of Hong Kong, 2012., Includes bibliographical references (leaves 101-119)., also in Chinese., p.i, 摘要 --- p.vi, DECLARATION OF ORIGINALITY --- p.ix, ACKNOWLEDGEMENTS --- p.x, PUBLICATIONS AND PRESENTATIONS --- p.xii, LIST OF ABBREVIATIONS --- p.xiv, LIST OF TABLES --- p.xvii, LIST OF FIGURES --- p.xviii, TABLE OF CONTENTS --- p.xx, Chapter CHAPTER 1 --- INTRODUCTION AND LITERATURE REVIEW --- p.1, Chapter 1.1 --- An overview of large artery occlusive disease in ischemic stroke --- p.1, Chapter 1.2 --- Vulnerable plaque and plaque morphology in ischemic stroke --- p.3, Chapter 1.2.1 --- Definition of vulnerable plaque and plaque morphology --- p.4, Chapter 1.2.2 --- Imaging of vulnerable plaques --- p.5, Chapter 1.2.3 --- Factors affecting plaque stability or arterial luminal narrowing --- p.7, Chapter 1.2.3.1 --- Stenosis severity --- p.7, Chapter 1.2.3.2 --- Thickness and length of plaque --- p.8, Chapter 1.2.3.3 --- Mechanical stress, shear stress and hemodynamics on plaque stability --- p.9, Chapter 1.2.3.4 --- Plaque eccentricity --- p.10, Chapter 1.2.3.5 --- Plaque surface morphology --- p.11, Chapter 1.2.4 --- Morphological characteristics of symptomatic plaque --- p.11, Chapter 1.2.4.1 --- Carotid plaque morphology --- p.11, Chapter 1.2.4.2 --- Intracranial plaque morphology --- p.12, Chapter 1.2.5 --- Treatment of vulnerable intracranial stenosis --- p.12, Chapter 1.3 --- Occlusive radiation vasculopathy (ORV) --- p.13, Chapter 1.3.1 --- Epidemiology of ORV --- p.13, Chapter 1.3.2 --- Pathogenesis and Pathophysiology of ORV --- p.14, Chapter 1.3.3 --- Imaging and angiographic characteristics of ORV --- p.16, Chapter 1.3.4 --- Collateralization in ORV --- p.18, Chapter 1.3.5 --- Angioplasty and stenting for ORV --- p.19, Chapter CHAPTER 2 --- OBJECTIVES --- p.22, Chapter CHAPTER 3 --- RECRUITMENT OF STUDY PARTICIPANTS --- p.24, Chapter CHAPTER 4 --- REGRESSION OF SYMPTOMATIC INTRACRANIAL PLAQUE BY INTENSIVE RISK FACTOR CONTROL: A LONGITUDIANL STUDY ON PLAQUE MORPHOLOGY BY 3D-ROTATIONAL ANGIOGRAPHY --- p.28, Chapter 4.1. --- Background and objectives --- p.28, Chapter 4.2 --- Methods --- p.32, Chapter 4.2.1 --- Participants --- p.32, Chapter 4.2.2 --- Risk factors and intensive control --- p.33, Chapter 4.2.3 --- Evaluation of intracranial stenosis --- p.33, Chapter 4.2.3.1 --- DSA and 3D-RA protocol --- p.33, Chapter 4.2.3.2 --- Severity of stenosis --- p.34, Chapter 4.2.3.3 --- Analysis of morphological characteristics on 3D-RA --- p.34, Chapter 4.2.3.4 --- Plaque regression --- p.35, Chapter 4.2.4 --- Statistical analysis --- p.36, Chapter 4.3 --- Results --- p.36, Chapter 4.4 --- Discussion --- p.54, Chapter CHAPTER 5 --- ANGIOGRAPHY DISTINCTIONS AND COLLATERALIZATION IN SYMPTOMATIC CRANIO-CERVICAL OCCLUSIVE RADIATION VASCULOPATHY: A CASE-REFERENT STUDY --- p.58, Chapter 5.1 --- Background and objectives --- p.58, Chapter 5.2 --- Methods --- p.59, Chapter 5.2.1 --- ORV and referent patients --- p.60, Chapter 5.2.2 --- Evaluation of vascular lesions and collateral status --- p.61, Chapter 5.2.3 --- Statistical analysis --- p.64, Chapter 5.3 --- Results --- p.64, Chapter 5.4 --- Discussion --- p.81, Chapter CHAPTER 6 --- SAFETY AND CLINICAL OUTCOME OF CAROTID ARTERY STENTING IN STROKE PATIENTS WITH OCCLUSIVE RADIATION VASCULOPATHY --- p.86, Chapter 6.1. --- Background and objectives --- p.86, Chapter 6.2 --- Methods --- p.87, Chapter 6.2.1 --- Participants --- p.87, Chapter 6.2.2 --- Baseline clinical assessment --- p.87, Chapter 6.2.3 --- Carotid artery stenting (CAS) --- p.88, Chapter 6.2.4 --- Follow-up and end-points --- p.89, Chapter 6.2.5 --- Statistical analysis --- p.89, Chapter 6.3. --- Results --- p.90, Chapter 6.4. --- Discussion --- p.96, Chapter CHAPTER 7 --- CONCLUSIONS --- p.98, REFERENCES --- p.101, http://library.cuhk.edu.hk/record=b5549535, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

鈣粘蛋白是一類通過影響細胞粘附和細胞信號通路在腫瘤發生中起重要作用的細胞間粘附分子。鈣粘蛋白超家族包括經典鈣粘蛋白和非經典鈣粘蛋白，其中非經典鈣粘蛋白包含了原鈣粘蛋白。啟動子CpG甲基化調控下的基因沉默或表達下調是腫瘤發生中一個關鍵事件的觀點現已得到廣泛認可。一些鈣粘蛋白家族成員，如鈣粘蛋白-1/4/13（CDH1，CDH4，CDH13）被已有研究報導是受擬遺傳學調控沉默的功能性腫瘤抑制基因。本研究主要針對兩個鈣粘蛋白家族成員鈣粘蛋白-11（CDH11）和原鈣粘蛋白-10（PCDH10）進行腫瘤發生相關功能和機制的研究。, CDH11位於雜合性缺失（LOH）經常發生而預示可能存在抑癌基因的染色體16q21-22區域，我們實驗室先前通過基因組芯片雜交技術（aCGH）對腫瘤細胞系的研究已發現它是該區域一個可能的抑癌基因。我們通過進一步的半定量反轉錄聚合酶鏈反應（RT-PCR）發現CDH11在正常組織和永生化正常上皮細胞中廣泛表達，但在各腫瘤細胞系中表達下降。甲基化特異性聚合酶鏈反應（MSP）和亞硫酸氫鹽處理的基因組測序（BGS）檢測到CDH11啟動子甲基化常發生于腫瘤細胞和腫瘤組織中。在CDH11表達缺失的腫瘤細胞中重新導入該基因的表達可顯著減少細胞克隆的形成，誘導細胞凋亡并抑制腫瘤細胞的遷移。通過更深入的機制研究，我們發現CDH11通過抑制Wnt/β-catenin信號通路發揮功能。, 本研究的另一個鈣粘蛋白家族成員是PCDH10。之前我們實驗室的工作已經證實了PCDH10是一個在鼻咽癌和食管癌中受啟動子甲基化調控的抑癌基因,這裡我們主要研究它在大腸癌發病中的功能和機制。我們發現在PCDH10表達缺失的大腸癌細胞中重新導入PCDH10表達可顯著抑制腫瘤細胞的克隆形成，細胞遷移和幹細胞性。機制研究顯示PCDH10抑制Wnt/β-catenin和RhoA信號轉導通路，并進一步抑制腫瘤上皮細胞-間充質轉化（EMT）的過程，誘導幹細胞標記的下調。, 綜上所述，本研究顯示CDH11和PCDH10兩個鈣粘蛋白家族成員在多種腫瘤中廣泛受甲基化調控失活，它們是重要的Wnt/β-catenin信號通路拮抗因素，可抑制腫瘤細胞的克隆形成和細胞遷移, Cadherins are an important group of cell-cell adhesion molecules, which play crucial roles in tumorigenesis by affecting cell adhesion and cell signaling. Cadherin superfamily consists of classical cadherins and non-classical cadherins including protocadherins. It has been well recognized that silencing or downregulation of tumor suppressor genes (TSGs) by promoter CpG methylation is a critical event in human tumorigenesis. Some cadherin family members, such as CDH1, CDH4, CDH13, have been reported as functional TSGs silenced through epigenetic regulation. In this study, I mainly focus on the function and mechanism studies of two cadherin members-Cadherin 11(CDH11) and Protocadherin 10 (PCDH10)., CDH11 is located in 16q21-22, a region with frequent loss of heterozygosity (LOH), indicating the presence of candidate TSG. Previously, our lab also identified CDH11 as a candidate TSG through array-CGH of tumor cell lines. I further found by semi-quantitative RT-PCR that CDH11 was broadly expressed in normal tissues while frequently downregulated in multiple tumor cell lines, but not in immortalized normal epithelial cells. Methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS) detected frequent promoter methylation of CDH11 in tumor cell lines and primary tumor samples. Ectopic expression of CDH11 dramatically reduced tumor cell clonogenecity, induced tumor cell apoptosis and inhibited tumor cell migration. By further mechanism study, I found that CDH11 is a negative inhibitor of Wnt/β-catenin signaling pathway., Another cadherin family protein which I chose to study is PCDH10. Previously our lab identified PCDH10 as a TSG by promoter methylation in nasopharyngeal and esophageal carcinomas. I studied the function and mechanism of PCDH10 in the pathogenesis of colon cancer. I found ectopic expression of PCDH10 strongly suppressed colon tumor cell clonogenecity, migration and stemness. Moreover, I found that PCDH10 repressed Wnt/β-catenin and RhoA signaling, thus further inhibited the epithelial-to-mesenchymal transition (EMT) of tumor cells and downregulated stem cell markers., In summary, this study demonstrates two cadherin family members CDH11 and PCDH10, as important antagonists to Wnt/β-catenin signaling pathway, suppress tumor cell clonogenecity, migration, and are also frequently inactivated by epigenetic mechanism in multiple tumors., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Zhang, Yanjiao., Thesis (M.Phil.)--Chinese University of Hong Kong, 2012., Includes bibliographical references (leaves 84-95)., s also in Chinese., p.i, Acknowledgements --- p.iii, Table of Contents --- p.vi, List of Tables --- p.x, List of Figures --- p.xi, List of Abbreviations --- p.xiii, List of Publications --- p.xvi, Chapter Chapter 1 --- Introduction and Literature Review --- p.1, Chapter 1.1 --- Cancer --- p.1, Chapter 1.1.1 --- General introduction about cancer --- p.1, Chapter 1.1.2 --- Oncogenes and TSGs --- p.3, Chapter 1.1.3 --- Cancer mechanism models --- p.3, Chapter 1.2 --- Cancer Epigenetics --- p.5, Chapter 1.2.1 --- DNA methylation --- p.6, Chapter 1.2.2 --- DNA methylation and gene silencing --- p.7, Chapter 1.2.3 --- DNA methylation and cancer --- p.7, Chapter 1.2.4 --- Clinical implications of DNA methylation --- p.8, Chapter 1.3 --- Cadherins --- p.10, Chapter 1.3.1 --- General introduction of cadherin superfamily --- p.10, Chapter 1.3.2 --- Cadherin classification --- p.10, Chapter 1.3.3 --- Cadherin and cancers --- p.12, Chapter 1.3.4 --- Cadherin switch and EMT in cancer --- p.16, Chapter 1.4 --- Wnt/β-catenin signaling pathway and cancer --- p.16, Chapter 1.4.1 --- Wnt/β-catenin signaling pathway --- p.16, Chapter 1.4.2 --- Wnt/β-catenin signaling pathway in cancer --- p.18, Chapter 1.4.3 --- Epigenetic silencing of Wnt/β-catenin signaling --- p.20, Chapter 1.4.4 --- Wnt/β-catenin signaling pathway in CRC --- p.21, Chapter Chapter 2 --- Aims of the Study --- p.22, Chapter Chapter 3 --- Materials and Methods --- p.25, Chapter 3.1 --- Cell lines and tissue samples --- p.25, Chapter 3.1.1 --- Cell lines --- p.25, Chapter 3.1.2 --- Maintenance of cell lines --- p.25, Chapter 3.1.3 --- Drug treatment of cell lines --- p.26, Chapter 3.1.4 --- Normal and primary tissues --- p.26, Chapter 3.1.5 --- Total RNA extraction --- p.27, Chapter 3.1.6 --- Genomic DNA extraction --- p.28, Chapter 3.2 --- Gene expression analysis --- p.29, Chapter 3.2.1 --- Reverse transcription (RT) --- p.29, Chapter 3.2.2 --- Semi-quantitative RT-PCR --- p.30, Chapter 3.3 --- Methylation Analysis --- p.32, Chapter 3.3.1 --- CpG island prediction --- p.32, Chapter 3.3.2 --- Sodium bisulfite treatment of genomic DNA --- p.33, Chapter 3.3.3 --- Methylation-specific PCR (MSP) --- p.33, Chapter 3.3.4 --- Bisulfite Genomic Sequencing (BGS) --- p.34, Chapter 3.4 --- Construction of expression plasmids --- p.36, Chapter 3.4.1 --- Construction of CDH11 expression vector --- p.36, Chapter 3.4.2 --- Construction of PCDH10 expression plasmid --- p.38, Chapter 3.4.3 --- Plasmid extraction --- p.39, Chapter 3.5 --- Plasmid transfection --- p.41, Chapter 3.6 --- Subcellular localization --- p.42, Chapter 3.7 --- Function analyses --- p.43, Chapter 3.7.1 --- Colony formation assay --- p.43, Chapter 3.7.2 --- Wound healing assay --- p.44, Chapter 3.8 --- Mechanism exploration --- p.44, Chapter 3.8.1 --- Protein extraction and western-blot --- p.44, Chapter 3.8.2 --- Dual-luciferase reporter assay --- p.47, Chapter 3.9 --- Statistical analysis --- p.48, Chapter Chapter 4 --- CDH11 functions as a tumor suppressor via modulating Wnt/β-catenin signaling and is frequently downregulated by promoter methylation --- p.49, Chapter 4.1 --- The CpG island of CDH11 gene promoter --- p.50, Chapter 4.2 --- CDH11 expression profile in normal tissues --- p.50, Chapter 4.3 --- Frequent silencing of CDH11 by promoter methylation in multiple tumors --- p.51, Chapter 4.4 --- Restoration of CDH11 expression by pharmacologic demethylation --- p.53, Chapter 4.5 --- Frequent CDH11 methylation in primary tumors --- p.54, Chapter 4.6 --- Function studies --- p.56, Chapter 4.6.1 --- Ectopic expression of CDH11 inhibited tumor cell clonogenecity --- p.57, Chapter 4.6.2 --- CDH11 induced tumor cell apoptosis --- p.57, Chapter 4.6.3 --- CDH11 inhibited tumor cell migration --- p.58, Chapter 4.7 --- CDH11 antagonized Wnt/β-catenin signaling --- p.59, Chapter 4.8 --- Discussion --- p.60, Chapter Chapter 5 --- Epigenetic inactivated tumor suppressor PCDH10 exerts tumor suppressive functions through modulating Wnt/β-catenin signaling and cell stemness in colon cancer --- p.66, Chapter 5.1 --- PCDH10 was broadly expressed in normal tissues and frequently silenced in CRC cell lines --- p.67, Chapter 5.2 --- Promoter methylation mediated PCDH10 silencing in CRC cell lines --- p.68, Chapter 5.3 --- Frequent PCDH10 methylation in CRC primary tumors --- p.69, Chapter 5.4 --- PCDH10 was located in cytoplasm and membrane --- p.70, Chapter 5.5 --- Function Studies --- p.71, Chapter 5.5.1 --- PCDH10 inhibited clonogenicity of tumor cells --- p.71, Chapter 5.5.2 --- PCDH10 suppressed tumor cell mobility --- p.72, Chapter 5.6 --- Mechanism exploration of PCDH10 in CRC --- p.72, Chapter 5.6.1 --- PCDH10 antagonized Wnt/β-catenin signaling --- p.72, Chapter 5.6.2 --- PCDH10 negatively regulated EMT and stemness of tumor cells --- p.74, Chapter 5.6.3 --- PCDH10 inhibited RhoA signaling --- p.75, Chapter 5.7 --- Discussion --- p.75, Chapter Chapter 6 --- Conclusions and Future studies --- p.80, Chapter 6.1 --- Conclusions --- p.80, Chapter 6.2 --- Future studies --- p.82, Reference List --- p.84, http://library.cuhk.edu.hk/record=b5549114, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

儘管心力衰竭的診斷和治療已取得了長足進步，但是心力衰竭依然是目前主要的致殘和致死病因。而且，隨著人口的老齡化，心力衰竭的發病率不斷上升。然而心力衰竭的快速診斷、心功能評價以及患者的危險分層依然面臨眾多挑戰。Acoustic cardiography 是一項經濟簡單的新技術。憑藉獨有的雙功能感測器，這項技術可以同時評估收縮間期（systolic time intervals）以及舒張期心音（diastolic heart sounds）。這項技術提供的主要參數包括：第三心音分數（S3 score；第三心音存在的可能性），電機械時間（EMAT, electromechanical activation time；從心電圖Q 波到心音圖第一心音的時間）及電機械時間比例（%EMAT；電機械時間占整個心動週期的比例），收縮障礙指數（SDI, systolic dysfunctionindex）。本論文主要涵蓋Acoustic cardiography 在心力衰竭患者中如下三個方面 的應用：, 一、心力衰竭的診斷和不同亞型的識別, 本研究入組了 94 名高血壓但無心力衰竭患者、109 名射血分數正常的心力衰竭患者以及89 名射血分數減低的心力衰竭患者，我們發現%EMAT 可以鑒別射血分數正常的心力衰竭和高血壓患者。另一方面，SDI 是鑒別分射血分數正常和射血分數減低患者的最好指標。, 二、心力衰竭患者心功能障礙嚴重程度評估, 此研究共招募 94 名高血壓患者和127 名射血分數減低的心力衰竭患者。結果顯示：SDI 可以鑒別射血分數減低的心力衰竭和高血壓患者。亞組分析顯示：SDI 可以區分射血分數嚴重減低和中度減低的心力衰竭患者；S3 score 可以識別伴舒張功能嚴重障礙的心力衰竭患者。, 三、心力衰竭患者的危險分層, 共計 474 名心力衰竭患者被納入此研究，平均隨訪時間484±316 天，169名患者死亡，其中125 名死於心臟病。SDI 和S3 score 都是全因死亡率的獨立預測因數；Kaplan Meier 分析顯示：SDI ≥ 5 或S3 score ≥ 4.12 的心力衰竭患者的生存率顯著降低。, 通過以上三個方面的研究，我們發現這項新技術有助於（1）心力衰竭的診斷和不同亞型的識別；（2）評估心力衰竭患者的心功能障礙嚴重程度，進而發現其中的高危人群；（3）心力衰竭患者的危險分層。因此，這項新技術有望在心力衰竭患者的管理中扮演早期診斷、評估以及危險分層的重要角色。, Despite recent advances in its management, heart failure remains a major cause of disability and death and its prevalence is still increasing as the population ages. However, rapid and accurate bedside diagnosis, evaluation as well as risk stratification of heart failure still remain challenging., Acoustic cardiography (AUDICOR, Inovise Medical, Inc., Portland, OR, USA) is a novel and user friendly equipment which can be used in a wide variety of clinical conditions. With proprietary dual-functional sensors, this technology permits simultaneous acquisition of detailed information regarding systolic time intervals and diastolic heart sounds and provides a computerized interpretation of the findings. Major acoustic cardiographic parameters include S3 score (probability that the third heart sound exists), electromechanical activation time (EMAT, interval from Q wave to the first heart sound; %EMAT is the proportion of cardiac cycle that EMAT occupies), and systolic dysfunction index (SDI= exp [S3 score/10] x QRS interval x QR interval x %EMAT).This thesis will cover 3 aspects of clinical application of acoustic cardiography in heart failure patients., I. Identification of heart failure and its phenotypes, We performed one study involving 94 patients with hypertension without heart failure, 109 patients with heart failure with normal ejection fraction (HFNEF, EF > 50%) and 89 patients with heart failure and reduced ejection fraction (HFREF, EF < 50%). We found that %EMAT significantly differentiated HFNEF from hypertension. Whereas SDI out-performed the other acoustic cardiographic parameters in differentiating HFREF from HFNEF., II. Assessment of HFREF patients at high risk by evaluating the severity of left ventricular (LV) systolic and diastolic dysfunction, Ninety-four hypertensive patients without heart failure and 127 HFREF patients (EF < 50%) were consecutively recruited for the study. SDI significantly differentiated HFREF from hypertension. In subgroup analysis, SDI discriminated HFREF patients with severely impaired EF (EF ≤ 35%) from those with moderately impaired EF (35% < EF <50%). S3 score > 4.67 identified HFREF patients with restrictive LV filling pattern., III. Risk stratification in patients with heart failure, A total of 474 patients hospitalized for heart failure were enrolled into our study. During a mean follow-up time of 484±316 days, 169 (35.7%) patients died and 125 (26.4%) of them died of cardiac causes. After controlling for other potential confounders, we found that S3 score ≥ 4.12, and SDI ≥ 5 were both independent predictors for all-cause mortality. Kaplan-Meier analysis showed that heart failure patients with SDI ≥ 5 or S3 score ≥ 4.12 had a significantly lower survival rate than those with lower SDI or S3 score., In summary, this bedside technology offers a wide variety of clinical applications in (1) identification of heart failure and its phenotypes; (2) assessmet of HFREF patients at high risk by evaluating the severity of LV systolic and diastolic dysfunction; (3) risk stratification in patients with heart failure. Thus, acoustic cardiography is likely to be helpful in the management of heart failure patients, acting as an early detection, evaluation and risk-stratification tool., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Wang, Shang., Thesis (Ph.D.)--Chinese University of Hong Kong, 2012., Includes bibliographical references (leaves 123-135)., also in Chinese., DECLARATION OF ORIGINALITY --- p.i, ACKNOWLEDGEMENTS --- p.ii, PUBLICATIONS RELATED TO THIS THESIS --- p.iv, Full publications --- p.iv, Conference presentations --- p.v, TABLE OF CONTENTS --- p.vi, LIST OF TABLES --- p.xi, LIST OF FIGURES --- p.xiii, LIST OF ABBREVIATIONS --- p.xv, p.xviii, 論文摘要 --- p.xx, Chapter PART I --- LITERATURE REVIEW --- p.1, Chapter Chapter 1 --- Introduction to Acoustic Cardiography --- p.2, Chapter 1.1 --- History of auscultation, phonocardiography --- p.2, Chapter 1.2 --- STIs --- p.3, Chapter 1.2.1 --- Conventional STIs --- p.3, Chapter 1.1.2 --- Echocardiographic STI --- p.5, Chapter 1.3 --- Acoustic cardiography --- p.7, Chapter 1.3.1 --- ECG parameters of acoustic cardiography --- p.11, Chapter 1.3.2 --- Systolic parameters of acoustic cardiography --- p.12, Chapter 1.3.3 --- Diastolic Parameters of acoustic cardiography --- p.13, Chapter 1.4 --- Comparison between acoustic cardiography and traditional phonocardiography --- p.19, Chapter Chapter 2 --- Clinical Application of Acoustic Cardiography --- p.27, Chapter 2.1 --- Mechanism of generation of S3 and S4 --- p.27, Chapter 2.2 --- Prevalence of S3 and S4 --- p.28, Chapter 2.3 --- Clinical auscultation of S3 and S4 problems --- p.29, Chapter 2.4 --- Rapid identification of heart failure or LV dysfunction --- p.32, Chapter 2.4.1 --- S3 and S4 --- p.32, Chapter 2.4.2 --- EMAT --- p.33, Chapter 2.4.3 --- SDI --- p.34, Chapter 2.4.5 --- Other derived acoustic cardiographic parameters --- p.34, Chapter 2.5 --- Predicting elevated LV filling pressure --- p.35, Chapter 2.6 --- Improving diagnostic utility of BNP in detection of heart failure or LV dysfunction --- p.36, Chapter 2.7 --- Hemodynamic correlations of acoustic cardiographic parameters --- p.37, Chapter 2.8 --- Prognostic value of acoustic cardiography --- p.38, Chapter 2.9 --- Cardiac resynchronization therapy --- p.39, Chapter 2.10 --- Detection of ischemia --- p.40, Conclusions --- p.42, Chapter PART II --- STUDIES ON APPLICATION OF ACOUSTIC CARDIOGRAPHY --- p.48, Chapter Chapter 3 --- Acoustic Cardiography Helps to Identify Heart Failure and Its Phenotypes --- p.49, Introduction --- p.49, Methods --- p.50, Participants and study design --- p.50, Echocardiography --- p.51, Acoustic cardiography --- p.52, Assessment of reproducibility --- p.55, Statistical analysis --- p.55, Results --- p.56, Characteristics of study subjects --- p.56, Acoustic cardiographic and echocardiographic characteristics --- p.59, Diagnostic characteristics of acoustic cardiography --- p.64, Analysis of covariance results --- p.68, Inter-operator reproducibility --- p.68, Discussion --- p.68, Chapter Chapter 4 --- Rapid Bedside Identification of High-Risk Population in Heart Failure with Reduced Ejection Fraction by Acoustic Cardiography --- p.72, Introduction --- p.72, Methods --- p.73, Study population --- p.73, Echocardiography --- p.73, Acoustic cardiography --- p.74, Assessment of reproducibility --- p.74, Statistical analysis --- p.74, Results --- p.75, Baseline characteristics of study subjects --- p.75, Acoustic cardiographic and echocardiographic characteristics --- p.78, Diagnostic test characteristics of acoustic cardiography --- p.84, Analysis of covariance results --- p.89, Inter-operator reproducibility --- p.89, Discussion --- p.89, Chapter Chapter 5 --- Prognostic value of Acoustic Cardiography in Risk Stratification of Patients With Heart Failure --- p.93, Introduction --- p.93, Methods --- p.94, Study population --- p.94, Acoustic cardiography --- p.94, Echocardiography --- p.94, Endpoint --- p.95, Assessment of reproducibility --- p.95, Statistical analysis --- p.95, Results --- p.96, Study population --- p.96, All-cause mortality --- p.100, Cardiac death --- p.100, Subgroup analysis in 232 patients undergoing echocardiography --- p.107, Inter-operator reproducibility --- p.107, Discussion --- p.114, Strengths and potential limitations --- p.115, Chapter PART III --- CONCLUSIONS --- p.117, Chapter Chapter 6 --- Summary of the Present Studies --- p.118, Chapter I. --- Identification of heart failure and its phenotypes --- p.118, Chapter II. --- Assessment of HFREF patients at high risk by evaluating the severity of LV systolic and diastolic dysfunction --- p.119, Chapter III. --- Risk stratification in patients with heart failure --- p.119, Chapter Chapter 7 --- Future Research Directions --- p.121, References --- p.123, http://library.cuhk.edu.hk/record=b5549431, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

抑制腫瘤的基因（又稱抑癌基因）之表達失活，是導致癌變的重要機制之一。除了基因突變之外，越來越多研究證明抑癌基因的關閉轉錄，是由於抑癌基因啟動子區的CpG島甲基化所致。本論文的研究確定了兩個候選抑癌基因ADAMTS8和TUSC14，在多種腫瘤細胞株中經常因動子區的CpG島甲基化而下調或停止表達，這有別於它們在正常組織中廣泛表達的情況。沉默細胞株在脫氧核糖核酸甲基轉移酶的抑製劑5-氮-2'-脫氧胞苷(5-aza-2′-deoxycytidine; Aza) 或與組蛋白去乙酰化酶抑製劑曲古抑菌素A (trichostatin A, TSA)的去甲基化作用下，能恢復這兩個抑癌基因的表達，因而證明了啟動子甲基化是直接導致其表達下調及沉默的機制。, 論文的第一部分，主要調查ADAMTS8啟動子區在原發腫瘤樣本被甲基化的比率，並研究其腫瘤抑制功能。含血小板凝血酶敏感蛋白基序的解整聯蛋白金屬蛋白酶 (ADAMTSs) ，在各種癌症中的表達異常已有報導。然而，它們在腫瘤的職能作用仍然模糊不清。本研究發現，異位表達ADAMTS8誘導細胞凋亡，因而顯著抑制腫瘤細胞克隆形成的能力,。這些都突顯其抑制腫瘤的功能。此外，作為分泌蛋白酶的ADAMTS8，能夠透過減少表皮生長因子受體(EGFR) 蛋白的磷酸化，抑制EGFR / MEK / ERK信號通路，並進一步破壞肌動蛋白應力纖維的組織，抑制腫瘤細胞的遷移性。, 論文的第二部分，集中於研究一個未知功能的基因TUSC14，這基因的蛋白質編碼具有氨基末端蛋白質相互作用域 (BTB/POZ domain)及C₂H₂乙炔鋅指結構。TUSC14的異位表達能抑制腫瘤細胞克隆的形成，但這種抑制作用會在删除蛋白中的BTB/POZ或C₂H₂乙炔鋅指結構功能域後消失。因此證實了TUSC14蛋白同時需要BTB / POZ和C₂H₂乙炔鋅指結構兩個功能域來抑制腫瘤生長。此外，TUSC14具有抑制NF-kB轉錄的功能，其功能不但依賴於组蛋白去乙酰基酶(HDAC)，並且與c-MYC和cIAP-2等NF-κB靶基因下調表達相關。TUSC14的抑癌功能，包括抑制腫瘤生長與增加細胞凋亡，與其減少c-MYC及抗凋亡基因cIAP-2的表達，效果一致。進一步的分析發現，TUSC14與HDAC1和P65於蛋白質複雜免疫共沉澱實驗中，有物理相互作用。此外，染色質免疫沉澱實驗顯示TUSC14透過與c-MYC和cIAP-2的相互作用，抑制其基因啟動子區的轉錄功能。結果表明，TUSC14是通過招募HDAC至NF-κB靶基因的啟動子區這機制，來抑制NF-kB靶基因的轉錄，以達至抑制癌細胞生長和誘導癌細胞凋亡的效果。因此，TUSC14的沉默是破壞癌細胞中NF-kB信號通路負調控(negative regulation)的重要因素。, 綜上所述，本研究鑒定了兩個在多種腫瘤細胞因表觀遺傳沉默效應而表達下調或沉默的抑癌基因ADAMTS8和TUSC14，並證實它們具有抑癌功能。, Inactivation of tumor suppressor genes (TSGs) is one of the critical mechanisms leading to carcinogenesis. Apart from genetic mutations, a growing number of TSG has been shown to be silenced through promoter CpG methylation. In this thesis, we identified two candidate TSGs: ADAMTS8 and TUSC14 that are frequently downregulated or silenced in multiple carcinoma cell lines by promoter methylation while broadly expressed in normal tissues. Expression of these two genes was restored after treatment with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (Aza) or in combination with a histone deacetylase inhibitor trichostatin A (TSA), suggesting promoter-methylation directly contributes to their silencing., In the first part of the thesis, prevalence silencing of ADAMTS8 was detected in primary tumor samples. Expression of many disintegrins and metalloproteinases with thrombospondin motifs (ADAMTSs) was reported to be dysregulated in various cancers. However, their functional roles in tumorigenesis remain obscure. This study revealed that ectopic expression of ADAMTS8 markedly inhibits tumor cell clonogenicity by inducing apoptosis, underscoring its function as a tumor suppressor. Furthermore, ADAMTS8, as a secreted protease, inhibits EGFR/MEK/ERK signaling pathway by reducing their phosphorylation, further resulting in the disruption of actin stress fiber organization and suppression of tumor cell motility., The second part of the thesis focused on a novel gene TUSC14 which encodes a protein with BTB/POZ domain and C₂H₂zinc-fingers. Ectopic expression of TUSC14 suppresses colony formation of cancer cells but this inhibitory effect is abolished with the deletion of BTB/POZ domain or C₂H₂ zinc-fingers. This suggested that both BTB/POZ domain and C₂H₂ zinc-fingers are required for inhibiting tumor cell clonogenecity. In addition, TUSC14 functions as a transcriptional repressor of NF-kB pathway that is dependent on HDAC. Suppression of NF-κB transcriptional activity by TUSC14 expression correlates with the downregulation of NF-κB target genes including c-MYC and cIAP-2. Reduction of c-MYC and anti-apoptotic cIAP-2 agrees well with the consequent growth suppression and enhanced apoptosis following the ectopic expression of TUSC14. Further analyses showed TUSC14 physically interacts with HDAC1 and p65 via co-immunoprecipitation assay. Preliminary ChIP assay showed that TUSC14 associates with gene promoters of c-MYC and cIAP-2 for their transcription repressions. These results revealed that TUSC14 represses NF-kB activity through recruiting HDAC to the NF-kB target genes; and transcription repression of NF-kB represents a mechanism for TUSC14 to mediate its growth inhibitory and apoptosis-inducing effects in cancer. Hence, silencing of TUSC14 contributes to the lost of negative regulation on NF-kB signaling in cancer., In summary, this study demonstrated that ADAMTS8 and TUSC14 are functional tumor suppressors that are epigenetically silenced in multiple tumors., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Choi, Ching Gee., Thesis (Ph.D.)--Chinese University of Hong Kong, 2012., Includes bibliographical references (leaves 140-153)., also in Chinese., p.i, Chinese abstract --- p.iv, AcknowledgEments --- p.vii, List of Figures --- p.ix, List of Tables --- p.xi, LIST OF ABBREVIATIONS --- p.xii, List of PUBLICATIONs --- p.xiv, Chapter CHAPTER 1 --- Introduction --- p.1, Chapter 1.1 --- Overview of cancer epigenetics --- p.1, Chapter 1.2 --- Epigenetic events --- p.2, Chapter 1.2.1 --- DNA methylation --- p.2, Chapter 1.2.2 --- Histone modifications --- p.5, Chapter 1.2.3 --- The interdependence of DNA methylation and histone modifications --- p.8, Chapter 1.3 --- Epigenetic alterations in cancer --- p.9, Chapter 1.3.1 --- Genome-wide DNA hypomethylation --- p.9, Chapter 1.3.2 --- CpG island promoter hypermethylation silencing of tumour suppressor genes in tumorigenesis --- p.10, Chapter 1.3.3 --- Aberrations of histone modifications --- p.11, Chapter 1.4 --- Causes for epigenetic deregulation in cancer --- p.14, Chapter 1.5 --- The interplay of genetic and epigenetic aberration in cancer progression --- p.21, Chapter 1.6 --- Epigenetic inactivation of tumor suppressor genes in cancer --- p.23, Chapter 1.7 --- Clinical implications of epigenetic research --- p.27, Chapter 1.7.1 --- Epigenetic modifications as biomarker for cancer diagnosis --- p.27, Chapter 1.7.2 --- Targeting epigenetic modifications as therapeutics towards cancers --- p.29, Chapter 1.8 --- Roles of ADAMTS proteins in cancer --- p.32, Chapter 1.8.1 --- Introduction on ADAMTS metalloproteases --- p.32, Chapter 1.8.2 --- Deregulation of ADAMTS protein in cancer --- p.34, Chapter 1.9 --- Roles of BTB/POZ-ZF family of transcription factors in cancer --- p.36, Chapter 1.9.1 --- Introduction on BTB/POZ-ZF Family --- p.36, Chapter 1.9.2 --- BTB/POZ-ZF functions as transcription repressors --- p.37, Chapter 1.9.3 --- Many BTB/POZ-ZF proteins are important players in tumorigenesis --- p.39, Chapter 1.9.4 --- The role of BTB/POZ-ZF in tumor initiation and progression --- p.40, Chapter CHAPTER 2 --- Aims of this study --- p.44, Chapter CHAPTER 3 --- General Methodology --- p.46, Chapter 3.1 --- Cell Culture --- p.46, Chapter 3.1.1 --- Growth and maintenance of cells --- p.46, Chapter 3.1.2 --- Mammalian cell transfection --- p.46, Chapter 3.1.3 --- Drug and stress treatments --- p.47, Chapter 3.2 --- DNA and RNA extraction --- p.47, Chapter 3.3 --- Semi-quantitative RT-PCR and Real-time PCR --- p.48, Chapter 3.4 --- CpG island and Transcription factor binding sites analysis --- p.49, Chapter 3.5 --- Methylation-specific PCR (MSP) and Bisulfite genomic sequencing (BGS) --- p.49, Chapter 3.6 --- Bacterial transformation and Plasmid extraction --- p.50, Chapter 3.6.1 --- Heat-shock transformation --- p.50, Chapter 3.6.2 --- Mini-scale preparation of plasmid DNA --- p.51, Chapter 3.6.3 --- Preparation of endotoxin-free plasmids --- p.52, Chapter 3.7 --- DNA cycle sequencing --- p.52, Chapter 3.8 --- Indirect immunofluorescence for subcellular localization study --- p.54, Chapter 3.9 --- Colony formation assay --- p.54, Chapter 3.10 --- Cell cycle analysis --- p.55, Chapter 3.11 --- Apoptosis assay --- p.56, Chapter 3.12 --- Co-immunoprecipitation and Western blot --- p.56, Chapter 3.13 --- Chromatin immunoprecipitation (ChIP) --- p.58, Chapter 3.14 --- Dual Firefly and Renilla luciferase reporter gene assay --- p.59, Chapter 3.15 --- Statistical analysis --- p.60, Chapter CHAPTER 4: --- Characterization of the Tumor Suppressive Functions of ADAMTS8 --- p.61, Chapter 4.1 --- Introduction --- p.61, Chapter 4.2 --- Materials and Methods --- p.64, Chapter 4.2.1 --- Tumor samples --- p.64, Chapter 4.2.2 --- Expression of ADAMTS8 --- p.64, Chapter 4.2.3 --- Immunofluorescence staining of ADAMTS8 --- p.64, Chapter 4.2.4 --- Detection of secreted ADAMTS8 in culture medium --- p.65, Chapter 4.2.5 --- Collection of conditioned medium and Western Blotting --- p.66, Chapter 4.2.6 --- Wound healing assay --- p.66, Chapter 4.3 --- Result and Discussion --- p.69, Chapter 4.3.1 --- Frequent ADAMTS8 methylation in primary carcinomas --- p.69, Chapter 4.3.2 --- ADAMTS8 is a secreted protease --- p.70, Chapter 4.3.3 --- ADAMTS8 inhibits phosphorylation of pEGFR --- p.73, Chapter 4.3.4 --- ADAMTS8 suppresses cell migration --- p.77, Chapter 4.4 --- Summary --- p.81, Chapter CHAPTER 5: --- Epigenetic Alterations of TUSC14 Gene in multiple carcinomas --- p.83, Chapter 5.1 --- Introduction --- p.83, Chapter 5.2 --- Materials and Methods --- p.84, Chapter 5.2.1 --- Cell lines --- p.84, Chapter 5.2.2 --- Normal and primary tumor tissues --- p.85, Chapter 5.3 --- Results and Discussion --- p.86, Chapter 5.3.1 --- Expression profiling of TUSC14 in normal tissues and tumor cell lines --- p.86, Chapter 5.3.2 --- Frequent inactivation of TUSC14 by promoter CpG methylation --- p.90, Chapter 5.3.3 --- Pharmacologic and genetic demethylation restores TUSC14 expression --- p.94, Chapter 5.3.4 --- Frequent TUSC14 methylation in primary tumors --- p.96, Chapter 5.4 --- Summary --- p.98, Chapter CHAPTER 6 --- Characterization of the Tumor Suppressive Functions of TUSC14 --- p.99, Chapter 6.1 --- Introduction --- p.91, Chapter 6.2 --- Materials and Methods --- p.100, Chapter 6.2.1 --- Gene cloning and plasmids construction of TUSC14 --- p.100, Chapter 6.2.2 --- Drug and stress treatments of cells --- p.100, Chapter 6.3 --- Results and Discussion --- p.102, Chapter 6.3.1 --- TUSC14 localizes to nuclear speckles --- p.102, Chapter 6.3.2 --- TUSC14 inhibits clonogenicity --- p.107, Chapter 6.3.3 --- Expression of TUSC14 induces apoptosis in tumor cells --- p.110, Chapter 6.3.4 --- TUSC14 alters cell cycle progression --- p.112, Chapter 6.3.5 --- TUSC14 acts as a transcriptional repressor of multiple genes --- p.114, Chapter 6.3.6 --- TUSC14 represses NF-кB activity through an HDAC-dependent mechanism --- p.118, Chapter 6.3.7 --- The effect of TUSC14 on the expression of downstream targets of NF-κB Signaling --- p.120, Chapter 6.3.8 --- TUSC14 co-immunoprecipitates with HDAC1 and p65 --- p.124, Chapter 6.3.9 --- ChIP analysis of promoters of TUSC14-regulated genes --- p.127, Chapter 6.4 --- Summary --- p.130, Chapter CHAPTER 7 --- General Discussion --- p.133, Chapter CHAPTER 8 --- Conclusions --- p.138, References --- p.140, http://library.cuhk.edu.hk/record=b5549491, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

微小RNA是内源性小非编码RNA，在肿瘤生成中扮演重要角色。Brucein D(BD)是一种B. javanica果实提取物，已被报道在胰腺癌中具有抗肿瘤作用。在此研究中，我们证明了BD在体内和体外均可抑制肝癌细胞生长。为了研究BD是否通过调节微小RNA来执行其抗肿瘤功能，我们进行了一个肿瘤微小RNA定量PCR阵列谱分析。此阵列包括95个已被报道与肿瘤有关的微小RNA。通过对比BD处理前后微小RNA谱的变化，我们发现微小RNA-95在BD处理后被显著下调了。其后促凋亡的CUGBP2被确定为微小RNA-95的下游靶基因。, 胰腺癌是一种预后很差的恶性肿瘤，常常在确诊时已发生转移。为了找出在胰腺癌转移过程中发挥决定性作用的基因，我们进行了全基因组范围的RNA干扰筛选。一个包含针对全部人类基因的shRNA文库被导入胰腺癌细胞系capan-2.然后将这些细胞移植到裸鼠的胰腺中来建立一个原位胰腺癌小鼠模型。我们的假设是下调某个基因会促使低转移潜力的capan-2细胞转移到肝脏。通过从肝转移结节中回收shRNA模板，我们找到了几个推定的转移抑制基因。其中之一，SOX9，通过体内实验验证，证明下调SOX9基因的表达可促进胰腺癌转移。, 化疗适用于进展期胰腺癌病人。然而他们对一线化疗药吉西他滨的反应并不乐观，这进一步使胰腺癌的预后变差。我们展开了一个全基因组范围的RNA干扰筛选来确定一些在化疗耐药过程中起关键作用的基因。携带上述shRNA文库的capan-2细胞被用于吉西他滨药物处理之下的筛选。通过微阵列分析，一些基因被筛选成为可影响癌细胞对药物敏感性的潜在的靶基因。通过进一步验证，LLGL1基因被确定为在调节癌细胞对化疗敏感性过程中起重要作用的基因。, MicroRNAs (miRNAs) are endogenous small non-coding RNAs that have been shown to play important roles in tumorigenesis. Brucein D (BD), a chemical compound isolated from Brucea javanica fruit, has previously been reported to have anti-cancer effect in pancreatic cancer. In this study, we showed that BD also inhibited the growth of liver cancer cells both in vitro and in vivo. To investigate whether BD exerts its anti-cancer effect through regulation of miRNAs, we performed a cancer miRNA qPCR array profiling. From the profiling, miR-95 was found to be significantly down-regulated after BD treatment. Subsequently, a pro-apoptotic gene CUGBP2 was identified as a direct downstream target of miR-95. These findings suggested BD suppressed liver cancer cell growth through down-regulation of miR-95 and reinforcing CUGBP2., Pancreatic cancer is an aggressive malignancy with extremely poor prognosis. It is usually diagnosed when metastases are already present. To identify genes that play critical roles in the processes of pancreatic cancer metastasis, a whole genome RNAi screening was performed. An shRNA library targeting all human genes was introduced into a human pancreatic cancer cell line capan-2. The infected cells were then transplanted into the pancreas of nude mice. Because capan-2 is of low metastatic potential, we hypothesized that knocking down of metastasis suppressor genes would facilitate capan-2 cells to spread to the liver. By retrieving shRNA templates from the liver metastatic nodules, several candidate genes were found. One of them, SOX9, has been validated as metastasis suppressor gene in vivo, implying that loss of expression of SOX9 promotes pancreatic cancer metastasis., Chemotherapy is recommended for patients of pancreatic cancer in advanced stage. However, their response to the first-line chemotherapy drug gemcitabine is not satisfactory. A genome-wide RNAi screening was conducted to identify genes that were critical in chemotherapy resistance. Capan-2 cells containing the above shRNA library were applied for the screening under gemcitabine treatment. Through microarray analysis, a number of genes were screened as potential gemcitabine sensitivity genes. Validation experiments implied that the gene LLGL1 may play an important role in modulating pancreatic cancer cells’ sensitivity to gemcitabine., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Xia, Tian., Thesis (M.Phil.)--Chinese University of Hong Kong, 2012., Includes bibliographical references (leaves 125-134)., s also in Chinese., Chapter Abstract --- p.I, Chapter Acknowledgements --- p.V, Chapter Abbreviations --- p.VI, Chapter List of Figures --- p.XV, Chapter List of Tables --- p.XVI, Chapter Part I: --- Brucein D-modulated microRNA-95 expression inhibits hepatocellular carcinoma cell growth --- p.1, Chapter 1 --- Introduction --- p.1, Chapter 1.1 --- Hepatocellular carcinoma --- p.1, Chapter 1.1.1 --- Definition and classification --- p.1, Chapter 1.1.2 --- Epidemiology --- p.1, Chapter 1.1.3 --- Etiology --- p.3, Chapter 1.1.4 --- Molecular pathogenesis of HCC --- p.4, Chapter 1.1.4.1 --- Genomic instability --- p.4, Chapter 1.1.4.2 --- Deregulation of key signaling pathways --- p.5, Chapter 1.1.4.3 --- Epigenetic changes of HCC --- p.6, Chapter 1.1.4.4 --- Two models of HCC pathogenesis --- p.7, Chapter 1.1.5 --- Therapeutic methods and prognosis of HCC --- p.8, Chapter 1.2 --- Apoptosis --- p.9, Chapter 1.2.1 --- Types of cell death --- p.9, Chapter 1.2.2 --- Apoptosis --- p.10, Chapter 1.2.3 --- Morphological features of apoptosis --- p.10, Chapter 1.2.4 --- Molecular mechanisms of apoptosis --- p.11, Chapter 1.2.5 --- Apoptosis and cancer --- p.13, Chapter 1.2.5.1 --- Imbalance of pro-apoptotic proteins and anti-apoptotic proteins --- p.13, Chapter 1.2.5.2 --- Impaired caspases activity --- p.14, Chapter 1.2.5.3 --- Deregulated death receptor signaling --- p.15, Chapter 1.2.6 --- Cancer therapy targeting apoptotic defects --- p.15, Chapter 1.3 --- microRNA --- p.16, Chapter 1.3.1 --- Overview --- p.16, Chapter 1.3.2 --- Biogenesis and maturation of microRNA --- p.17, Chapter 1.3.3 --- Gene silencing by microRNA --- p.18, Chapter 1.3.4 --- MicroRNA and cancers --- p.19, Chapter 1.3.5 --- MicroRNA’s involvement in HCC --- p.21, Chapter 1.3.6 --- Involvement of miR-95 in cancer --- p.22, Chapter 1.4 --- Brucein D --- p.22, Chapter 1.5 --- Aims of study --- p.23, Chapter 2 --- Materials and Methods --- p.25, Chapter 2.1 --- Cell Culture --- p.25, Chapter 2.1.1 --- Mammalian Cell Culture --- p.25, Chapter 2.1.2 --- Preparation of cell stock --- p.25, Chapter 2.1.3 --- Cell recovery from liquid nitrogen stock --- p.26, Chapter 2.1.4 --- Preparation of drugs for treatments --- p.26, Chapter 2.1.5 --- Drug treatment --- p.26, Chapter 2.1.6 --- Transfection of siRNA --- p.27, Chapter 2.1.7 --- MTT Assay --- p.28, Chapter 2.1.8 --- Luciferase reporter assays --- p.28, Chapter 2.1.9 --- Annexin V Assay --- p.29, Chapter 2.2 --- In vivo mouse model --- p.29, Chapter 2.3 --- Tunel Assay (Terminal deoxynucleotide transferase dUTP Nick End Labeling Assay) --- p.30, Chapter 2.4 --- RNA manipulation --- p.31, Chapter 2.4.1 --- RNA Isolation --- p.31, Chapter 2.4.2 --- Synthesis of cDNA from miRNA --- p.32, Chapter 2.4.3 --- Synthesis of cDNA from RNA and quantitative PCR --- p.33, Chapter 2.4.4 --- miRNA qPCR array --- p.34, Chapter 2.5 --- DNA manipulation --- p.34, Chapter 2.5.1 --- Agarose gel electrophoresis and purification of DNA --- p.34, Chapter 2.5.2 --- Restriction enzymes digestion --- p.35, Chapter 2.5.3 --- Ligation of DNA fragments --- p.36, Chapter 2.5.4 --- Polymerase chain reaction --- p.36, Chapter 2.5.5 --- Preparation of competent E. coli cells --- p.37, Chapter 2.5.6 --- Transformation of E. coli cells --- p.37, Chapter 2.5.7 --- Small scale plasmid isolation from E. coli (mini-prep) --- p.38, Chapter 3 --- Results --- p.39, Chapter 3.1 --- Brucein D inhibited the growth of HCC cells both in vitro and in vivo --- p.39, Chapter 3.2 --- BD induced apoptosis in HCC cells --- p.43, Chapter 3.3 --- miR-95 is an target of BD to modulate cell growth --- p.46, Chapter 3.4 --- Identification of CUGBP2 as a downstream target of miR-95 --- p.55, Chapter 4 --- Discussion --- p.60, Chapter Part II: --- Genome-wide RNAi screening identifies tumor metastasis suppressor genes and drug sensitivity genes in pancreatic cancer --- p.65, Chapter 1 --- Introduction --- p.65, Chapter 1.1 --- Pancreatic cancer --- p.65, Chapter 1.1.1 --- Overview --- p.65, Chapter 1.1.2 --- Pancreatic ductal adenocarcinoma (PDAC) --- p.67, Chapter 1.1.3 --- Molecular basis of PDAC --- p.67, Chapter 1.1.3.1 --- KRAS --- p.67, Chapter 1.1.3.2 --- TP53 --- p.68, Chapter 1.1.3.3 --- CDKN2A --- p.69, Chapter 1.1.4 --- Gemcitabine treatment in PDAC --- p.69, Chapter 1.2 --- Metastasis --- p.71, Chapter 1.2.1 --- Overview --- p.71, Chapter 1.2.2 --- The stepwise process of metastasis --- p.72, Chapter 1.2.3 --- Metastasis of pancreatic cancer --- p.74, Chapter 1.3 --- SOX9 --- p.75, Chapter 1.4 --- Aims of study --- p.77, Chapter 2 --- Materials and Method --- p.78, Chapter 2.1 --- Cell culture --- p.78, Chapter 2.1.1 --- Mammalian Cell Culture --- p.78, Chapter 2.1.2 --- MTT Assay --- p.78, Chapter 2.1.3 --- Colony formation assay --- p.79, Chapter 2.1.4 --- Wound healing assay --- p.79, Chapter 2.1.5 --- Transwell migration chamber assay --- p.80, Chapter 2.1.6 --- Immunocytochemistry --- p.80, Chapter 2.1.7 --- Transient transfection of siRNA --- p.81, Chapter 2.2 --- Establishment of in-vivo and in-vitro models --- p.82, Chapter 2.2.1 --- shRNA library introduction --- p.82, Chapter 2.2.2 --- Establishment of the orthotopic pancreatic cancer mouse model --- p.82, Chapter 2.2.3 --- Package of lentivirus expressing shRNA --- p.83, Chapter 2.2.4 --- Generation of stable cell line expressing shRNA --- p.84, Chapter 2.3 --- DNA manipulation --- p.84, Chapter 2.3.1 --- Large scale plasmid isolation from E. coli (maxi-prep) --- p.84, Chapter 2.4 --- Analysis of Protein --- p.85, Chapter 2.4.1 --- Preparation of protein cell lysates --- p.85, Chapter 2.4.2 --- Protein concentration determination --- p.86, Chapter 2.4.3 --- SDS-PAGE --- p.86, Chapter 2.4.4 --- Immunoblotting (Western blotting) --- p.87, Chapter 2.5 --- RNA manipulations --- p.88, Chapter 2.5.1 --- RNA Isolation --- p.88, Chapter 2.5.2 --- Synthesis of cDNA from RNA and quantitative PCR --- p.89, Chapter 2.6 --- Analysis of Clinical Samples --- p.90, Chapter 2.6.1 --- Clinical specimens --- p.90, Chapter 2.6.2 --- Immunohistochemistry --- p.90, Chapter 3 --- Results --- p.92, Chapter 3.1 --- Genome-wide RNAi screening identifies genes as metastasis suppressors in an orthotopic pancreatic cancer mouse model --- p.92, Chapter 3.2 --- SOX9 is a metastasis suppressor gene in pancreatic cancer --- p.97, Chapter 3.3 --- Investigation into cellular functions of SOX9 --- p.102, Chapter 3.3.1 --- SOX9’s effect on cell growth --- p.102, Chapter 3.3.2 --- SOX9’s effect on cell migration --- p.105, Chapter 3.4 --- Implication of SOX9 in human pancreatic cancer samples --- p.109, Chapter 3.5 --- Genome-wide RNAi screening for the identification of gemcitabine sensitivity genes --- p.113, Chapter 4 --- Discussion --- p.120, Chapter General conclusions --- p.125, http://library.cuhk.edu.hk/record=b5549124, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

近年來，腸促胰島素類藥物胰高血糖素樣肽－1受體（GLP-1R）激動劑（如liraglutide，exenatide）和二肽基肽酶-4（DPP-4）抑制劑（如sitagliptin，vildagliptin）在2型糖尿病治療中得到廣泛應用。然而，2型糖尿病患者中腸促胰島素效應嚴重受損。研究表明，2型糖尿病患者的腸促胰島素激素（GLP-1和GIP）分泌並不顯著降低，因此腸促胰島素效應受損主要是由於2型糖尿病患者中β細胞對腸促胰島素激素反應性降低。GIP在2型糖尿病患者中刺激胰島素分泌的功能幾乎完全消失。相比較，GLP-1刺激胰島素分泌功能在2型糖尿病患者中部分保留。2型糖尿病中腸促胰島素效應受損的具體機制目前仍不清楚。本論文主要從2型糖尿病患者的腸促胰島素效應受損的機理及對腸促胰島素類藥物療效的影響上進行相關研究。, 我們較早的研究發現高血糖能降低胰島β細胞GLP-1R受體的表達，從而損傷胰島β細胞GLP-1R信號通路是2型糖尿中腸促胰島素受損的部分原因。由於高血脂和高血糖都是糖尿病的主要特徵，我進一步研究了高血脂對β細胞的腸促胰島素信號通路的影響。體外實驗表明，棕櫚酸能降低胰島β細胞中GLP-1R的表達，並且抑制了GLP-1刺激的cAMP產生及CREB的磷酸化；在β細胞中外源性表達GLP-1R能部分恢復棕櫚酸損傷的GLP-1刺激cAMP產生及CREB的磷酸化。此外，在db/db小鼠和HFD誘導的肥胖及糖尿病小鼠模型中，降脂藥bezafibrate和niacin 能顯著提高腸促胰島素類藥物sitagliptin 和exendin-4的降糖效果，並且伴隨著對胰島形態結構的改善以及增加胰島β細胞質量。, 另一方面，2型糖尿病患者胰島β細胞的功能和品質持續性的減少。其中慢性的高血糖和高血脂是兩個主要因素。臨床研究發現sitagliptin的降糖效果隨著糖尿病持續的時間增加而降低，而具體機理並不清楚。我們以前研究發現高血糖損傷胰島β細胞GLP-1R的表達及其信號通路是2型糖尿病腸促胰島素效應受損的重要原因。我進一步探討了exendin-4在STZ-HFD 誘導的較輕程度糖尿病（MH）小鼠（相對較輕的高血糖以及β細胞質量減少）和重度糖尿病(SH)小鼠（嚴重高血糖以及β細胞質量減少）的治療效果。研究發現， exendin-4只在MH小鼠中顯著降低血糖，改善糖耐量，恢復正常胰島結構及增加β細胞質量。而在兩組小鼠中exendin-4 都能降低體重，增加胰腺重量，但對食都沒影響。儘管exendin-4能顯著降低MH小鼠中胰島素耐量實驗葡萄糖水平，但HORM-IR無顯著差異。此外，exendin-4處理對胰島素刺激的肝臟及肌肉組織中磷酸化AKT和GSK水準在兩組小鼠中都無明顯改變。, 總之，我的研究強調了血糖血脂的控制在2型糖尿病患者中的重要作用。我也發現高血糖、高血脂導致2型糖尿病患者β細胞功能持續受損的同時也損傷了GLP-1R信號通路，以至腸促胰島素類藥物療效的降低。我們的研究對腸促胰島素類藥物在2型糖尿病患者中的合理使用提供了重要資訊。, Incretin-based drugs, such as glucagon-like peptide-1 (GLP-1) receptor agonists (e.g. liraglutide and exenatide) and dipeptidyl peptidase-4 (DPP-4) inhibitors (e.g. sitagliptin and vildagliptin), which inhibit degrading intact GLP-1, have been a novel therapeutics for the treatment of type 2 diabetes. Type 2 diabetes mellitus (T2DM) is associated with reduced incretin effects. The underlying mechanism, however, is not well understood., Our previous studies showed that hyperglycemia downregulates glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) which potentially contributes to the impaired incretin responses in cells. Whereas the effects of hyperlipidemia, another common clinical feature of T2DM, on GLP-1 response is largely unknown. In this study, I investigated the effects of free fatty acids (FFA) on incretin receptor signalings, and examined the glucose-lowering efficacy of incretin-based drugs in combination with lipid-lowering agents. I found that palmitate treatment decreased GLP-1R expression in rodent insulinoma cell lines, which was associated with impaired GLP-1 stimulated cAMP production and phosphorylation of CREB. Over-expression of GLP-1R restored the cAMP production and the phosphorylation of CREB. Treatment with bezafibrate or niacin in combination with des-fluoro-sitagliptin or exendin-4 produced more robust glycemic control associated with improved pancreatic islet morphology and islet cell function in db/db mice and HFD-fed mice., On the other hand, chronic hyperglycemia and hyperlipidemia can cause a progressive deterioration in pancreatic beta-cell function and mass in patients with type 2 diabetes mellitus. It has been reported that the efficacy of incretin-based therapeutics is attenuated with the duration of diabetes. In our previous study, we have shown that hyperglycemia downregulates GLP-1 receptor which in turn may contribute to the impaired incretin effect in type 2 diabetes. In this study, I further investigated the efficacy of GLP-1 based drug exendin-4 in a rodent model of type 2 diabetes with different degrees of hyperglycemia and reduction of beta cell mass. I found that in moderate hyperglycemia (MH) group but not in severe hyperglycemia (SH) group, exendin-4 treatment significantly reduced fed glucose levels and plasma lipid profiles, improved glucose tolerance and glucose stimulated insulin secretion, and was associated with restored islets morphology and increased beta cell mass. Exendin-4 significantly decreased body weight gain and increased pancreatic mass both in MH and SH group. Although glucose levels were significantly reduced in MH group with exentin-4 treatment during insulin tolerance test, exendin-4 had no effects on HORM-IR, food intake, and insulin stimulated p-AKT/p-GSK in liver and muscle in both MH and SH group., In summary, my findings highlight the importance of comprehensive lipid and glycemic control in type 2 diabetes mellitus. I found that factors including hyperglycemia and hyperlipidemia that cause progressive decline in beta cell failure impaired beta cell GLP-1R signaling as well as the efficacy of incretin-based therapies. These results add to our knowledge regarding the mechanism of incretin-based therapy in improving glycemic control in type 2 diabetic patients and provide new insights in designing treatment strategies including incretin-based therapy for type 2 diabetic patients., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Kang, Zhanfang., Thesis (Ph.D.)--Chinese University of Hong Kong, 2012., Includes bibliographical references (leaves 103-123)., also in Chinese., 論文摘要 --- p.viii, Impaired Incretin Effects in Type 2 Diabetes: Mechanism and Therapeutic Implication --- p.1, DECLARATION --- p.i, ACKNOWLEGEMENTS --- p.ii, ABBREVIATIONS --- p.iii, PUBLICATIONS AND AWARDS --- p.v, Chapter 1 --- Abstract --- p.vi, Chapter 2 --- Chapter : Introduction --- p.1, Chapter 2.1 --- The history of incretin discovery --- p.1, Chapter 2.2 --- The incretin hormones:GLP-1 and GIP --- p.1, Chapter 2.3 --- Gene structure and regulation of incretin hormone gene expression --- p.2, Chapter 2.3.1 --- Gene structure and regulation of GLP-1 gene expression --- p.2, Chapter 2.3.2 --- Gene structure and regulation of GIP gene expression --- p.5, Chapter 2.4 --- Secretion and metabolism of GLP-1 and GIP --- p.5, Chapter 2.4.1 --- Regulation of GLP-1 and GIP secretion --- p.5, Chapter 2.4.2 --- Degradation of GLP-1 and GIP by DPP-4 enzyme --- p.8, Chapter 2.5 --- GLP-1 and GIP receptor --- p.10, Chapter 2.6 --- biological actions of GLP-1 and GIP --- p.11, Chapter 2.6.1 --- Actions of GLP-1 in peripheral tissues --- p.12, Chapter 2.6.2 --- Actions of GIP in peripheral tissues --- p.17, Chapter 2.7 --- Impaired incretin effect in type 2 diabetes patients --- p.17, Chapter 2.7.1 --- Secretion of incretin hormones in patients with type 2 diabetes --- p.18, Chapter 2.7.2 --- Impaired the responsiveness to GLP-1 and GIP in pancreatic beta cells --- p.19, Chapter 2.8 --- Incretin-based drugs --- p.19, Chapter 2.9 --- Type 2 diabetes and beta cell failure --- p.21, Chapter 2.9.1 --- Natural history of type 2 diabetes --- p.21, Chapter 2.9.2 --- Decline of beta cell function and mass in type 2 diabetes --- p.22, Chapter 2.9.3 --- Factors for progressive loss of beta cell function and mass --- p.24, Chapter 3 --- Chapter: Pharmacological reduction of free fatty acids restores the efficacy of incretin-based therapies in diabetic mouse models through beta cell GLP-1 receptor signalings --- p.30, Chapter 3.1 --- Summary --- p.30, Chapter 3.2 --- Introduction --- p.32, Chapter 3.3 --- Materials and Methods --- p.35, Chapter 3.3.1 --- Chemicals and Reagents --- p.35, Chapter 3.3.2 --- Preparation of 8 mM sodium palmitate solution with 10.5% BSA --- p.35, Chapter 3.3.3 --- Construct of an adenoviral vector for expressing mouse GLP-1R --- p.36, Chapter 3.3.4 --- Cell culture and treatment --- p.37, Chapter 3.3.5 --- RNA extraction and quantitative RT-PCR --- p.37, Chapter 3.3.6 --- Analysis of phosphorylation of CREB --- p.38, Chapter 3.3.7 --- Measurement of insulin secretion --- p.39, Chapter 3.3.8 --- RT-PCR analysis of mouse GLP-1R expression --- p.39, Chapter 3.3.9 --- Measurement of cAMP production --- p.40, Chapter 3.3.10 --- Animals and experimental protocols --- p.40, Chapter 3.3.11 --- Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) --- p.41, Chapter 3.3.12 --- Acute glucose-lowering actions of Ex-4 and D-GIP in db/db Mice --- p.41, Chapter 3.3.13 --- Lipid levels measurement --- p.42, Chapter 3.3.14 --- Histological analysis --- p.42, Chapter 3.3.15 --- Statistical analysis --- p.42, Chapter 3.4 --- Results --- p.43, Chapter 3.4.1 --- Reduced expression of GLP-1R in palmitate-treated b cells and islets of hyperlipedemic db/db mice. --- p.43, Chapter 3.4.2 --- Palmitate impairs GLP-1 stimulated cAMP production and p-CREB in rodent insulinoma cell lines --- p.45, Chapter 3.4.3 --- Palmitate reduced GLP-1 and GIP stimulated insulin secretion in rat INS-1E cells --- p.46, Chapter 3.4.4 --- Mouse GLP-1R mRNA is expressed in rat INS-1E cells after infected with Ad-GLP-1R --- p.47, Chapter 3.4.5 --- Expression of exogenous GLP-1R restores GLP-1 stimulated cAMP production and p-CREB in palmitate-treated rodent insulinoma cell lines --- p.48, Chapter 3.4.6 --- Lipid lowering enhances the efficacy of DPP-4 inhibitor des-fluoro-sitagliptin in db/db mice --- p.50, Chapter 3.4.7 --- Lipid-lowering enhances the efficacy of DPP-4 inhibitor des-flouro-sitagliptin in HFD-fed mice --- p.56, Chapter 3.4.8 --- Lipid lowering enhances the efficacy of an agonist to GLP-1R (Ex-4) but not to GIPR (D-GIP) in db/db mice --- p.59, Chapter 3.5 --- Discussion --- p.67, Chapter 4 --- Chapter : Further study on the impairment of incretin effect by hyperglycemia in a rodent model of type 2 diabetes --- p.71, Chapter 4.1 --- Summary --- p.71, Chapter 4.2 --- Introduction --- p.73, Chapter 4.3 --- Materials and Methods --- p.76, Chapter 4.3.1 --- Animals and treatment --- p.76, Chapter 4.3.2 --- Oral glucose tolerance test and insulin tolerance test --- p.76, Chapter 4.3.3 --- Plasma parameters --- p.77, Chapter 4.3.4 --- Histological staining and quantification of beta cell mass --- p.77, Chapter 4.3.5 --- Insulin signaling analysis --- p.78, Chapter 4.3.6 --- Western blot analysis --- p.78, Chapter 4.3.7 --- Statistical analysis --- p.79, Chapter 4.4 --- Results --- p.80, Chapter 4.4.1 --- Exendin-4 reduced fed glucose levels in MH mice but not in SH mice --- p.80, Chapter 4.4.2 --- Exendin-4 reduced body weight gain and did not affect food intake in both MH mice and SH mice --- p.81, Chapter 4.4.3 --- Exendin-4 improved glucose tolerance and glucose stimulated insulin secretion in MH mice but not in SH mice --- p.83, Chapter 4.4.4 --- Effects of exendin-4 on insulin sensitivity in MH and SH mice --- p.84, Chapter 4.4.5 --- Effects of exendin-4 on lipid profiles in MH and SH mice --- p.86, Chapter 4.4.6 --- Effects of exendin-4 on tissues weight in MH and SH mice --- p.87, Chapter 4.4.7 --- Pancreatic islet morphology and analysis of beta cell mass --- p.88, Chapter 4.4.8 --- Exendin-4 had no significant effects on insulin signaling pathway in liver and muscle --- p.91, Chapter 4.5 --- Discussion --- p.94, Chapter 5 --- Chapter : Summary --- p.100, Chapter 6 --- References --- p.103, http://library.cuhk.edu.hk/record=b5549514, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

雖然錯誤調控微小核糖核酸 (miRNA) 引起肝細胞癌 (HCC) 發生發展的生物途徑得到了廣泛的研究，但是對於上游的調控機制卻知之甚少。以往的研究表明，組蛋白甲基化轉移酶 (EZH2) 介導的組蛋白3上27位賴氨酸三甲基化（H3K27me3）是一類通過沉默腫瘤抑制基因而誘發癌症的機制，並且與脫氧核糖核酸 (DNA) 啟動子甲基化機制獨立存在。另一方面，基因抑制也與 H3K27和DNA甲基化相關聯。盡管如此，miRNA沉默機制，特別是在肝癌中，仍然是知之甚少。, 在這項研究中，我們使用整合全基因組定位和表達分析方法，以探討在肝癌細胞中miRNA表達的表觀遺傳和轉錄控制。通過染色質免疫沉澱偶聯人類啟動子芯片的方法，我們發現在Hep3B和HKCI - 8肝癌細胞中分別有8.4和12.2%的審問miRNA有豐富的H3K27me3。另一方面，在甲基結合域捕捉偶聯芯片實驗中，我們發現在Hep3B和HKCI-8肝癌細胞中分別有15.5和14.7% 的miRNA出現DNA超甲基化。與以往的蛋白質編碼基因結果相同，大多數 H3K27me3豐富的miRNA沒有被檢測到DNA超甲基化，並且反之亦然。 敲除EZH2基因引起H3K27me3水平廣泛下降,並且恢復 H3K27me3抑制的 miRNA表達，而DNA去甲基化劑5-氮雜 -2'-脫氧胞苷 (5-aza-dC) 卻不能重新啟動他們的表達，進一步顯示EZH2基因介導的H3K27me3引發miRNA沉默的機制是獨立存在的。然而，一些過往研究證明有腫瘤抑制功能的miRNA，包括 miR-9-1，miR-9-2和 miR-9-3 被發現同時被 H3K27me3和DNA甲基化調節。我們進一步發現，在肝瘤細胞中，miR-9 特異性調控致癌性的基因結合核因 (NF-κB) 信號通路，並且與配對的非腫瘤肝組織相比，miR-9 的表達在大約一半的原發性肝癌腫瘤（五十九分之三十零）中顯著被壓抑。, 為了調查在H3K27me3介導的miRNA基因沉默中參與的轉錄因子，我們應用轉錄因子結合位點分析的方法檢查H3K27me3結合蛋白編碼和miRNA基因的調控區域。在包括miR-9亞型的miRNA中，滎陽 1（YY1）的結合位點在這些調控區域中反覆出現並有很高的代表性。定量芯片聯合聚合酶鏈反應結果顯示，在Hep3B細胞中，敲除YY1不僅大大降低了自身的結合力，同時在 miR-9-1，miR-9-2和 miR-9-3 的啟動子中，EZH2基因和H3K27me3結合也大大降低了。尤其重要的是，敲除YY1可以顯著地重新激活他們的表達，表明在肝癌細胞中YY1在EZH2基因介導的的miR-9 表觀遺傳沉默中發揮重要作用。功能研究證明，下調YY1能夠抑制肝癌細胞的增殖，增加細胞凋亡和減少體內的腫瘤生長。定量實時聚合酶鏈反應進一步證實在miR-9 被下調的子集肝癌腫瘤中，有超過85的樣本顯示YY1和EZH2基因同時過量表達，表明我們的研究結果具有臨床相關性。, 總之，我們完整的分析表明miRNA的調控在肝癌上的獨特表觀遺傳模式。 H3K27me3介導的miRNA沉默可由擁有致癌功能的YY1誘發，它亦可能代表一個可能公認的肝癌癌基因。綜合表觀遺傳和miRNA表達的轉錄控制的結果，能夠提高我們對肝癌發生發展的認識和闡明利用表觀遺傳方法針對性治療肝癌的新的發展方向。, Although the biological pathways by which mis-regulated microRNAs (miRNAs) contribute to the development of hepatocellular carcinoma (HCC) have been extensively investigated, little is known about the upstream regulatory mechanisms. Previous studies demonstrated that EZH2-mediated histone H3 lysine 27 trimethylation (H3K27me3) is a mechanism of tumor-suppressor gene silencing in cancer that is potentially independent of promoter DNA methylation. On the other hand, gene repression can be associated with both H3K27 and DNA methylation. However, the mechanisms underlying miRNA silencing, particularly in HCC, are poorly understood., In this study, we used an integrated genome-wide location and expression analysis to investigate the epigenetic and transcriptional controls of miRNA expression in HCC cells. Chromatin immunoprecipitation (ChIP) coupled with human promoter microarrays revealed that 8.4 and 12.2% of the interrogated miRNA were enriched with H3K27me3 in Hep3B and HKCI-8 HCC cells, respectively. On the other hand, Methyl-binding domain capture coupled with microarray (MethylCap-chip) uncovered that 15.5 and 14.7% of miRNA were hypermethylated in Hep3B and HKCI-8 HCC cells, respectively. Consistent with previous observation on protein-coding genes, most of the miRNAs enriched with H3K27me3 had no detectable DNA hypermethylation and vice versa. Knockdown of EZH2 decreased global H3K27me3 level and restored expression of the H3K27me3-targeted miRNAs while the DNA demethylating agent 5-aza-2’-deoxycytidine (5-aza-dC) did not reactivate their expression, further suggesting the independence of EZH2-mediated H3K27me3 in miRNA silencing. Nevertheless, a few miRNAs reported to exhibit tumor-suppressive functions including miR-9-1, miR-9-2 and miR-9-3 were found to be regulated by both H3K27me3 and DNA methylation. We further found that miR-9 targeted the oncogenic NF-κB signaling pathway in HCC cells and were significantly repressed in approximately half of the primary HCC tumors (30/59) compared to the paired non-tumor liver tissues., To investigate the involvement of transcription factors in H3K27me3-mediated gene silencing of miRNAs, the regulatory regions of H3K27me3-bound protein-coding and miRNA genes were submitted to transcription factor binding site analysis. The binding sites for Ying Yang 1 (YY1) were recurrently over-represented in these loci including the miR-9 isoforms. Quantitative ChIP-PCR demonstrated that knockdown of YY1 in Hep3B cells not only significantly reduced its own binding, but also the EZH2 and H3K27me3 promoter occupancy at miR-9-1, miR-9-2 and miR-9-3. Importantly, their expression levels were significantly reactivated by YY1 knockdown, suggesting that YY1 plays part in the EZH2-mediated epigenetic silencing of miR-9 in HCC cells. Functionally, down-regulation of YY1 was shown to inhibit HCC cell proliferation, increase cell apoptosis and reduce tumor growth in vivo. Quantitative RT-PCR further demonstrated that YY1 and EZH2 were concordantly over-expressed in over 85% of the same subset of HCC tumors that exhibited miR-9 down-regulation, demonstrating the clinical relevance of our findings., In conclusion, our integrated analysis demonstrated differential epigenetic patterns of miRNA regulation in HCC. H3K27me3-mediated silencing of miRNAs may be initiated by YY1, which possesses oncogenic functions and may represent a putative HCC oncogene. The findings of combinatorial epigenetic and transcriptional control of miRNA expression enhance our understanding of hepatocarcinogenesis and shed light on the development of novel epigenetic targeted therapy of HCC., Detailed summary in vernacular field only., Tsang, Pui Fong., Thesis (M.Phil.)--Chinese University of Hong Kong, 2012., Includes bibliographical references (leaves 111-121)., s also in Chinese., (English version) --- p.i, (Chinese version) --- p.iv, Acknowledgements --- p.vi, table of contents --- p.vii, List of tables --- p.x, List of Figures --- p.xi, Abbreviations --- p.xiii, Chapter CHAPTER 1 --- INTRODUCTION4, Chapter 1.1 --- Hepatocellular carcinoma --- p.1, Chapter 1.1.1 --- Epidemiology --- p.1, Chapter 1.1.2 --- Etiology --- p.2, Chapter 1.2 --- Epigenetic mechanisms --- p.3, Chapter 1.2.1 --- Epigenetic silencing by DNA hypermethylation --- p.3, Chapter 1.2.2 --- Epigenetic silencing by Polycomb group protein --- p.5, Chapter 1.2.3 --- Interplay between H3K27me3 and DNA hypermethylation --- p.7, Chapter 1.3 --- microRNA --- p.10, Chapter 1.3.1 --- Transcriptional gene silencing by miRNA --- p.11, Chapter 1.3.2 --- miRNA and cancers --- p.12, Chapter 1.3.3 --- miRNA and liver cancer --- p.13, Chapter 1.4 --- Signal transduction pathway and cancers --- p.14, Chapter 1.5 --- Aims of study --- p.15, Chapter CHAPTER 2 --- MATERIALS AND METHODS, Chapter 2.1 --- Cell lines --- p.16, Chapter 2.2 --- Clinical samples --- p.16, Chapter 2.3 --- Plasmid DNA transfection --- p.16, Chapter 2.4 --- Small interfering RNA transfection --- p.17, Chapter 2.5 --- Extraction of total RNA --- p.19, Chapter 2.6 --- Western blot analysis --- p.19, Chapter 2.7 --- Quantitative RT-PCR --- p.20, Chapter 2.8 --- miRNA Real Time RT-PCR --- p.22, Chapter 2.9 --- ChIP-chip assay --- p.24, Chapter 2.10 --- MethylCap-chip --- p.27, Chapter 2.11 --- miRNA microarray --- p.28, Chapter 2.12 --- ChIP Assay and Quantitative ChIP-PCR Assay --- p.28, Chapter 2.13 --- Colony formation assay --- p.33, Chapter 2.14 --- Cell proliferation assay --- p.33, Chapter 2.15 --- Annexin V apoptosis assay --- p.34, Chapter 2.16 --- Cancer 10-pathway reporter array --- p.34, Chapter 2.16.1 --- Transfection of siEZH2 with 5-aza-dC treatment --- p.34, Chapter 2.16.2 --- Transfection of siYY1 and pcDNA3-miR9 plasmid --- p.35, Chapter 2.16.3 --- Luciferase reporter array --- p.35, Chapter 2.17 --- Animal Studies --- p.36, Chapter 2.18 --- Statistical Analysis --- p.36, Chapter CHAPTER 3 --- Results, Chapter 3.1 --- Occupancy of miRNA genes by epigenetic marks in HCC cells --- p.37, Chapter 3.1.1 --- Identification of H3K27me3-occupied miRNAs --- p.37, Chapter 3.1.2 --- Identification of DNA methylation-occupied miRNAs --- p.41, Chapter 3.1.3 --- Relationship between H3K27me3 and DNA methylation occupancy of miRNAs in HCC cells --- p.45, Chapter 3.2 --- Regulation of miRNA expression by H3K27me3 and DNA methylation in HCC cells --- p.51, Chapter 3.3 --- Epigenetic regulation and molecular function of miR-9 in HCC cells --- p.56, Chapter 3.3.1 --- Confirmation of H3K27me3 and DNA methylation occupancy in miR-9 genes --- p.59, Chapter 3.3.2 --- Synergistic reactivation of miR-9 upon removal of epigenetic marks --- p.62, Chapter 3.3.3 --- Effect of miR-9 re-expression on NFKB1 (p50) expression and NF-κB signaling in HCC cells --- p.66, Chapter 3.4 --- Role of the transcription factor YY1 in the epigenetic regulation of miR-9 --- p.72, Chapter 3.4.1 --- Identification of transcription factors involved in the regulation of H3K27me3-bound genes --- p.72, Chapter 3.4.2 --- Occupancy of YY1 on miR-9 in HCC cells --- p.75, Chapter 3.4.3 --- Effects of YY1 on EZH2/H3K27me3 occupancy and expression of miR-9 --- p.78, Chapter 3.4.4 --- Effects of YY1 on p50/p65 expression and NF-κB signaling in HCC cells --- p.81, Chapter 3.5 --- Functional significance of YY1 in HCC --- p.84, Chapter 3.5.1 --- Effect of YY1 on HCC cell growth --- p.84, Chapter 3.5.2 --- Effect of YY1 on HCC cell apoptosis --- p.87, Chapter 3.5.3 --- Effect of YY1 on HCC cell growth in vivo --- p.90, Chapter 3.5.4 --- Expressions of YY1, EZH2 and miR-9 on clinical HCC samples --- p.92, Chapter CHAPTER 4 --- DISCUSSION, Chapter 4.1 --- Independence of EHZ2-mediated H3K27me3 and DNA methylation --- p.97, Chapter 4.2 --- Concordant H3K27 and DNA methylation-mediated silencing of miR-9 --- p.101, Chapter 4.3 --- Ectopic expression of miR-9 inhibits NF-kB signaling in HCC cells --- p.102, Chapter 4.4 --- YY1 is involved in the regulation of H3K27me3-bound genes --- p.103, Chapter 4.5 --- Knockdown of YY1 inhibits NF-kB signaling in HCC --- p.105, Chapter 4.6 --- Clinical relevance and therapeutic significance of miR-9 silencing by YY1-mediated recruitment of EZH2 --- p.106, Chapter 4.7 --- Limitations and future studies --- p.109, REFERENCES --- p.111, PUBLICATION --- p.122, http://library.cuhk.edu.hk/record=b5549094, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

腫瘤發生過程中，遺傳和/或表觀遺傳異常都可導致抑癌基因(TSG)的失活。新TSG的鑒定和研究對理解癌症發展至關重要，并能提供潛在的治療靶點和腫瘤標誌物。本研究的目標是在人類腫瘤中鑒定新的被表觀遺傳異常沉默的TSG并進一步研究其抑癌的分子機理。, 表觀遺傳修飾因子是重要的腫瘤相關基因。這裡，我研究了兩個作為功能性TSG的表觀遺傳修飾基因。首先，我發現一個在腫瘤中被甲基化和下調的候選TSG, PRDM5, 可通過抑制TCF/LEF依賴的轉錄和引起多個癌基因的表觀遺傳下調而抑制癌細胞增殖。其次，通過檢測24個表觀遺傳修飾基因在癌細胞系中的表達，我發現一個頻繁下調的新候選TSG，TUSC12。TUSC12在正常組織中普遍表達，但在腫瘤細胞系中被啟動子區甲基化下調，且原發腫瘤中也存在高頻TUSC12甲基化。TUSC12顯著抑制癌細胞克隆形成能力，但這種抑制效果因其PHD功能域的失活被部份削弱。TUSC12與轉錄抑制因子KAP1在細胞核中共定位，并可能通過招募HDAC相關複合體而抑制基因轉錄。, 另外，我研究了一個通過以前鼻咽癌(NPC) aCGH鑒定得到的新3p14.2 TSG，ZNF312。啟動子區甲基化導致ZNF312在NPC細胞系和原發癌中的沉默。恢復ZNF312表達可抑制NPC細胞生長，并引起細胞週期阻滯和凋亡。作為一個轉錄抑制因子，ZNF312靶向EZH2和MDM2的啟動子區并下調它們的表達。, 之前的基因數字表達分析已篩選出一些可能在腫瘤中低表達的基因。我研究了一個通過這種方法分離出的新TSG，TUSC45。相比正常組織，TUSC45表達在癌組織中顯著下降，而且TUSC45低表達與病人的低存活相關。TUSC45在多個腫瘤細胞系中也被下調，但它的基因組缺失卻不多見。啟動子區甲基化導致TUSC45在多數細胞系中的沉默，而且藥物或遺傳去甲基化能顯著激活它的表達。特別的，TUSC45在腫瘤組織而不在正常組織中被高頻率甲基化。誘導TUSC45表達可抑制細胞增殖，引起細胞凋亡、週期阻滯、和衰老，並上調一個關鍵抑癌基因p53。TUSC45以p53依賴的方式激活p53的靶基因，而TUSC45對p53缺失的H1299和HCT116/p53KO細胞沒有生長抑制作用。TUSC45與p53/MDM2複合物結合并正向調節p53蛋白穩定性。 TUSC45表達導致MDM2蛋白半衰期縮短，提示一種可能的TUSC45調節p53通路的機制。, 本研究表明癌變過程中四個抑癌基因腫瘤特異的甲基化和沉默引起了多個細胞信號通路紊亂，並可作為潛在的腫瘤檢測標誌物。, Tumor suppressor genes (TSGs) can be inactivated by genetic and/or epigenetic alterations during carcinogenesis. Identification and characterization of novel TSGs are critical for the understanding of cancer development, and provide potential targets for clinical treatment and biomarkers for tumor diagnosis. In this study, I aimed to identify novel TSGs epigenetically silenced in human cancers and further characterize the molecular basis of their anti-tumorigenic functions., Emerging evidence highlights the importance of epigenetic modifiers as cancer genes. Here, I characterized two epigenetic modifier genes as functional TSGs. First, I found PRDM5, a candidate TSG methylated and downregulated in multiple cancers, suppressed tumor cell proliferation through inhibiting TCF/LEF-dependent transcription and inducing epigenetic repression of multiple oncogenes. Second, through expression profiling of 24 epigenetic modifiers, I identified a novel candidate TSG, TUSC12, showing frequent silencing in tumor cell lines. TUSC12 was broadly expressed in human normal tissues, but downregulated by promoter CpG methylation in tumor cell lines. Frequent TUSC12 methylation was detected in primary tumors as well. TUSC12 dramatically inhibited tumor cell clonogenicity, but this growth inhibitory effect was partially impaired by disrupting its PHD domain. TUSC12 colocalized with the transcription corepressor KAP1 in the nucleus and is likely to repress gene transcription through recruit HDAC-associated complex., I also studied a novel 3p14.2 TSG, ZNF312, identified from previous aCGH profiling of nasopharyngeal carcinoma (NPC). Frequent promoter methylation silenced ZNF312 in NPC cell lines and tissues. Restored ZNF312 expression strongly suppressed NPC cell growth through inducing cell cycle arrest and apoptosis. Further, ZNF312 acted as a transcription repressor targeting the promoter regions of EZH2 and MDM2 and downregulating their expression., Moreover, previous digital gene expression subtraction from cDNA libraries revealed a list of genes possibly downregulated in tumors compared to normal tissues. I characterized a novel TSG, TUSC45, initially isolated from this strategy. The expression of TUSC45 was significantly reduced in tumor tissues compared to normal tissues, with lower TUSC45 expression associated with poorer patient survival. Downregulation of TUSC45 in multiple tumor cell lines was also observed, while only infrequent genomic deletion was detected. In contrast, promoter methylation was responsible for TUSC45 silencing in most cell lines, in which pharmacologic or genetic demethylation can dramatically restore its expression. Remarkably, TUSC45 was frequently methylated in primary tumors but not in normal tissues. Further, TUSC45 suppressed anchorage-dependent and -independent tumor cell growth. Induced TUSC45 expression inhibited cell proliferation, induced apoptosis, cell cycle arrest and senescence, and lead to the upregulation of a key tumor suppressor p53. Moreover, TUSC45 activated p53 target genes in a p53-dependent manner. Forced TUSC45 expression in p53-null H1299 and HCT116/p53KO (knock out) cells showed no inhibitory effect on cell growth. Finally, TUSC45 interacted with p53/MDM2 complex and positively regulated p53 protein stability. The protein half-life of MDM2 was shortened by TUSC45, indicating a possible mechanism for TUSC45 modulation on p53 signaling., In conclusion, this study showed the tumor-specific methylation and silencing of the four TSGs lead to the epigenetic disruption of multiple cell signalings during tumorigenesis and could potentially be used as biomarkers for cancer detection., Detailed summary in vernacular field only., Shu, Xingsheng., Thesis (Ph.D.)--Chinese University of Hong Kong, 2012., Includes bibliographical references (leaves 121-140)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., p.i, Acknowledgements --- p.vi, Table of Contents --- p.vii, List of Tables --- p.xi, List of Figures --- p.xii, List of Publications --- p.xiv, Abbreviations --- p.xv, Chapter Chapter 1 --- Introduction and Literature Reviews --- p.1, Chapter 1.1 --- Tumor suppressor genes (TSGs) and the pathways they control --- p.2, Chapter 1.1.1 --- Basic concepts about TSG --- p.2, Chapter 1.1.1.1 --- The discovery of TSG --- p.2, Chapter 1.1.1.2 --- Knudson’s “two-hit“ hypothesis --- p.3, Chapter 1.1.1.3 --- New insights of the “two-hit“ model --- p.4, Chapter 1.1.2 --- Key TSGs and their cellular pathways --- p.5, Chapter 1.1.2.1 --- p53 pathway --- p.6, Chapter 1.1.2.2 --- Rb pathway --- p.9, Chapter 1.1.2.3 --- APC and Wnt/β-catenin pathway --- p.10, Chapter 1.1.2.4 --- Chromatin regulators with tumor suppressive properties --- p.12, Chapter 1.1.3 --- Methods for TSG identification --- p.17, Chapter 1.2 --- The epigenetic abnormalities of TSGs in cancer --- p.19, Chapter 1.2.1 --- Promoter CpG methylation --- p.20, Chapter 1.2.1.1 --- Molecular basis of DNA methylation --- p.20, Chapter 1.2.1.2 --- Silencing of TSGs by promoter methylation --- p.22, Chapter 1.2.1.3 --- Mechanism of methylation-induced transcription repression --- p.23, Chapter 1.2.1.4 --- Abnormal DNA methylation contributes to early stages of tumorigenesis --- p.25, Chapter 1.2.2 --- Aberrant histone modification and chromatin remodeling --- p.26, Chapter 1.2.3 --- Clinical applications of epigenetic biomarkers and therapeutic targets --- p.28, Chapter 1.2.3.1 --- DNA methylation and histone modification as biomarkers for cancer diagnosis and prognosis --- p.28, Chapter 1.2.3.2 --- Epigenetic targets for cancer treatment --- p.29, Chapter Chapter 2 --- Aims and Design of This Study --- p.32, Chapter Chapter 3 --- Materials and Methods --- p.34, Chapter 3.1 --- Cell lines --- p.34, Chapter 3.2 --- Human normal and cancer tissues --- p.35, Chapter 3.3 --- DNA and RNA extraction --- p.35, Chapter 3.4 --- Semi-quantitative and quantitative RT-PCR --- p.36, Chapter 3.5 --- DNA methylation analysis --- p.37, Chapter 3.5.1 --- Bisulfite modification of genomic DNA --- p.37, Chapter 3.5.2 --- CpG island analysis --- p.37, Chapter 3.5.3 --- Methylation-specific PCR (MSP) --- p.38, Chapter 3.5.4 --- Bisulfite genomic sequencing (BGS) --- p.38, Chapter 3.5.5 --- Pharmacologic demethylation treatment of cell lines --- p.38, Chapter 3.6 --- Luciferase assay of gene promoter activity --- p.39, Chapter 3.7 --- Multiplex genomic-DNA PCR --- p.39, Chapter 3.8 --- Construction of expression plasmids and PCR-mediated mutagenesis --- p.40, Chapter 3.9 --- Plasmid mini- and midi-preparation --- p.41, Chapter 3.10 --- Creation of stable cell line for inducible gene expression --- p.42, Chapter 3.11 --- Monolayer-culture and soft-agar colony formation assay --- p.42, Chapter 3.12 --- Cell proliferation assay --- p.43, Chapter 3.13 --- Flow cytometry analysis of cell cycle --- p.43, Chapter 3.14 --- Apoptosis assay --- p.44, Chapter 3.15 --- Senescence cell staining --- p.44, Chapter 3.16 --- Western blotting --- p.45, Chapter 3.17 --- Co-immunoprecipitation (Co-IP) --- p.46, Chapter 3.18 --- Transcription factor activity assay --- p.46, Chapter 3.19 --- Immunofluorescence microscopy --- p.47, Chapter 3.20 --- Chromatin Immunoprecipitation (ChIP) --- p.47, Chapter 3.21 --- Gene expression and copy number analysis using Oncomine database --- p.48, Chapter 3.22 --- Protein half-life assay --- p.48, Chapter 3.23 --- In vivo ubiquitination assay --- p.48, Chapter 3.24 --- Statistical analysis --- p.49, Chapter Chapter 4 --- Two Epigenetic Modifying Genes, PRDM5 and TUSC12, Suppress Tumor Cell Growth and are Silenced by Promoter Methylation in Human Cancers --- p.50, Chapter 4.1 --- PRDM5 --- p.50, Chapter 4.1.1 --- PRDM5 is a candidate TSG downregulated and methylated in multiple carcinomas --- p.50, Chapter 4.1.2 --- PRDM5 is a stress responsive gene but its response is disrupted by promoter methylation --- p.53, Chapter 4.1.3 --- PRDM5 suppresses cancer cell growth and proliferation --- p.54, Chapter 4.1.4 --- PRDM5 inhibits TCF/LEF-dependent transcription and induced epigenetic repression of multiple oncogenes --- p.55, Chapter 4.2 --- TUSC12 --- p.59, Chapter 4.2.1 --- mRNA expression profiling of epigenetic modifying genes in tumor cell lines --- p.59, Chapter 4.2.2 --- Promoter CpG methylation contributes to TUSC12 silencing in tumor cells --- p.61, Chapter 4.2.3 --- Demethylation of TUSC12 promoter restores its expression --- p.63, Chapter 4.2.4 --- TUSC12 methylation in primary tumor tissues --- p.65, Chapter 4.2.5 --- TUSC12 expression inhibits anchorage-dependent and -independent tumor cell growth --- p.66, Chapter 4.2.6 --- TUSC12 is an epigenetic modifier repressing transcription --- p.68, Chapter 4.3 --- Discussion --- p.69, Chapter Chapter 5 --- The Human Zinc Finger Protein 312 is a Novel Tumor Suppressor for Nasopharyngeal Carcinoma --- p.73, Chapter 5.1 --- Identification of ZNF312 as a candidate 3p14.2 TSG --- p.74, Chapter 5.2 --- Silencing of ZNF312 by promoter methylation in NPC cell lines --- p.75, Chapter 5.3 --- ZNF312 is frequently downregulated and methylated in primary NPC tissues --- p.78, Chapter 5.4 --- ZNF312 suppresses tumor cell clonogenicity --- p.79, Chapter 5.5 --- ZNF312 is a transcription repressor --- p.80, Chapter 5.6 --- ZNF312 regulates cell cycle progression, induces apoptosis, and inhibits cell stemness --- p.83, Chapter 5.7 --- ZNF312 represses oncogene expression --- p.85, Chapter 5.8 --- Discussion --- p.87, Chapter Chapter 6 --- Identification of a Novel Tumor Suppressor Regulating p53 Signaling and Frequently Methylated in Multiple Tumors --- p.91, Chapter 6.1 --- TUSC45 is broadly expressed in human normal tissues but frequently downregulated in tumor cell lines --- p.91, Chapter 6.2 --- Reduced TUSC45 expression in primary tumors is associated with poor survival of patients --- p.94, Chapter 6.3 --- TUSC45 is mainly silenced by promoter CpG methylation in tumor cell lines --- p.96, Chapter 6.4 --- Pharmacologic or genetic demethylation reactivates TUSC45 in silenced cell lines --- p.98, Chapter 6.5 --- TUSC45 is frequently methylated in primary tumors --- p.99, Chapter 6.6 --- TUSC45 suppresses anchorage-dependent and -independent tumor cell growth --- p.101, Chapter 6.7 --- Induction of TUSC45 expression inhibits tumor cell growth through inducing apoptosis, cell cycle arrest and senescence --- p.104, Chapter 6.8 --- TUSC45 tumor-suppressive function is dependent on p53 signaling --- p.108, Chapter 6.9 --- TUSC45 positively regulates p53 protein stability --- p.110, Chapter 6.10 --- Discussion --- p.112, Chapter Chapter 7 --- General Discussion --- p.116, Chapter 7.1 --- General discussion --- p.116, Chapter 7.2 --- Future perspectives --- p.118, Reference List --- p.121, http://library.cuhk.edu.hk/record=b5549510, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

目的：結直腸癌是世界上第三常見惡性腫瘤，結腸鏡檢查是診斷的金標准。但其創傷性、昂貴的設備以及人力的需求阻礙了廣泛應用。本研究評估了糞便miRNA作為非損傷性分子生物標記物篩查結直腸腺瘤和腫瘤的可行性，並深入探究了致癌miRNA的基因靶點。, 方法：我們評估了糞便miRNAs的穩定性以及檢測的可重復性。糞便樣本收集自88例結腸直腸癌患者，57例結直腸息肉患者和101名健康對照，用實時定量逆轉錄PCR檢測miRNA水平。所有候選miRNA標記物在配對的癌及癌旁組織中進行驗証。我們共測試了糞便中7種miRNAs，包括前期報道在結直腸癌中上調的miR-21和miR-92a(第一部分)，以及在667個miRNA中在結直腸癌上調最高的5個miRNA(第二部分)。我們研究了它們的水平與腫瘤分期及位置的關系。並隨訪了病人經腫瘤或腺瘤切除術后其糞便miRNA水平，從而証實它們是否與腫瘤相關。我們應用了彗星試驗、細胞活力試驗、集落形成試驗，以及細胞凋亡分析試驗研究了miR-18a在腫瘤的發展過程中的作用(第三部分)。, 結果：第一部分，我們確定糞便miRNA的穩定性，能被實時定量逆轉錄PCR檢測並顯示高重復性。糞便miR-92a標記物的靈敏度和特異性分別為71.6%和73.3%，miR-21分別為55.7%和73.3%。MiR-92a水平顯示遠端結直腸癌比近端結直腸癌的檢測具有更高靈敏度，晚期腺瘤比小息肉更具靈敏度。腫瘤切除后，miR-21和miR-92a水平顯著下降。, 第二部分，基於結直腸腫瘤miRNA的表型，我們發現糞便miR-18a, miR-20a, miR-135b和miR-221能作為標記物鑒別結直腸癌，miR-18a (敏感度: 51.1%, 特異性: 90.1%); miR-20a (72.7%, 81.2%) ; miR-135b (81.8%, 68.3%); miR-221 (69.3%, 77.2%)。腫瘤切除后，這四種標記物會顯著下降。MiR-135b和miR-221也能鑒別腺瘤。四種標記物對遠近端結腸癌的檢測無顯著差異。, 第三部分，通過程序和熒光素酶報告基因活性預測和驗証，我們發現一種重要的DNA修復蛋白---共濟失調毛細血管擴張突變(ATM)是miR-18a的靶蛋白。MiR-18a的異位表達減弱細胞DNA雙鏈損傷修復機制，導致腫瘤發生的易感性。, 結論：我們發現糞便中miR-21, miR-92a, miR-18a, miR-20a, miR-135b 和miR-221標記物能夠鑒別結直腸癌。MiR-92a, miR-135b 和miR-221能鑒別結直腸腺瘤。MiR-18a抑制共濟失調毛細血管擴張突變基因表達並減弱細胞DNA雙鏈損傷修復機制。糞便miRNA是結直腸癌篩查的有效生物標記物。, Objective: Colorectal cancer (CRC) is the third most common cancer worldwide. Colonoscopy is the current gold standard for diagnosing CRC. However, its invasive nature, the cost of equipment and the demand for manpower have hampered the wide application of this procedure. This study evaluated the feasibility of using stool-based miRNA as non-invasive biomarkers for the screening of colorectal adenoma and cancer, and investigated the gene target of a candidate oncogenic miRNA., Methods: The reproducibility of detection and stability of stool-based miRNAs were first evaluated. Stool samples were collected from 88 CRC patients, 57 patients with colorectal polyp and 101 healthy controls MiRNA levels were detected by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). AII candidate miRNA markers were validated in a cohort of paired tumor and adjacent normal tissues. In total, we tested 7 miRNAs in the stool, including miR-21 and miR-92a which were reported to be up-regulated in CRC in previous studies (part one), and 5 miRNAs which were found to be the most up-regulated in colorectal tumor based on the profiling of 667 miRNAs (part two). Their levels with tumor stage and location were evaluated. Their change in level was followed up in a subset of patients after the removal of tumor or adenoma. We investigated miR-18a for its function in cancer development using comet assay, cell viability assay, colony formation assay, and analysis on apoptosis (part three)., Results: In part one, we found stool miRNAs stable and detectable with high reproducibility by qRT-PCR. In detecting CRC, stool miR-92a had a sensitivity of 71.6% and a specificity of 73.3%, stool miR-21 had a sensitivity of 55.7% and a specificity of 73.3%. Stool miR-92a level had higher sensitivity for distal CRC than proximal CRC, and a higher sensitivity for advanced adenoma than minor polyps. The removal of tumor resulted in reduced stool miR-21 and miR-92a levels., In part two, based on miRNA profiling of CRC tumors, we found that stool-based miR-18a, miR-20a, miR-135b, and miR-221 can discriminate colorectal cancer patients from healthy individuals: miR-18a (sensitivity: 51.1%, specificitiy: 90.1%); miR-20a (72.7%, 81.2%); miR-135b (8 1.8%, 68.3%); miR-221 (69.3%, 77.2%). Levels of these 4 stool-based markers dropped after removal of tumors. Stool-based miR-135b and miR-221 also discriminated patients with adenoma from healthy individuals. MiR-18a, miR-20a, miR-135b and miR-221 showed no desparity in detecting proximal or distal colon cancer., In part three, based on in silico prediction and validation with luciferase reporter activity, we identified Ataxia Telangiectasia Mutated (ATM ), a protein crucial to DNA repair, as a target of miR-18a. Ectopic expression miR-18a attenuates DNA double strand break repair mechanism, creating a genetic predisposition to the development of cancer., Conclusion: Stool-based miR-21, miR-92a, miR-18a, miR-20a, miR-135b and miR-221 can discriminate patients with CRC from healthy individuals. Notably, a subset of these miRNAs (miR-92a, miR-135b, and miR-221) can discriminate patients with colorectal adenoma from healthy individuals MiR-18a suppressed ATM gene expression and attenuated cellular repair mechanism to DNA double strand breaks. Stool-based miRNAs are useful CRC screening biomarkers., Detailed summary in vernacular field only., Wu, Chung Wah., Thesis (Ph.D.)--Chinese University of Hong Kong, 2012., Includes bibliographical references (leaves 80-92)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., Chapter CHAPTER ONE --- INTRODUCTION --- p.1, Chapter 1.1 --- Colorectal cancer --- p.1, Chapter 1.2 --- Current screening methods --- p.3, Chapter 1.2.1 --- Colonoscopy --- p.3, Chapter 1.2.2 --- Sigmoidoscopy --- p.4, Chapter 1.2.3 --- Stool-based tests --- p.4, Chapter 1.2.3.1 --- Fecal occult blood test --- p.5, Chapter 1.2.3.2 --- Stool-based DNA test --- p.6, Chapter 1.2.3.3 --- Stool-based RNA and protein test --- p.7, Chapter 1.3 --- MiRNA and its role in cancer --- p.8, Chapter 1.4 --- Aims of study --- p.9, Chapter CHAPTER TWO --- METHODOLOGY --- p.10, Chapter 2.1 --- Subjects and sample collection --- p.10, Chapter 2.2 --- MiRNA extraction in tissue and stool samples --- p.13, Chapter 2.3 --- MiRNA quantitation by quantitative reverse transcription quantitative reverse transcription polymerase chain reaction --- p.14, Chapter 2.4 --- Determining the stability of miRNA in stool samples --- p.15, Chapter 2.5 --- Determining the reproducibility of miRNA quantitation in stool samples --- p.16, Chapter 2.6 --- Reverse transcription for miRNA array --- p.16, Chapter 2.7 --- Quantitative polymerase chain reaction for miRNA array --- p.16, Chapter 2.8 --- Cell culture, miRNA precursors and transfection --- p.17, Chapter 2.9 --- Dual-luciferase reporter assay --- p.17, Chapter 2.10 --- Quantitative reverse transcription polymerase chain reaction for mRNA --- p.19, Chapter 2.11 --- Western blot analysis --- p.19, Chapter 2.12 --- Comet assay --- p.19, Chapter 2.13 --- Colony formation and cell viability assay --- p.20, Chapter 2.14 --- Annexin V apoptosis assay --- p.21, Chapter 2.15 --- Statistics --- p.21, Chapter CHAPTER THREE --- RESULTS --- p.23, Chapter 3.1 --- PART 1 --- p.23, Chapter 3.1.1 --- Stability of miRNA detection in stool samples --- p.23, Chapter 3.1.2 --- Reproducibility of miRNA quantitation in stool samples --- p.23, Chapter 3.1.3 --- Detection and normalization of miRNA levels --- p.25, Chapter 3.1.4 --- Expression of miR-21 and miR-92a in CRC tissue samples --- p.28, Chapter 3.1.5 --- Levels of stool-based miR-21 and miR-92a in CRC and polyp patients --- p.30, Chapter 3.1.6 --- Sensitivity of stool-based miR-21 and miR-92a towards colorectal cancer and polyps --- p.32, Chapter 3.1.7 --- Association of stool-based miR-21 and miR-92a with clinicopathological features --- p.34, Chapter 3.1.8 --- Follow-up on stool miR-21 and miR-92a levels after removal of lesion --- p.37, Chapter 3.2 --- Part 2 --- p.39, Chapter 3.2.1 --- MiRNA profiling in colorectal tumors --- p.39, Chapter 3.2.2 --- Validation of miRNA profiling results --- p.41, Chapter 3.2.3 --- Candidate miRNA levels in stool samples of CRC and adenoma patients --- p.44, Chapter 3.2.4 --- Sensitivities and specificities of miRNA candidates for adenoma and CRC --- p.47, Chapter 3.2.5 --- Sensitivties of miRNA candidates based on tumor location --- p.49, Chapter 3.2.6 --- Follow-up on stool miRNA levels after removal of lesion --- p.51, Chapter 3.2.7 --- Association of stool-based miRNAs with nodal involvement in CRC --- p.53, Chapter 3.2.8 --- Establishing the miRNA marker panel --- p.55, Chapter 3.3 --- Part 3 --- p.57, Chapter 3.3.1 --- In Silico prediction of miR-18a target and validation by luciferase assay --- p.57, Chapter 3.3.2 --- Expression and correlation between miR-18a and ATM in paired colorectal tumor tissue, cell lines and normal colon biopsies --- p.60, Chapter 3.3.3 --- Regulation of double strand DNA damaga recovery by miR-18a --- p.62, Chapter 3.3.4 --- Cell sensitization to genotoxin by miR-18a --- p.64, Chapter 3.3.5 --- Effect of miR-18a on genotoxin induced apoptosis --- p.66, Chapter CHAPTER FOUR --- DISCUSSION --- p.68, Chapter 4.1 --- Stability and detection reproducibility of stool-based miRNA --- p.68, Chapter 4.2 --- Stool-based miRNAs for screening colorectal cancer and polyps/adenomas --- p.69, Chapter 4.2.1 --- MiR-21 --- p.69, Chapter 4.2.2 --- MiR-18a, miR-20a and miR-92a --- p.70, Chapter 4.2.3 --- MiR -135b and miR -221 --- p.71, Chapter 4.2.4 --- MiR -31 --- p.72, Chapter 4.3 --- Discriminating proximal and distal CRC --- p.73, Chapter 4.4 --- Evaluation of stool-based miRNA level after removal of lesions --- p.74, Chapter 4.5 --- MiRNA marker panel --- p.74, Chapter 4.6 --- Advantages of stool-based miRNA tests --- p.75, Chapter 4.7 --- Ataxia telangiectasia mutated as the direct target of miR -18a --- p.75, Chapter 4.8 --- Future directions for study --- p.78, Chapter 4.9 --- Conclusion --- p.78, REFERENCES --- p.80, PUBLICATIONS --- p.93, http://library.cuhk.edu.hk/record=b5549509, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

研究介紹：炎症與纖維化是糖尿病腎病（DN）的主要特徵。研究發現生長轉化因子TGF-β/Smad信號在糖尿病所致炎症與纖維化中均起重要作用。我們認為TGF-β/Smad信號通路失調是導致糖尿病腎損傷的主要機制，恢復信號通路或有治療價值。為此我們通過以下研究證實：（1）研究Smad7基因在DN中的作用，及評估Smad7基因治療效果；（2）研究miR-29在DN中的作用，及評估miR-29基因治療效果；（3）研究C反應蛋白（CRP）在DN中的作用及機制。, 研究方法：（1）利用Smad7基因敲除（KO）小鼠建立糖尿病小鼠，並研究Smad7基因在DN的作用，並在链脲佐菌素（STZ）誘導的糖尿病大鼠上利用微泡導入Smad7基因治療觀察其療效；（2）在10週齡db/db小鼠上利用微泡導入可誘導的miR-29b基因，觀察miR-29b在糖尿病腎病中的作用，並用miR-29敲除或高表達細胞株研究其機制；（3）利用CRP轉基因小鼠誘導糖尿病，觀察CRP在DN中的作用，及以高糖和/或CRP刺激腎小管細胞研究CRP的致病機制。, 研究結果：我們發現（1）糖尿病Smad7 KO小鼠出現更嚴重的腎損傷，包括蛋白尿增加，腎臟炎症及纖維化加重。進一步研究發現Smad7下調所致TGF-β/Smad和NF-kB信號過度活化是導致腎臟炎症及纖維化加重的重要原因。運用基因治療恢復糖尿病大鼠的Smad7水平，發現能夠減輕蛋白尿增加，及抑制TGF-β/Smad引起的纖維化和NF-kB所致炎症反應；（2）我們發現miR-29b在20週齡db/db小鼠比10週齡的顯著降低，並伴隨有蛋白尿加重，腎臟纖維化和炎症反應增加，及TGF-β/Smad，NF-kB，T-bet信號上調，而miR-29b基因治療能減輕蛋白尿，及減輕腎臟纖維化和炎症反應增加，及TGF-β/Smad，NF-kB，T-bet信號上調。體外實驗證實AGEs刺激miR-29敲除細胞株增加纖維化，伴隨有TGF-β/Smad3及炎症因子上調，而刺激高表達細胞株能抑制纖維化，及TGF-β/Smad和炎症因子下調；（3）糖尿病CRP轉基因小鼠出現更嚴重的腎損傷，出現蛋白尿和腎損傷分子-1上升、巨噬細胞和T細胞侵潤、炎症和纖維化增加，並伴有CRP受體（CD32a）上調、TGF-β/Smads及NFκB/p65信號過度活化。體外實驗進一步證實CRP通過其受體CD32a/CD64增加炎症和纖維化。另外證實CRP與高糖有協同作用。, 結論：TGF-β/Smad信號通路是糖尿病腎病的重要致病機制。糖尿病腎病導致Smad7、miR-29b下調，運用基因治療恢復其表達能減輕糖尿病腎損傷。, Diabetic nephropathy (DN) is characterized by renal fibrosis and inflammation. Increasing evidence shows that TGF-β/Smad signaling plays a critical role in DN. This thesis tested a hypothesis that TGF-β/Smad signaling may play a central role in diabetic kidney injury and targeting this pathway may represent a novel therapy for DN. The hypothesis was tested in a type-1 model of diabetes induced in Smad7 knockout (KO) or CRP transgene, and the therapeutic potential for DN was examined by overexpressing renal Smad7 or miR-29b in both type-1 or type-2 models of diabetes., As described in Chapter Three, the protective role and therapeutic potential of Smad7 in diabetic kidney disease was investigated in streptozotocin-induced diabetic model in Smad7 KO mice and in diabetic rats given Smad7 gene transfer using an ultrasound-microbubble-mediated technique. Results showed that Smad7 KO mice developed more severe diabetic kidney injury than wild type (WT) mice as evidenced by a signicant increase in microalbuminuria, renal brosis, and renal inammation, which was mediated by enhanced activation of both TGF-β/Smads and NF-κB signaling pathways. To develop a therapeutic potential for diabetic kidney disease, Smad7 gene was transferred into the kidney, which results in high levels renal Smad7, thereby blocking microalbuminuria, TGF-β/Smad3-mediated renal brosis and NF-κB/p65-driven renal inammation in diabetic rats., To test a novel hypothesis that TGF-β/Smad3-mediated DN via the Smad3-dependent miR-29, in Chapter Four, the role and mechanisms of miR-29b in DN were examined in vitro in a stable mesangial cell line with overexpression or knockdown of miR-29b and the therapeutic effect of miR-29b on DN was developed by delivering a Dox-inducible miR-29b into 10-week db/db mice. Results showed that addition of AGEs induced a loss of miR-29b with increased fibrosis and inflammation in mesangial cells, which was further enhanced with miR-29b knockdown, but inhibited by overexpressing miR-29b. In db/db mice, reduction of renal miR-29b over the 20 week time was associated with a marked increase in microabluminuria, renal fibrosis and inflammation. Restoring miR-29b resulted in inhibition of kidney injuries by blocking TGF-β/Smad3-mediated renal fibrosis, NF-kB/p65-driven renal inflammation, and importantly, the Th1-dependent immune response, revealing a critical role and therapeutic potential for miR-29b in the pathogenesis of DN., Finally, diabetic kidney injury was also assessed in under high inflammation conditions in CRP transgenic (Tg) mice. As shown in Chapter Five, CRP Tg mice developed more severe diabetic kidney injury than WT mice, as evidenced by a significant increase in microalbuminuria and kidney injury molecule-1, macrophages and T cells, and upregulation of pro-inflammatory cytokines and extracellular matrix. CRP-mediated DN was associated with upregulation of CRP receptor, CD32a, and over-activation of the TGF-β/Smads and NFκB/p65 signaling pathways. These findings were further confirmed in vitro under high levels of CRP. In addition, CRP was induced by high glucose, which synergistically promoted high glucose-mediated renal inflammation and fibrosis, suggesting a positive feedback-loop between CRP and high glucose under diabetic conditions., In conclusion, TGF-β/Smads play critical roles in the pathogenesis of DN. Loss of renal Smad7 and miR-29b may be a key mechanism of DN. Thus, over-expression of Smad7 or miR-29b may represent novel therapeutic strategies for diabetic kidney complications., Detailed summary in vernacular field only., Chen, Haiyong., Thesis (Ph.D.)--Chinese University of Hong Kong, 2012., Includes bibliographical references (leaves 202-236)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., p.ii, DECLARATION --- p.vi, ACKNOWLEDGEMENT --- p.vii, PUBLICATIONS --- p.ix, PRESENTATIONS/AWARDS --- p.xi, TABLE OF CONTENTS --- p.xii, LIST OF ABBREVIATIONS --- p.xxii, LIST OF FIGURES/TABLES --- p.xxiv, Chapter CHAPTER ONE --- INTRODUCTION --- p.1, Chapter 1.1 --- TGF-β superfamily --- p.2, Chapter 1.2 --- TGF-β/Smad signaling pathway --- p.3, Chapter 1.2.1 --- TGF-β --- p.3, Chapter 1.2.1.1 --- TGF-β structure --- p.3, Chapter 1.2.1.2 --- Activation of latent TGF-β --- p.4, Chapter 1.2.1.3 --- Latent TGF-β receptors --- p.6, Chapter 1.2.2 --- TGF-β signaling pathway --- p.7, Chapter 1.2.2.1 --- Receptors --- p.7, Chapter 1.2.2.2 --- Smads --- p.10, Chapter 1.2.2.3 --- Smad-dependent TGF-β signaling pathways --- p.13, Chapter 1.2.2.4 --- Smad-independent TGF-β signaling pathways --- p.14, Chapter 1.3 --- Diabetes nephropathy --- p.15, Chapter 1.3.1 --- Diabetes Mellitus --- p.15, Chapter 1.3.2 --- Type 1 and type 2 diabetes --- p.16, Chapter 1.3.3 --- Diabetic complications --- p.16, Chapter 1.3.4 --- Cellular and molecular mechanisms in diabetic complications --- p.17, Chapter 1.3.4.1 --- Increased polyol pathway flux --- p.17, Chapter 1.3.4.2 --- Increased advanced glycation end-products (AGEs) formation --- p.18, Chapter 1.3.4.3 --- Activation of protein kinase C (PKC) isoforms --- p.20, Chapter 1.3.4.4 --- Increased hexosamine pathway flux --- p.22, Chapter 1.3.4.5 --- Increased Reactive Oxygen Species --- p.23, Chapter 1.3.5 --- Diabetic kidney injuries --- p.24, Chapter 1.3.5.1 --- Exacerbation of renal structure and function --- p.24, Chapter 1.3.5.2 --- Fibrosis in diabetic nephropathy --- p.25, Chapter 1.3.5.3 --- Inflammation in diabetic nephropathy --- p.26, Chapter 1.4 --- Role of TGF-β/Smad signaling pathway in diabetic nephropathy --- p.28, Chapter 1.4.1 --- Smad-depedent signaling in diabetic nephropathy --- p.28, Chapter 1.4.2 --- Cross talk between Smads and other signaling pathways in diabetic nephropathy --- p.30, Chapter 1.4.3 --- TGF-β/Smads and MicroRNA regulation in diabetic nephropathy --- p.32, Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.35, Chapter 2.1 --- Materials --- p.36, Chapter 2.1.1 --- Regents and equipment --- p.36, Chapter 2.1.1.1 --- Reagents and equipment for cell culture --- p.36, Chapter 2.1.1.2 --- Reagents and equipment for real-time RT-PCR --- p.37, Chapter 2.1.1.3 --- Reagents and equipment for western blotting --- p.38, Chapter 2.1.1.4 --- Reagents and equipment for immunohistochemistry --- p.39, Chapter 2.1.1.5 --- Reagents and equipment for in situ hybridization --- p.40, Chapter 2.1.1.6 --- Reagents and equipment for plasmid purification --- p.40, Chapter 2.1.1.7 --- Reagents and equipment for genotyping --- p.41, Chapter 2.1.1.8 --- Other reagents --- p.41, Chapter 2.1.2 --- Buffers --- p.42, Chapter 2.1.2.1 --- Western blotting buffer --- p.42, Chapter 2.1.2.2 --- Immunohistochemistry buffer --- p.45, Chapter 2.1.2.3 --- ELISA buffers --- p.47, Chapter 2.1.2.4 --- In Situ hybridization buffer --- p.48, Chapter 2.2.2 --- Antibodies --- p.49, Chapter 2.2.3 --- Primer sequences --- p.49, Chapter 2.2 --- Methods --- p.56, Chapter 2.2.1 --- Animal model --- p.56, Chapter 2.2.1.1 --- Animals --- p.56, Chapter 2.2.1.2 --- Diabetic animal models --- p.57, Chapter 2.2.2 --- Sample Collection --- p.59, Chapter 2.2.2.1 --- Urine collection --- p.59, Chapter 2.2.2.2 --- Plasma collection --- p.59, Chapter 2.2.2.3 --- Tissue collection --- p.60, Chapter 2.2.2.4 --- Paraffin embedding --- p.60, Chapter 2.2.3 --- Ultrasound-microbubble-mediated gene transfer system --- p.61, Chapter 2.2.3.1 --- Smad7 gene therapy --- p.61, Chapter 2.2.3.2 --- miR-29 gene therapy --- p.62, Chapter 2.2.4 --- Microalbumin and renal function --- p.63, Chapter 2.2.4.1 --- Microalbuminuria --- p.63, Chapter 2.2.4.2 --- Creatinine measurement --- p.63, Chapter 2.2.5 --- Enzyme-Linked Immunosorbent Assay (ELISA) --- p.64, Chapter 2.2.6 --- Histology and immunohistochemistry --- p.64, Chapter 2.2.6.1 --- Tissue process --- p.64, Chapter 2.2.6.2 --- Periodic Acid-Schiff Staining (PAS) --- p.64, Chapter 2.2.6.3. --- Immunohistochemistry (IHC) --- p.65, Chapter 2.2.6.4 --- In Situ hybridization --- p.66, Chapter 2.2.6.5 --- Quantitation of histology and IHC --- p.67, Chapter 2.2.7 --- Cell culture --- p.67, Chapter 2.2.8 --- Real-time polymerase chain reaction (PCR) --- p.69, Chapter 2.2.9 --- Western Blotting --- p.70, Chapter 2.3 --- Statistical analysis --- p.71, Chapter CHAPTER THREE --- THE PROTECTIVE ROLE OF SMAD7 IN DIABETIC NEPHROPATHY --- p.72, Chapter 3.1 --- Introduction --- p.73, Chapter 3.2 --- Materials and methods --- p.74, Chapter 3.2.1 --- Animal models --- p.74, Chapter 3.2.2 --- Ultrasound-mediated gene transfer of inducible Smad7 gene-bearing microbubbles into the kidney --- p.74, Chapter 3.2.3 --- Real-time PCR --- p.75, Chapter 3.2.4 --- Western blotting --- p.75, Chapter 3.2.5 --- Microalbuminuria and urinary creatinine analysis --- p.76, Chapter 3.2.6 --- Histology and immunohistochemistry --- p.76, Chapter 3.2.7 --- Statistical analysis --- p.77, Chapter 3.3 --- Results --- p.77, Chapter 3.3.1 --- Genotyping for Smad7 KO and WT mice --- p.77, Chapter 3.3.2 --- Disruption of Smad7 enhances diabetic kidney injury --- p.78, Chapter 3.3.3 --- Disruption of Smad7 enhanced fibrosis in diabetic kidney --- p.80, Chapter 3.3.3.1 --- Collagen I is enhanced in diabetic Smad7 KO mice --- p.81, Chapter 3.3.3.2 --- Collagen IV is enhanced in diabetic Smad7 KO mice --- p.82, Chapter 3.3.3.3 --- Fibronectin is enhanced in diabetic Smad7 KO mice --- p.84, Chapter 3.3.4 --- Disruption of Smad7 exacerbates inflammation in diabetic kidney --- p.85, Chapter 3.3.4.1 --- Disruption of Smad7 increases IL-1β in diabetic kidney --- p.85, Chapter 3.3.4.2 --- Disruption of Smad7 increases TNF-α in diabetic kidney --- p.86, Chapter 3.3.4.3 --- Disruption of Smad7 Increases MCP-1 in diabetic kidney --- p.87, Chapter 3.3.4.4 --- Disruption of Smad7 increases ICAM-1 in diabetic kidney --- p.88, Chapter 3.3.4.5 --- Disruption of Smad7 increases macrophage infiltration in diabetic kidney --- p.90, Chapter 3.3.5 --- Enhanced activation of TGF-β/Smad3 and NF-κB Signaling is a central mechanism by which disruption of Smad7 promotes diabetic renal fibrosis and inflammation --- p.91, Chapter 3.3.5.1 --- Smad7 decreases in diabetic kidney --- p.91, Chapter 3.3.5.2 --- Enhanced activation of TGF-β/Smad3 signaling pathway contributes to fibrosis in diabetic kidney --- p.92, Chapter 3.3.5.3 --- Enhanced activation of NF-κB/p65 signaling pathway contributes to inflammation in diabetic kidney --- p.93, Chapter 3.3.6 --- Smad7 transfection rate by gene therapy in diabetic rats --- p.94, Chapter 3.3.7 --- Restoring Smad7 attenuates kidney injury in diabetic rats --- p.96, Chapter 3.3.8 --- Restoring Smad7 attenuates renal fibrosis in diabetic rats --- p.98, Chapter 3.3.8.1 --- Restoring Smad7 attenuates collagen I in diabetic kidney --- p.98, Chapter 3.3.8.2 --- Restoring Smad7 attenuates collagen IV in diabetic kidney --- p.100, Chapter 3.3.8.3 --- Restoring Smad7 attenuates collagen III in diabetic kidney --- p.101, Chapter 3.3.9 --- Restoring Smad7 attenuates renal inflammation in diabetic rats --- p.104, Chapter 3.3.9.1 --- Restoring Smad7 attenuates IL-1b in diabetic kidney --- p.104, Chapter 3.3.9.2 --- Restoring Smad7 attenuates TNF-α in diabetic kidney --- p.106, Chapter 3.3.9.3 --- Restoring Smad7 Attenuates MCP-1 in diabetic kidney --- p.107, Chapter 3.3.9.4 --- Restoring Smad7 attenuates ICAM-1 in diabetic kidney --- p.109, Chapter 3.3.9.5 --- Restoring Smad7 attenuates macrophage infiltration in diabetic kidney --- p.111, Chapter 3.3.10 --- Blockade of activation of TGF-β/Smad3 and NF-κB signaling is a key mechanism by which overexpression of smad7 inhibits diabetic renal injury --- p.113, Chapter 3.3.10.1 --- Restoring Smad7 inhibits activation of TGF-β/Smad3 signaling --- p.113, Chapter 3.3.10.2 --- Restoring Smad7 inhibits activation of NF-κB signaling --- p.115, Chapter 3.3 --- Discussion --- p.117, Chapter CHAPTER FOUR --- THE PROTECTIVE ROLE OF MICRORNA-29B IN DIABETIC NEPHROPATHY --- p.121, Chapter 4.1 --- Introduction --- p.122, Chapter 4.2 --- Materials and methods --- p.123, Chapter 4.2.1 --- Animal model --- p.123, Chapter 4.2.2 --- Ultrasound-microbubble-mediated-miR-29 gene transfer --- p.124, Chapter 4.2.3 --- Real-time polymerase chain reaction (PCR) --- p.124, Chapter 4.2.4 --- Western Blotting --- p.125, Chapter 4.2.5 --- Albumin excretion measurement --- p.126, Chapter 4.2.6 --- ELISA --- p.126, Chapter 4.2.7 --- Histology and immunohistochemistry --- p.126, Chapter 4.2.8 --- In Situ hybridization --- p.127, Chapter 4.2.9 --- Cell culture --- p.128, Chapter 4.2.10 --- Statistical analysis --- p.129, Chapter 4.3 --- Results --- p.129, Chapter 4.3.1 --- Over-expression of miR-29b attenuates, but knockdown of miR-29b enhances fibrosis in vitro --- p.129, Chapter 4.3.1.1 --- Over-expression of miR-29b attenuates fibrosis --- p.129, Chapter 4.3.1.2 --- Knockdown of miR-29b enhances fibrosis --- p.132, Chapter 4.3.2 --- Restoring miR-29b attenuates kidney injury in db/db mice --- p.134, Chapter 4.3.3 --- Restoring miR-29b attenuates renal fibrosis in db/db mice --- p.139, Chapter 4.3.3.1 --- Restoring miR-29b attenuates collagen IV in db/db mice --- p.139, Chapter 4.3.3.2 --- Restoring miR-29b attenuates collagen I in db/db mice --- p.141, Chapter 4.3.3.3 --- Restoring miR-29b attenuates fibronectin in db/db mice --- p.144, Chapter 4.3.4 --- Restoring miR-29b inhibits renal fibrosis via TGF-β/Smad3 dependent pathway --- p.146, Chapter 4.3.5 --- Restoring miR-29b inhibits th1 immune response in diabetic kidney --- p.148, Chapter 4.3.6 --- Restoring miR-29b inhibits inflammation in diabetic kidney --- p.151, Chapter 4.4 --- Discussion --- p.154, Chapter 4.5 --- Conclusion --- p.161, Chapter CHAPTER FIVE --- THE PATHOGENIC ROLE OF C-REACTIVE PROTEIN IN DIABETIC NEPHROPATHY --- p.162, Chapter 5.1 --- Introduction --- p.163, Chapter 5.2 --- Materials and methods --- p.164, Chapter 5.2.1 --- Mouse model of STZ induced diabetes --- p.164, Chapter 5.2.2 --- Measurement of blood glucose, urinary albumin excretion, and creatinine clearance --- p.165, Chapter 5.2.3 --- Histology and immunohistochemistry --- p.165, Chapter 5.2.4 --- Cell culture --- p.166, Chapter 5.2.5 --- Real-time PCR --- p.166, Chapter 5.2.6 --- Western blotting --- p.167, Chapter 5.2.7 --- Statistical analyses --- p.168, Chapter 5.3 --- Results --- p.168, Chapter 5.3.1 --- Diabetic renal injury is exacerbated in CRP Tg mice --- p.168, Chapter 5.3.2 --- Renal inflammation is exacerbated in diabetic CRP Tg mice --- p.172, Chapter 5.3.2.1 --- F4/80+ macrophage infiltration is enhanced in diabetic CRP Tg mice --- p.172, Chapter 5.3.2.2 --- CD3+ T cell infiltration is enhanced in diabetic CRP Tg mice --- p.173, Chapter 5.3.2.3 --- TNF-α expression is enhanced in diabetic CRP Tg mice --- p.173, Chapter 5.3.2.4 --- IL-1β expression is enhanced in diabetic CRP Tg mice --- p.174, Chapter 5.3.3 --- Renal fibrosis is enhanced in diabetic CRP Tg mice --- p.175, Chapter 5.3.3.1 --- Collagen I is enhanced in Diabetic CRP Tg mice --- p.175, Chapter 5.3.3.2 --- Collagen IV is enhanced in diabetic CRP Tg mice --- p.176, Chapter 5.3.4 --- Enhanced CRP signaling and activation of NF-κB and TGF-β/Smad3 signaling are key mechanism by which CRP promotes diabetic renal injury --- p.177, Chapter 5.3.4.1 --- Enhanced CRP signaling via upregulation of CD32a expression --- p.177, Chapter 5.3.4.2 --- enhanced activation of NF-κB signaling is key mechanism by which CRP promotes renal inflammation --- p.179, Chapter 5.3.4.3 --- Enhanced activation of TGF-β/Smad3 signaling is key mechanism by which CRP promotes renal inflammation --- p.181, Chapter 5.4 --- Discussion --- p.194, Chapter 5.5 --- Conclusion --- p.197, Chapter CHAPTER SIX --- SUMMARY AND CONCLUSION --- p.198, REFERENCES --- p.202, http://library.cuhk.edu.hk/record=b5549653, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

研究背景, 系統性紅斑狼瘡（SLE）是一種較常見的累及多系統多器官的自身免疫性疾病，由於細胞和體液免疫功能障礙，產生多種自身抗體，確切病因不明。研究一般認為是在遺傳、環境等諸多因素的共同作用下導致了機體固有免疫及獲得性免疫系統功能紊亂，從而發病。作為機體抵抗病原體入侵的第一道防線，固有免疫系統通過模式識別受體（PRRs），不僅可以識別結合外源性的病原體相關模式分子（PAMPs），也可識別機體自身細胞所釋放的內源性危險信號又稱破壞相關模式分子（DAMPs），從而啟動信號傳導途徑，激活天然免疫細胞，從而導致一系列免疫應答的發生。本文將初步探討三類PRRs在SLE發病機制及病毒感染中的作用：（i）胞質中識別細胞壁肽聚糖的NOD樣受體（NLR），（ii）識別危險信號分子（DAMP）的膜結合型晚期糖基化終末產物受體（RAGE）及（iii）識別核酸的胞內Toll樣受體（TLR）。, NLR是一種新發現的PAMP識別受體，在配體識別及信號傳導方面有別於膜型PRRs，在固有免疫應答中發揮重要作用。目前報道中NLR至少有23個成員，其中最有代表性的是NOD1和NOD2，通過特異性識別細菌胞壁肽聚糖產物從而參與固有免疫應答並誘導炎症反應和細胞凋亡。前期研究多集中於NODs與SLE基因易感性的探討，而SLE患者體內免疫細胞是否功能性表達NOD2及其下遊 的效應如何，仍有待進一步探討。, RAGE是一種多配體受體，廣泛分布於上皮細胞、血管及炎症細胞表面，低表達於正常組織細胞，但與其配體結合後可啟動激活細胞內部各種信號傳導機制從而產生相應的生物學效應。HMGB1作為RAGE的重要配體在細胞和組織中分佈十分廣泛，近年研究證實，胞外高表達的HMGB1為一種重要的內源性危險信號分子，通過RAGE受體通路，可促進趨化作用，並通過激活NF-κB途徑誘導炎症反應。越來越多的證據表明HMGB1在自身免疫性疾病中起積極作用， RAGE-HMGB1軸在SLE的炎症反應及組織損傷中的重要作用值得研究。, RAGE基因可因RNA的選擇性剪接而分為：全長RAGE（flRAGE）、截去N端的RAGE及截去C端的可溶性RAGE（sRAGE）。sRAGE有兩種來源，其中由細胞分泌而來的又稱為內分泌性RAGE（esRAGE）。sRAGE通過與flRAGE競爭性與結合配體從可抑制RAGE誘導的細胞信號傳導途徑，故又稱為作為“誘餌受體，作為潛在的治療靶點，對疾病的進展有保護作用。因此，評估sRAGE/esRAGE與flRAGE及配體HMGB1在SLE患者體內的動態平衡具有顯著臨床意義。, 人類乳頭狀病毒（HPV）感染是子宮頸癌的致病因素，高危型HPV感染的持續存在是子宮頸癌的重要風險因素之一。早期病例對照研究已提示伴高炎症狀態及長期多重高危型HPV感染的SLE患者其子宮頸巴氏塗片異常和宮頸癌的發病率顯著高於對照人群，但在前瞻性隊列研究中其致高危致病因素及預測因子並未得到證實。TLR家族在早期固有免疫中對入侵病原微生物的識別發揮重要作用，胞內TLR3、TLR7、TLR8、TLR9通過識別病毒核酸成分通過介導下遊信號轉導誘導免疫反應。是否固有免疫系統異常參與SLE患者體內HPV持續及高危感染，仍有待進一步探討。, 研究目的, 1.研究NOD2受體通路及RAGE-HMGB1軸在SLE發病機制中的作用；, 2.闡明sRAGE/esRAGE作為“保護因子與flRAGE及其配體HMGB1在SLE患者體內的動態平衡及評估與疾病活動相關性；, 3.探討TLR受體通路在宿主SLE抗 HPV感染中的作用。, 研究方法, 本文通過三項臨床病例對照研究分別探討NLR、RAGE、TLR在SLE發病機制及病毒感染中的作用。, 研究結果, 1.SLE外周高表達於單核細胞內的NOD2可通過特異性識別配體誘導外周血單個核細胞的異常活化及促炎細胞因子的產生；而免疫抑制治療可下調CD8+ T細胞及抗原體提呈細胞內NOD2表達及抗炎細胞因子的產生；, 2.FlRAG高表達於SLE患者外周血單核細胞；血漿sRAGE作為獨立風險因素，與SLE疾病活動指數呈負相關；HMGB1單獨或與TLR9配體協同作用可刺激單核細胞分泌促炎細胞因子並激活信號轉導通路；, 3.TLR拮抗劑（羥氯喹）及強的松治療作為獨立風險因素可下調SLE患者子宮頸上皮細胞中TLR7和TLR9的表達；腫瘤相關的高危型HPV細胞株內核酸識別受體TLR及幹擾素刺激基因（ISGs）表達明顯下調伴功能異常。, 研究結論, 一方面，異常活化的PRRs通過識別結合外源及內源性病原體相關分子從而啟動固有免疫應答，激活一系列信號轉導通路，參與SLE的自身免疫反應：, 1.胞內受體NOD2可能通過特異性識別細菌胞壁酰二肽及誘導炎症反應，參與固有免疫應答反應抵禦外源性病原體侵襲，為感染因素導致SLE發病的假說尋找進一步理論依據；, 2.膜表面受體RAGE與配體HMGB1結合可激活細胞內多信號轉導機制，參與固有免疫應答反應抵禦內源性病原體侵襲，為胞內危險信號分子释放導致SLE無菌性炎症的假說提供初步理論依據；, 3.可溶性“誘餌受體RAGE作為潛在治療靶點可抑制SLE體內高炎症反應。, 另一方面，多重因素交叉作用可引起SLE患者體內PRR轉錄及表達下調，從而抑制固有免疫應答，導致病毒逃避宿主免疫系統的監視及清除而長期潛伏：, 1.TLR拮抗劑（羥氯喹）和強的松治療可能引起SLE患者體內TLR7和TLR9表達下調，從而抑制固有免疫系統對外源侵入性病原體HPV的識別；, 2.腫瘤相關的高危型HPV細胞株亦可通過抑制TLR7和TLR9轉錄、下調受體表達及功能致 HPV逃避宿主免疫防禦而長期潛伏。, Introduction, The pathogenesis of systemic lupus erythematosus (SLE) is a complicated process caused by genetic and environmental factors resulting in abnormalities of both the innate and the adaptive immune system. Sensing the presence of a pathogen is the first step for the immune system to mount an effective response to eliminate invading microorganisms and establish protective immunity. The innate immune system constitutes an important defense system to respond rapidly to both endogenous and exogenous molecules, in which the pathogen associated molecular patterns (PAMPs) and danger associated molecular patterns (DAMPs) can interact with the pattern recognition receptors (PRRs) and then activate the antigen presenting cells (APCs), T, B cells., Effective sensing of endogenous and exogenous molecules promotes autoreactivity via immune activation and antigen presentation. In lupus, these molecules may have a special role in the pathogenesis since they can serve as targets of autoreactivity as well as inducers. In this series of experiments, we focused on the roles of various PRRs including nucleic acid sensing toll-like receptors (TLRs), bacterial peptidoglycans sensing NOD-like receptors (NLRs) and dangerous signals sensing receptor for advanced glycation end products (RAGE), which are involved in the recognition of PAMPs and DAMPs sharing between microbes and the host in the pathogenesis of SLE., In contrast to the well elucidated membrane-bound TLRs, cytoplasmic NLRs are a new family of PRRs for the recognition of extracellular PAMPs. NLRs can participate in the signaling events triggered by host recognition of specific motifs of bacterial peptidoglycans and, upon activation, induce the production of proinflammatory mediators. Apart from the putative link between genetic mutations of NOD2 and SLE, little is known regarding the expression and function of NOD2 in SLE., RAGE is a transmembrane cell-surface receptor on a variety of immune effector cells, which is expressed at low levels in normal tissues and vasculature, but is upregulated wherever the accumulation of its proinflammatory ligands, especially the key ligand, high mobility group box protein 1 (HMGB1). Both endogenous secretory RAGE (esRAGE) as well as soluble RAGE (sRAGE) can be detected in blood serum and are able to bind the circulating ligands, neutralizing their actions. In those conditions characterized by high concentrations of the circulating ligands, the decoy receptors are reduced drastically, revealing the system function. Therefore, the relationship between the upregulation of full-length (fl) RAGE/RAGE ligands and the levels of “protective“ esRAGE/sRAGE in SLE is of obvious clinical interest., Apart from inducing and perpatating autoreactivity, abnormal innate response may also be responsible for the increased risk of infection in patients with lupus. The prevalence of abnormal Papanicolaou (Pap) smear was significantly increased in lupus patients in cross-sectional studies, associated with a higher prevalence of high-risk and multiple human papillomavirous (HPV) infections. However, none of the clinical, lifestyle, gynecological and treatment parameters was predictive of persistent HPV infection. Innate immune recognition of viral infection triggers antiviral immune responses. Whether the abnormal host innate immune response in lupus patients may play a role in enhancing HPV persistence remained unknown., Hypothesis, 1.Aberrant activation of NLR and RAGE pathways by endogenous or exogenous ligands lead to the initation and/or perpetuation of autoimmune responses in SLE, 2.HPV infection suppresses the host immune response by deregulating the TLRs transcript, leading to increased viral persistence in SLE., Aims, 1.To evaluate the role of NOD2 pathway in the pathogenesis of SLE, 2.To elucidate the relationship and regulatory mechanisms among members of the RAGE axis in the pathogenesis of SLE, 3.To investigate the role of TLR in the defense against HPV infection in SLE., Methods, The present thesis comprised of three cross-sectional studies in Chinese patients with SLE and controls in Hong Kong. Clinical assessments and review of medical records were performed to obtain information regarding disease status., Results, 1.Over-expression of NOD2 in monocytes was observed in immunosuppressant naive SLE patients, and was positively associated with longer disease duration. Immunosuppressive therapy was an independent explanatory variable for downregulating NOD2 expression in CD8⁺ T, monocytes and dendritic cells (DCs). Ex vivo basal productions of cytokines [Interleukin (IL)-6, IL-8 and IL-10] were significantly increased in immunosuppressant naive patients and patients with active disease despite immunosuppressants compared with healthy controls. Upon muramyl dipeptide (MDP) stimulaiton, relative induction (%) of cytokines (IL-1β) from peripheral blood mononuclear cells (PBMCs) was significantly increased in immunosuppressant naive patients with inactive disease, and patients with active disease despite immunosuppressant treatment compared with healthy controls. Immunosuppressant usage was associated with a decreased basal production and MDP induced relative induction (%) of IL-10 in patients with inactive disease compared with immunosuppressant naive patients and healthy controls., 2.Plasma sRAGE level was negatively correlated with SLE disease activities. The reduction in sRAGE levels in SLE patients with flare indicates that sRAGE may play a regulatory role on disease activity. HMGB1 alone could only mildly induce IL-6 production, which resulted in a transient phosporylation of intracellular p38 mitogen activated protein kinase (MAPK), c-Jun NH2- terminal protein kinase (JNK) and nuclear factor (NF)-κB. On the other hand, CpG-oligodeoxynucleotides (ODN) (TLR9 ligand) together with HMGB1 not only had a additive effect on IL-6 and IL-12p70 secretions compared with each agent alone, but also activated the phosphorylation of p38 MAPK and NF-κB., 3.TLR inhibitor (hydroxychloroquine) and prednisolone may down-regulate protein levels of TLRs 7 and 9 in cervical epithelial cells of lupus patients. In the cervical cell lines, TLRs 3, 7, 8, 9 protein levels and antiviral interferon-stimulated genes (ISG) 15 and myxovirus resistance (Mx) 1 gene expressions were inhibited in two oncogenic HPV types. Functional data showed that the induction of pro-inflammatory cytokines by TLR ligands [R837, single stranded (ss) RNA and CpG-ODN] was greatly impaired in CaSki and HeLa than C33A cells., Conclusions, Aberrant activation of PRR pathways by endogenous or exogenous molecules triggers the initation and/or perpetuation of autoimmune responses as follows, 1.NOD2 may participate in the pathogenesis of lupus via the recognition of MDP and induction of proinflammatory effects, implicating the innate immune response for endogenous pathogens in the immunopathological mechanisms in SLE, 2.Over-expression of RAGE may amplify the pro-inflammatory effects of DAMP such as HMGB1, while soluble RAGE may serve as a decoy receptor to suppress inflammation in patients with lupus nephritis., 3.Upregulated HMGB1 may act alone or in combine with TLR9 ligand through the phosphorylation of p38 MAPK and NF-κB to promote inflammation in lupus., On the other hand, the immune evasion strategy via avoidance of stimulation and downregulation of PRRs may promote establishment of persistent infection as listed below, 1.TLR inhibitor (hydroxychloroquine) and prednisolone may down-regulate protein levels of TLRs 7 and 9 in lupus patients, thereby decreasing the innate immune response against HPV infection., 2.Upon infection, HPV further down-regulate TLRs 7 and 9 levels for viral persistence., 3.Reduction of TLRs 7, 8 and 9 in carcinogenic HPVs ensures that the expression of inducible pro-inflammatory cytokines is minimized to prevent the expression of antiviral ISGs on a biologically relevant antiviral response., Detailed summary in vernacular field only., Yu, Shuilian., Thesis (Ph.D.)--Chinese University of Hong Kong, 2012., Includes bibliographical references (leaves 114-141)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., p.i, 摘要 --- p.v, ACKNOWLEDGEMENTS --- p.viii, LIST OF PUBLICATIONS --- p.ix, LIST OF AWARDS AND GRANDT RECORD --- p.x, CONTENTS --- p.viii, LIST OF TABLES --- p.xiii, LIST OF FIGURES --- p.xv, LIST OF APPENDIXS --- p.xvi, ABBREVIATIONS --- p.xvii, Chapter CHAPTER 1 --- REVIEW OF THE LITERATURE --- p.1, Chapter 1.1 --- What is systemic lupus erythematosus (SLE)? --- p.1, Chapter 1.2 --- Epidemiology of SLE --- p.1, Chapter 1.3 --- Etiology and pathogenesis of SLE --- p.3, Chapter 1.3.1 --- Genetic factors --- p.5, Chapter 1.3.2 --- Environmental triggers --- p.5, Chapter 1.3.3 --- Cellular abnormalities --- p.6, Chapter 1.3.3.1 --- APCs and cell debris clearance --- p.6, Chapter 1.3.3.2 --- B cell and the production of autoantibodies --- p.8, Chapter 1.3.3.3 --- T cell and the regulation of the immune responses --- p.9, Chapter 1.3.4 --- Disturbance of the innate immune response --- p.10, Chapter 1.3.4.1 --- PAMPs and DAPMs: all we need to know about danger in SLE --- p.10, Chapter 1.3.4.2 --- Sensors of innate immunity --- p.12, Chapter 1.3.4.2.1 --- TLRs sensing nucleic acids --- p.14, Chapter 1.3.4.2.2 --- NLRs sensing bacterial peptidoglycans --- p.16, Chapter 1.3.4.2.3 --- RAGE sensing dangerous signals --- p.16, Chapter 1.3.5 --- Dysregulation of cytokine networks --- p.19, Chapter 1.3.5.1 --- Anti-inflammatory cytokines --- p.20, Chapter 1.3.5.2 --- Proinflammatory cytokines --- p.20, Chapter 1.3.6 --- Abnormal signaling transduction --- p.22, Chapter 1.4 --- Clinical features of SLE --- p.22, Chapter 1.5 --- Laboratory features of SLE --- p.26, Chapter 1.6 --- Assessing disease activity and damage of SLE --- p.28, Chapter 1.7 --- Treatment of SLE --- p.28, Chapter 1.7.1 --- Current immunosuppressive therapy --- p.28, Chapter 1.7.2 --- Novel biologic therapies --- p.30, Chapter 1.8 --- Human papillomavirus (HPV) infection in SLE --- p.32, Chapter 1.8.1 --- Are women with lupus at higher risk of HPV infection? --- p.32, Chapter 1.8.2 --- Abnormalities of TLR-IFN axis potentially increases HPV risk --- p.32, Chapter 1.8.3 --- TLR suppressing mediciation potentially increases HPV risk --- p.34, Chapter CHAPTER 2 --- HYPOTHESISS AND AIMS --- p.35, Chapter CHAPTER 3 --- METHODOLOGIES --- p.36, Chapter 3.1 --- Materials --- p.36, Chapter 3.1.1 --- Selection of patients and controls --- p.36, Chapter 3.1.2 --- Blood and cervical samples --- p.36, Chapter 3.1.3 --- Cervical epithelial cell lines --- p.37, Chapter 3.1.4 --- Culture medium and serum supplement --- p.37, Chapter 3.1.5 --- Culture ligands --- p.37, Chapter 3.1.6 --- Reagents for flow cytometric analysis (FCM) --- p.37, Chapter 3.1.7 --- Antibodies for FCM --- p.38, Chapter 3.1.8 --- Quantative assay kits --- p.39, Chapter 3.1.9 --- Membrane array of phosphorylated intracellular kinases --- p.39, Chapter 3.1.10 --- Primers for qPCR --- p.40, Chapter 3.2 --- Methods --- p.40, Chapter 3.2.1 --- Study design and patient assessment --- p.40, Chapter 3.2.2 --- Isolation of PBMCs --- p.41, Chapter 3.2.3 --- Isolation of monocytes --- p.42, Chapter 3.2.4 --- Cell culture --- p.42, Chapter 3.2.5 --- Sampling procedure of cervical epithelial cells --- p.42, Chapter 3.2.6 --- HPV identification --- p.43, Chapter 3.2.7 --- Flow cytometry gating of target cells --- p.43, Chapter 3.2.8 --- FCM of target molecules and phosphorylated signaling molecules --- p.45, Chapter 3.2.9 --- Membrane array of phosphorylated intracellular kinases --- p.46, Chapter 3.2.10 --- Cytokine cytometric bead array --- p.46, Chapter 3.2.11 --- Enzyme-linked immunosorbent assay --- p.46, Chapter 3.2.12 --- Real-time qPCR --- p.46, Chapter 3.2.13 --- Statistical analysis --- p.47, Chapter CHAPTER 4 --- DOWN-REGULATED NOD2 BY IMMUNOSUPPRESSANTS IN PERIPHERAL BLOOD CELLS IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS REDUCES THE MURAMYL DIPEPTIDE-INDUCED IL-10 PRODUCTION --- p.48, Chapter 4.1 --- INTRODUCTION --- p.48, Chapter 4.2 --- METHODS --- p.49, Chapter 4.2.1 --- Patient selection and assessment --- p.49, Chapter 4.2.2 --- FCM of NOD2 expression in T, B cells, monocytes and DCs --- p.49, Chapter 4.2.3 --- Cell culture --- p.49, Chapter 4.2.4 --- Quantitative assay --- p.49, Chapter 4.2.5 --- Statistical analyses --- p.49, Chapter 4.3 --- RESULTS --- p.50, Chapter 4.3.1 --- Characteristics of lupus patients and control subjects --- p.50, Chapter 4.3.2 --- Identification of DCs, T, B lymphocytes and monocytes --- p.50, Chapter 4.3.3 --- Protein level of NOD2 in DCs, T, B lymphocytes and monocytes --- p.53, Chapter 4.3.4 --- Potential explanatory variables associated with NOD2 levels in lupus patients --- p.55, Chapter 4.3.5 --- Induction of inflammatory cytokines by NOD2 ligand --- p.61, Chapter 4.4 --- DISCUSSION --- p.63, Chapter 4. --- 5 CONCLUSIONS --- p.67, Chapter CHAPTER 5 --- MEMBERS OF THE RECEPTOR FOR ADVANCED GLYCATION ENDPRODUCTS AXIS AS POTENTIAL THERAPEUTIC TARGETS IN SYSTEMIC LUPUS ERYTHEMATOSUS --- p.68, Chapter 5.1 --- INTRODUCTION --- p.68, Chapter 5.2 --- METHODS --- p.68, Chapter 5.2.1 --- Patients selection and assessment --- p.68, Chapter 5.2.2 --- FCM of monocyte-surface flRAGE --- p.69, Chapter 5.2.3 --- Cell culture --- p.69, Chapter 5.2.4 --- Quantitative assay --- p.69, Chapter 5.2.5 --- Membrane array of phosphorylated of intracellular kinases --- p.69, Chapter 5.2.6 --- FCM of activated intracellular signaling molecules --- p.69, Chapter 5.2.7 --- Statistical analysis --- p.69, Chapter 5.3 --- RESULTS --- p.69, Chapter 5.3.1 --- Characteristics of SLE patients --- p.69, Chapter 5.3.3 --- Relationships between RAGE isoforms and HMGB1 --- p.75, Chapter 5.3.4 --- Potential explanatory variables associated with levels of RAGE isoforms and HMGB1 in LN patients --- p.77, Chapter 5.3.5 --- Activity of HMGB1 alone or in combine with TLR9 ligand --- p.81, Chapter 5.4 --- DISCUSSION --- p.84, Chapter 5.5 --- CONCLUSIONS --- p.88, Chapter CHAPTER 6 --- ANTAGONIST-MEDIATED DOWN-REGULATION OF TOLL-LIKE RECEPTOR INCREASES THE PREVALENCE OF HUMAN PAPILLOMAVIRUS INFECTION IN SYSTEMIC LUPUS ERYTHEMATOSUS --- p.89, Chapter 6.1 --- INTRODUCTION --- p.89, Chapter 6.2 --- METHODS --- p.90, Chapter 6.2.1 --- Patient selection and assessment --- p.90, Chapter 6.2.2 --- HPV sampling procedure and identification --- p.90, Chapter 6.2.3 --- FCM of TLRs 3, 7, 8 and 9 in cervical epithelial cells --- p.90, Chapter 6.2.4 --- Cell culture --- p.90, Chapter 6.2.5 --- Quantitative assay --- p.90, Chapter 6.2.6 --- Real-time qPCR of Interferon-stimulated genes (ISGs) --- p.90, Chapter 6.2.7 --- Statistical analysis --- p.91, Chapter 6.3 --- RESULTS --- p.91, Chapter 6.3.1 --- Pap smear findings, socio-demographic and clinical characteristics --- p.91, Chapter 6.3.2 --- Identification of cervical epithelial cells --- p.95, Chapter 6.3.3 --- Protein level of TLRs 3, 7, 8 and 9 in cervical epithelial cells --- p.96, Chapter 6.3.4 --- Potential explanatory variables associated with TLR levels in lupus patients --- p.98, Chapter 6.3.5 --- TLRs and ISGs expressions are inhibited by oncogenic HPVs --- p.102, Chapter 6.3.6 --- Induction of inflammatory cytokines by TLR agonists was impaired in oncogenic HPVs --- p.103, Chapter 6.4 --- DISCUSSION --- p.105, Chapter 6.5 --- CONCLUSIONS --- p.107, Chapter CHAPTER 7 --- CONCLUSIONS OF THE THESIS --- p.108, Chapter 7.1 --- Answers to the hypotheses --- p.108, Chapter 7.2 --- Conclusions and implications --- p.109, Chapter 7.3 --- Liminations and future plan --- p.110, Chapter 7.3.1 --- Liminations of study design --- p.110, Chapter 7.3.2 --- Liminations of methodology --- p.111, Chapter 7.3.3 --- Liminations of CHAPTER 4 and future plan --- p.111, Chapter 7.3.4 --- Liminations of CHAPTER 5 and future plan --- p.112, Chapter 7.3.5 --- Liminations of CHAPTER 6 and future plan --- p.112, Chapter CHAPTER 8 --- REFERENCES --- p.114, Chapter CHAPTER 9 --- APPENDIX --- p.142, http://library.cuhk.edu.hk/record=b5549572, Use of this resource is 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血管紧张素II(Ang II)在慢性肾脏病中起重要的致病作用，尽管体外研究证实TGF-β/Smad3起正调控，Smad7起负调控作用，但Smad3在Ang II 诱导的肾脏损害中的作用仍不清楚。因此，本论文在Smad3基因敲除的小鼠中通过Ang II诱导的高血压肾损伤模型研究TGF-β/Smad3通路的作用及机制。如第三章所述，敲除Smad3的小鼠不发生Ang II诱导的高血压肾损伤如尿白蛋白，血肌酐升高，肾脏炎症（如IL-1, TNFα上调，F4/80+ 巨噬细胞浸润）及肾脏纤维化（包括α-SMA+肌成纤维细胞聚集，和胶原基质沉积）。敲除Smad3对高血压肾病起保护作用是因为抑制了肾脏TGF-β1表达及Smurf2 依赖的Smad7泛素化降解，从而抑制TGF-β/Smad3介导的肾脏纤维化和NF-B介导的炎症。, 越来越多的证据显示Ang II产生和降解的平衡在高血压肾病的发展中起重要作用。在这篇论文中，我们假设ACE2的降解可能会引起Ang II代谢通路的失衡，从而加重其介导的高血压肾病。这一假设在第四章得到验证，在单侧输尿管梗阻小鼠模型敲除ACE2加重肾内Ang II介导的肾脏纤维化和炎症。这一变化与肾内高水平的Ang II和降低的血管紧张素1-7，上调的血管紧张素受体1，及激活的TGF-β/Smad3 和 NF-κB 信号通路有关。另外，升高的Smurf2介导的Smad7泛素化降解加重了敲除ACE2 基因后Ang II介导的肾脏纤维化和炎症。, 因为Smad7 是TGF-β/Smad和NF-κB通路的负调控因子，因此论文进一步提出假设过表达Smad7能够阻止Ang II介导的肾脏纤维化炎症。如第五章所述，ACE2基因敲除的小鼠肾内升高的Smurf2介导了肾脏Smad7 的泛素化降解， 加重了Ang II 介导的肾脏损伤如白蛋白尿，血肌酐的升高，及肾脏纤维化和炎症，这与激活的Ang II/TGF-β/Smad3/NF-κB信号有关。相反，过表达Smad7能够阻断TGF-β/Smad3 介导的肾脏纤维化和 NF-κB介导的肾脏炎症以缓解ACE2敲除小鼠中Ang II诱导的肾脏损伤。, 总之，Smad3在Ang II诱导的高血压肾脏病中起关键作用，Smad7具有肾脏保护作用。 ACE2敲除引起Ang II产生和降解的失衡从而增加肾内Ang II的产生，加重TGF-β/Smad3介导的肾脏纤维化和NF-κB介导的肾脏炎症，而这可以被Smad7缓解。 本论文得出结论针对TGF-β/Smad3 和NF-κB通路，通过过表达Smad7可能为高血压肾脏病和慢性肾脏病提供新的治疗策略。, Angiotensin II (Ang II) plays a pathogenic role in chronic kidney disease (CKD). Although in vitro studies find that Ang II mediates renal fibrosis via the Smad3-dependent mechanism, the functional role of Smad3 in Ang II-mediated kidney disease remains unclear. Therefore, this thesis examined the pathogenesis role and mechanisms of TGF-β/Smad3 in Ang II-mediated hypertensive nephropathy in Smad3 Knockout (KO) mice. As described in Chapter III, Smad3 deficiency protected against Ang II-induced hypertensive nephropathy as demonstrated by lowering levels of albuminuria, serum creatinine, renal inflammation such as up-regulation of pro-inflammatory cytokines (IL-1β, TNFα) and infiltration of CD3+ T cells and F4/80+ macrophages, and renal fibrosis including α-SMA+ myofibroblast accumulation and collagen matrix deposition (all p<0.01). Inhibition of hypertensive nephropathy in Smad3 KO mice was associated with reduction of renal TGF-β1 expression and Smurf2-associated ubiquitin degradation of renal Smad7, thereby blocking TGF-β/Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation., Increasing evidence shows that the balance between the generation and degradation of Ang II is also important in the development of hypertensive nephropathy. In this thesis, we also tested a hypothesis that enhanced degradation of ACE2 may result in the imbalance between the Ang II generation and degradation pathways, therefore enhancing Ang II-mediated hypertensive nephropathy and CKD. This hypothesis was examined in a mouse model of unilateral ureteral obstructive nephropathy (UUO) induced in ACE2 KO mice. As described in Chapter IV, loss of ACE2 increased intrarenal Ang II-mediated renal fibrosis and inflammation in the UUO kidney. These changes were associated with higher levels of intrarenal Ang II, reduced Ang 1-7, up-regulated AT1R, and activation of TGF-β/Smad3 and NF-κB signalling. In addition, enhanced Smurf2-associated ubiquitin degradation of Smad7 was another mechanism by which loss of ACE2 promoted Ang II-mediated renal fibrosis and inflammation., Because Smad7 is a negative regulator for TGF-β/Smad and NF-κB signalling, this thesis also examined a hypothesis that overexpression of renal Smad7 may be able to prevent Ang II-induced, TGF-β/Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation in ACE2 KO mice. As described in Chapter V, mice null for ACE2 resulted in degradation of renal Smad7 via the Smurf2 -- dependent mechanism (all p<0.01). Enhanced Ang II-mediated renal injury in ACE2 KO mice such as albuminuria, serum creatinine, and renal fibrosis and inflammation was associated with enhanced activation of Ang II/TGF-β/Smad3/NF-κB signalling. In contrast, overexpression of Smad7 was able to rescue AngII-induced progressive renal injury in ACE2 KO mice by blocking TGF-β/Smad3 and NF-κB-dependent renal fibrosis and inflammation. In conclusion, Smad3 plays an essential role in Ang II-induced hypertensive nephropathy, while Smad7 is reno-protective. Loss of ACE2 results in the imbalance between the Ang II generation and degradation pathways and thus enhances intrarenal Ang II-induced, TGF-β/Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation, which can be rescued by Smad7. Results from this thesis indicate that targeting TGF-β/Smad3 and NF-κB pathways by overexpressing Smad7 may represent a novel therapy for hypertensive nephropathy and CKD., Detailed summary in vernacular field only., Liu, Zhen., Thesis (Ph.D.)--Chinese University of Hong Kong, 2012., Includes bibliographical references (leaves 189-209)., s also in Chinese., p.i, DECLARATION --- p.v, ACKNOWLEDGEMENTS --- p.vi, LIST OF PUBLICATION --- p.viii, TABLE OF CONTENTS --- p.ix, LIST OF ABBREVIATIONS --- p.xiv, LIST OF FIGURES AND TABLES --- p.xvii, CHAPTER I --- p.1, INTRODUCTION --- p.1, Chapter 1.1 --- RAS (Renin-Angiotensin system) --- p.2, Chapter 1.1.1 --- Circulating RAS --- p.2, Chapter 1.1.2 --- Tissue RAS --- p.5, Chapter 1.1.2.1 --- Angiotensinogen --- p.6, Chapter 1.1.2.2 --- Renin Receptors --- p.7, Chapter 1.1.2.3 --- ACE and ACE2 --- p.9, Chapter 1.1.2.4 --- Angiontensin II and Its Receptors --- p.10, Chapter 1.1.2.5 --- AT2 Receptors --- p.11, Chapter 1.1.2.6 --- Chymase-Alternative Pathways of Ang II Generation --- p.13, Chapter 1.1.2.7 --- Ang (1-7) Receptor (MAS) --- p.13, Chapter 1.2 --- Ang II and Renal Injury --- p.15, Chapter 1.2.1 --- Pressure Dependent Renal Injury Induced by Ang II --- p.15, Chapter 1.2.2 --- Ang II induces production of cytokines and growth factors --- p.16, Chapter 1.2.3 --- Ang II and Renal Fibrosis --- p.17, Chapter 1.2.4 --- Signalling Mechanisms Involved in Ang II-Induced Renal Fibrosis --- p.18, Chapter 1.2.5 --- Ang II in Renal Inflammation --- p.22, Chapter 1.3 --- TGF-β/Smad Signalling Pathway in Renal Disease --- p.24, Chapter 1.3.1 --- Mechanisms of TGF-β/Smad Activation --- p.24, Chapter 1.3.1.1 --- Cross-talk Between Smads and Other Signalling Pathways in Renal Fibrosis --- p.26, Chapter 1.3.1.2 --- Activation of R-Smads (Smad2 and Smad3) --- p.28, Chapter 1.3.2 --- Inhibitory Role of Smad7 in Renal Fibrosis and Inflammation --- p.30, Chapter CHAPTER II --- p.32, MATERIALS AND METHODS --- p.32, Chapter 2.1 --- MATERIALS --- p.33, Chapter 2.1.1 --- Regents and Equipments --- p.33, Chapter 2.1.1.1 --- Regents and Equipments for Cell Culture --- p.33, Chapter 2.1.1.2 --- General Reagents and Equipments for Real-time PCR --- p.34, Chapter 2.1.1.3 --- General Reagents and Equipments for Masson Trichrome Staining --- p.34, Chapter 2.1.1.4 --- General Reagents and Equipments for Immunohistochemistry --- p.35, Chapter 2.1.1.5 --- General Reagents and Equipments for Western Blot --- p.35, Chapter 2.1.1.6 --- General Reagents and Equipments for ELISA --- p.37, Chapter 2.1.1.7 --- Measurement of Blood Pressure in Mice --- p.37, Chapter 2.1.1.8 --- Reagents and Equipment for Genotyping --- p.37, Chapter 2.1.2 --- Buffers --- p.38, Chapter 2.1.2.1 --- Immunohistochemistry Buffers --- p.38, Chapter 2.1.2.2 --- Buffers for Western Blotting --- p.40, Chapter 2.1.2.3 --- ELISA Buffers --- p.44, Chapter 2.1.2.4 --- Primer Sequences --- p.46, Chapter 2.1.2.5 --- Primary Antibodies --- p.47, Chapter 2.1.2.6 --- Secondary Antibodies --- p.48, Chapter 2.2 --- METHODS --- p.49, Chapter 2.2.1 --- Animal --- p.49, Chapter 2.2.1.1 --- Genotypes of Gene KO Mice --- p.49, Chapter 2.2.1.2 --- Animal Model of Unilateral Ureteral Obstruction (UUO) --- p.50, Chapter 2.2.1.3 --- Animal Model of Angiotensin II (Ang II)-Induced Hypertensive Nephropathy --- p.50, Chapter 2.2.1.4 --- Measurement of Ang II and Ang 1-7 --- p.51, Chapter 2.2.2 --- Cell Culture --- p.51, Chapter 2.2.3 --- Microalbuminuria and Renal Function --- p.51, Chapter 2.2.3.1 --- Urine Collection --- p.51, Chapter 2.2.3.2 --- Plasma Collection --- p.52, Chapter 2.2.3.3 --- Microalbuminuria --- p.52, Chapter 2.2.3.4 --- Creatinine Measurement --- p.52, Chapter 2.2.4 --- Real-time PCR --- p.53, Chapter 2.2.4.1 --- Total RNA Extraction --- p.53, Chapter 2.2.4.2 --- Reverse Transcription --- p.53, Chapter 2.2.4.3 --- Real-time PCR --- p.54, Chapter 2.2.4.4 --- Analysis of Real-time PCR --- p.54, Chapter 2.2.5 --- Western Blot --- p.55, Chapter 2.2.5.1 --- Protein Preparation --- p.55, Chapter 2.2.5.2 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.56, Chapter 2.2.5.3 --- Protein Transfer (Wet Transfer) --- p.56, Chapter 2.2.5.4 --- Incubation of Antibodies --- p.56, Chapter 2.2.5.5 --- Scanning and Analysis --- p.57, Chapter 2.2.5.6 --- Stripping --- p.57, Chapter 2.2.6 --- Histochemistry --- p.57, Chapter 2.2.6.1 --- Tissue Fixation --- p.57, Chapter 2.2.6.2 --- Tissue Embedding and Sectioning --- p.58, Chapter 2.2.6.3 --- Preparation of Paraffin Tissue Sections for PAS Staining --- p.58, Chapter 2.2.6.4 --- PAS Staining --- p.58, Chapter 2.2.7 --- Immunohistochemistry --- p.59, Chapter 2.2.7.1 --- Tissue Embedding and Sectioning --- p.59, Chapter 2.2.7.2 --- Antigen-Antibody Reaction and Immunostaining --- p.59, Chapter 2.2.7.3 --- Semi-quantification of Immunohistochemistry --- p.60, Chapter 2.2.8 --- Statistical Analysis --- p.60, Chapter CHAPTER III --- p.62, ROLE OF SMAD3 IN ANGIOTENSIN II-INDUCED RENAL FIBROSIS AND INFLAMMATION --- p.62, Chapter 3.1 --- INTRODUCTION --- p.63, Chapter 3.2 --- MATERIALS AND METHODS --- p.64, Chapter 3.2.1 --- Generation of Smad3 KO Mice --- p.64, Chapter 3.2.2 --- Mouse Model of Ang II-Induced Hypertension --- p.64, Chapter 3.2.3 --- Histology and Immunohistochemistry --- p.65, Chapter 3.2.4 --- Renal Function and Proteinuria --- p.65, Chapter 3.2.5 --- Western Blot Analysis --- p.65, Chapter 3.2.6 --- Real-time RT-PCR --- p.65, Chapter 3.2.7 --- In Vitro Study of Mesangial Cells from Smad3 WT and KO Mice --- p.66, Chapter 3.2.8 --- Statistical Analysis --- p.66, Chapter 3.3 --- RESULTS --- p.66, Chapter 3.3.1 --- Smad3 KO Mice Prevents Ang II-induced Renal Injury Independent of Blood Pressure --- p.66, Chapter 3.3.2 --- Smad3 KO Mice Are Resistant to Renal Fibrosis in a Mouse Model of Ang II -Induced Hypertension --- p.70, Chapter 3.3.3 --- Smad3 KO Mice Are Resistant to Renal Inflammation in a Mouse Model of Ang II-Induced Hypertension --- p.76, Chapter 3.3.4 --- Smad3 Deficiency Inhibits Ang II-induced Renal Fibrosis and Inflammation In Vitro --- p.82, Chapter 3.3.5 --- Smad3 Mediates Ang II-Induced Renal Fibrosis by the Positive Feedback Mechanism of TGF-β/Smad Signalling --- p.87, Chapter 3.3.6 --- Enhancing NF-κB Signalling via the Smurf2-associated Ubiquitin Degradation of Smad7 In Vivo and In Vitro --- p.92, Chapter 3.4 --- DISCUSSION --- p.101, Chapter 3.5 --- CONCLUSION --- p.106, Chapter CHAPTER IV --- p.107, LOSS OF ANGIOTENSIN-CONVERTING ENZYME 2 ENHANCES TGF-β/SMAD-MEDIATED RENAL FIBROSIS AND NF-κB-DRIVEN RENAL INFLAMMATION IN A MOUSE MODEL OF OBSTRUCTIVE NEPHROPATHY --- p.107, Chapter 4.1 --- INTRODUCTION --- p.108, Chapter 4.2 --- MATERIALS AND METHODS --- p.109, Chapter 4.2.1 --- Generation of ACE2 KO Mice --- p.109, Chapter 4.2.2 --- Mouse Model of Unilateral Ureteral Obstruction (UUO) --- p.109, Chapter 4.2.3 --- Histology and Immunohistochemistry --- p.110, Chapter 4.2.4 --- Western Blot Analysis --- p.110, Chapter 4.2.5 --- Real-time RT-PCR --- p.110, Chapter 4.2.6 --- Measurement of Ang II and Ang 1-7 --- p.110, Chapter 4.2.7 --- Statistical Analysis --- p.111, Chapter 4.3 --- RESULTS --- p.111, Chapter 4.3.1 --- ACE2 KO Mice Accelerate Renal Fibrosis and Inflammation Independent of Blood Pressure in the UUO Nephropathy --- p.111, Chapter 4.3.2 --- Loss of ACE2 Enhances Ang II, Activation of TGF-β/Smad and NF-κB Signalling Pathways --- p.128, Chapter 4.3.3 --- Loss of Renal Smad7 Is an Underlying Mechanism Accounted for the Progression of TGF-β/Smad-mediated Renal Fibrosis and NF-κB-Driven Renal Inflammation in the UUO Nephropathy in ACE2 KO Mice --- p.140, Chapter 4.4 --- DISCUSSION --- p.143, Chapter 4.5 --- CONCLUSION --- p.147, CHAPTER V --- p.148, PROTECTIVE ROLE OF SMAD7 IN HYPERTENSIVE NEPHROPATHY IN ACE2 DEFICIENT MICE --- p.148, Chapter 5.1 --- INTRODUCTION --- p.149, Chapter 5.2 --- MATERIALS AND METHODS --- p.151, Chapter 5.2.1 --- Generation of ACE2 KO Mice --- p.151, Chapter 5.2.2 --- Mouse Model of Ang II-Induced Hypertension --- p.151, Chapter 5.2.3 --- Smad7 Gene Therapy --- p.151, Chapter 5.2.4 --- Histology and Immunohistochemistry --- p.152, Chapter 5.2.5 --- Western Blot Analysis --- p.153, Chapter 5.2.6 --- Real-time RT-PCR --- p.153, Chapter 5.2.7 --- Measurement of Ang II and Ang 1-7 --- p.153, Chapter 5.2.8 --- Statistical Analysis --- p.153, Chapter 5.3 --- RESULTS --- p.154, Chapter 5.3.1 --- Deletion of ACE2 Accelerates Ang II-Induced Renal Injury --- p.154, Chapter 5.3.2 --- Renal Fibrosis and Inflammation are Enhanced in ACE2 KO Mice with Ang II-Induced Renal Injury --- p.156, Chapter 5.3.3 --- Enhanced Activation of TGF-β/Smad3 and NF-κB Signalling Pathways are Key Mechanism by Which Deletion of ACE2 Promotes Ang II-Induced Renal Injury --- p.163, Chapter 5.3.4 --- Loss of Renal Smad7 Mediated by Smurf2-ubiquintin Degradation Pathway Contributes to Ang II-Induced Hypertensive Nephropathy in ACE2 KO Mice --- p.166, Chapter 5.3.5 --- Overexpression of Smad7 is able to Rescue Ang II-induced Renal Injury in ACE2 KO Mice by Blocking Both TGF-β/Smad3 and NF-κB-dependent Renal Fibrosis and Inflammation --- p.168, Chapter 5.4 --- DISCUSSION --- p.180, Chapter 5.5 --- CONCLUSION --- p.182, Chapter CHAPTER VI --- p.183, SUMMARY AND DISCUSSION --- p.183, Chapter 6.1 --- Smad3 Plays a Key Role in Ang II-Induced Hypertensive Nephropathy --- p.185, Chapter 6.2 --- The Intrarenal Ang II Plays a Key Role in the Progress of Ang II-Mediated Renal Injury --- p.185, Chapter 6.3 --- A Novel Finding of Ang II-Smad3-TGF-β-Smad3 amplification loop in Ang II-mediated Renal Fibrosis --- p.186, Chapter 6.4 --- Smurf2-associated Ubiquitin-Proteasome Degradation of Smad7 Contributes to the Progression of Ang II-mediated Renal Injury in ACE2 KO Mice --- p.187, Chapter 6.5 --- Smad7 Protects against Ang II-Mediated Hypertensive Kidney Disease by Negatively Regulating TGF-β/Samd and NF-κB Signalling --- p.187, REFERENCE --- p.189, http://library.cuhk.edu.hk/record=b5549544, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

腎細胞癌是一種成人惡性腫瘤，治療效果不理想且常發生腫瘤轉移。目前對腎細胞癌的研究主要集中於鑒定並驗證可用於癌癥早期診斷和預後判斷的新型潛在生物標誌物。擬遺傳學變化尤其是啟動子CpG二核苷酸甲基化所導致的抑癌基因功能喪失已被廣泛認為是腫瘤發生的一個主要機理。迄今為止，已有許多抑癌基因在腎細胞癌中被報道出現啟動子甲基化。這些發現為腎癌發生的分子機制及潛在生物標誌物提供了新的思路。本課題旨在探索ZNF382和BCL6B這兩個鋅指蛋白抑癌基因在腎細胞癌中的啟動子甲基化情況，及其與腫瘤抑制有關的生物學功能和可能的分子機制。, 鋅指蛋白轉錄抑制子ZNF382已在多種癌癥中被報道為功能性抑癌基因， 且常伴隨有啟動子甲基化導致的基因沈默，但其在腎細胞癌中尚未被報道。我們發現ZNF382在腎癌細胞系中由於啟動子CpG甲基化而致表達下調或基因沈默，並且其表達下調或沈默可被去甲基化藥物逆轉，在正常細胞系中則觀察不到這一現象。腎癌原發腫瘤組織中也廣泛檢測到ZNF382異常甲基化。在ZNF382沈默的腎細胞癌細胞系中，外源表達的ZNF382顯著地抑制了腫瘤細胞集落形成和細胞遷移，並且誘導細胞發生雕亡。而且，我們發現ZNF382在腎癌細胞系中可抑制多種致瘤基因和幹細胞標誌基因的表達。因此，本研究證明ZNF382通過抑制下遊致癌基因和幹細胞標誌基因的表達從而發揮抑制腫瘤的功能，並且其在腎細胞癌中常因啟動子高度甲基化而導致基因失活。, 另一個鋅指蛋白基因BCL6B（ZNF62）已被證實可通過招募組蛋白去乙酰化酶抑制靶基因的轉錄，但其在腎細胞癌中的表達情況和生物學功能尚不清楚。我們發現，BCL6B基因在正常腎組織和正常細胞系中穩定表達， 但在腎癌細胞系中由於啟動子甲基化其表達下調或沈默。去甲基化藥物可以重新激活BCL6B的表達，同時伴隨其啟動子的去甲基化。BCL6B甲基化在腎癌原發腫瘤組織中也被頻繁檢測到。在腎癌細胞系中，外源表達BCL6B顯著抑制了腫瘤細胞集落形成和細胞遷移，並且誘導腫瘤細胞雕亡。我們進一步發現，BCL6B作為功能性轉錄抑制子在腎癌細胞系中抑制多種致癌基因和幹細胞標誌基因的表達。這些結果表明BCL6B是腎細胞癌的一個抑癌基因且其在腎癌中常被甲基化。, 綜上所述，本課題從擬遺傳學和生物學功能兩個方面分別鑒定了腎癌中的兩個鋅指蛋白抑癌基因，ZNF382 和BCL6B。此研究可以幫助更好地了解腎癌發生的分子機理，並且為發展新的腎癌標誌物提供了更多思路。, Renal cell carcinoma (RCC) is a malignant cancer in adults, often with poor outcome and frequent metastasis. Recent studies on this disease focus on the identification and verification of novel potential biomarkers for early detection and prognostic prediction of cancer. Epigenetic alterations, especially promoter CpG methylation, leading to the loss of tumor suppressor gene (TSG) function have been widely recognized as a major cause for tumor pathogenesis. To date, a number of TSGs with aberrant promoter methylation have been reported in RCC, which provides new insights into the molecular mechanism of renal cancer and the potential as biomarkers. The aim of this study is to characterize promoter methylation of two zinc finger tumor suppressors, ZNF382 and BCL6B, their biological functions and underlying molecular mechanisms in RCC., Transcription repressor ZNF382 (zinc finger protein 382) was reported as a functional TSG with frequent inactivation by promoter methylation in multiple carcinomas, but not studied in RCC yet. I found that ZNF382 was silenced or downregulated in RCC cell lines due to promoter CpG methylation which could be reversed by pharmacologic demethylation treatment, but not in normal renal cell lines. Aberrant methylation of ZNF382 was also frequently detected in the RCC primary tumors. Ectopic expression of ZNF382 in the silenced RCC cells strongly inhibited their clonogenicity and migration, as well as promoted cell apoptosis. Moreover, I found that ZNF382 repressed the expression of multiple oncogenes and stem cell markers in RCC cells. Therefore, my results demonstrate ZNF382 exerts the tumor suppressive function through repressing the downstream target oncogenes and stem cell markers, and is often epigenetically inactivated by promoter methylation in RCC., Another zinc finger protein, B cell CLL/lymphoma 6 member B (BCL6B, ZNF62) has been identified to repress transcription of its target genes by recruiting histone deacetylases, but its expression and biological function in RCC remain largely unknown. BCL6B was readily expressed in normal kidney tissue and renal cell line. BCL6B was silenced or downregulated by promoter CpG methylation in RCC cell lines. Pharmacologic demethylation reactivated BCL6B expression along with concomitant promoter demethylation. BCL6B methylation was also frequently detected in RCC primary tumors. Ectopic expression of BCL6B in RCC cells significantly inhibited tumor clonogenicity and migration of RCC cells, and induced tumor cell apoptosis. We further found that BCL6B as functional repressor suppressed the expression of multiple oncogenes and stem cell markers. These data indicated BCL6B was a functional tumor suppressor frequently methylated in RCC., In summary, my study identified two zinc finger tumor suppressors, ZNF382 and BCL6B, in RCC from both epigenetical and functional aspects. This work may contribute to a better understanding of the molecular mechanisms of renal cancer pathogenesis and also give more clues to the discovery of novel biomarkers for RCC., Detailed summary in vernacular field only., Rong, Rong., Thesis (M.Phil.)--Chinese University of Hong Kong, 2012., Includes bibliographical references (leaves 110-128)., s also in Chinese., in English --- p.i, in Chinese --- p.iii, Acknowledgements --- p.v, Table of Content --- p.vi, List of abbreviations --- p.xi, List of Figures --- p.xv, List of Tables --- p.xvii, List of Publications --- p.xviii, Chapter Chapter 1 --- Literature Reviews --- p.1, Chapter 1.1 --- Molecular basis of cancer --- p.1, Chapter 1.1.1 --- Oncogenes and TSGs --- p.2, Chapter 1.1.2 --- Cancer genetics --- p.3, Chapter 1.1.3 --- Cancer epigenetics --- p.4, Chapter 1.1.4 --- DNA methylation --- p.4, Chapter 1.1.4.1 --- Mechanism of DNA methylation --- p.5, Chapter 1.1.4.2 --- DNA methylation and gene transcription --- p.6, Chapter 1.1.4.3 --- Types of DNA methylation in human cancers --- p.7, Chapter 1.1.4.3.1 --- Hypomethylation in cancer genome --- p.8, Chapter 1.1.4.3.2 --- Hypermethylation of TSGs in cancer --- p.8, Chapter 1.1.5 --- The link between cancer genetics and cancer epigenetics --- p.9, Chapter 1.2 --- Renal cell carcinoma (RCC) --- p.10, Chapter 1.2.1 --- Epidemiology of RCC --- p.10, Chapter 1.2.2 --- Histopathology of RCC --- p.14, Chapter 1.2.3 --- Genetic and epigenetic alterations in RCC --- p.16, Chapter 1.2.3.1 --- Genetic alterations --- p.17, Chapter 1.2.3.2 --- Epigenetic alterations --- p.22, Chapter 1.2.3.2.1 --- Aberrant DNA hypermethylation in RCC --- p.22, Chapter 1.2.3.2.2 --- Histone and chromatin regulations in RCC --- p.24, Chapter 1.2.4 --- Signaling pathways associated with RCC --- p.25, Chapter 1.2.4.1 --- VHL/HIF signaling in RCC --- p.26, Chapter 1.2.4.2 --- PI3K/AKT/mTOR signaling in RCC --- p.27, Chapter 1.2.4.3 --- Wnt/β-catenin signaling in RCC --- p.28, Chapter 1.2.4.4 --- HGF/MET signaling in RCC --- p.31, Chapter 1.3 --- Transcription factor family of zinc finger proteins --- p.32, Chapter 1.3.1 --- Zinc Finger Protein 382 (ZNF382) --- p.33, Chapter 1.3.2 --- B cell CLL/lymphoma 6, member B (BCL6B) --- p.34, Chapter Chapter 2 --- Aim of Study --- p.36, Chapter 2.1 --- Identify two zinc finger repressors as TSGs for RCC --- p.37, Chapter 2.2 --- Study their tumor suppressor roles in RCC --- p.37, Chapter 2.3 --- Explore the mechanisms of their tumor suppressor function --- p.38, Chapter Chapter 3 --- Materials and Methods --- p.39, Chapter 3.1 --- Cell lines and tissue samples --- p.39, Chapter 3.1.1 --- Cell lines, tumors and normal tissue samples --- p.39, Chapter 3.1.2 --- Maintenance of cell lines --- p.39, Chapter 3.1.3 --- Drug treatment of cell lines --- p.40, Chapter 3.1.4 --- Total RNA extraction --- p.40, Chapter 3.1.5 --- Genomic DNA extraction --- p.41, Chapter 3.2 --- General techniques --- p.42, Chapter 3.2.1 --- Agarose gel electrophoresis --- p.42, Chapter 3.2.2 --- TA cloning --- p.43, Chapter 3.2.3 --- Transformation of cloning vectors into E. coli competent cells --- p.43, Chapter 3.2.4 --- Plasmid DNA extraction --- p.44, Chapter 3.2.4.1 --- Mini-prep of plasmid DNA --- p.44, Chapter 3.2.4.2 --- Midi-prep of plasmid DNA --- p.45, Chapter 3.2.5 --- Measurement of DNA and RNA concentrations --- p.45, Chapter 3.2.6 --- Preparation of reagents and medium --- p.46, Chapter 3.2.6.1 --- Reagents for agarose gel electrophoresis --- p.46, Chapter 3.2.6.2 --- Reagents for mini-prep of plasmid DNA --- p.46, Chapter 3.2.6.3 --- LB medium and LB plates --- p.46, Chapter 3.3 --- Semi-quantitative Reverse transcription (RT)-PCR --- p.47, Chapter 3.3.1 --- Reverse Transcription --- p.47, Chapter 3.3.2 --- Semi-quantitative PCR --- p.48, Chapter 3.3.2.1 --- Primer design --- p.48, Chapter 3.3.2.2 --- PCR reaction --- p.49, Chapter 3.4 --- Real-time PCR --- p.49, Chapter 3.5 --- Methylation analysis --- p.50, Chapter 3.5.1 --- Bisulfite treatment of genomic DNA --- p.50, Chapter 3.5.2 --- Bioinformatical analysis of CpG island --- p.51, Chapter 3.5.3 --- Methylation-specific PCR (MSP) --- p.51, Chapter 3.5.3.1 --- Primers design --- p.51, Chapter 3.5.3.2 --- PCR reaction --- p.53, Chapter 3.5.4 --- Bisulfite genomic sequencing (BGS) --- p.53, Chapter 3.5.4.1 --- Primers design --- p.53, Chapter 3.5.4.2 --- PCR amplification and TA-cloning --- p.54, Chapter 3.6 --- Construction of expression plasmids for studied genes --- p.54, Chapter 3.6.1 --- Construction of the ZNF382-expressing vector --- p.54, Chapter 3.6.2 --- Construction of the BCL6B-expressing vector --- p.55, Chapter 3.7 --- Functional Study --- p.56, Chapter 3.7.1 --- Colony formation assay on monolayer culture --- p.56, Chapter 3.7.2 --- Wound healing assay --- p.57, Chapter 3.7.3 --- TUNEL assay --- p.58, Chapter 3.8 --- Western blot --- p.58, Chapter 3.9 --- Statistical analysis --- p.58, Chapter Chapter 4 --- Results --- p.60, Chapter 4.1 --- Epigenetic and Functional study of ZNF382 in RCC --- p.60, Chapter 4.1.1 --- Expression profiling of ZNF382 in human adult tissues --- p.60, Chapter 4.1.2 --- Expression profiling of ZNF382 in RCC cell lines --- p.61, Chapter 4.1.3 --- Dense promoter CpG methylation of ZNF382 correlated with its reduced expression in RCC --- p.62, Chapter 4.1.4 --- Restoration of ZNF382 expression by pharmacologic demethylation --- p.65, Chapter 4.1.5 --- Frequent methylation of ZNF382 in RCC primary tumors --- p.67, Chapter 4.1.6 --- Functional study of ZNF382 in RCC --- p.68, Chapter 4.1.6.1 --- Ectopic expression of ZNF382 inhibits clonogencity of RCC cells --- p.68, Chapter 4.1.6.2 --- Ectopic expression of ZNF382 inhibits migration of RCC cells --- p.71, Chapter 4.1.7 --- ZNF382 induces apoptosis of RCC cells --- p.72, Chapter 4.1.8 --- ZNF382 represses the expression of multiple oncogenes and stem cell markers in RCC --- p.73, Chapter 4.1.9 --- Discussion --- p.76, Chapter 4.2 --- Epigenetic and Functional study of BCL6B in RCC --- p.82, Chapter 4.2.1 --- Expression profiling of BCL6B in human adult tissues --- p.82, Chapter 4.2.2 --- Expression profiling of BCL6B in RCC cell lines --- p.83, Chapter 4.2.3 --- Correlation of the methylation status of ZNF382 promoter CpG island with its aborted expression in RCC --- p.84, Chapter 4.2.4 --- Restoration of BCL6B expression by pharmacological demethylation --- p.87, Chapter 4.2.5 --- Frequent BCL6B methylation in RCC primary tumors --- p.88, Chapter 4.2.6 --- Functional study of BCL6B in RCC --- p.90, Chapter 4.2.6.1 --- Ectopic expression of BCL6B inhibits clonogencity of RCC cells --- p.90, Chapter 4.2.6.2 --- Ectopic expression of ZNF382 inhibits migration of RCC cells --- p.92, Chapter 4.2.7 --- BCL6B induces apoptosis of RCC cells --- p.93, Chapter 4.2.8 --- BCL6B represses the expression of multiple oncogenes and stem cell markers in RCC --- p.94, Chapter 4.2.9 --- Discussion --- p.97, Chapter Chapter 5 --- General discussion --- p.103, Chapter Chapter 6 --- Summary --- p.106, Chapter Chapter 7 --- Future Study --- p.108, Chapter 7.1 --- Identification of key responsive elements in gene promoter --- p.108, Chapter 7.2 --- Study of genetic alterations leading to gene inactivation --- p.109, Chapter 7.3 --- Elucidation of the transcription-repressor activity in RCC --- p.109, Reference List --- p.110, http://library.cuhk.edu.hk/record=b5549093, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

目的： 转化生长因子－1（TGF-β1）通过与II型受体结合而引起I型受体活化，进一步激活其下游信号分子蛋白Smad2 和Smad3，它们与Smad4（Co-Smad）结合后形成Smad复合体并发生核转移，从而发挥广泛的生物学效应。同时，整个TGF-β信号通路又受到其抑制因子Smad7的负反馈调节。研究结果显示Smad3是肾脏炎症和纤维化中重要的致病分子，相反，Smad7在多种肾脏疾病中起保护作用。然而，由于转化生长因子II型受体（TβRII），Smad2 或Smad4基因敲除的小鼠无法存活，这些分子在TGF-β1介导的肾脏炎症和纤维化中的功能尚未见报道。因此，本研究旨在剖析TβRII、Smad2 和Smad4 在肾脏疾病发生发展中的作用及机制。, 方法：本研究利用Cre/LoxP系统分别靶向敲除小鼠肾小管上皮细胞的TβRII、Smad2 或者Smad4，通过结扎小鼠单侧输尿管建立梗阻性肾病模型，观察这些分子对肾脏炎症和纤维化的影响，并用体外实验进行验证。具体实验结果请参见本论文第III,IV, V章。, 结果：通过分析，本论文取得以下新的发现：, (1) TβRII在TGF-β1介导的肾脏炎症和纤维化的双向调节中起到了决定性的作用：研究结果显示条件性敲除TβRII明显抑制TGF-β/Smad3介导的肾脏纤维化，同时增强NF-κB引起的肾脏炎症反应。由此可见，TRII不仅仅是TGF-β/Smad信号通路的启动因子，更决定了TGF-β1对肾脏炎症和纤维化的双向性调节。(参见第III章), (2)尽管Smad2和Smad3结构相似并共同介导了TGF-β1的生物学效应，本研究意外发现Smad2可反向调节Smad3引起的纤维化。体内和体外实验共同证实，敲除Smad2基因增强了Smad3的磷酸化，核转位及其转录子活性，并能促进Smad3与I型胶原转录子的结合，进而加重肾脏纤维化（参见第IV章）。, (3)我们还发现Smad4不仅作为TGF-β/Smad信号通路的共有蛋白，它在TGF-β1介导肾脏炎症和纤维化中起到了重要的双向性调节作用：条件敲除Smad4显著降低了Smad7对NF-κB介导肾脏炎症的抑制作用，同时在转录水平（而非磷酸化水平）抑制Smad3的功能，从而减轻纤维化。（参见第V章）, 结论：TβRII和Smad4 在TGF-β1介导肾脏炎症和纤维化中起到了重要的双向性作用；Smad2通过抑制Smad3信号传导和功能，在肾脏纤维化中起保护作用。, Objectives: TGF-β1 binds its receptor II (TβRII) and then activates receptor I to initiate the downstream Smad signaling, called Smad2 and Smad3 which bind a common Smad4 to form the Smad complex and then translocate to nucleus to exert its biological activities. This process is negatively regulated by an inhibitory Smad7. While the pathogenic role of Smad3 and the protective role of Smad7 in renal fibrosis and inflammation are clearly understood, the functional role of TβRII, Smad2 and Smad4 in kidney diseases remains largely unexplored due to the lethality of these knockout mice. Therefore, the aim of present study is to dissect the functional role of these TGF-β/Smad signaling molecules in renal inflammation and fibrosis., Methods: Kidney conditional knockout (KO) mice for TβRII, Smad2 and Smad4 were generated by crossing the FloxFlox mice with the kidney specific promoter driven Cre (KspCre) mice, in which TβRII, Smad2 or Smad4 were specifically deleted from the kidney tubular epithelial cells (TEC) respectively. Then, a well-characterized progressive renal inflammation and fibrosis mouse model of Unilateral ureteral obstructive (UUO) nephropathy was induced in these conditional KO mice and the specific roles for TβRII, Smad2, and Smad4 in renal inflammation and fibrosis were investigated in vivo and in vitro as described in the Chapter III, IV and V of this thesis., Results: There were several novel findings through this thesis, 1. TGF-β1 signals through its TβRII to diversely regulate renal fibrosis and inflammation. We found that disrupted TRII suppressed Smad3-dependent renal fibrosis while enhancing NF-κB-driven renal inflammation. Thus, TβRII not only acts as a binding receptor for initiating the TGF-β signaling, but also determines the diverse role of TGF-β1 in inflammation and fibrosis, which was described in the Chapter III., 2. As shown in the Chapter IV, an unexpected finding from this thesis was that although Smad2 and Smad3 were homologically similar and bound together in response to TGF-β1 stimulation, Smad2 counter-regulated Smad3-mediated renal fibrosis. This was evidenced by the findings that conditional deletion of Smad2 enhanced Smad3 signaling including phosphorylation, nuclear translocation, the Smad3 responsive promoter activity, and the binding of Smad3 to Col1A2 promoter. Thus, disrupted Smad2 from the kidney significantly enhanced Smad3-mediated renal fibrosis in the UUO kidney and in cultured TEC., 3. Finally, we also showed that that Smad4 acted not only as a common Smad in TGF-β signaling, but exerted its regulatory role in determining the diverse role of TGF-β1 in renal inflammation and fibrosis. Disruption of Smad4 significantly enhanced renal inflammation by impairing inhibitory effect of Smad7 on NF-κB-driven renal inflammation. In contrast, disrupted Smad4 inhibited renal fibrosis by blocking Smad3 functional activity without influencing Smad3 signaling. Because deletion of Smad4 inhibited TGF-β1-induced Smad3 responsive promoter activity and the binding of Smad3 to the Col1A2 promoter without altering the phosphorylation and nuclear translocation of Smad3 (Chapter V)., Conclusions: TβRII and Smad4 may function as key regulators of TGF-β signaling and diversely regulate the renal inflammation and fibrosis. Smad2 plays a protective role in renal fibrosis by counter-regulating Smad3 signaling., Detailed summary in vernacular field only., Meng, Xiaoming., Thesis (Ph.D.)--Chinese University of Hong Kong, 2012., Includes bibliographical references (leaves 202-231)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., p.i, Declaration --- p.viii, Acknowledgement --- p.ix, Table of Contents --- p.xii, List of Abbreviations --- p.xxvii, List of Figures/Tables --- p.xxix, Chapter CHAPTER I --- INTRODUCTION --- p.1, Chapter 1.1 --- TGF-β signaling pathway --- p.2, Chapter 1.1.1 --- TGF-β superfamily --- p.2, Chapter 1.1.2 --- TGF-β signaling transduction --- p.3, Chapter 1.1.2.1 --- Smad-dependent TGF-β signaling --- p.4, Chapter 1.1.2.2 --- Smad-independent TGF-β signaling --- p.10, Chapter 1.2 --- Chronic Kideny disease (CKD) --- p.12, Chapter 1.2.1 --- Epidemiology of CKD --- p.12, Chapter 1.2.2 --- Pathophysiology of CKD --- p.12, Chapter 1.3 --- TGF-β signaling in renal diseases --- p.13, Chapter 1.3.1 --- Role of TGF-β1 in renal diseases --- p.13, Chapter 1.3.2 --- Potential role of TβRII in renal diseases --- p.15, Chapter 1.3.3 --- Potential role of Smad2 in renal diseases --- p.17, Chapter 1.3.4 --- Potential role of Smad4 in renal diseases --- p.20, Chapter 1.3.5 --- Role of Smad7 in renal diseases --- p.23, Chapter 1.3.6 --- Role of Smad-independent TGF-β signaling in renal disease --- p.24, Chapter CHAPTER II --- MATERIALS AND METHODS --- p.26, Chapter 2.1 --- MATERIALS --- p.27, Chapter 2.1.1 --- Reagents and Equipments --- p.27, Chapter 2.1.1.1 --- General reagents and equipments for cell culture --- p.27, Chapter 2.1.1.2 --- General reagents and equipments for real-time RT-PCR --- p.28, Chapter 2.1.1.3 --- General reagents and equipments for Masson Trichrome Staining --- p.28, Chapter 2.1.1.4 --- General reagents and equipments for Immunohistochemistry --- p.29, Chapter 2.1.1.5 --- General reagents and equipments for Immunofluorescence --- p.29, Chapter 2.1.1.6 --- General reagents and equipments for Western Blot --- p.29, Chapter 2.1.1.7 --- General reagents and equipments for Promoter assay --- p.31, Chapter 2.1.1.8 --- General reagents and equipments for ChIP assay --- p.32, Chapter 2.1.2 --- Buffers --- p.32, Chapter 2.1.2.1 --- Buffers for Immunohistochemistry --- p.32, Chapter 2.1.2.2 --- Buffers for Western blot --- p.35, Chapter 2.1.3 --- Sequences of Primers and siRNAs --- p.40, Chapter 2.1.4 --- Antibodies --- p.42, Chapter 2.2 --- METHODS --- p.44, Chapter 2.2.1 --- Animal model of Unilateral Ureteral Obstruction (UUO) --- p.44, Chapter 2.2.2 --- Cell culture --- p.44, Chapter 2.2.2.1 --- NRK52E cell line --- p.44, Chapter 2.2.2.2 --- Smad2 WT/KO mouse embryonic fibroblasts (MEFs) --- p.45, Chapter 2.2.2.3 --- Primary culture of kidney fibroblasts --- p.45, Chapter 2.2.2.4 --- Primary culture of peritoneal macrophages --- p.46, Chapter 2.2.3 --- PAS staining --- p.47, Chapter 2.2.3.1 --- Tissue Handling and Fixation --- p.47, Chapter 2.2.3.2 --- Tissue embedding and sectioning --- p.47, Chapter 2.2.3.3 --- Preparation of Paraffin Tissue Sections for PAS staining --- p.48, Chapter 2.2.3.4 --- PAS staining --- p.48, Chapter 2.2.4 --- Real-time RT-PCR --- p.48, Chapter 2.2.4.1 --- Total RNA isolation --- p.48, Chapter 2.2.4.2 --- Reverse Transcription --- p.49, Chapter 2.2.4.3 --- Real-time PCR --- p.50, Chapter 2.2.4.4 --- Analysis of Real-time PCR --- p.50, Chapter 2.2.5 --- Masson Trichrome Staining --- p.51, Chapter 2.2.6 --- Immunohistochemistry --- p.52, Chapter 2.2.6.1 --- Preparation of Paraffin Tissue Sections for IHC --- p.52, Chapter 2.2.6.2 --- Antigen-Antibody Reaction --- p.52, Chapter 2.2.6.3 --- Signal Detection --- p.53, Chapter 2.2.6.4 --- Semi-quantification of Immunohistochemistry --- p.53, Chapter 2.2.7 --- Immunofluorescence --- p.54, Chapter 2.2.8 --- Western blot analysis --- p.54, Chapter 2.2.8.1 --- Protein preparation --- p.55, Chapter 2.2.8.2 --- SDS-PAGE --- p.56, Chapter 2.2.8.3 --- Transmembrane of protein --- p.56, Chapter 2.2.8.4 --- Incubation of first and second antibody --- p.57, Chapter 2.2.8.5 --- Signal capture and analysis --- p.57, Chapter 2.2.8.6 --- Stripping --- p.57, Chapter 2.2.9 --- Promoter assay --- p.58, Chapter 2.2.10 --- ChIP assay --- p.61, Chapter 2.2.11 --- Statistical analysis --- p.62, Chapter CHAPTER III --- THE DIVERSE ROLE OF TGF-BETA RECEPTOR II IN RENAL INFLAMMATION AND FIBROSIS --- p.63, Chapter 3.1 --- INTRODUCTION --- p.64, Chapter 3.2 --- AIMS --- p.64, Chapter 3.3 --- MATERIALS AND METHODS --- p.66, Chapter 3.3.1 --- Generation and characterization of TβRII conditional Knockout mice --- p.66, Chapter 3.3.2 --- Generation and characterization of TβRII disrupted tubular epithelial cell line (NRK52E) and kidney interstitial fibroblasts --- p.67, Chapter 3.3.3 --- Animal model of Unilateral Ureteral Obstruction --- p.67, Chapter 3.3.4 --- Cell culture --- p.67, Chapter 3.3.5 --- Real-time RT-PCR --- p.68, Chapter 3.3.6 --- Masson Trichrome Staining --- p.68, Chapter 3.3.7 --- Immunohistochemistry --- p.68, Chapter 3.3.8 --- PAS staining --- p.69, Chapter 3.3.9 --- Immunofluorescence --- p.69, Chapter 3.3.10 --- Western blot analysis --- p.70, Chapter 3.3.11 --- Promoter assay --- p.70, Chapter 3.3.12 --- Statistical analysis --- p.70, Chapter 3.4 --- RESULTS --- p.71, Chapter 3.4.1 --- Characterization of TβRII conditional Knockout mice and TβRII disrupted cells --- p.71, Chapter 3.4.2 --- Disruption of TβRII suppresses renal interstitial damage in the UUO kidney --- p.72, Chapter 3.4.3 --- Disruption of TβRII suppresses renal fibrosis in UUO kidney and TGF-β1-induced fibrotic response in vitro --- p.76, Chapter 3.4.3.1 --- Conditional knockout of TβRII from the kidney decreases the collagen I level in UUO kidney --- p.76, Chapter 3.4.3.2 --- Disruption of TβRII inhibits TGF-β1 induced collagen I level in vitro --- p.79, Chapter 3.4.3.3 --- Conditional knockout of TβRII from the kidney decreases the α-SMA positive cells infiltration in vivo --- p.81, Chapter 3.4.3.4 --- Disruption of TβRII inhibits TGF-β1-induced α-SMA expression in vitro --- p.83, Chapter 3.4.3.5 --- Conditional knockout of TβRII from the kidney decreases the FN level in UUO nephropathy --- p.85, Chapter 3.4.3.6 --- Disruption of TβRII decreases TGF-β1-induced FN expression in vitro --- p.87, Chapter 3.4.4 --- Disruption of TβRII impairs the TGF-β/Smad signaling in vivo in the UUO kidney and in vitro in TGF-β1 treated tubular epithelial cells and kidney fibroblasts --- p.89, Chapter 3.4.4.1 --- Conditional knockout of TβRII decreases the UUO induced TGF-β1 expression in vivo and the TGF-β1 auto-induction in vitro --- p.89, Chapter 3.4.4.2 --- Disrupted TβRII decreases CTGF level in the UUO nephropathy in vivo and the TGF-β1 induced CTGF mRNA level in vitro --- p.91, Chapter 3.4.4.3 --- Conditional knockout of TβRII impairs the Smad3 signaling in the injured kidney --- p.93, Chapter 3.4.4.4 --- Disrupted TβRII inhibits TGF-β1-induced Smad3 phosphorylation, P-Smad3 nuclear translocation and Smad3 responsive promoter activity in vitro --- p.95, Chapter 3.4.4.5 --- Conditional knockout of TβRII doesn’t alter the activation of ERK and P38 signaling in the UUO kidney --- p.97, Chapter 3.4.4.6 --- Disrupted TβRII inhibits TGF-β1-induced ERK and P38 phosphorylation in vitro --- p.99, Chapter 3.4.5 --- Disruption of TβRII enhances inflammatory cytokines expression in the UUO kidney and impairs the anti-inflammatory effect of TGF-β1 in response to IL-1β triggered inflammatory response in the TEC cells --- p.101, Chapter 3.4.5.1 --- Conditional knockout of TβRII increases the TNF-α expression in the UUO nephropathy --- p.101, Chapter 3.4.5.2 --- Conditional knockout of TβRII increases the IL-1β expression in the UUO nephropathy --- p.103, Chapter 3.4.5.3 --- Conditional knockout of TβRII doesn’t enhance the MCP-1 expression and macrophages infiltration in the UUO nephropathy --- p.104, Chapter 3.4.5.4 --- Disruption of TβRII in TECs decreases the anti-inflammatory effect of TGF-β1 in response to IL-1β --- p.106, Chapter 3.4.6 --- Disruption of TβRII enhances NFκB activation in vivo and in vitro --- p.108, Chapter 3.5 --- DISCUSSION --- p.110, Chapter 3.6 --- CONCLUSION --- p.114, Chapter CHAPTER IV --- Smad2 protects against TGF-β/Smad3 mediated renal fibrosis --- p.115, Chapter 4.1 --- INTRODUCTION --- p.116, Chapter 4.2 --- AIMS --- p.117, Chapter 4.3 --- MATERIALS AND METHODS --- p.117, Chapter 4.3.1 --- Generation and characterization of Smad2 conditional Knockout mice --- p.117, Chapter 4.3.2 --- Generation and characterization of Smad2 KO MEFs and Smad2 knockdown/overexpression tubular epithelial cell line (NRK52E) --- p.118, Chapter 4.3.3 --- Animal model of Unilateral Ureteral Obstruction --- p.118, Chapter 4.3.4 --- Cell culture --- p.118, Chapter 4.3.5 --- Real-time RT-PCR --- p.119, Chapter 4.3.6 --- Western blot analysis --- p.119, Chapter 4.3.7 --- Immunohistochemistry --- p.119, Chapter 4.3.8 --- Masson Trichrome Staining --- p.119, Chapter 4.3.9 --- Immunofluorescence --- p.120, Chapter 4.3.10 --- Promoter assay --- p.120, Chapter 4.3.11 --- ChIP assay --- p.120, Chapter 4.3.12 --- Statistical analysis --- p.120, Chapter 4.4 --- RESULTS --- p.121, Chapter 4.4.1 --- Characterization of Smad2 disrupted mice and cells --- p.121, Chapter 4.4.1.1 --- Characterization of Smad2 conditional Knockout mice --- p.121, Chapter 4.4.1.2 --- Characterization of Smad2 knockout MEFs, Smad2 knockdown/overexpression TECs --- p.123, Chapter 4.4.2 --- Disruption of Smad2 further enhances renal fibrosis in vivo and in vitro --- p.124, Chapter 4.4.2.1 --- Conditional knockout of Smad2 increases total collagen deposition and Col.I level in the UUO kidney --- p.124, Chapter 4.4.2.2 --- Disruption of Smad2 in MEFs and TECs increases Col.I production in a time- and dosage-dependent manner in response to TGF-β1 --- p.126, Chapter 4.4.2.3 --- Conditional knockout of Smad2 increases Col.III level in the UUO kidney --- p.128, Chapter 4.4.2.4 --- Disruption of Smad2 in MEFs and TECs increases Col.III production in a time- and dosage-dependent manner in response to TGF-β1 --- p.130, Chapter 4.4.3 --- Disruption of Smad2 further enhances renal fibrosis by suppressing the collagen degradation system in vivo and in vitro --- p.132, Chapter 4.4.3.1 --- Conditional knockout of Smad2 inhibits the MMP2 mRNA while enhances TIMP-1 production in UUO kidney --- p.132, Chapter 4.4.3.2 --- Disruption of Smad2 in MEFs and TECs decreases the MMP2 level while enhances TIMP-1 production in response to TGF-β1 --- p.133, Chapter 4.4.4 --- Disruption of Smad2 further increases renal fibrosis by increasing TGF-β1 auto-induction and CTGF level in vivo and in vitro --- p.135, Chapter 4.4.4.1 --- Disruption of Smad2 increases TGF-β1 auto-induction in vivo and in vitro --- p.135, Chapter 4.4.4.2 --- Disruption of Smad2 increases CTGF synthesis in vivo and in vitro --- p.137, Chapter 4.4.5 --- Disruption of Smad2 further increases renal fibrosis by enhancing Smad3 signaling in vivo and in vitro --- p.139, Chapter 4.4.5.1 --- Conditional knockout of Smad2 further enhances Smad3 phosphorylation and nuclear translocation --- p.139, Chapter 4.4.5.2 --- Disruption of Smad2 in MEFs and TECs further enhances Smad3 phosphorylation, nuclear translocation, Smad3 responsive promoter activity and the binding to the Col1A2 promoter --- p.141, Chapter 4.4.6 --- Overexpression of Smad2 suppresses Smad3 signaling therefore ameliorates the TGF-β1-induced fibrotic response in TECs --- p.144, Chapter 4.4.6.1 --- Overexpression of Smad2 ameliorates the TGF-β1- induced fibrotic response in TECs --- p.144, Chapter 4.4.6.2 --- Overexpression of Smad2 suppresses Smad3 phosphorylation --- p.146, Chapter 4.5 --- DISCUSSION --- p.147, Chapter 4.6 --- CONCLUSION --- p.150, Chapter CHAPTER V --- THE DISTINCT ROLE OF SMAD4 IN RENAL INFLAMMATION AND FIBROSIS --- p.151, Chapter 5.1 --- INTRODUCTION --- p.152, Chapter 5.2 --- AIMS --- p.152, Chapter 5.3 --- MATERIALS AND METHODS --- p.153, Chapter 5.3.1 --- Generation and characterization of Smad4 conditional Knockout mice --- p.153, Chapter 5.3.2 --- Generation and characterization of Smad4 disrupted kidney interstitial fibroblasts and peritoneal macrophages --- p.153, Chapter 5.3.3 --- Animal model of Unilateral Ureteral Obstruction (UUO) --- p.154, Chapter 5.3.4 --- Cell culture --- p.154, Chapter 5.3.5 --- Real-time RT-PCR --- p.155, Chapter 5.3.6 --- Western blot analysis --- p.155, Chapter 5.3.7 --- Immunohistochemistry --- p.155, Chapter 5.3.8 --- Masson Trichrome Staining --- p.155, Chapter 5.3.9 --- Promoter assay --- p.156, Chapter 5.3.10 --- ChIP assay --- p.156, Chapter 5.3.11 --- Statistical analysis --- p.156, Chapter 5.4 --- RESULTS --- p.157, Chapter 5.4.1 --- Characterization of Smad4 conditional Knockout mice and Smad4 disrupted cells --- p.157, Chapter 5.4.2 --- Disruption of Smad4 suppresses renal fibrosis in the UUO nephropathy in vivo and TGF-β1-induced fibrotic response in vitro --- p.160, Chapter 5.4.2.1 --- Conditional knockout of Smad4 from the kidney decreases the total collagen deposition in the UUO nephropathy --- p.160, Chapter 5.4.2.2 --- Conditional knockout of Smad4 from the kidney decreases the Col.I production in the UUO nephropathy --- p.161, Chapter 5.4.2.3 --- Disruption of Smad4 inhibits TGF-β1-induced Col.I production in vitro --- p.163, Chapter 5.4.3 --- Disruption of Smad4 impairs the Smad3 function in vivo and in vitro --- p.164, Chapter 5.4.3.1 --- Conditional knockout of Smad4 doesn’t decrease Smad3 phosphorylation and P-Smad3 nuclear translocation in vivo and in vitro --- p.164, Chapter 5.4.3.2 --- Disruption of Smad4 inhibits TGF-β1 induced Smad3 promoter activity and the Smad3 binding to Col1A2 promoter --- p.166, Chapter 5.4.3.3 --- Disruption of Smad4 has minimal effect on the activation of ERK signaling in vivo and in vitro --- p.167, Chapter 5.4.4 --- Disruption of Smad4 enhances renal inflammation and impairs the anti-inflammatory effect of TGF-β1 in response to IL-1β triggered inflammatory response in vitro --- p.169, Chapter 5.4.4.1 --- Conditional knockout of Smad4 increases the inflammatory cells infiltration --- p.169, Chapter 5.4.4.2 --- Conditional knockout of Smad4 increases the TNFα expression in the UUO nephropathy --- p.171, Chapter 5.4.4.3 --- Conditional knockout of Smad4 increases the IL-1β expression in the UUO nephropathy --- p.172, Chapter 5.4.4.4 --- Conditional knockout of Smad4 increases the MCP-1 expression in the UUO nephropathy --- p.173, Chapter 5.4.4.5 --- Conditional knockout of Smad4 increases the ICAM-1 level in the UUO nephropathy --- p.174, Chapter 5.4.4.6 --- Time and dosage dependent experiments in response to IL-1β in macrophages --- p.175, Chapter 5.4.4.7 --- Disruption of Smad4 in macrophages decreases the anti-inflammatory effect of TGF-β1 in response to IL-1β --- p.176, Chapter 5.4.5 --- Disruption of Smad4 impairs the inhibitory effect of Smad7 on NFκB activation in vivo and in vitro --- p.178, Chapter 5.4.5.1 --- Conditional knockout of Smad4 largely inhibits Smad7 level in UUO kidney --- p.178, Chapter 5.4.5.2 --- Conditional knockout of Smad4 suppresses IκBα and further increases NF-κB p65 activation in UUO kidney --- p.180, Chapter 5.4.5.3 --- Disruption of Smad4 inhibits Smad7 synthesis in macrophages --- p.182, Chapter 5.4.5.4 --- Conditional knockout of Smad4 impair the inhibition effect of TGF-β1 on the activation of NFκB p65 in macrophages --- p.184, Chapter 5.5 --- DISCUSSION --- p.186, Chapter 5.6 --- CONCLUSION --- p.189, Chapter CHAPTER VI --- SUMMARY AND DISCUSSION OF THE MAJOR FINDINGS --- p.190, Chapter 6.1 --- SUMMARY AND DISCUSSION --- p.192, Chapter 6.1.1 --- The diverse role of TβRII in renal inflammation and fibrosis both in vivo and in vitro --- p.192, Chapter 6.1.2 --- Smad2 protects renal fibrosis by counter-regulating Smad3 signaling --- p.192, Chapter 6.1.3 --- Disruption of Smad4 increased renal inflammation while suppressed the renal fibrosis in vivo and in vitro --- p.194, Chapter 6.1.4 --- Comparative analysis of functions and related mechanisms between TβRII and Smad4 in renal disease --- p.195, Chapter 6.1.5 --- Inadequacies of current work and future plan --- p.197, Chapter 6.1.6 --- Perspectives (1) : The balance within the TGF-b/Smad signaling may determine the fate of renal diseases --- p.197, Chapter 6.1.7 --- Perspectives(2)：The balance within the TGF-β/Smad signaling may determine the fate of renal diseases --- p.198, Chapter 6.2 --- CONCLUSION --- p.201, REFERENCES --- p.202, PUBLICATION LIST --- p.232, HONORS AND AWARDS --- p.237, http://library.cuhk.edu.hk/record=b5549453, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

糖尿病是一种呈现流行趋势的代谢紊乱综合症，现如今，全球大约有3.46亿糖尿病患者， 这庞大的数字给各国的公共健康安全支出带来了严重的财政负担。 其中，二型糖尿病（T2DM）占90%。其特点是周围组织的胰岛素抵抗以及后期损伤的胰岛β细胞的功能。在饮食后，小肠会分泌两种肠促胰岛素，葡萄糖依赖性促胰岛素多肽（GIP）和胰高血糖素样肽-1（GLP-1）。两种多肽的主要功能是促进餐后胰岛细胞中胰岛素的分泌，另外他们还可以通过其自身的G蛋白偶联受体，GIPR和GLP-1R发挥其他作用，如葡萄糖依赖性的刺激胰岛素的生成，刺激胰岛β细胞的增殖，抑制细胞的凋亡等。这些功能也使肠促胰岛素成为糖尿病治疗的一种手段，比如Exendin-4和DPP4抑制剂。 然而，除了在胰岛中的作用，肠促胰岛还可能和脂质代谢相关，其中GIP和脂质代谢的报导研究的更加深入。在肥胖的状态下，血液中GIP含量高于正常水平；GIPR基因敲除老鼠和GIPR的抑制剂喂养的小鼠可以抵抗高脂饮食诱导的肥胖和2型糖尿病；GIP还可以直接调节脂肪细胞的脂肪生成和脂解。这些数据表明GIP在肥胖和糖尿病的发生过程中可能存在促进作用,这使得GIP治疗药物的开发需要谨慎的对待。, 为了进一步研究GIP在脂肪细胞中发挥的生物学效应，在本研究中，我们利用腺病毒介导技术通过在脂肪细胞中过表达GIPR来增加GIP的活性，然后检查GIP在脂肪细胞中所起的作用。实验结果表明,GIP可以通过cAMP-PKA信号通路迅速并且长期的刺激脂肪细胞的炎症反应，增强IKKβ-NFκB信号通路和增加炎症基因的表达。更深入的机制研究表明，JNK 信号通路也参与GIP诱导的炎症反应，抑制JNK通路可以大部分恢复GIP增加的炎症因子的表达和IKKβ的磷酸化水平。由于长期的炎症反应，脂肪细胞的胰岛素信号通路受到GIP的损伤，在GIPR过表达的脂肪细胞中，胰岛素刺激的AKT磷酸化水平和葡萄糖吸收能力都被GIP降低，葡萄糖转运蛋白4（Glut-4）的表达水平也同时减少。因此，本研究结果表明GIP可能在肥胖的发展过程中，通过诱导脂肪细胞的炎症反应来损伤胰岛素敏感性而最终导致2型糖尿病的发生。, Diabetes mellitus is a type of metabolic syndrome that has prevailed all over the world with the development of economic and over-nutrient lifestyle. It is estimated to 346 million diabetes patients in the worldwide most recently. The huge population put a major burden on the cost of public health care to all the countries. Among the types of diabetes, type 2 diabetes (T2DM) makes up 90% of recorded cases. The characteristics of T2DM are insulin resistance of peripheral tissues and impaired pancreatic cell function and mass. Two major incretins GIP (glucose-dependent insulinotropic peptide) and GLP-1 (glucagon-like peptide 1) are secreted from gut in response to food ingestion. The prominent role of GIP and GLP-1 is to stimulate glucose-dependent insulin release in pancreatic β cell. In addition, they both exert multiple biological effects via their relative G-protein coupled receptors, GIPR and GLP-1R, including glucose-stimulated insulin production, cell proliferation and anti-apoptosis in pancreatic β cells. The beneficent effects of incretins potentiate them as targets for the treatment of diabetes. GLP-1 analog, exendin-4 and DDP4 (dipeptidyl peptidase-4) inhibitors (to prevent GIP and GLP-1 from degradation) have been already used in clinical research. However, in addition to their effects on pancreatic β cell, both peptides are also related to lipid metabolism. The role of GIP has been studied more extensively. In obese state, the circulating level of GIP is elevated. GIPR knockout (KO) mice are resistant to high fat diet (HFD) induced obesity, a similar phenotype is found in GIPR antagonist administrated HFD-mice. Moreover, GIP also directly promotes lipogenesis and lipolysis in adipocytes. The rising evidence suggests a potential role of GIP in adipocyte biology and lipid metabolism, which diminishes the enthusiasm of GIP as a candidate therapeutic reagent for T2DM., In order to further understand the biological effects of GIP in adipocytes, here, we over-expressed GIPR in 3T3-L1 CAR adipocytes via adenovirus-mediated gene transfer technology to enhance the activity of GIP. The results demonstrate that GIP impairs the physiological functions of adipocytes as a consequence of increasing the production of inflammatory cytokines, chemokines, and phosphorylation of IkB kinase (IKK) β through activation of the cyclic AMP-protein kinase A (cAMP-PKA) pathway. Activation of Jun N-terminal Kinase (JNK) pathway is also observed in GIP-induced inflammatory responses in adipocytes. An inhibitor of JNK blocks GIP-stimulated secretion of inflammatory cytokines and chemokines, as well as phosphorylation of IKKβ. The chronic inflammatory response eventually impairs insulin signaling in adipocytes, as demonstrated by reduction of protein kinase B (PKB/AKT) phosphorylation. The subsequently physiological analysis also indicates that GIP inhibits insulin-stimulated glucose uptake, and gene expression analysis reveals a decrease of glucose transporter 4 (Glut-4) in the meanwhile. The results suggest that GIP may be one of stimuli attributable to obesity induced insulin resistance via induction of adipocyte inflammation., Detailed summary in vernacular field only., Nie, Yaohui., Thesis (Ph.D.)--Chinese University of Hong Kong, 2012., Includes bibliographical references (leaves 95-111)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., p.i, 摘要 --- p.iii, Acknowledgements --- p.v, INTRODUCTION --- p.1, Chapter Part 1 --- Obesity and Type 2 diabetes --- p.1, Chapter 1.1 --- Introduction to diabetes --- p.1, Chapter 1.1.2 --- Physiology of adipocyte --- p.4, Chapter 1.1.3 --- Mechanism of obesity induced diabetes --- p.10, Chapter Part 2 --- Incretins and T2DM --- p.12, Chapter 2.1 --- History of incretins --- p.12, Chapter 2.2 --- Physiological actions of incretins --- p.14, Chapter 2.3 --- Molecular mechanism of incretin actions in pancreas --- p.16, Chapter 2.4 --- Incretins and T2DM --- p.19, Chapter Part 3 --- Incretins and lipid metabolism --- p.23, Objective --- p.26, Methods and materials --- p.28, Chapter 1 --- Cell culture --- p.28, Chapter 1.1 --- 3T3-L1 culture and differentiation --- p.28, Chapter 1.2 --- 3T3-L1 CAR culture and differentiation --- p.29, Chapter 2 --- Cloning and recombinant adenovirus construction --- p.30, Chapter 2.1 --- Plasmid construct --- p.30, Chapter 2.2 --- Construct of recombinant adenoviruses --- p.30, Chapter 2.3 --- Generation and infection of the adenoviruses --- p.31, Chapter 3 --- Physiological and morphological assays --- p.32, Chapter 3.1 --- Lipolysis assay --- p.32, Chapter 3.2 --- TUNEL assay --- p.32, Chapter 3.3 --- Glucose uptake --- p.33, Chapter 3.4 --- Glut-4 localization --- p.33, Chapter 4 --- Gene expression analysis --- p.35, Chapter 4.1 --- Quantitative real-time PCR --- p.35, Chapter 4.2 --- Immunoblot analysis --- p.35, Chapter 4.3 --- ELISA assay --- p.36, Chapter 5 --- Isolation of primary adipocytes --- p.37, Results --- p.38, Chapter Part 1 --- Role of GIP in 3T3-L1 cells --- p.38, Chapter 1.1 --- Differentiation of 3T3-L1 adipocytes --- p.38, Chapter 1.2 --- GIP slightly stimulates phosphorylation of p-CREB and lipolysis in 3T3-L1 cells. --- p.40, Chapter 1.3 --- Analysis of gene expression in GIP-treated adipocytes --- p.42, Chapter 1.4 --- Discussion --- p.44, Chapter Part 2 --- Role of GIP in GIPR over-expressing 3T3-L1 CAR adipocytes --- p.46, Chapter 2.1 --- Differentiation of 3T3-L1 CAR adipocytes --- p.46, Chapter 2.2 --- Functional tests in GIPR over-expressing 3T3-L1 CAR adipocytes. --- p.48, Chapter 2.3 --- Effect of GIP on cell viability --- p.50, Chapter 2.4 --- Analysis of gene expression in GIP-treated adipocytes --- p.52, Chapter 2.5 --- GIP activates inflammatory responses in GIPR over-expressing adipocytes --- p.54, Chapter 2.6 --- Inhibition of IKKb pathway restores GIP-induced inflammatory responses --- p.56, Chapter 2.7 --- Effects of GIP on adipocytes are partially dependent on the cAMP-PKA pathway --- p.58, Chapter 2.8 --- Activation of cAMP-PKA pathway induces adipocyte inflammation. --- p.60, Chapter 2.9 --- cAMP-Epac pathway is not involved in GIP-induced inflammation --- p.62, Chapter 2.10 --- GIP stimulates cell stress activated kinases --- p.64, Chapter 2.11 --- JNK partially mediates GIP-induced adipocyte inflammation --- p.65, Chapter 2.12 --- Inhibition of JNK pathway partially restores GIP-induced inflammatory responses --- p.67, Chapter 2.13 --- GIP impairs insulin signaling in GIPR over-expressing 3T3-L1 CAR adipocytes via inducing inflammatory response --- p.69, Chapter 2.14 --- GIP enhances basal glucose uptake but impairs insulin stimulated glucose uptake in 3T3-L1 CAR GIPR over-expressing adipocytes --- p.71, Chapter 2.15 --- Discussion --- p.73, Chapter Part 3 --- Role of GIP in primary adipocytes --- p.78, Chapter 3.1 --- GIPR expression level in primary adipocytes --- p.78, Chapter 3.2 --- Analysis of gene expression in primary adipocytes after GIP treatment --- p.80, Chapter 3.3 --- Discussion --- p.81, SUMMARY --- p.82, Chapter Future investigation --- p.83, Chapter Appendix 1: --- Abbreviations --- p.86, Chapter Appendix 2: --- Protocols --- p.90, Preparation of competent cells --- p.90, Outlines of recombinant adenovirus preparation --- p.91, Virus titering (TCID50) --- p.92, Primers for real-time PCR --- p.93, Chapter Publications and Scientfic activities --- p.94, Thesis related publication: --- p.94, Other pubiliations: --- p.94, Scientific activities: --- p.94, References --- p.95, http://library.cuhk.edu.hk/record=b5549650, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

痴呆是导致中风后存活人群自理能力降低的重要因素。中风后痴呆（PSD）包括发生在中风后的所有类型的痴呆，不论其痴呆的原因为何，如血管性痴呆(VD)，阿尔茨海默氏症(AD)以及混合型痴呆。由于中风致死率的降低和人口老龄化的到来，有可能在未来几十年，全世界范围内中风后痴呆的发病率将大幅增加。在此，我们将报告中风后早期出现的痴呆及长期的认知功能下降的发病率和危险因素，并通过使用PIB正电子扫描确定中风后痴呆中合并脑部淀粉样变的发病率。了解中风后痴呆的发病机制和危险因素，将有利于我们寻求治疗和预防措施，从而减低中风后痴呆的发生。, 研究目的, 研究1：中风后早期痴呆的发病率及危险因素, 早期PSD及长期认知功能下降的发病机制甚为复杂。我们在中国中风存活者中建立了名为 STroke Registry IVEstigating COGnitive decline (STRIVE-COG) 的研究。此研究将报告早期PSD的危险因素。, 研究2：中风后长期（15-18个月）认知功能下降的发病率及预测因素中风会增加远期痴呆的发生。但是，中风后远期认知功能下降的发病机制还未确定。我们的研究旨在观察中风后远期认知功能下降的危险因素。, 研究方法, 研究1：我们连续纳入一年的在我院中风中心留院的中风及短暂性缺血发作病人。在病人中风发生后的3-6个月后，对其进行多个认知领域的神经心理学检查。我们观察了患者的临床特征及结构影像学改变与早期PSD的相关性。我们还对部分早期PSD的病人进行了PIB正电子发射扫描（PIB-PET）检查。, 研究2：在完成中风后3-6个月认知检查后的1年，即中风后的15-18个月，我们完成了认知心理学的随访检查。我们将认知功能下降定义为简易精神状态评分降低3分及以上，或者临床痴呆评分增加1分及以上。我们观察了认知功能下降和基线期临床、认知心理学和影像学特征（包括白质病变严重程度、陈旧性腔梗、全脑萎缩、额叶萎缩、顶叶萎缩、中颞叶萎缩）。在一组（n=18）早期PSD患者中，我们观察了脑部有类似阿尔茨海默氏病变表现的患者的认知功能下降的发病率。, 结果, 研究1：在所有入组的患者中（n=549），早期PSD的发病率为15.3%（n=84）。多因素回归分析显示，除了年龄和性别，早期PSD的危险因素包括急性腔隙性梗死灶(危险比[OR] 2.725, 95% 可信区间[CI] 1.364-5.434, P=0.004)及急性非腔隙性梗死灶(OR 2.809, 95%CI 1.124-6.410, P=0.014)比上无急性梗死灶的病人，还包括白质病变严重度（OR 1.120, 95% CI 1.037-1.210, p=0.004），额叶萎缩（OR 2.596, 95% CI 1.080-6.241, p=0.033），由脑室-大脑比表示的全脑萎缩（4th 四分卫区间 vs 1st区间, OR 3.096, 95% CI 1.374-6.993, p=0.006）。在19个完成了PIB-PET扫描的病人中，6人（31.6%）具有类似AD的脑部淀粉样物聚集的表现。, 研究2：在452（82.3%）个完成了中风后15-18个月随访检查的病人中，认知功能下降的患者有73个（16.2%）。而年龄、受教育年限、多发陈旧性腔隙性梗死是其独立性预测因素。随访过程中中风复发的患者只有5.1%并且与认知功能下降无相关性。进展性的认知功能下降在PIB阴性（n=12）和PIB阳性（n=6）的患者中分别为41.7%和33.3%，而两者之间显著差别（p=0.731）。, 结论, 研究 1: 早期PSD的危险因素除了包括年龄、性别及脑部急性梗死灶之外，还包括脑部的慢性改变，包括白质病变、全脑萎缩、额叶萎缩及合并AD样病变特征。, 研究 2: 年龄、受教育水平和多发陈旧性腔隙梗死是中风后15-18个月认知功能下降的独立危险因素。而合并AD样病变并不是导致中风后远期认知功能下降的必要因素。, Dementia is a main cause of dependency in stroke survivors. Poststroke dementia (PSD) includes any dementia after a stroke, irrespective of its causes, e.g. vascular dementia (VD), Alzheimer’s disease (AD) or mixed dementia. A huge increase in prevalence and burden of PSD is likely to happen because of the decline in mortality after stroke and ageing of populations in the coming decades worldwide. In this thesis, we reported the risk factors for early PSD and delayed poststroke cognitive decline, and the prevalence of concurrent amyloid pathology as identified by Pittsburgh compound B (PIB) positron emission tomography (PET) in PSD. Understanding the risk factors of PSD will help to devise preventive and treatment strategies that may reduce the burden of PSD., Objectives, Study1: Risk factors of early PSD, Mechanisms explaining poststroke early and delayed cognitive decline are complex. We set up a STroke Registry IVEstigating COGnitive decline (STRIVE-COG) among Chinese stroke survivors. Study 1 reported the findings on risk factors for early PSD., Study 2: Prevalence and predictors for delayed (15-18 months) cognitive decline after stroke, Having a stroke increases the risk of delayed dementia. However, mechanisms accounting for the cognitive decline are uncertain. We investigated the predictors for delayed poststroke cognitive decline., Subjects and Methods, Study 1：We recruited consecutive stroke or transient ischemic attack (TIA) patients admitted to our acute stroke unit over 1 year. We performed neuropsychological assessment 3-6 months poststroke. We investigated the association between clinical and structural neuroimaging features with early PSD. We performed PIB positron PET among a subset of subjects with early PSD., Study 2: We performed neuropsychological assessment at baseline (i.e. 3-6 months poststroke) and at 15-18 months poststroke. We defined cognitive decline as a drop of ≥ 3 points in the mini-mental state examination and/or increment in ≥ 1 grading of the clinical dementia rating scale. We investigated the association between cognitive decline with baseline clinical, neuropsychological, and neuroimaging features (white matter changes [WMC] severity, old lacunar infarct, global atrophy, frontal lobe atrophy [FLA], parietal lobe atrophy, medial temporal lobe atrophy). Among a subset of subjects (n=18) with PSD at baseline, we investigated the influence of AD-like PIB retention upon the rate of cognitive decline., Results, Study 1: Prevalence of early PSD among all recruited subjects (n=549) was 15.3% (n=84). Apart from age and female gender, multivariate regression analyses showed that risk factors for early PSD were presence of acute lacunar (odds ratio [OR] 2.725, 95% confidence interval [CI] 1.364-5.434, P=0.004) or non-lacunar infarct (OR 2.809, 95%CI 1.124-6.410,P=0.014) over no acute infarct apparent on neuroimaging, WMC severity (OR 1.120, 95% CI 1.037-1.210, p=0.004), FLA (OR 2.596, 95% CI 1.080-6.241, p=0.033), and global brain atrophy (4th quartile vs 1st quartile, OR 3.096, 95% CI 1.374-6.993, p=0.006). Among 19 subjects with early PSD who had PIB PET, 6 (31.6%) had AD-like PIB retention., Study 2: Among 452(82.3%) subjects who had completed the study, cognitive decline occurred in 73 (16.2%) subjects. Age, education, and multiple old lacunar infarcts independenty predicted cognitive decline. Recurrent stroke occurred only in 5.1% and was not associated with cognitive decline. Progressive cognitive decline occurred in 41.7% and 33.3% of PIB negative (n=12) and PIB positive (n=6) PSD patients, respectively (p=0.731)., Conclusion, Study 1: Apart from age, female gender, and presence of acute infarct evident in neuroimaging, chronic brain changes (WMC, global brain atrophy, FLA, and concurrent AD pathology) are associated with early PSD., Study 2: Age, education, and multiple old lacunar infarcts predicted cognitive decline at 15-18 months poststroke. Concurrent AD-like lesion is not necessary associated with a rapid cognitive decline., Detailed summary in vernacular field only., Yang, Jie., Thesis (Ph.D.)--Chinese University of Hong Kong, 2012., Includes bibliographical references (leaves 93-109)., also in Chinese., Chapter PART I --- LITERATURE REVIEW --- p.1, Chapter Chapter 1 --- Introduction --- p.2, Chapter 1.1 --- An overview of stroke --- p.2, Chapter 1.2 --- Introduction of poststroke dementia --- p.5, Chapter Chapter 2 --- Poststroke Dementia --- p.6, Chapter 2.1 --- Defining poststroke dementia --- p.6, Chapter 2.2 --- Classification of poststroke dementia --- p.7, Chapter 2.3 --- Frequency, incidence, and clinical determinants of poststroke dementia --- p.20, Chapter 2.4 --- Imaging methods and imaging features in poststroke dementia --- p.22, Chapter 2.5 --- Mechanisms of stroke-associated dementia --- p.24, Chapter 2.6 --- Influence of poststroke dementia on stroke outcome --- p.28, Chapter PART II --- STUDIES ON EARLY POSTSTROKE DEMENTIA AND DELAYED COGNITIVE DECLINE --- p.31, Chapter Chapter 3: --- Stroke Registry Investigating Cognitive decline (STRIVE-COG): Risk Factors for Early Poststroke Dementia (Study 1) --- p.32, Chapter 3.1 --- Abstract --- p.32, Chapter 3.2 --- Introduction --- p.33, Chapter 3.3 --- Methods --- p.35, Chapter 3.4 --- Results --- p.45, Chapter 3.5 --- Discussion --- p.48, Chapter Chapter 4 --- Stroke Registry Investigating Cognitive decline (STRIVE-COG): Predictors for Delayed Poststroke Cognitive decline (Study 2) --- p.63, Chapter 4.1 --- Abstract --- p.63, Chapter 4.2 --- Introduction --- p.64, Chapter 4.3 --- Methods --- p.66, Chapter 4.4 --- Results --- p.76, Chapter 4.5 --- Discussion --- p.78, Chapter PART III --- CONCLUSION --- p.88, Chapter Chapter 5 --- Strengths and Limitations --- p.89, Chapter Chapter 6 --- Summary and Future Directions --- p.91, References --- p.93, http://library.cuhk.edu.hk/record=b5549579, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

近年，研究發現許多心臟衰竭患者的左室射血分數在正常範圍內。這種類型的心臟衰竭，已被稱為“射血分數保持的心臟衰竭（HFPEF）。研究還發現，HFPEF患者往往是老年女性，有高血壓病史，其預後比射血分數降低的心衰更好。, 然而，很少人研究過中國人中HFPEF患者的死亡率。同時，經治療后HFPEF患者長期的生活質量是否改善沒有得到很好的研究，特別是在老年HFPEF患者中。此外，到目前為止，一直沒有一個風險評分系統用於預測HFPEF患者的預後。, 我們從2006年至2010年在一所大學附屬醫院建立的心臟衰竭注冊研究中，前瞻性納入了847 名HFPEF的患者進行研究。此外，我們通過國際疾病分類第九版臨床修正（ICD-9- CM）代碼428進行數據檢索，回顧性分析了2001年至2005年入住我院的心臟衰竭的患者。其中170名射血分數超過50的患者納入本研究。爲了消除兩組病人基線差異對臨床終點的影響，我們計算出傾向性得分。在建立風險評分方面，所有HFPEF患者隨機分為推導組和驗證組。從推導組中，我們得到了風險評分，然後我們再在驗證組中測試評分系統是否可行。本研究中，生活質量是通過明尼蘇達州心力衰竭問卷（MLHFQ）進行評估。, 我們研究的主要發現包括：, 1、與2001-2005年納入的HFPEF患者比，2006-2010年納入的HFPEF患者，一年生存率有顯著提高（76.9%比65.5%，P = 0.001），心臟衰竭的再次住院率也顯著下降（33.3%比50.6%，P <0.001）。傾向得分匹配調整後1年生存率提高（78.9%比68.1%，P = 0.02）和心衰再次住院率降低（34.3&比51.2%，P = 0.002）仍然顯著。, 2、各個年齡組基線（32±16比30±15比34±11，P = 0.12）和12個月（16±14比16±12比19±13，P = 0.62）的MLHFQ得分均沒有顯著。HFPEF患者12個月時生活質量得到改善的比例在年齡組之間相似（84.0%比80.2%比87.5%，P = 0.68）。, 3、我們通過Cox多因素回歸分析得到了了6個獨立的預測HFPEF患者1年死亡率的預後因素。每個因素根據其回歸系統獲得一個分數：低蛋白血症（5分），不使用鈣通道阻滯劑（3分），充血性心臟衰竭病史（2.5分），腦血管疾病病史（2.5分），尿素氮> 10mmol / L（2.5分），年齡> 78歲（2分）。每一個患者根據風險分數而被分為三個危險人群：低風險（0至5.5分），中等風險（10.5分）和高風險（11至17.5分）。在推導隊列，這三組的1年死亡率分別為10.5%，22.3%和48.7%分別。在驗證隊列，相應的死亡率分別為15.4%，25.3&和39%。, 4、低蛋白血症為HFPEF患者1年死亡率的最有力的預測指標。, 綜上所述，我們發現，近年來，HFPEF患者一年的死亡率和心臟衰竭再次住院率有所下降。與相對年輕的HFPEF患者相比，老年HFPEF患者經歷了類似的生活質量的改善。從臨床常用的變量得到的風險評分可用於預測HFPEF患者1年死亡率。低蛋白血症為HFPEF患者1年死亡率的最有力的預測指標。, Recently, many studies have found that many patients presenting with clinical heart failure (HF) had a left ventricular ejection fraction in the normal range. This entity has been termed “heart failure with preserved ejection fraction (HFPEF). Previous studies have indicated that patients who have HFPEF tend to be older, female, and to have a history of hypertension., However, little was known about the clinical outcome and related predictors of HFPEF patients in Chinese population. Long term quality of life (QOL) after treatment in HFPEF patients have not been well studied, especially in very elderly HFPEF. Furthermore, there has been no a risk score used HFPEF patients., We studied 847 HFPEF patients who were prospectively enrolled into a HF Registry from 2006 to 2010 at a teaching hospital. In addition, a historical cohort of patients admitted in our hospital from 2001 to 2005 was retrospectively retrieved and data searched using the ICD-9-CM code 428. Among this, 170 with HFPEF were selected for study. To adjust for the impact of baseline differences between the 2 cohorts on clinical outcomes, we calculated a propensity score. To establish a risk score, HFPEF patients were randomly divided into derivation group and validation group. We got a risk score from the derivation group and then checked in the validation one. QOL was assessed by the Minnesota Living with Heart Failure Questionnaire (MLHFQ) instruments., Main findings of our study included, 1. 1-year survival rates improved (65.5% vs. 76.9%, p=0.001) and HF re-hospitalization rates decreased (50.6% vs. 33.3%, p<0.001 in HFPEF patients admitted between 2001-2005 and 2006-2010, respectively). The improvement in 1-year survival (68.1% vs. 78.9%, p=0.02) and HF re-hospitalization (51.2% vs. 34.3%, p=0.002) remained significant after propensity score matching., 2. Baseline (30±16 vs. 28±16 vs. 29±16, p=0.87) and 12-months (15±14 vs. 16±14 vs. 15±12, p=0.92) MLHFQ score showed no significant differences with advancing age. Proportion of patients who experienced improvement in QOL at 12-months were similar among age groups (84.0% vs. 80.2% vs. 87.5%, p=0.68)., 3. Six independent prognostic factors were identified, and each was assigned a number of points proportional to its regression coefficient: hypoalbuminemia (5 points), not use of CCB (3 points), history of HF (2.5 points), history of CVD (2.5 points), BUN>10mmol/L (2.5 points), age>78 years (2 points). Wecalculated risk scores for each patient and defined three risk groups: low risk (0 to 5.5 points), intermediate risk (6 to 10.5 points) and high risk (11 to 17.5 points). In the derivation cohort, the 1-year mortality rates for these three groups were 10.5%, 22.3%, and 48.7% respectively. In the validation cohort, the corresponding mortality rates were 15.4%, 25.3% and 39%., 4. Hypoalbuminemia was the most powerful predictor of 1 year mortality for HFPEF patients., In summary, we found that the mortality of HFPEF patients in the first year decreased over time. Elderly HFPEF patients experienced similar improvements in QOL compared to younger ones. The clinical based risk score can be used to predict mortality of HFPEF patients. Hypoalbuminemia was the most powerful predictor of 1 year mortality for HFPEF patients., Detailed summary in vernacular field only., Liu, Ming., Thesis (Ph.D.)--Chinese University of Hong Kong, 2012., Includes bibliographical references (leaves 124-150)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., Declaration of originality --- p.i, Acknowledgement --- p.ii, List of abbreviations --- p.iv, Publications --- p.vii, Full paper --- p.vii, s --- p.viii, p.ix, 中文摘要 --- p.xii, Table of Contents --- p.xiv, List of Tables --- p.xx, List of Figures --- p.xxi, Chapter SECTION I --- LITERATURE REVIEW --- p.1, Chapter CHAPTER 1 --- DEFINITION, PATHOPHYSIOLOGY AND DIAGNOSIS OF HFPEF --- p.1, Chapter 1.1 --- Definition of HFPEF --- p.2, Chapter 1.2 --- Pathophysiology of HFPEF --- p.3, Chapter 1.2.1 --- Structure abnormality in HFPEF --- p.3, Chapter 1.2.2 --- Diastolic dysfunction in HFPEF --- p.10, Chapter 1.2.3 --- Systolic function in HFPEF --- p.12, Chapter 1.2.4 --- Left atrial dysfunction in HFPEF --- p.14, Chapter 1.2.5 --- Peripheral factors in HFPEF --- p.15, Chapter 1.3 --- Diagnosis of HFPEF --- p.16, Chapter 1.3.1 --- Clinical features --- p.17, Chapter 1.3.2 --- Echocardiographic features of HFPEF patients --- p.18, Chapter 1.3.3 --- BNP AND N-pro BNP assays --- p.18, Chapter CHAPTER 2 --- EPIDEMIOLOGY OF HFPEF --- p.28, Chapter 2.1 --- Prevalence of HFPEF among HF patients --- p.28, Chapter 2.2 --- Demographic features and comorbid conditions --- p.29, Chapter 2.2.1 --- Age --- p.30, Chapter 2.2.2 --- Gender --- p.31, Chapter 2.2.3 --- Hypertension --- p.31, Chapter 2.2.4 --- Coronary artery disease --- p.32, Chapter 2.2.5 --- Atrial fibrillation --- p.33, Chapter 2.2.6 --- Diabetes Mellitus --- p.34, Chapter 2.2.7 --- Renal Dysfunction --- p.34, Chapter 2.2.8 --- Body Mass Index --- p.35, Chapter 2.2.9 --- Anemia --- p.35, Chapter 2.2.10 --- Chronic Obstructive Pulmonary Disease --- p.35, Chapter 2.3 --- Mortality of HFPEF patients --- p.36, Chapter 2.3.1 --- Mortality rates --- p.36, Chapter 2.3.2 --- Pattern of death --- p.37, Chapter 2.4 --- Prognostic predictors --- p.38, Chapter 2.5 --- Health related quality of life in HFPEF patients --- p.40, Chapter CHAPTER 3 --- TREATMENT OF HFPEF PATIENTS --- p.42, Chapter 3.1 --- Non-pharmacologic Therapy --- p.42, Chapter 3.2 --- Medical and Surgical Therapy --- p.43, Chapter 3.2.1 --- Clinical Studies --- p.43, Chapter 3.2.2 --- Randomized Controlled Clinical Trials --- p.43, Chapter 3.2.3 --- Current Therapeutic Recommendations --- p.45, Conclusions --- p.46, Chapter SECTIONS II --- STUDIES ABOUT HFPEF --- p.47, Chapter CHAPTER 4 --- OBJECTIVES AND HYPOTHESIS --- p.47, Chapter 4.1 --- Objectives of the study --- p.47, Chapter 4.2. --- Hypothesis --- p.48, Chapter CHAPTER 5 --- METHODOLOGY --- p.49, Chapter 5.1 --- Patient population --- p.49, Chapter 5.2 --- Definition of HFPEF patients --- p.49, Chapter 5.3 --- Baseline patient data --- p.50, Chapter 5.4 --- Echocardiogram --- p.50, Chapter 5.5 --- Health related quality of life assessment --- p.51, Chapter 5.6 --- Follow-up and clinical outcome --- p.51, Chapter 5.7 --- Statistical analysis --- p.52, Chapter CHAPTER 6 --- IMPROVED 12 MONTH SURVIVAL OF PATIENTS ADMITTED WITH HFPEF OVER THE LAST DECADE --- p.54, Chapter 6.1 --- Introduction --- p..54, Chapter 6.2 --- Methods --- p.54, Chapter 6.2.1 --- Patient population --- p.54, Chapter 6.2.2 --- Baseline patient data --- p.55, Chapter 6.2.3 --- Study endpoints --- p.56, Chapter 6.2.4 --- Statistical analysis --- p.56, Chapter 6.3 --- Results --- p.57, Chapter 6.3.1 --- Baseline patient characteristics --- p.57, Chapter 6.3.2 --- Unadjusted clinical outcomes --- p.57, Chapter 6.3.3 --- Propensity score adjusted clinical outcomes --- p.58, Chapter 6.4 --- Discussion --- p.58, Chapter 6.5 --- Conclusions --- p.61, Chapter CHAPTER 7 --- QUALITY OF LIFE IN ELDERLY PATIENTS WITH HFPEF --- p.67, Chapter 7.1 --- Introduction --- p.67, Chapter 7.2 --- Methods --- p.68, Chapter 7.2.1 --- Patient population --- p.68, Chapter 7.2.2 --- Health related quality of life assessment --- p.69, Chapter 7.2.3 --- Follow-up --- p.69, Chapter 7.2.4 --- Statistical analysis --- p.69, Chapter 7.3 --- Results --- p.70, Chapter 7.3.1 --- Baseline patient characteristics --- p.70, Chapter 7.3.2 --- Mortality --- p.71, Chapter 7.3.3 --- Health-related quality of life --- p.71, Chapter 7.3.4 --- Therapy --- p.71, Chapter 7.3.5 --- Predictors of HRQoL improvement in HFPEF patients --- p.72, Chapter 7.4 --- Discussions --- p.72, Chapter 7.5 --- Conclusions --- p.75, Chapter CHAPTER 8 --- A RISK SCORE TO PREDICT 1 YEAR MORATALITY IN PATIENTS WITH HFPEF --- p.83, Chapter 8.1 --- Introduction --- p.83, Chapter 8.2 --- Methods --- p.84, Chapter 8.2.1 --- Patient population --- p.84, Chapter 8.2.2 --- Candidate Predictor Variables --- p.84, Chapter 8.2.3 --- Statistical analysis --- p.85, Chapter 8.3 --- Results --- p.86, Chapter 8.3.1 --- Patient Characteristics and Outcomes --- p.86, Chapter 8.3.2 --- Predictors of Mortality --- p.87, Chapter 8.3.3 --- Generation of the Risk score --- p.87, Chapter 8.3.4 --- Validation of the risk score --- p.88, Chapter 8.4 --- Discussions --- p.88, Chapter 8.5 --- Conclusions --- p.91, Chapter CHAPTER 9 --- ALBUMIN LEVELS PREDICT SURVIVAL IN PATIENTS WITH HFPEF --- p.97, Chapter 9.1 --- Introduction --- p.97, Chapter 9.2 --- Methods 97 --- p.xviii, Chapter 9.2.1 --- Patient population --- p.97, Chapter 9.2.2 --- Baseline measurement --- p.98, Chapter 9.2.3 --- End points --- p.99, Chapter 9.2.4 --- Statistical analysis --- p.99, Chapter 9.3 --- Results --- p.100, Chapter 9.3.1 --- Baseline patient characteristics --- p.100, Chapter 9.3.2 --- Hypoalbuminemia and Cardiac Events --- p.101, Chapter 9.3.3 --- Albumin and body mass index (BMI) --- p.102, Chapter 9.3.4 --- Causes of hypoaluminemia in HFPEF patients --- p.102, Chapter 9.4 --- Discussion --- p.103, Chapter 9.4.1 --- Liver dysfunction --- p.104, Chapter 9.4.2 --- Hemodilution --- p.105, Chapter 9.4.3 --- BMI and hypoalbuminemia --- p.105, Chapter 9.4.4 --- Renal failure --- p.106, Chapter 9.4.5 --- B-type Natriuretic Peptides and albumin --- p.107, Chapter 9.5. --- Conclusions --- p.109, Chapter CHAPTER 10 --- GENERAL SUMMARY --- p.117, Chapter 10.1 --- Main findings of our study --- p.117, Chapter 10.2 --- Clinical implications --- p.119, Chapter 10.3 --- Potential for final development of research --- p.120, Chapter CHAPTER 11 --- CONCLUSIONS --- p.123, References --- p.124, http://library.cuhk.edu.hk/record=b5549503, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

越來越多證據表明了煙酰胺腺嘌呤核苷酸磷酸（nicotinamide adenine dinucleotide phosphate, NADPH）代謝對腫瘤細胞增殖和生存的重要性。最近一項研究證實了一個p53調節基因，TP53誘導的糖酵解和凋亡調節因數（TP53-inducible glycolysis and apoptosis regulator, TIGAR），在抑制凋亡和誘導NADPH產生方面的作用。抗凋亡蛋白B細胞淋巴瘤2（B-cell lymphoma 2, Bcl-2）之前被報導能夠通過還未確定的機制誘導NADPH的產生。在這篇論文研究中，我們假設Bcl-2與TIGAR耦合從而調節NADPH的產生和細胞生存，並且TIGAR可能成為一個促進細胞生長的新生存因數。這篇論文研究的目的是：1）檢測在NADPH代謝中Bcl-2與TIGAR間的生物聯繫；2）探究在哺乳動物系統中TIGAR調節的分子機制；3）研究TIGAR在體外和體內調節正常及腫瘤細胞生存的作用。, 這篇論文的第一部分結果顯示在正常小鼠胚胎成纖維細胞（MEFs）和缺少功能性p53的人非小細胞肺癌（NSCLC）細胞中存在Bcl-2/TIGAR/NADPH這樣一個新的信號通路軸，重要抗凋亡蛋白Bcl-2與TIGAR耦合後，以一個特有並持久的方式調節NADPH代謝和細胞生長。用特異的小干擾RNA（siRNA）靶向抑制Bcl-2能夠在NSCLC細胞中抑制TIGAR/NADPH/生長信號軸。用小分子抑制劑ABT-737對Bcl-2進行藥理學抑制也能夠顯示相似的作用，而這個作用能夠通過過表達TIGAR被部分逆轉。而且，敲除TIGAR能夠降低Bcl-2誘導的NADPH產生和細胞生長，表明了TIGAR在介導TIGAR/NADPH/生長信號軸中的關鍵作用。, 為了第二個目的，我們研究了Bcl-2對TIGAR的機制調節。我們發現過表達Bcl-2不能改變TIGAR的mRNA水平，但是能誘導轉錄後的TIGAR蛋白累積。有趣的是，TIGAR似乎能通過一個正反饋回路誘導Bcl-2的表達，這進一步促進了由Bcl-2誘導的TIGAR表達上調。除了Bcl-2，許多致癌基因或生長調節因數也被證實參與了TIGAR的調節，包括信號轉導和轉錄啟動因數3（signal transducer and activator of transcription 3, STAT3, Bcl-2的上有調節因數），表皮生長因數受體（epidermal growth factor receptor, EGFR）和肝細胞生長因數受體（hepatocyte growth factor receptor, c-Met）。, 這篇論文的第三部分證實了TIGAR在缺少功能性p53細胞模型中的致癌作用。TIGAR過表達能夠誘導一些癌症的標誌性特徵，包括細胞代謝調節異常，細胞生長增加和凋亡減少。重要的是，TIGAR的過表達在MEFs和缺少功能性p53的NSCLC細胞中直接促成了腫瘤形成，促進了腫瘤的生長。但是功能性p53的存在無論在體外還是體內都能夠消除TIGAR這種生長促進和成瘤的作用。這些發現表明當p53功能消失時TIGAR能成為一個致癌基因，促進腫瘤形成。, 總的來說，我們發現了在腫瘤中Bcl-2/TIGAR/NADPH這樣一條新的信號通路。Bcl-2通過增加TIGAR蛋白累積的轉錄後機制調節TIGAR。我們也證實了TIGAR受到多種致癌基因或生長調節因數的調節，包括STAT3，EGFR和c-Met。並且，我們闡明了當p53功能消失時TIGAR能成為一個致癌基因，促進細胞轉化和腫瘤形成。, Emerging evidences reveal the importance of nicotinamide adenine dinucleotide phosphate (NADPH) metabolism for cancer cell proliferation and survival. A recent study demonstrated the role of a p53-regulated gene, TP53-inducible glycolysis and apoptosis regulator (TIGAR), in inhibiting apoptosis and inducing NADPH production. The anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) has previously been reported to induce NADPH generation through undefined mechanism. In this thesis study, we hypothesized that Bcl-2 is coupled to TIGAR for regulation of NADPH production and cell survival, and that TIGAR may serve as a novel survival factor contributing to cell growth. The objectives of the thesis study are: 1) to examine the biologic relationship between Bcl-2 and TIGAR in NADPH metabolism; 2) to investigate the molecular mechanisms of TIGAR regulation in mammalian systems; and 3) to examine the role of TIGAR in regulating normal and cancer cell survival in vitro and in vivo., Findings in first part of the thesis revealed a novel signaling axis of Bcl-2/TIGAR/NADPH in normal mouse embryonic fibroblasts (MEFs) and human non-small cell lung cancer (NSCLC) cells lacking functional p53, coupling the major anti-apoptotic protein Bcl-2 to TIGAR regulation for NADPH metabolism and cell growth in a specific and sustained manner. Targeting of Bcl-2 by specific siRNA inhibited TIGAR/NADPH/growth axis in NSCLC cells. Pharmacologic inhibition of Bcl-2 by small molecule inhibitor ABT-737 also exhibited similar effects, which was partially reversed by the overexpression of TIGAR. In addition, TIGAR knockdown reduced Bcl-2-induced NADPH production and cell growth, implicating a critical role of TIGAR in mediating Bcl-2/NADPH/growth signaling axis., For the second objective, we studied the mechanistic regulation of TIGAR by Bcl-2. We found that overexpression of Bcl-2 did not alter TIGAR mRNA expression but induced TIGAR protein accumulation post-transcriptionally. Interestingly, TIGAR seemed to induce Bcl-2 expression via a positive feedback loop, which may further contribute to the upregulation of TIGAR expression induced by Bcl-2. In addition to Bcl-2, a number of oncogene/growth regulators were demonstrated to be involved in TIGAR regulation, including signal transducer and activator of transcription 3 (STAT3, an upstream regulator of Bcl-2), epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-Met)., The third part of the thesis demonstrated the oncogenic role of TIGAR in cell models lacking functional p53. TIGAR overexpression induced several hallmark features of cancer including deregulated cell metabolism, increased cell growth and inhibited apoptosis. Strikingly, overexpression of TIGAR directly drove tumor formation and promoted tumor growth in MEFs and NSCLC cells lacking functional p53. The growth stimulatory and tumorigenic activities of TIGAR were abolished in the presence of functional p53 both in vitro and in vivo. These findings revealed TIGAR as an oncogene capable of driving tumorigenesis when p53 function is lost., In summary, we have identified a novel signaling pathway of Bcl-2/TIGAR/NADPH in cancer. TIGAR is regulated by Bcl-2 via a post-transcriptional mechanism by enhancing TIGAR protein accumulation. We also demonstrated the regulation of TIGAR by multiple oncogene/growth regulators including STAT3, EGFR and c-Met. Furthermore, we have demonstrated TIGAR as an oncogene capable of driving cell transformation and tumorigenesis when p53 function is lost., Detailed summary in vernacular field only., Lam, Kai Yee., Thesis (Ph.D.)--Chinese University of Hong Kong, 2012., Includes bibliographical references (leaves 158-176)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., p.i, 摘要 --- p.iv, Declaration --- p.vi, Acknowledgements --- p.vii, Publications --- p.viii, Table of Content --- p.ix, List of Illustrations --- p.xii, List of Abbreviations --- p.xv, Chapter Chapter 1: --- Introduction, Chapter 1.1 --- Overview of tumor metabolism --- p.1, Chapter 1.1.1 --- Glucose metabolism --- p.4, Chapter 1.1.2 --- Glutamine metabolism --- p.6, Chapter 1.1.3 --- De novo fatty acid synthesis --- p.7, Chapter 1.1.4 --- Advantages of metabolic reprogramming in tumor cells --- p.8, Chapter 1.2 --- Mechanisms regulating metabolic reprogramming in cancers --- p.11, Chapter 1.2.1 --- Tumor suppressor p53 --- p.11, Chapter 1.2.2 --- Hypoxia-inducible factor 1 (HIF-1) --- p.13, Chapter 1.2.3 --- PI3K/Akt/mTOR signaling pathway --- p.15, Chapter 1.2.4 --- Oncogenic Myc --- p.16, Chapter 1.2.5 --- Oncogenic Ras --- p.18, Chapter 1.3 --- TP53-Inducible Glycolysis and Apoptosis Regulator (TIGAR) --- p.22, Chapter 1.3.1 --- TIGAR and oxidative stress --- p.25, Chapter 1.3.2 --- TIGAR and carcinogenesis --- p.29, Chapter 1.3.3 --- TIGAR and other diseases --- p.32, Chapter 1.4 --- Hypotheses and Aims --- p.36, Chapter Chapter 2: --- Materials and Methods, Chapter 2.1 --- Materials --- p.38, Chapter 2.1.1 --- Chemicals and reagents --- p.38, Chapter 2.1.2 --- Drugs --- p.41, Chapter 2.1.3 --- Commercial detection kits --- p.41, Chapter 2.1.4 --- Antibodies --- p.42, Chapter 2.2 --- Cell culture --- p.43, Chapter 2.3 --- Plasmids --- p.44, Chapter 2.4 --- Transfection --- p.46, Chapter 2.5 --- Retrovirus infection --- p.47, Chapter 2.6 --- Drug treatment --- p.47, Chapter 2.7 --- Cell viability assay --- p.48, Chapter 2.8 --- Trypan blue exclusion assay --- p.49, Chapter 2.9 --- NADPH assay --- p.49, Chapter 2.10 --- Cell death detection ELISA --- p.50, Chapter 2.11 --- Western blotting --- p.50, Chapter 2.12 --- Reverse TranscriptionPolymerase Chain Reaction (RT-PCR) --- p.51, Chapter 2.13 --- Cell cycle analysis --- p.52, Chapter 2.14 --- Transwell migration and matrigel invasion assays --- p.53, Chapter 2.15 --- In vivo studies --- p.54, Chapter 2.16 --- Histology and immunohistochemistry --- p.55, Chapter 2.17 --- Statistical analysis --- p.56, Chapter Chapter 3: --- Indentification of Novel Bcl-2/TIGAR/NADPH Signaling Axis, Chapter 3.1 --- Introduction --- p.57, Chapter 3.2 --- Results --- p.60, Chapter 3.2.1 --- Bcl-2-induced NADPH production and cell growth is associated with TIGAR upregulation in p53-null MEFs --- p.60, Chapter 3.2.2 --- Identification of Bcl-2/TIGAR/NADPH signaling axis in p53-negative NSCLC cells --- p.63, Chapter 3.2.3 --- Bcl-2 targeting reduces TIGAR expression and NADPH production in MEFs and NSCLC cells --- p.66, Chapter 3.2.4 --- Pharmacologic intervention of Bcl-2 alters TIGAR levels in MEFs and NSCLC cells --- p.71, Chapter 3.2.5 --- Bcl-2 targeting inhibits TIGAR expression, NADPH production and cell growth in Bcl-2-amplified small cell lung cancer (SCLC) cells --- p.76, Chapter 3.2.6 --- Bcl-2-induced TIGAR expression is highly specific compared to other pro-survival proteins --- p.78, Chapter 3.2.7 --- Bcl-2/TIGAR/NADPH signaling axis is functionally intact in p53-positive NSCLC cells --- p.80, Chapter 3.3 --- Discussion --- p.83, Chapter Chapter 4: --- Mechanisms of TIGAR Regulation, Chapter 4.1 --- Introduction --- p.88, Chapter 4.2 --- Results --- p.90, Chapter 4.2.1 --- Bcl-2 regulates TIGAR by post-transcriptional mechanism --- p.90, Chapter 4.2.2 --- Bcl-2-induced TIGAR expression is associated with upregulation of other pro-survival proteins --- p.94, Chapter 4.2.3 --- STAT3 activation can serve as a biological signal for TIGAR induction --- p.97, Chapter 4.2.4 --- Tyrosine kinase receptors contributes to TIGAR regulation --- p.100, Chapter 4.2.5 --- The existence of TIGAR/Bcl-2 positive feedback loop --- p.102, Chapter 4.3 --- Discussion --- p.104, Chapter Chapter 5: --- TIGAR is Oncogenic, Chapter 5.1 --- Introduction --- p.107, Chapter 5.2 --- Results --- p.109, Chapter 5.2.1 --- Overexpression of TIGAR alters several hallmark features of cancer --- p.109, Chapter 5.2.2 --- TIGAR drives tumor formation in vivo --- p.114, Chapter 5.2.3 --- Tumorigenic activity of TIGAR is functionally counteracted by functional p53 --- p.123, Chapter 5.3 --- Discussion --- p.134, Chapter Chapter 6: --- Summary --- p.138, Chapter Chapter 7: --- Future Prospective --- p.143, Appendices --- p.148, References --- p.158, http://library.cuhk.edu.hk/record=b5549393, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Detailed summary in vernacular field only., Zhao, Junhong., also in Chinese., p.i, Acknowledgements --- p.vi, Publications --- p.vii, Research articles --- p.vii, Conference abstracts --- p.viii, Table of contents --- p.x, List of tables --- p.xiii, List of figures --- p.xiv, List of abbreviations --- p.xvi, Chapter Chapter 1 --- Introduction --- p.1, Chapter 1.1 --- General introduction --- p.1, Chapter 1.2 --- Gastric Cancer --- p.3, Chapter 1.2.1 --- Eipdemiology of Gastric Cancer --- p.4, Chapter 1.2.2 --- Pathology of Gastric Cancer --- p.8, Chapter 1.2.3 --- Risk Factors for Gastric Cancers --- p.11, Chapter 1.3 --- Epstein-Barr Virus-associated Gastric Cancer (EBVaGC) --- p.15, Chapter 1.3.1 --- Historical Discovery and Harm of EBV --- p.15, Chapter 1.3.2 --- Molecular Biology of EBV --- p.16, Chapter 1.3.3 --- Latent and Lytic Infection of EBV --- p.18, Chapter 1.3.4 --- EBV Products --- p.18, Chapter 1.3.5 --- EBV-associated gastric cancer (EBVaGC) --- p.28, Chapter 1.4 --- EBV-induced Epigenetic Alteration in Gastric Carcinogenesis --- p.36, Chapter 1.4.1 --- Cytosine Methylation and CpG Island --- p.36, Chapter 1.4.2 --- DNA Methylation in Gastric Cancer --- p.39, Chapter 1.5 --- How to identify EBV-induced promoter methylation in gastric cancer --- p.45, Chapter 1.5.1 --- Methylated DNA Immunoprecipitation (MeDIP) --- p.48, Chapter 1.5.2 --- Combined Bisulfite Restriction Analysis (COBRA) --- p.49, Chapter 1.5.3 --- Bisulfite Genomic Sequencing --- p.49, Chapter 1.5.4 --- Pyrosequencing --- p.49, Chapter Chapter 2 --- Materials and Methods --- p.51, Chapter 2.1 --- Materials --- p.51, Chapter 2.1.1 --- Cancer Cell Lines and Culture Condition --- p.51, Chapter 2.1.2 --- Primary GC Samples --- p.52, Chapter 2.2 --- EBV Encoded Nuclear RNA (EBER) in situ Hybridization (EBER-ISH) --- p.52, Chapter 2.3 --- Western Blot Analysis --- p.53, Chapter 2.4 --- Plasmid and Transfection --- p.56, Chapter 2.4.1 --- Plasmid Construction and Extraction --- p.56, Chapter 2.4.2 --- Plasmid Transfection --- p.59, Chapter 2.5 --- Gene Expression Analysis --- p.59, Chapter 2.5.1 --- Purification of Total RNA (RNeasy Kit, Qiagen) --- p.59, Chapter 2.5.2 --- cDNA Reverse Transcription --- p.60, Chapter 2.5.3 --- Semi-Quantitative PCR --- p.62, Chapter 2.5.4 --- Quantitative Real-Time PCR (qRT-PCR) --- p.64, Chapter 2.6 --- DNMT1 and 3b Activity Assay --- p.64, Chapter 2.7 --- DNA Methylation Analysis --- p.64, Chapter 2.7.1 --- Genomic DNA Extraction --- p.64, Chapter 2.7.2 --- Genome-wide Profiling of EBV-associated DNA Methylation by MeDIP-chip --- p.65, Chapter 2.7.3 --- Bioinformatics Analysis --- p.66, Chapter 2.7.4 --- CpG Island Prediction and Analysis of the Targets' Promoter Region --- p.66, Chapter 2.7.5 --- DNA Sodium Bisulfite Modification --- p.67, Chapter 2.7.6 --- Target Gene Methylation in GC Cell Lines --- p.67, Chapter 2.7.7 --- Bisulfite Pyrosequencing Analysis in GC Tissue Samples --- p.70, Chapter 2.8 --- Biological Function Analysis of SSTR1 --- p.72, Chapter 2.8.1 --- Cell Proliferation Assay for Stable Transfection --- p.72, Chapter 2.8.2 --- Colony Formation Assay --- p.72, Chapter 2.8.3 --- Cell Cycle Analysis Assay --- p.72, Chapter 2.8.4 --- Cell Migration Analysis --- p.73, Chapter 2.8.5 --- Invasion Analysis --- p.73, Chapter 2.8.6 --- Human Cancer Pathway Finder RT2 Profiler PCR Array Analysis --- p.74, Chapter 2.9 --- Statistical Analysis --- p.76, Chapter Charpter 3 --- results --- p.77, Chapter 3.1 --- EBV Infection in AGS-EBV Cell Model --- p.77, Chapter 3.2 --- Activation of DNMT3b in AGS-EBV Cells --- p.80, Chapter 3.3 --- LMP2A Induced DNMT3b Activity in AGS Cells --- p.82, Chapter 3.4 --- Genome-wide Profiling of DNA Methylation Associated with EBV Infection Using MeDIP-chip --- p.84, Chapter 3.5 --- EBV-associated Cancer Pathways Defined by EBV-associated Promoter Methylated Genes --- p.86, Chapter 3.6 --- CpG Hypermethylation and Transcriptional Silencing of EBV-associated Methylated Genes in AGS-EBV Cells --- p.88, Chapter 3.7 --- Bioinformatics Analysis of SSTR1 Using University of California Santa Cruz Genome Bioinformatics (UCSC) Database and CpG Island Searcher --- p.93, Chapter 3.8 --- COBRA Analysis of SSTR1 Promoter Methylation in GC Cell Lines --- p.93, Chapter 3.9 --- Frequent SSTR1 Hypermethylation was Associated with EBV Positive Primary Gastric Cancer --- p.96, Chapter 3.10 --- SSTR1 was Down-regulated in GC Cell Lines through RNA Interference --- p.103, Chapter 3.11 --- SSTR1 Knockdown Induced Cell Proliferation in GC Cell Lines --- p.105, Chapter 3.12 --- SSTR1 Knock-down Promoted Cells to Enter into S Phase --- p.108, Chapter 3.13 --- SSTR1 Knock-down Increased the Migration Ability of GC --- p.110, Chapter 3.14 --- SSTR1 Knock-down Promoted Cell Invasion --- p.112, Chapter 3.15 --- Ectopic Expression of SSTR1 Inhibited Proliferation and Clonogenicity in PANC1 Cancer Cells --- p.114, Chapter 3.16 --- Identification of Genes Modulated by SSTR1 --- p.116, Chapter Chapter 4 --- Discussion --- p.119, Chapter Chapter 5 --- Limitation of the study --- p.127, ConclusionS --- p.128, Reference --- p.129, Http://library.cuhk.edu.hk/record=b5549615, Use of this resource is governed by the terms and conditions of the Creative Commons "Attribution-NonCommercial-NoDerivatives 4.0 International" License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Leung, Wan Lun Fiona., Thesis (M.Phil.)--Chinese University of Hong Kong, 2011., Includes bibliographical references (leaves 179-204)., s in English and Chinese., p.i, 中文摘要 --- p.v, Acknowledgement --- p.viii, List of Abbreviations and Symbols x --- p.vi, List of Tables --- p.xx, List of Figures --- p.xxi, Chapter CHAPTER ONE --- Introduction --- p.1, Chapter 1.1 --- General Overview --- p.1, Chapter 1.2 --- Necrotizing Enterocolitis (NEC) --- p.3, Chapter 1.2.1 --- Epidemiology of NEC --- p.3, Chapter 1.2.2 --- "Clinical Presentation, Diagnosis and Management of NEC" --- p.5, Chapter 1.2.3 --- Pathophysiology of NEC --- p.9, Chapter 1.2.3.1 --- Prematurity --- p.9, Chapter 1.2.3.2 --- Bacterial Colonization --- p.12, Chapter 1.2.3.3 --- Enteral Feeding --- p.15, Chapter 1.2.3.4 --- Hypoxia and Ischemia --- p.16, Chapter 1.2.3.5 --- Genetic Polymorphism --- p.17, Chapter 1.2.3.6 --- Inflammatory Mediators --- p.20, Chapter 1.3 --- Spontaneous Intestinal Perforation (SIP) --- p.24, Chapter 1.3.1 --- Epidemiology of SIP --- p.24, Chapter 1.3.2 --- "Clinical Presentation, Diagnosis and Management of SIP" --- p.26, Chapter 1.3.3 --- Risk Factors of SIP --- p.28, Chapter 1.3.3.1 --- Prematurity --- p.29, Chapter 1.3.3.2 --- Use of Drugs --- p.30, Chapter 1.4 --- Comparison between NEC and SIP --- p.32, Chapter 1.5 --- Role of Cytokines in Pathogenesis of NEC and SIP --- p.38, Chapter 1.6 --- Immunoregulatory Molecules of Interest in This Study --- p.46, Chapter 1.6.1 --- Angiopoietin-2 (Ang-2) --- p.46, Chapter 1.6.2 --- v-erb-b2 Erythroblastic Leukemia Viral Oncogene Homolog 2 (avian) (ErbB3) --- p.48, Chapter 1.6.3 --- Type II Interleukin-1 Receptor (IL-1RII) --- p.52, Chapter 1.6.4 --- Urokinase Plasminogen Activator Receptor (uPAR) --- p.54, Chapter CHAPTER TWO --- Objectives --- p.57, Chapter CHAPTER THREE --- Materials and Methodology --- p.58, Chapter 3.1 --- Overview of the Experimental Procedures --- p.58, Chapter 3.1.1 --- Investigation on the Profile of Circulatory Immunoregulatory Proteins in Plasma of NEC and SIP High Risk Neonates --- p.58, Chapter 3.1.2 --- Investigation on the mRNA Expression Level of Targeted Immunoregulatory Molecules on Resected Intestinal Tissues in NEC and SIP Neonates --- p.58, Chapter 3.1.3 --- Investigation on the mRNA and Protein Expression Levels of Targeted Immunoregulatory Molecules in Human Intestinal Cell Lines --- p.60, Chapter 3.2 --- Reagents and Lab-wares with Their Sources --- p.61, Chapter 3.3 --- Study Population --- p.63, Chapter 3.4 --- Collection of Neonatal Whole Blood Samples --- p.65, Chapter 3.5 --- Cytokine Antibody Array Analyses --- p.67, Chapter 3.6 --- Enzyme-linked Immunosorbant Assays (ELISA) --- p.69, Chapter 3.6.1 --- Angiopoietin-2 --- p.69, Chapter 3.6.2 --- sErbB3 --- p.71, Chapter 3.6.3 --- sIL-lRII --- p.72, Chapter 3.6.4 --- suPAR --- p.74, Chapter 3.7 --- Collection of Neonatal Resected Intestinal Tissues --- p.76, Chapter 3.8 --- Resected Intestinal Tissue RNA Isolation --- p.78, Chapter 3.9 --- Purity Assessment of the Purified Tissue RNA Samples --- p.80, Chapter 3.10 --- Integrity Assessment of the Purified Tissue RNA Samples --- p.81, Chapter 3.11 --- In vitro Stimulation of Human Enterocytes by Lipopolysaccharides (LPS) and/or Platelet Activating Factor (PAF) --- p.84, Chapter 3.12 --- mRNA Expression Level Assessment of Selected Target Genes in Resected Intestinal Tissues and Human Intestinal Cell Lines --- p.86, Chapter 3.12.1 --- Synthesis of First Strand cDNA --- p.86, Chapter 3.12.2 --- Quantitative Polymerase Chain Reaction (qPCR) --- p.87, Chapter 3.13 --- Statistical Analysis --- p.89, Chapter CHAPTER FOUR --- Screening of Immunoregulatory Target Protein Molecules in Plasma of NEC and SIP Patients by Cytokine Array Analyses --- p.104, Chapter 4.1 --- Results --- p.104, Chapter 4.1.1 --- Screening of Detectable Immunoregulatory Target Molecules --- p.104, Chapter 4.1.2 --- Selection of Target Molecules Based on the Fold Change in NEC or SIP Compared with Control Samples --- p.105, Chapter 4.1.2.1 --- Similar Regulation of Target Molecules in Both NEC and SIP patients --- p.105, Chapter 4.1.2.2 --- Differential regulation of Target Molecules in NEC and SIP Patients --- p.106, Chapter 4.1.2.3 --- "Relative Normalized Expressions of Selected Circulatory Immunoregulatory Protein Molecules in NEC, SIP and Control Neonates" --- p.108, Chapter 4.1.2.3.1 --- Anti-inflammation --- p.108, Chapter 4.1.2.3.2 --- Pro-inflammation --- p.109, Chapter 4.1.2.3.3 --- Cell Growth --- p.110, Chapter 4.1.2.3.4 --- Wound Healing --- p.110, Chapter 4.1.2.3.5 --- Angiogenesis --- p.111, Chapter 4.1.2.3.6 --- "Anti-apoptosis, Cell Adhesion and Extracellular Matrix Organization" --- p.112, Chapter 4.1.3 --- Further Selection of Novel Target Molecules Based on Statistical Significance and Fold Change of NEC versus SIP --- p.113, Chapter 4.2 --- Discussion --- p.115, Chapter CHAPTER FIVE --- Validation of Target Proteins in Plasma of NEC and SIP Patients by Enzyme-linked Immunosorbant Assay --- p.132, Chapter 5.1 --- Results --- p.133, Chapter 5.1.1 --- Demographic Data of the Study Group --- p.133, Chapter 5.1.2 --- "Comparison of Plasma Levels of Target Proteins between NEC, SIP and Respective Controls" --- p.134, Chapter 5.1.3 --- Longitudinal Study of the Pre- and Post-operative Target Proteins Levels in Plasma --- p.136, Chapter 5.2 --- Discussion --- p.138, Chapter CHAPTER SIX --- Investigation on mRNA Expression Levels of Target Immunoregulatory Protein Molecules in Intestinal Tissue and Intestinal Cell Lines --- p.151, Chapter 6.1 --- Results --- p.152, Chapter 6.1.1 --- mRNA Expression Levels of Target Molecules in the Diseased Margin of Resected Intestinal Tissues of NEC and SIP patients --- p.152, Chapter 6.1.2 --- mRNA Expression Levels of Target Molecules in the Macroscopically Normal and Diseased Margin of Resected Intestinal Tissues of NEC and SIP patients --- p.154, Chapter 6.1.3 --- mRNA Expression Levels of Target Molecules in Human Intestinal Cell Lines upon LPS and PAF Challenge --- p.156, Chapter 6.1.3.1 --- FHs-74 Int Cell Line --- p.156, Chapter 6.1.3.2 --- Caco-2 Cell Line --- p.157, Chapter 6.2 --- Discussion --- p.158, Chapter CHAPTER SEVEN --- General Discussion --- p.171, Chapter 7.1 --- Overall Findings --- p.171, Chapter 7.2 --- Limitations of Study --- p.174, Chapter 7.3 --- Future Investigations --- p.177, References --- p.179, http://library.cuhk.edu.hk/record=b5894836, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)