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在世界範圍內，肝細胞癌(HCC)是其中一種惡性程度很高並且預後很差的疾病。和其他的癌症一樣，肝癌是一种基因疾病，基因異常的積累在肝癌的生成中扮演著重要的角色。近年來，第二代測序技術（NGS）的迅速發展顯現了前所未見的能力揭示癌癥基因組中分子的複雜性，這為癌癥的生物學，診斷和药物治療提供了一個嶄新的思路。, 非酒精脂肪性肝炎（NASH）是一種與代謝有關的疾病，在發達國家地區例如美國，歐洲，日本和加拿大，這是其中一種越來越常見的HCC 的病因。在本論文的第一部份，三個NASH 相關的HCC 以及其配對的血DNA 進行了全基因組測序（WGS）。另外還有一個來源于這三個NASH 相關的HCC其中之一的細胞株也進行了全基因組測序。在全部樣品中，測序深度範圍介乎29.1X 到102.2X，序列覆蓋度均大於99%。結果顯示發現的新的單核苷酸變異（SNVs）數量介乎于6,898 至17,129，平均值是11,133。根據這些找到的SNV，隨機選出56 個體細胞SNV 進行Sanger 測序，其中92.9%可以被確認。基因突變譜顯示有頻繁的A:T>G:C 和C：G>A:T 體細胞突變，而C：G>T:A 則在CpG 位點頻繁出現。在眾多的非同義體細胞突變基因中，我們選擇了CTNNB1，PNLIP 和MLL2 這三個基因進行進一步研究，此三個基因都在多於一個病例中發現有突變。在額外的一組44 對NASH 相關HCC及50 對HBV 相關的HCC 的癌變組織和其臨近非腫瘤組織中，我們進一步對這三個基因和TP53 的編碼區域進行了測序，而TP53 是在HBV 相關HCC中被報導有高頻率突變的。在NASH 相關的HCC 中，這些基因都只在腫瘤組織中發現重複出現的錯義突變，在臨近的非腫瘤肝組織沒有發現有突變。在NASH 相關的HCC 中，CTNNB1 的突變率（36.4%）明顯高於在HBV相關的HCC 中的突變率（12.0%，P=0.007）。PNLIP 和MLL2 的突變只在NASH相關的HCC 中發現，其突變率分別為12.1%和7.1%，而在HBV 相關的HCC中，則沒有發現突變。然而，TP53 的突變率在NASH 相關的HCC 及HBV相關的HCC 中差別不明顯（P>0.1）。在功能性研究的實驗中，我们发现在HKCI-10（有PNLIP 突變D396N）細胞株中，PNILP 的活性比在正常肝細胞L02 細胞株（野生型PNLIP）中要低。在永生性肝細胞L02 細胞株中，CTNNB1的突變引起了TOPFLASH 活性的提高以及增加了細胞群落形成的能力。HKCI-10 是一條我們實驗室建立的NASH 相關的肝細胞癌細胞株，在HKCI-10細胞株中，抑制CTNNB1 表達引起了細胞生長和增殖的減少。另外，在DEN誘導肝癌的有代謝失衡的db/db 轉基因鼠中，發現了一個CTNNB1 的突變T41A。據報導，CTNNB1 發生的致癌突變會引起蛋白的穩定並且因此激活經典的Wnt/β-catenin 信號通路，從而引致特定基因的轉錄。對於CTNNB1中最常見的突變S45P(在發現的CTNNB1 突變中占31.3%)，我們做了ChIP-array 實驗，結果顯示，在HKCI-10（CTNNB1 有S45P 突變）中，CTNNB1比在Hep3B 中（野生型CTNNB1）有著更緊密的啟動子結合能力（P<0.001）。Gene ontology 分析結果表明，被S45P 富集的生物過程涉及有RNA 代謝調節，轉錄因子活性和凋亡。MYC，E2F1 和ZFX 被ChIP-PCR 證實是與CTNNB1突變子S45P 有著更緊密結合能力的轉錄因子。, 在本論文的第二部份，爲了進一步闡明致癌性的CTNNB1 突變S45P在HCC 中的角色，我們研究了一個此前未在HCC 報導過的基因ZFX。ZFX是一個在脊椎動物中高度保守，屬於Zfy 家族的zinc finger 蛋白。有報導指出ZFX 對於胚胎和造血幹細胞的自我更新有著重要的作用。另外亦有文獻報導ZFX 在一系列人類癌癥病例中有過度表達，例如食道癌，胃癌，前列腺癌和神經膠質瘤，並且顯現出致癌基因的特性。在本研究中，qRT-PCR結果提示ZFX 在HCC 腫瘤中的表達明顯高於正常肝組織。ZFX 的表達在51.8%的HCC 腫瘤中明顯高於其配對的鄰近非腫瘤肝組織。功能性研究表明，在MTT 實驗中，細胞的生存力在ZFX 缺失的穩定克隆中明顯減弱（P<0.0001）。在細胞群落形成實驗中，ZFX 缺失的穩定克隆顯示出明顯減弱的群落生長能力（P<0.0001）。在單細胞克隆生成實驗中，ZFX 缺失的HCC穩定克隆展示出數量更少，體積更小的細胞群落。另外，ZFX 基因抑制或者cisplatin 單獨處理均顯示細胞生存力的抑制，其抑制效率分別是24.0%和30.9%。當ZFX 基因抑制合併cisplatin 處理時，細胞生存力的抑制效率顯著地提高至65.2%，這個結果提示ZFX 基因抑制和cisplatin 處理兩者有協同增效作用（P<0.0001）。ZFX 基因的缺失會引起兩個為人熟知的胚胎幹細胞（ESCs）標誌物SOX-2 和NANOG 表達的明顯降低。, 綜上所述，通過進行全基因測序，本論文的研究結果為NASH 相關的HCC 分子層面上的異常提供了一個高解析度的視覺角度。在NASH 相關的HCC 中，一些可能對於肝細胞癌變有重要作用並且重複出現突變的基因被確定，例如CTNNB1 和PNLIP。CTNNB1 的突變體S45P 的其中一個下游目標基因ZFX，在HCC 中被證實有幹細胞和腫瘤啟動細胞特性。闡明在HCC發展過程中的分子改變以及機制，對於為肝癌病人探索新的治療手段有著重要意義。, Hepatocellular Carcinoma (HCC) is one of the most malignant diseases worldwide with poor prognosis. Like other human cancers, HCC is a genetic disease, where accumulation of genetic aberration plays important role in the liver carcinogenesis. The rapid advances in Next Generation Sequencing (NGS) technology in recent years have allowed unprecedented ability to unravel the molecular complexity of the cancer genome, providing new insights into the cancer biology, diagnosis and therapeutic drug development., Non-alcoholic steatohepatitis (NASH), which is related to metabolic disorder, is an increasing common etiological factor of HCC, especially in developed countries such as United States, Europe, Japan and Canada. In first part of this thesis, Whole Genome Sequencing (WGS) was performed on three NASH-associated HCCs and their matched lymphocytic DNA. A cell line developed from one of the three NASH-associated HCCs was also subjected to WGS. The sequencing depth ranged from 29.1X to 102.2X with the coverage >99% shown in all samples. Novel SNVs identified ranged from 6,898 to 17,129 with an average of 11,133. Based on the SNVs found, the validation rate was 92.9% in 56 randomly selected somatic SNVs by Sanger sequencing. Mutational spectrum showed frequent somatic substitution of A:T>G:C and C:G>A:T while C:G>T:A transition exhibited a predominant somatic mutation rate in CpG sites. Among the non-synonymous somatic mutated genes, we selected CTNNB1, PNLIP and MLL2 which were mutated in more than one tumor for further study. In additional cohort of 44 NASH-associated and 50 HBV-associated HCC tumors and adjacent non-tumoral tissues, further sequencing all the coding regions of these three genes and TP53, which has been reported to be highly mutated in HBV-associated HCCs, were carried out. In NASH-associated HCCs, all genes harboured recurrent missense mutations exclusive in tumor but none in adjacent ,non-tumoral liver. The prevalence of CTNNB1 mutations was significantly higher in NASH-associated HCCs (36.4%) when compared to HBV-associated HCCs (12.0%, P=0.007). Mutations of PNLIP and MLL2 were detected only in NASH-associated HCCs with rates of 12.1% and 7.1%, respectively, but none in HBV-associated HCCs. The mutation rate of TP53, however, did not differ much between NASH-associated and HBV-associated HCCs (P>0.1). In functional study, for PNLIP, a loss-of-function in PNLIP activity was found in HKCI-10 harbouring D396N mutation as compared to normal liver cell, L, In the second part of the thesis, we aimed to further understand the role of oncogenic CTNNB1 S45P mutant in HCC. Zinc finger protein X-linked (ZFX) gene which is previously undescribed in HCC was studied. ZFX is a zinc finger protein of Zfy family that is highly conserved among vertebrates. It has been reported that ZFX is required for self-renewal of embryonic and hematopoietic stem cells. Also, ZFX is suggested to be overexpressed in a number of human cancers such as esophageal carcinoma, gastric cancer, prostate cancer and glioma, conferring oncogenic characteristics. In this study, qRT-PCR analysis showed a significant higher ZFX expression in HCC tumors compared to normal livers. 51.8% of HCC tumors showed significant up-regulations of ZFX when compared to paired adjacent non-tumoral livers. Functional studies showed significant reduction in in-vitro cell proliferation in HCC by MTT assay (P<0.0001) and colony formation ability by colony formation assay (P<0.0001) in ZFX-deficient stable clones. In single-cell clonogenic assay, ZFX-depleted HCC exhibited fewer and smaller colonies. In addition, while ZFX knockdown or cisplatin treatment alone showed an inhibitory effect on cell viability by 24.0% and 30.9%, respectively, the reduction efficiency of cell viability increased dramatically to 65.2% when combine ZFX-knockdown and cisplatin treatment, indicating a synergistic effect between them (P<0.0001). Also, significant reduced expressions of SOX-2 and NANOG, both well-known embryonic stem cells (ESCs) markers, were observed as a result of ZFX depletion., In summary, findings from this thesis provided high-resolution insight into the molecular aberrations in NASH-associated HCCs by performing WGS. Recurrent mutated genes which may be of great importance in hepatocellular carcinogenesis induced by NASH were identified, such as CTNNB1 and PNLIP. One of the S45P CTNNB1 downstream targets ZFX was demonstrated to confer stemness and tumor-initiating cells (TICs) characteristics in HCC. The understanding of molecular changes and mechanisms underlying HCC development will thus facilitate the exploration of new therapeutic options in patients with this deadly disease., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Chen, Jiawei., Thesis (Ph.D.) Chinese University of Hong Kong, 2014., Includes bibliographical references (leaves 166-190)., s also in Chinese., http://library.cuhk.edu.hk/record=b6115447, 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/)

Kan, Hoi Lam., Thesis (M.Phil.)--Chinese University of Hong Kong, 2009., Includes bibliographical references (leaves 103-124)., s in English and Chinese., p.i, 摘要 --- p.iv, ACKNOWLEDGEMENTS --- p.vi, TABLE OF CONTENTS --- p.viii, LIST OF TABLES --- p.xii, LIST OF FIGURES --- p.xiii, LIST OF ABBREVIATIONS --- p.xiv, PUBLICATION --- p.xvi, Chapter SECTION I: --- BACKGROUND --- p.1, Chapter Chapter 1: --- Hepatocellular Carcinoma (HCC) --- p.2, Chapter 1.1. --- Epidemiology of HCC --- p.3, Chapter 1.2. --- Etiology of HCC --- p.3, Chapter 1.2.1. --- Cirrhosis --- p.4, Chapter 1.2.2. --- Hepatitis virus --- p.4, Chapter 1.2.3. --- Plant carcinogens --- p.5, Chapter 1.2.4. --- Miscellaneous factors --- p.6, Chapter 1.3. --- Clinical presentation of HCC --- p.6, Chapter 1.4. --- Existing diagnostic tests for HCC --- p.6, Chapter 1.4.1. --- Alpha-fetoprotein (AFP) --- p.7, Chapter 1.4.2. --- Imaging --- p.7, Chapter 1.5. --- Treatment of HCC --- p.8, Chapter 1.5.1. --- Surgical Resection and Transplantation --- p.8, Chapter 1.5.2. --- Tumor Ablation or Embolization --- p.8, Chapter 1.5.3. --- Chemotherapy and Radiotherapy --- p.9, Chapter 1.6. --- Tumor marker development for HCC detection --- p.10, Chapter 1.6.1. --- Oncofetal antigens and glycoprotein antigens --- p.11, Chapter 1.6.2. --- Enzymes and isoenzymes --- p.12, Chapter 1.6.3. --- Growth factors --- p.12, Chapter 1.6.4. --- Genetics and epigenetics - mRNA and methylation --- p.13, Chapter Chapter 2: --- Hypermethylation of tumor suppressor genes in cancer --- p.14, Chapter 2.1. --- Cancer epigenetics --- p.14, Chapter 2.2. --- DNA methylation in normal cells --- p.15, Chapter 2.3. --- Physiological role of DNA methylation in normal cells --- p.18, Chapter 2.4. --- Aberrant DNA methylation in cancer --- p.19, Chapter 2.4.1. --- DNA hypomethylation in cancer --- p.20, Chapter 2.4.2. --- DNA hypermethylation in cancer --- p.20, Chapter 2.5. --- Development of methylation markers in tumor diagnosis --- p.21, Chapter 2.5.1. --- Methods for the analysis of DNA methylation markers --- p.22, Chapter 2.5.2. --- Detection of tumor-associated methylated DNA in the circulation of cancer patients, Chapter 2.6. --- Aim of thesis --- p.27, Chapter SECTION II: --- MATERIALS AND METHODS --- p.28, Chapter Chapter 3: --- Methods for detecting DNA methylation --- p.29, Chapter 3.1. --- Subject recruitment --- p.29, Chapter 3.2. --- Sample collection and processing --- p.29, Chapter 3.2.1. --- Tumor tissue samples --- p.29, Chapter 3.2.2. --- Peripheral blood samples --- p.29, Chapter 3.3. --- DNA extraction --- p.30, Chapter 3.3.1. --- Plasma samples --- p.30, Chapter 3.3.2. --- Blood cells --- p.33, Chapter 3.3.3. --- Tumor tissue --- p.33, Chapter 3.4. --- Quantitative analysis of methylated DNA using methylation-sensitive restriction enzyme-mediated real-time quantitative PCR (MSRE-qPCR) --- p.34, Chapter 3.4.1. --- Methylation-sensitive restriction enzyme-mediated real-time quantitative PCR --- p.34, Chapter 3.4.3. --- Real-time PCR primer design --- p.36, Chapter 3.4.4. --- Duplex real-time PCR --- p.40, Chapter 3.4.5. --- "Real-time detection of GSTP1, SOCS1, A PC, pl6 and ACTB sequences" --- p.41, Chapter 3.4.6. --- Statistical analysis of real-time PCR results --- p.41, Chapter 3.5. --- "Methylation study of GSTP1, SOCS1, APC, pl6 and ACTB in tumor tissues and blood cells using bisulfite sequencing" --- p.46, Chapter 3.5.1. --- Principle of bisulfite modification --- p.46, Chapter 3.5.2. --- Bisulfite conversion --- p.47, Chapter 3.5.3. --- Sequencing primer design --- p.47, Chapter 3.5.4. --- Conventional PCR after bisulfite treatment --- p.49, Chapter 3.5.5. --- Cloning and bisulfite genomic sequencing --- p.53, Chapter 3.5.6. --- Data acquisition and interpretation --- p.54, Chapter SECTION III: --- DEVELOPMENT OF METHYLATION MARKERS IN HCC DETECTION, Chapter Chapter 4: --- Evaluation of the real-time PCR assay for quantification of methylated tumor suppressor genes --- p.57, Chapter 4.1. --- Development of real-time PCR assays --- p.57, Chapter 4.2. --- Methylation analyses by bisulfite sequencing were concordant with the real-time quantification results --- p.61, Chapter Chapter 5: --- Clinical application of methylated markers in the detection of hepatocellular carcinoma --- p.69, Chapter 5.1. --- Demographics of HCC patients and HB V carriers --- p.69, Chapter 5.2. --- Quantitative analysis of hypermethylated tumor suppressor genes in tumor and plasma samples --- p.71, Chapter 5.3. --- Effect of cirrhosis on the plasma methylated tumor suppressor gene concentrations --- p.77, Chapter 5.4. --- Changes in the concentration of the tumor suppressor genes one month after surgical resection of the cancer --- p.81, Chapter 5.5. --- Concurrent use of serum AFP level and plasma methylated markers for HCC diagnosis --- p.84, Chapter 5.6. --- Prognostic value of plasma methylated TSGs --- p.86, Chapter SECTION IV: --- DISCUSSION --- p.90, Chapter Chapter 6: --- Discussion --- p.91, Chapter 6.1. --- Tumor and plasma detection of hypermethylated tumor suppressor genes --- p.92, Chapter 6.2. --- No effect of cirrhosis on plasma methylated DNA level --- p.94, Chapter 6.3. --- Clearance of methylated TSG sequences after tumor resection --- p.95, Chapter 6.4. --- Concurrent use of serum AFP level and the presence of methylated markers in the plasma in HCC diagnosis --- p.95, Chapter 6.5. --- Prognostic significance of circulating methylated tumor markers --- p.96, Chapter SECTION V: --- CONCLUDING REMARKS --- p.98, Chapter Chapter 7: --- Conclusions and future perspectives --- p.99, REFERENCES --- p.103, http://library.cuhk.edu.hk/record=b5893988, 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/)

Aim. In this study, we attempted to identify HCC-specific serum glycoproteins by using glycoproteomic technologies. We targeted at finding glycoforms with aberrant alpha-2,6-sialylation and alpha-1,6-fucosylation in sera of HCC patients. These glycoforms may have potentials to be used as tumor marker(s) in the diagnosis of HCC., Among the validated differential glycoproteins, Hp was the only glycoprotein, glycoforms of which were found to be significantly up-regulated in the HCC group when examining both the sialylated glycoprotein profiles and the fucosylated glycoprotein profiles. This glycoprotein was selected for further investigation. In the independent validation group, increased serum levels of Hp (total), alpha-2,6 sialylated Hp and alpha-1,6 fucosylated Hp was observed in the HCC patients. A unique pattern of Hp glycoforms comprising both hypersialylated fucosylated and hyposialytated fucosylated species was found in the HCC patients. Serum concentrations of these glycoforms were significantly higher in the HCC patients with advanced tumor, suggesting their tumor-specific nature. Besides, we have performed quantitative profiling of N-glycans of serum Hp in the HCC patients, CLD patients and normal subjects, and have attempted to identify HCC-associated N-glycans for HCC diagnosis. Combined used of serum alpha-fetoprotein, serum Hp and its N-glycans, we could achieve 84% sensitivity at 100% and 93% specificities when distinguishing the HCC patients from the CLD patients and from the normal subjects, respectively., Background. Hepatocellular carcinoma (HCC) often arises in patients with coexisting chronic liver disease (CLD). Since alpha-fetoprotein, a conventional biomarker, may also be raised in patients with uncomplicated CLD, the use of alpha-fetoprotein in early detection of HCC is limited. Identification of additional biomarkers may improve early detection. Previous studies have shown that levels of alpha-2,6-sialyltransferase and alpha-1,6-fucosyltransferase change in liver cancer, leading to aberrant glycosylations on some serum proteins., Conclusion. By undertaking glycoproteomic approach, we have identified a panel of potential biomarkers for diagnosis of HCC. These biomarkers were useful for classifying among normal healthy subjects, CLD patients, patients with early HCC and patients with advanced HCC. Some of them were validated with the independent cases. Finally, we have identified a unique pattern of Hp glycoforms comprising both hypersialylated fucosylated and hyposialylated fucosylated species in the HCC patients. Serum Hp and its N-glycans have been shown to have potential values for aiding the diagnosis of HCC., Methodology. There are four parts in this study. The first part is "method development". A method for obtaining quantitative profiles of serum glycoproteins with alpha-2,6-sialylation or alpha-1,6-fucosylation was developed by combined use of lectin affinity chromatography, two-dimensional polyacrylamide gel electrophoresis and enzyme-linked lectin assay. The second part is "biomarker discovery". The quantitative profiles of the serum glycoproteins from 20 HCC patients and 10 CLD patients (control) were compared by bioinformatic approaches to identify potential biomarkers for diagnosis of HCC. The protein identities of the potential targets were obtained by using MALDI-TOF/TOF mass spectrometry. The third part is "validation". An independent set of serum samples from 40 HCC patients, 30 CLD patients and 20 normal subjects was used to evaluate the diagnostic values of the potential biomarkers. The last part of this study was aimed to identify HCC-associated N-glycans on one of potential biomarkers found, and examine their values in diagnosis of HCC., Result. When analyzing alpha-2,6-sialylated glycoproteins, 53 glycoprotein spots were significantly different between the HCC and CLD groups, of which 44 spots belonged to 13 glycoproteins. Bioinformatic analyses revealed that these differential sialoglycoprotein profiles contained valuable information for differentiating the HCC patients from CLD patients, and classifying between early HCC and advanced HCC patients. When analyzing alpha-1,6-fucosylated proteins, 11 glycoprotein spots were significant different between the two study groups, of which 8 spots belonged to 1 glycoprotein. Majority of the protein identities were successfully obtained by MALDI-TOF/TOF mass spectrometry. Among the differential glycoproteins, we have identified a subgroup with a unique pattern of glycosylation. These glycoproteins were characterized by the presence of hypersialylated and fucosylated glycoforms. The differential patterns and the diagnostic values of some of these serum glycoproteins were confirmed in the independent validation group by measuring their serum levels with immunoassays. The results of the logistic regression analyses suggest that complement factor B and haptoglobin (Hp) can be used in combination with alpha-fetoprotein to improve the diagnosis of HCC., Ang, Ling., Adviser: Tereng Chuen Wai Poon., Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0947., Thesis (Ph.D.)--Chinese University of Hong Kong, 2007., Includes bibliographical references (leaves 215-238)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., School code: 1307., isbn: 9780549480891, 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/)

Focal liver lesions are commonly encountered in clinical practice. To be able to differentiate potentially life-threatening lesions from clinically insignificant lesions, and to be able to treat them effectively are the two basic problems of a clinician who comes across such lesions. Percutaneous intervention of the liver with a needle enables a clinician to solve the above two problems in a minimally invasive manner. To date, there is a diversity of needle-related percutaneous interventional procedures that are applicable to the clinical management of patients with liver lesions, such as biopsy of focal lesions, drainage of abscesses, and ablation of tumors. Despite a reasonable safety and efficacy associated with these procedures, there are always grounds of further improvement in techniques and technology of needle-related percutaneous procedures to achieve an even better outcome. It was hypothesized that the application of needle-related interventional radiology to clinical management of focal liver lesions could be facilitated and extended with advancement and refinement in needle-related techniques and technology. This thesis was based on a series of nine studies that aimed to explore the potential of needle-related percutaneous interventions in the clinical management of focal liver lesions and to study the effect of the introduction of innovations in needle-related techniques and technology on such clinical applications. It was concluded that the hypothesis was confirmed., Yu Chun Ho., "April 2006.", Adviser: Anil Ahuja., Source: Dissertation Abstracts International, Volume: 68-08, Section: B, page: 5176., Thesis (M.D.)--Chinese University of Hong Kong, 2006., Includes bibliographical references (p. 219-235)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web., School code: 1307., isbn: 9780549177807, 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/)