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Many autofluorescent cells, rich in yellow pigment are described in the rabbit's sacculus rotundus. This pigment stained positively with Sudan black, PAS and Masson-Hamperl techniques. The cells correspond to those described in rat and in mice lymphatic organs. The possible origin of the pigment is discussed.

It has been proposed that diverse anticancer drugs and radiation therapy may induce a mode of cell death with the characteristics of apoptosis. Since apoptosis is under the control of several oncogenes, we analyzed the expression of the protein encoded by the proto-oncogenes bcl-2 and p53. Furthermore, we studied cell proliferation [using PC-10 mAb to proliferating cell nuclear antigen (PCNA)] and vascularization [using the CD-31 mAb and by counting intratumoral microvessel density (IMD)] using immunocytochemistry. A series of 73 patients with clinical stage II-IV squamous cell invasive carcinoma of the head and neck (HandN) were treated with concurrent chemoradiation therapy (cisplatin, 80 mg/m2, versus carboplatin, 375 mg/m2, three times every 3 weeks and a total dose of radiation therapy of 64 Gy in 6-8 weeks). We correlated the expression of these markers, determined prior to treatment, with response to the therapy and prognosis. Bcl-2 protein was expressed in 37.4% of the carcinomas (25/67 evaluable), and it was not significantly associated with any other feature studied. Forty (56. 4%) of the 71 carcinomas evaluable for p53 were p53 positive; the median IMD was 38 microvessels/field at the hot spot (range, 18-80), and the median percentage of nuclei labeled by the PC-10 mAb was 50% (range, 0-95%). In the univariate analysis, regional lymph node negativity (P = 0.016), good performance status (PS) (PS >/= 90; P = 0.044), bcl-2 positivity (P = 0.070), and low vascularization (P = 0. 085) were significantly associated with a higher probability of complete remission. In the multivariate analysis (final model), only IMD (continuous variable; P = 0.045) and PS (P = 0.017) retained significance. As far as prognosis is concerned, in the univariate analysis, patients with tumors with low histological grading (grades 1-2; P = 0.006), p53 negative (P = 0.09), bcl-2 positive (P = 0.08), and high PCNA labeling (P = 0.06) had a significantly better disease-free survival. In the multivariate analysis, only grading (P = 0.003) and p53 (P = 0.04) retained significance for disease-free survival. For overall survival, in the univariate analysis, the following markers were significantly prognostic when only deaths due to progression are considered: response to therapy (P = 0.00001), PS (P = 0.04), nodal status (P = 0.028), PCNA (P = 0.04), p53 (P = 0. 08), and grading (P = 0.01). In the multivariate analysis, only patients who achieved complete response (P = 0.00002), high PCNA values (P = 0.002), and low histological grading (P = 0.01) retained a statistically significant probability of better overall survival. Our results suggest that in this series of HandN cancer patients the markers capable of predicting response to therapy are distinct from those associated with prognosis, once the remission has been achieved. This information is potentially useful to the clinician for developing a more rational therapeutic approach for HandN cancer patients eligible for concurrent chemoradiation therapy.

We previously cloned and characterized the tumor suppressor gene FHIT (fragile histidine triad) at chromosome 3p14.2 and found that this gene is altered by deletions in human tumors, including lung cancer. To assess the frequency and specificity of inactivation and its relevance in a clinical setting, we have produced antibodies against the Fhit protein and studied its expression in a series of non-small cell lung cancers and normal bronchial mucosa and a spectrum of preinvasive lesions by immunohistochemistry. The data indicate that the loss of Fhit protein is the most frequent alteration in non-small cell lung cancer (73%) and precancerous lesions (93%), is significantly higher in the tumors of smokers (75%) than in those of nonsmokers (39%; P < 0.0005), and is an independent and more frequent event than p53 overexpression in tumors and precancerous lesions (73 versus 46%). The percentage of cases lacking Fhit expression was higher in the squamous type compared to adenocarcinoma (87 versus 57%; P < 0.00001), whereas other histotypes (large cell, mucoepidermal) showed an intermediate value (69%). Loss of Fhit expression in a very high percentage of primary lung carcinomas and precancerous lesions supports the notion that FHIT alterations play an important role in the growth control of bronchial cells. FHIT inactivation is particularly important in squamous cell carcinomas that are often associated with precursor dysplastic lesions. The overall high frequency and precocity of Fhit loss in lung carcinogenesis and the development of the presently described immunohistochemical approach suggest a potential use of this gene in the early detection of lung cancer and in chemopreventive studies as an intermediate biomarker.

Genomic alterations and abnormal expression of the FHIT gene at 3p14.2 have been observed in cell lines and primary tumors of the lung. To correlate FHIT locus DNA and RNA lesions with effects on Fhit protein expression, we have analyzed 11 lung cancer cell lines, 15 small cell lung carcinomas, and 38 pairs of non-small cell primary tumors and bronchial mucosa specimens by molecular genetic and immunocytochemical methods. Using specific antibodies against the Fhit protein, we observed concordance between RNA abnormalities and lack of Fhit protein expression in lung tumors and cell lines. In addition, absence of Fhit protein in some precancerous dysplastic lesions suggested that FHIT inactivation may occur at an early phase of lung carcinogenesis.

Tumor-associated macrophages (TAMs) produce angiogenic factors and in breast cancer are associated with high vascular grade and poor survival. TAMs preferentially migrate to hypoxic areas within tumors and strongly express hypoxia-inducible factor (HIF)-2 alpha. This study examined whether HIF-2 alpha was involved in TAM angiogenic activation by correlating its expression with tumor microvessel density as a marker of angiogenesis, and other tumor variables, in a series of human primary invasive breast carcinomas. A correlation was found between high TAM HIF-2 alpha and high tumor vascularity (P < 0.0001), as well as high tumor grade (P = 0.007). The relation of HIF-2 alpha expression to a recently described oxygen-dependent pathway of angiogenesis was also studied, and an inverse relationship was found between TAM HIF-2 alpha and tumor thymidine phosphorylase expression (P = 0.02). These results suggest that TAM HIF-2 signaling may be a useful target for future antiangiogenic strategies but show that tumors use both oxygen-dependent and oxygen deficiency-regulated pathways for angiogenesis. Thus, combined blockade of pathways and careful assessment of these pathways in trials are necessary.

Many systems have already been designed and successfully used for sharing histology images over large distances, without transfer of the original glass slides. Rapid evolution was seen when digital images could be transferred over the Internet. Nowadays, sophisticated virtual microscope systems can be acquired, with the capability to quickly scan large batches of glass slides at high magnification and compress and store the large images on disc, which subsequently can be consulted through the Internet. The images are stored on an image server, which can give simple, easy to transfer pictures to the user specifying a certain magnification on any position in the scan. This offers new opportunities in histology review, overcoming the necessity of the dynamic telepathology systems to have compatible software systems and microscopes and in addition, an adequate connection of sufficient bandwidth. Consulting the images now only requires an Internet connection and a computer with a high quality monitor. A system of complete pathology review supporting biorepositories is described, based on the implementation of this technique in the European Human Frozen Tumor Tissue Bank (TuBaFrost).

Monoclonal antibodies (MAb) anti-HIV core and envelope proteins and in situ hybridization, using cDNA HIV probe, were employed to determine which lymph node cells in LAS patients express viral antigens and viral nucleic acids. The results have been correlated with the histologic phases of LAS and with the germinal center lysis detected using DRC-1 MAb directed against follicular dendritic reticulum cells (FDRC). Viral antigens occasionally were detected on high endothelial cells of paracortical venules and frequently on germinal center FDR accessory cells; this last finding correlates well with the extent of FDRC lysis and of CD4+ and CD8+ lymphocyte infiltration in germinal centers. HIV replication, detected by in situ hybridization, was observed in mononucleated cells present in T and B areas and, in one case, in flat endothelium.

We explore the quantum nature of black holes by introducing an effective framework that takes into account deviations from the classical results. The approach is based on introducing quantum corrections to the classical Schwarzschild geometry in a way that is consistent with the physical scales of the black hole and its classical symmetries. This is achieved by organizing the quantum corrections in inverse powers of a physical distance. By solving the system in a self-consistent way we show that the derived physical quantities, such as event horizons, temperature and entropy can be expressed in a well defined expansion in the inverse powers of the black hole mass. The approach captures the general form of the quantum corrections to black hole physics without requiring to commit to a specific model of quantum gravity., Comment: Revised version matching the published one

The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal haematopoietic stem cell (HSC) malignancies that are characterised by ineffective haematopoiesis leading to peripheral blood cytopenias at one or more lineages. There are few effective treatments for this disorder, and the identification of new therapeutic agents in MDS is urgently needed. Drug repurposing is a potential alternative to novel drug discovery by redirecting existing drugs for treating another disease, which dramatically reduces drug development time, cost, and safety risk. In the last few years, gene expression signatures have been identified and compared based on genome-wide similarity metrics, unveiling ‘connections’ among drugs, pathways and disease phenotype by matching corresponding signatures of disease and drug responses. Some bioinformatic database and tools, such as connectivity map (CMap), containing transcriptomics of cell lines treated with small molecules were developed, greatly facilitating the discovery of new therapeutic applications of existing compounds. In this study, we performed CMap on lists of differentially expressed genes (DEG) obtained from the analysis of RNA-seq or microarray-based gene expression profiling (GEP) data generated from bone marrow CD34+ cells of MDS patients with splicing factor mutations or with advanced MDS (Refractory anaemia with excess blasts-2 subtype, RAEB-2 subtype). Compounds inducing a transcriptional response opposite to that of MDS cases were identified, which might be capable of reverting the gene expression signature, and hence may change the disease phenotype. The compounds were ranked by the absolute value of connectivity scores and were primarily screened by comprehensive literature review based on their Mode of Action (MoA) and toxicity profiles in the context of leukaemia, cancer and other diseases. Several top-ranking candidate drugs for repositioning to MDS were selected for in vitro experimental validation in leukaemia cell lines, patient sample derived induced pluripotent stem cells (iPSCs), and patient primary bone marrow cells. Celastrol and Withaferin A were identified from the CMap analysis of RNA-seq data set of splicing factor mutant MDS patients, and Parbendazole and Fenbendazole were identified by mapping a microarray-based MDS-RAEB-2 gene signature to the CMap database. These drugs markedly inhibited proliferation, arrested cell cycle, and induced apoptosis of leukaemia cells. Notably, our study provided evidence of the involvement of splicing pathway modulation in the anti-proliferative activity of these drugs in MDS and leukaemia cells. Higher cytotoxicity induced by Celastrol, Withaferin A, Parbendazole, and Fenbendazole treatment was observed in splicing factor mutant cells, including HUDEP2-U2AF1Q157 cells, K562-SF3B1K700E cells and K562-U2AF1S34F cells. Taken together, our data show that these compounds are promising candidates for repurposing to MDS and other related myeloid malignancies.

The geometric properties of sigma models with target space a Jacobi manifold are investigated. In their basic formulation, these are topological field theories - recently introduced by the authors - which share and generalise relevant features of Poisson sigma models, such as gauge invariance under diffeomorphisms and finite dimension of the reduced phase space. After reviewing the main novelties and peculiarities of these models, we perform a detailed analysis of constraints and ensuing gauge symmetries in the Hamiltonian approach. Contact manifolds as well as locally conformal symplectic manifolds are discussed, as main instances of Jacobi manifolds., 36 pages, Latex file. Text enlarged with introduction and final discussion reviewed

We introduce a two-dimensional sigma model associated with a Jacobi manifold. The model is a generalisation of a Poisson sigma model providing a topological open string theory. In the Hamiltonian approach first class constraints are derived, which generate gauge invariance of the model under diffeomorphisms. The reduced phase space is finite-dimensional. By introducing a metric tensor on the target, a non-topological sigma model is obtained, yielding a Polyakov action with metric and B-field, whose target space is a Jacobi manifold., 22 pages. Latex2e. Corrections added. Reference added

Angiogenesis has long been considered an essential prerequisite for tumour growth and progression. However, this understanding is being overturned by studies showing that many human tumours can metastasise without angiogenesis, suggesting that their microcirculation may be provided by non – sprouting vessels. Vessel co-option, a mechanism whereby tumour cells may incorporate and or migrate along pre-existing vessels presents an alternative mode of tumour blood supply. Vessel co-option has been found to manifest in many malignancies, occurring with increased frequency in highly vascularised tissues, such as the brain, lung and liver. 10-30% of primary and metastatic lung cancer, in addition to liver metastasis originating from different primary sources, have been found to utilise this alternative mode of blood supply. Furthermore, owing to the fact anti angiogenic drugs were designed to target new blood vessel growth, co-option as a result has also been proposed as a mechanism that could drive drug resistance to anti - angiogenic therapy. However, vessel co-option in the context of lung metastases has not been investigated extensively and likewise it’s potential to induce drug resistance in lung metastases is not well established. The aim of the work described in this thesis was therefore to investigate and further understand the molecular underpinnings that determine whether a tumour will exhibit a co-optive phenotype or an angiogenic phenotype and thus shed light on co-optive drug resistance molecular mechanisms in the context of lung metastases. Using a lung metastasis mouse model of co-option established originally by Andy Reynolds at the ICR in London, a multi omics approach was utilised to profile the “in vitro” and in “vivo” settings and identify potential molecular candidates that discriminate between angiogenesis and vessel co - option. These studies revealed the potential role the cellular microenvironment plays in dictating metastatic potential in addition to metabolic cellular profiles, ultimately driving the decision to co – opt existing vessels or promote angiogenesis. Moreover, preliminary proteomic and metabolomic data points towards potential differential regulation of metabolic pathways, cell signalling pathways and proteins involved in adhesion that show distinct profiles within tumour cells with different metastatic potential and tissues from co – opting versus angiogenic models. Of particular interest is the possible role of Carbonic anhydrase 2, the Na+/H+ exchanger protein family and pH regulation in general towards controlling the cellular tumorigenic potential and possibly the fate of angiogenic versus co optive tumours. To further drill down on this, subsequent proteomic analysis carried out on laser capture micro dissected tumours and vessels have further confirmed the role of metabolism, with proteins involved in lipid metabolism and reactive oxidative species metabolism being found to be upregulated in co optive vessels when compared to their angiogenic counterparts. In addition to this, the differential abundance of Carbonic anhydrase 2 was similarly confirmed, as well as proteins implicated in both adhesion and migration processes. In summary, these studies have contributed to paving the necessary groundwork in relation to understanding co-option in a pre-clinical model of lung metastases. They have not only confirmed what has been already noted in the literature but have also highlighted potential new molecular targets and pathways, worthy of further study and investigation.

Background: Aryl Hydrocarbon Receptor Interacting Protein-Like 1 (AIPL1) interacts with NUB1 and restricts the entry of NUB1 protein into the nucleus. The interferon-induced NEDD8 ultimate buster (NUB1) protein causes degradation of neddylated and FAT10ylated proteins through the ubiquitin proteasome system. We observed AIPL1 were frequently down-regulated in various cancers compared to normal tissues. The mechanistic roles of AIPL1 and NUB1 protein in cancer cell cycle regulation remain unexplored. Results: Meta-analysis of cancer databases revealed that expression transcripts of chaperones, including AIPL1, were down-regulated in lung, pancreatic cancer and breast cancer relative to the adjacent normal tissues. Opposite levels of both AIPL1 and NUB1 transcripts were observed in the breast cancer. So it triggers the in vitro experiments using breast cancer cells. METABRIC breast cancer clinical cohort highlighted that patients with low NUB1 transcripts had poor survival in the ER-negative subgroup (but not in ER-positive) of breast cancer patients: hazard ratio (HR)=0.66, 95% confidence interval (CI)=0.5-0.87, p=0.003 and triple negative subgroup of breast cancer patients: HR=0.67, 95% CI=0.47-0.96, p=0.028. NUB1 silencing significantly inhibits in vitro cell growth in MDA-MB-231 and MCF7 under hypoxia. AIPL1 protein forms multimers in cancer cells. NUB1 protein moved into the nucleus in hypoxia (0.1% O2 48hrs) with final confluency at 80-90%. p21 (marker of senescence) & p27 (marker of cell cycle arrest) accumulated in NUB1-silent MDA-MB-231 and RCC4 cells. It suggested that low NUB1 nuclear localisation in hypoxia cause cancer cell cycle arrest. In MDA-MB-231 cell, upon hypoxia, neddylation inhibitor (MLN4924) treated and siNUB1 transfected cells showed decreased CUL1 and further accumulated p21 & p27. The evidence suggested lower neddylated CUL1 and reduced NUB1 cooperatively stabilise p21 and p27 as the substrate of CUL1-ubiquitin ligase. The neddylation inhibitor MLN4924 treated and NUB1 knockdown group exhibited more cells in sub-G1 stage as compared to the control group. In connection to higher p21/p27, it is associated with prolonged arrested cellular aging with depletion. After silencing of NUB1, the increases in cell death of cancer cells upon hypoxia happen through the neddylation-dependent CUL1-p27-p21 and CUL2-VHL axis. We then demonstrated that HIF1α protein could be both neddylated and FAT10ylated upon reoxygenation. In a tissue microarray study of breast cancer, lower cytoplasmic expression (n=57) had worse overall survival than higher cytoplasmic expression (n=57): HR=1.779, 95% CI=1.006-3.346, p=0.048. Conclusions: AIPL1 and NUB1 proteins exert a role in cell cycle regulation in breast cancer. Low cytoplasmic NUB1 levels are observed in the G1-S transition of cancer cells. NUB1 depletion causes G0/G1 phase arrest due to CUL1 and CUL2 ubiquitin E3 ligase-dependent pathways.

TRAP1 is a Heat Shock Protein (HSP) chaperone to retinoblastoma but also associated to the tumor necrosis factor receptor. HSPs are primarily up regulated in cancer. Work in our lab noted a down regulation of TRAP1 in some non-small cell lung cancers compared to normal lung. The first aim of this project was to evaluate the effect of the loss of TRAP1 on cell proliferation using a spheroid model. The presence of TRAP1 in spheroids promoted cell proliferation and a faster onset of hypoxia. This suggests an oncogenic role for TRAP1 since rapid hypoxia development equates to poor prognosis. Micro array analysis showed that TRAP1’s loss was associated with increased transcrpition of the Junctional Mediating and Regulatory protein (JMY). JMY possesses an oncogenic property due to its ability to facilitate cell motility. Additionally it has tumor suppressor activity in promoting p53 activation. The second aim of this project was to produce an anti-JMY antibody and use it to characterize JMY and additionally verify the association between TRAP1 and JMY. JMY was found to be widely expressed in normal tissues and in many types of tumors. In neoplastic tissues, comparing primary versus metastatic tumors, JMY was found to have significantly higher expression in the metastatic compared with the primary tumors. A pilot study showed that nuclear co-expression of JMY and P53 was associated with shorter overall survival suggesting that a possible tumorigenesis mechanism could be via a deregulation/mutation of JMY/p53 or both. Finally, using 3 dimensional constructions, I demonstrated the distinct morphological difference between an angiogenic tumor and a non-angiogenic tumor. Additionally, I showed a characteristic cytoplasmic p53 sequestration in the non-angiogenic phenotype that is absent in the angiogenic phenotype. This could be the mechanism that the non-angiogenic tumor uses to adapt to hypoxia. This would imply that there is a potential for cancers to escape therapy by switching between these 2 phenotypes.

MicroRNAs (miRNAs) have now been identified in a vast array of organisms and a great deal of research has been carried out to elucidate the role they play. The dysregulation of miRNA expression has been implicated in a number of disease states and their importance has been highlighted by the beginning of their utilisation as therapeutics. The focus of this study was to identify the role played by miRNAs encoded by the Marek’s disease vaccine viruses, Marek’s disease virus serotype 2 (MDV-2) and Herpesvirus of turkeys (HVT). In order to better understand the functions of these miRNAs we wanted to identify their targets within the host cell. Using a combination of bioinformatic and biochemical approaches we were able to build up a library of potential targets. Three viral miRNA targets; AKT3, RAP1A and DEK, were further validated using dual-luciferase assays to highlight the exact site of miRNA targeting, and western blots to demonstrate an effect of miRNA targeting on protein abundance. An attempt at using label-free proteomics to observe the viral miRNA mediated changes in the host proteome is also described, however this proved to be unsuccessful. Additionally the function of one particular MDV-2 miRNA, mdv2-miR-M21, was explored in more detail, describing its role as a potential ortholog of the host miRNA; gga-miR-29b. By using the observation that the viral miRNA contained an identical 'seed' region to the host miRNA, we were able to use the data collected from existing studies on miR-29b to search for targets of mdv2-miR-M21. We demonstrated that mdv2-miR-M21 targeted DNMT3B, crucial for epigenetic modification of the genome. The final part of this study aimed to understand the wider context the viral miRNAs played in the viral biology and protective ability of the vaccine viruses. The miRNAs were deleted from the viruses, and then the miRNA-deletion viruses were used to vaccinate birds before challenge with the oncogenic Marek's disease virus serotype 1 (MDV-1), survival rates to the 'wild-type' MDV-2 and HVT vaccine viruses were then compared.