Numerous large clinical trials of cardiovascular risk lowering agents have been conducted in the hope of reducing the excess cardiovascular risk found in patients with diabetes mellitus. However, the relationship between glucose and cardiovascular disease remains complex and various areas require further study. Even in patients with diabetes, an individual’s cardiovascular risk is highly variable depending on other clinical characteristics, the assumption that glucose is a continuous risk factor has often been based on weak evidence from relatively short studies, the effect of commonly used cardiovascular risk lowering agents often has unexpected effects on new-onset diabetes and statins have not yet been studied in detail, and whether glucose-lowering therapies actually reduce cardiovascular risk has remained a contentious issue despite the conduct of large clinical trials. Furthermore, the realisation that the combination of diabetes and chronic heart failure, a common complication of coronary disease, carries a particularly poor prognosis suggests that prediction of diabetes in this population may be clinically valuable. Aims: I aimed to address the following different, though related, questions regarding glucose and cardiovascular disease: 1. Are anticipated cardiovascular event rates in diabetes endpoint trials actually achieved? Is it possible to easily identify patients with diabetes that are at particular risk of events (information that is crucial to investigators who wish to design clinical trials)? 2. Is fasting glucose concentration independently and convincingly associated with increased risk of cardiovascular events in those without diabetes? 3. Do statins, the most commonly prescribed medications worldwide, have any influence on the risk of developing diabetes? 4. If statins do indeed affect new-onset diabetes, is there any evidence of a dose-dependent effect? 5. How effectively can clinicians predict the development of diabetes in chronic heart failure using commonly recorded clinical information? 6. Does intensive glucose-lowering therapy reduce the risk of cardiovascular events in patients with diabetes? Methods: To address these questions three approaches were used, namely (i) systematic review of previously published data from large cardiovascular endpoint trials conducted in patients with diabetes; (ii) analyses of existing datasets from two large clinical trials; (iii) meta-analyses of published and unpublished data from large clinical trials. Results and interpretation: 1. In a systematic review of 29 trials with 116,790 patients with diabetes, it was apparent that the majority of large cardiovascular endpoint trials conducted in patients with diabetes vastly overestimated the likely cardiovascular event rates in initial power calculations. Introduction of (i) previous history of cardiovascular disease and/or (ii) presence of proteinuria, as binary trial inclusion criteria, provides a simple and effective way to identify patients at high risk, something that is sought after for appropriate clinical trial power calculations. 2. In a population of 6,447 men without diabetes at baseline, impaired fasting glycaemia was not associated with increased risk of cardiovascular events over 15 years. Similarly, when baseline fasting glucose values <7.0mmol/L were split into quintiles, patients in the highest quintile were at similar risk of all vascular endpoints to those in the lowest. By contrast, impaired fasting glycaemia was a powerful risk factor for developing diabetes. 3. A meta-analysis of published and unpublished data from most large placebo- and standard care-controlled statin trials, which included data for 91,140 trial participants without diabetes at baseline, revealed that statin therapy is associated with a 9% higher risk for developing diabetes. 4. A subsequent meta-analysis of unpublished data from five large trials comparing intensive statin therapy with moderate dose therapy found that intensive statin therapy increases the risk of developing diabetes by 12% compared to moderate dosing, in keeping with a dose-dependent effect. While statin therapy remains effective at reducing cardiovascular risk it appears that patients on statin therapy, especially those on intensive regimens, should be considered for diabetes screening. 5. In an analysis of data for 1,620 patients with chronic heart failure and no diabetes at baseline studied for 2.8 years, the strongest predictors of new-onset diabetes were similar to those in the general population. In particular, the combination of HbA1c and body mass index provided a c-statistic of 0.79. 6. In a meta-analysis of published data for 33,040 patients with diabetes who participated in clinical trials comparing intensive glucose-lowering therapy with standard therapy, non-fatal myocardial infarctions were reduced by 17% on intensive therapy but no other cardiovascular endpoints were reduced. Death rates were similar in both groups. Conclusion: While diabetes is associated with excess cardiovascular risk, risk varies considerably depending on other risk factors. Glucose is, at best, a weak risk factor in those without diabetes, and glucose-lowering in patients with diabetes has only yielded a modest reduction in non-fatal myocardial infarctions but not other events; by contrast, measures of glycaemia are powerful predictors of new-onset diabetes in patients with and without chronic heart failure. Finally, the relationship between glucose and vascular disease is further complicated by the fact that numerous medications designed to reduce cardiovascular risk appear to have surprising effects on the risk of developing diabetes.

RNA Polymerase III (Poll III) contributes to about 10% of nuclear transcription and is essential for the synthesis of short untranslated transcripts, including tRNA and 5S rRNA. Pol III deregulation has been implicated in driving cellular proliferation and transformation, along with increased expression of a number of Pol III specific transcription factors and transcripts. The significance of Pol III in clinical pathology, including that of human malignancies, remains to be formally tested. Using prostate cancer as a model, two key components of the Pol III complex, namely Brf1 and tRNAiMet, were investigated. The expression patterns of Brf1 and tRNAiMet were studied in this thesis using immunohistochemistry (IHC) and in situ hybridisation (ISH) respectively. Brf1, a subunit of transcription factor IIIB (TFIIIB), was detected predominantly in the nucleus with heterogenous staining intensity, its presence ranging from weak to strong. Examination across a wide range of tissue types and organs, both normal and tumour tissues revealed high levels of Brf1 expression in the epithelium. In addition, Brf1 expression could also be detected in connective tissues and, to a lesser extent, in muscular and nervous tissues. A number of tumour types exhibited elevated expression of Brf1 protein relative to their normal control tissues. These included prostate adenocarcinoma, B-cell lymphoma and, interestingly, tumours arising from connective tissues (sarcoma, fibrosarcoma and chondrosarcoma). As expected, tRNAiMet expression, as revealed by ISH analysis, was mostly observed in the cytoplasm, although some nuclear staining was also present. Similar to Brf1, tRNAiMet expression was detected predominantly in epithelial tissues such as the skin epidermis, prostate gland and epithelial lining of the cervix. A number of tumours were found to overexpress tRNAiMet. These included breast ductal carcinoma, oesophageal carcinoma and melanoma. The clinical impact of Brf1 and tRNAiMet overexpression was examined using tissue microarrays (TMA) containing tissue samples obtained from patients with prostate cancer (PCa) and benign prostate hyperplasia (BPH). Collectively, data from two independent patient cohorts, Glasgow TMA: BPH (n=21), PCa (n=151) and Newcastle TMA: BPH (n=113), PCa (n=365), showed that Brf1 expression was upregulated in prostate cancer relative to BPH (p=0.0034). Brf1 expression was not found to be associated with the following clinical and biologic parameters: Gleason sum score (indicative of tumour differentiation and morphology, p=0.653); prostate specific antigen (PSA level, indicative of tumour bulk or volume, p=0.381) and Ki-67 expression (signifying cellular proliferation, p=0.034). However, and importantly, within the prostate cancer patient cohorts studied, high Brf1 expression was associated with a significantly less favourable survival outcome (Kaplan Meier analysis, p<0.001). Together, the data presented in this study support the relevance of Brf1 and tRNAiMet overexpression as part of Pol III deregulation in tumours, especially of epithelial origin. This study also suggests the potential application of Brf1 as a prognostic marker in cancer, however, this warrants a further study.

Collagen is the ligand for the discoidin domain receptor-1 (DDR1), a receptor tyrosine kinase that is over-expressed in Hodgkin lymphoma. However, the role of DDR1 in diffuse large B cell lymphoma (DLBCL) is not known. I showed that DDR1 is over-expressed in a subset of DLBCL where it positively correlates with expression of its collagen ligands, and negatively correlates with expression of mitotic spindle genes. DDR1 correlated genes also overlapped with three aneuploidy signatures and DDR1 expression correlated significantly with autosomal aneuploidy index. RNAseq analysis revealed that over-expression of DDR1 in primary germinal centre B cells down-regulated expression of CENPE, an essential component of the mitotic spindle checkpoint that when inactivated leads to chromosome mis-segregation and aneuploidy. CENPE expression was also significantly reduced in primary DLBCL. Moreover, I showed that the constitutive activation of DDR1 in an in vitro lymphoma model led to aneuploidy. Finally, I showed that DDR1 can be inhibited by three small molecules and established the basis for in vivo model to test these inhibitors in DLBCL xenograft. My data provide evidence that DDR1 can induce aneuploidy in B cells, and as such identify a mechanism to potentially explain the link between chronic inflammation and lymphomagenesis.

Hepatic stem cells are found in the liver at all stages of development, including adult, and have the potential to give rise to daughter cells of biliary, hepatocytic and pancreatic lineages. Hepatic stem cells contribute significantly to liver regeneration, but may be associated with development of primary liver cancer, and are being investigated as a cellular therapy for liver failure. This thesis describes the development of methods for the immunohistochemical quantification of hepatic stem cell activation, allowing assessment of the association between hepatic stem cell activation in needle biopsy tissue and subsequent development of HCC in a retrospectively identified cohort of cirrhotic patients. A murine dietary model of NASH and HCC was developed and characterised in detail demonstrating progressive hepatic stem cell activation with increasing injury severity. We then went on to describe and prospectively isolate a resident population of stromal stem/progenitor cells in adult, uninjured mouse liver with the potential to give rise to both myofibroblasts, and under selective conditions, epithelial stem/progenitor cells. Finally, we demonstrated the isolation of hepatic stem cell from explanted cirrhotic liver and normal common bile duct and assessed their utility as a source of hepatic stem cells for cellular or regenerative therapy.

The liver has a unique connection with the immune system; harbouring vast numbers of lymphocytes, able to instigate secondary lymphoid organ-independent naive T cell activation, and promoting potent immune tolerance. We set out to determine the effect of this unique microenvironment on the biology of CD4+ T cells at three key interaction points: following migration into the parenchyma, after short-term hepatocyte contact, and at long-term tissue-residency. Modelling transmigration through hepatocytes revealed intrinsic, disease-specific cytokine responses in blood-derived CD4+ T cells, not discernible through static co-culture. However, short-term co-culture did induce activation-independent CD69 upregulation, reliant upon cell-cell contact. This phenotype mimicked the similar hepatic CD4+ CD69INT cells that we discovered in liver tissue. Unlike CD69HI cells which represented the tissue-resident memory T cells (TRM) of the liver, CD69INT cells were the most activated population, likely able to migrate to many liver and gut niches, and singularly able to produce IL-4 and IL-10. By contrast, CD69HI TRM displayed a resting phenotype, marked for more restricted movement, and produced the best multifunctional TH1 responses following stimulation. These data demonstrate the importance of studying migration, and provide detailed characterisation of CD69HI TRM and novel CD69INT cells, along with their proposed roles and generation pathways.

Recent evidence indicates that immune cells within adipose tissues can drive the formation of ectopic lymphoid structures, known as Fat Associated Lymphoid Clusters (FALC). FALC support B-cell antibody production in response to infection and inflammation. This investigation explores the immune cell composition and role of different adipose tissues both in steady state and during immune responses in mice. Firstly, a detailed analysis of the immune cell composition of peritoneal adipose tissues was performed. To investigate the function and migratory properties of these tissue-resident cells, cytokine and chemokine receptor expression was then assessed. How immune cells in adipose tissues responded to infection was examined using an intestinal helminth infection with the parasite Heligmosomoides polygyrus. Adipose tissues predominantly contained regulatory T cells, invariant natural killer T cells and group 2 Innate Lymphoid Cells (ILC2s). Significant differences were observed in composition, cell surface markers and cytokine production of ILC2s between adipose depots and secondary lymphoid tissues, indicating that tissue specific signals can direct ILC2 responses. Finally, increases were observed in ILC2 and FALC numbers in the mesenteries of WT mice following parasite infection. These data indicate that immune cells within adipose tissues respond to infection and may contribute to immune responses.

Volume 1, the research component, includes a literature review examining the efficacy of interventions for parents of children with autism spectrum disorder (ASD) in relation to parent outcomes; an empirical paper, which presents findings of a quantitative study exploring parental illness perceptions, coping, wellbeing, parenting stress and parent perceptions of the child-parent relationship in parents of a child with pathological demand avoidance syndrome (PDA) and a public dissemination document. Volume 2, the clinical component, includes: a report detailing two psychological formulations; one from a cognitive behavioural perspective and one from a systemic perspective, for a 20 year old gentleman who was experiencing anxiety and depression, following removal of part of his bowel; a service evaluation report detailing an investigation of the extent to which a local respiratory service was addressing the psychological needs of COPD patients; a single case experimental design presenting an evaluation of a behavioural intervention for a 25 year old woman with a moderate learning disability, who presented with skin picking behaviours; a case study of a fourteen year old girl, who was under investigation for Crohn's disease and experienced anxiety and an abstract, reflecting on providing consultation within a looked after and adopted child's psychology service.

Intracellular Ca2+ and adenosine triphosphate (ATP) are the key elements needed for stimulant-evoked exocytotic enzyme secretion from pancreatic acinar cells. Physiological Ca2+ signals consist of repetitive spikes confined to the secretory granule region, which stimulate ATP production; whereas sustained global cytosolic Ca2+ elevations - toxic Ca2+ signals decrease ATP levels and cause necrosis leading to the inflammation of the pancreas - acute pancreatitis (AP), a life-threatening disease currently without specific therapy. The work presented in this thesis focuses on the mechanisms underlying the development of pancreatitis evoked by L-asparaginase and the potential ways to intervene asparaginase-associated pancreatitis (AAP). Asparaginase is an essential element of the chemotherapy regimen in the successful treatment of acute lymphoblastic leukaemia (ALL), the most common childhood cancer. But asparaginase treatment can lead to AAP, which is a side-effect of ALL treatment occurring in about 5–10% of cases. Following AP, further treatment with asparaginase is withheld to prevent recurrence of AP; however, withholding scheduled asparaginase is associated with a potential increase in ALL relapse. Understanding the pathogenesis of AAP could lead to effective therapies for this complication, potentially reducing toxicity and allowing re-exposure to continue treatment with asparaginase.

This thesis explores the impact of lifestyle factors on the development of metabolic syndrome (MS) in Saudi Arabian women. A survey of a snowball sample was used to recruit 258 female and explored factors influencing physical activity (PA) and food intake and their effects on BMI in women living in the KSA and the UK. Participants completed a self-reporting questionnaire relating to knowledge, attitudes, barriers and levels of PA, sedentary activity and eating habits. Excessive energy intake, physical inactivity and sedentary lifestyle were all prevalent in Saudi women, resulting in 80%, over the age of 35y, being overweight or obese. BMI was associated with both energy intake and PA, though the relationship with the former was stronger. The most common barriers to regular exercise were transportation and lack of time. Findings were generally similar between women living in Saudi Arabia and the UK. The efficacy of reducing energy intake, with or without increased PA, on risk factors associated with MS in overweight Saudi women was investigated in a pilot study. After a four-week program, incorporating dietary modification alone (D) or in combination with regular vigorous aerobic exercise (D+E), improvements were seen in body composition and a range of metabolic risk factors. Both groups lost weight, but, paradoxically, those in D lost significantly more than those in D+E (5.3 vs. 3.3%, p=0.016). Moreover, significant reductions were also found in blood pressure, plasma triacylglycerol, insulin, total and LDL cholesterol, with no significant differences between the two groups. Plasma glucose and HDL cholesterol remained unaltered. Overall, these changes led to a decline in the prevalence of MS from 20% to 5% and 21% to 7% for the D and D+E groups, respectively. Thus, reducing energy intake appears, at least in the short term, more important than increasing PA in reducing body weight and associated metabolic risk factors. These studies confirm that excessive dietary intake and physical inactivity both contribute to overweight and obesity in Saudi Arabian women. With appropriate support, it is possible to both reduce energy intake and increase PA, although, in the short -term, the former appears to be most important. It remains to be established whether longer-term improvements in PA would further improve metabolic health.

Prostate cancer is the most common male cancer in the UK. Although incidence is increasing, prostate cancer mortality is decreasing, mainly owing to the over diagnosis of disease that would not have become clinically apparent during the patient’s lifetime. The gold-standard for prostate cancer diagnosis is transrectal ultrasound guided biopsy of the prostate. Whilst prostate biopsy can inform on diagnosis, it’s prognostic ultiltiy is poor. Currently clinicians lack pathological biomarkers to differentiate between patients with prostate cancer who have indolent disease that can be safely managed with surveillance strategies, and those who will go onto develop aggressive disease which requires early radical curative treatment. Phosphorylation of the androgen receptor has been extensively investigated in relation to prostate cancer development and progression. Androgen receptor phosphorylation has been shown to regulate cellular localisation, transcriptional activity, cell growth and sensitivity to androgens in prostate cancer. However, only a small number of studies have investigated the prognostic significance of androgen receptor phosphorylation, and only consider a limited number of serine residues in clinical specimens. The research presented in this thesis sought to investigate the prognostic and predictive significance of AR phosphorylation at serine 578 in hormone-naïve prostate cancer. It was hypothesised that pARS578 would be associated with poor outcomes in prostate cancer and may be utilised as a prognostic marker at diagnosis in prostate cancer and predict response to drug treatment with a PKC inhibitor. It was also hypothesised that PKC, the putative kinase for phosphorylation at serine 578, would be associated with poor outcomes and may offer a potential therapeutic target in prostate cancer. In the current study, the phosphorylation site of primary interest was serine 578. Scansite 2.0, an online kinase search tool, predicted that PKC is the putative kinase mediating phosphorylation at serine 578 on the androgen receptor. Phosphorylation of the androgen receptor at serine 578 has been linked with increased AR transcriptional activity, cell growth, nuclear cytoplasmic shuttling, modulation of other AR phosphorylation sites and DNA-repair mechanisms. The prognostic significance of androgen receptor phosphorylation at serine 81 was also investigated in this study. Serine 81 is phosphorylated in response to DHT via an alternative pathway to that of serine 578. Serine 81 phosphorylation is associated with increased androgen receptor transcriptional activity and increased cell growth in prostate cancer. It was therefore hypothesised that androgen receptor phosphorylation at serine 578 and serine 81 would be associated with poor outcome measures in prostate cancer. Immunohistochemical analysis was performed in a cohort of 105 hormone-naïve prostate cancer patients undergoing active surveillance, representing a cohort of patients with low-risk disease, as defined by current clinical markers such as PSA and Gleason score at diagnosis. Nuclear PKC expression was significantly associated with pARS578 expression in the clinical specimens, supporting the prediction of Scnasite 2.0 that PKC is the kinase responsible for phosphorylation of the AR at this site. High cytoplasmic expression of pARS81 was associated with decreased time to intervention (HR 2.76 (95% CI 1.1-7.3), p=0.032). There was no association between pARS578 and time to intervention in this cohort. Analysis of combined expression of both phosphorylation sites revealed an association between high dual expression of cytoplasmic pARS81 and cytoplasmic pARS578 and decreased time to treatment intervention (HR 2.35 (95% CI 1.2-4.6), p=0.031). These results suggest a synergistic prognostic effect when these two phosphorylation sites are combined and identifies a sub-population of low-risk prostate cancer patients who are at increased risk of disease progression. A second study was conducted to investigate if these results could be replicated in a cohort of prostate cancer patients with all stages of disease at diagnosis. Immunohistochemical analysis in 90 hormone-naïve prostate cancer patients found that high expression of nuclear pARS81 (HR 2.1 (95% CI 1.1 – 4.2), p=0.030), nuclear pARS578 (HR 2.24 (95% CI 1.0-4.9), p=0.036) and cytoplasmic pARS578 (HR 4.54 (95% CI 2.0-10.4), p= < 0.001) was associated with decreased disease survival. Furthermore, high expression of cytoplasmic pARS578 was associated with decreased time to biochemical relapse (HR 2.1 (95% CI 1.0-4.2), p=0.034) and decreased disease-specific survival following biochemical relapse (HR 3.2 (95% CI 1.0-9.9), p=0.034). Dual expression of nuclear, cytoplasmic and total pARS81 and pARS578 were all associated with decreased-disease specific survival, suggesting that there is a sub-population of prostate cancer patients who may benefit from dual targeted therapy with androgen deprivation therapy and PKC inhibitors. A validation cohort of 243 hormone-naïve prostate cancer patients with all stages of disease was utilised to verify the results of the second cohort. Unfortunately, due to technical issues and time constraints, IHC could not be completed for the phosphorylation sites of interest in all patients. Despite this, high expression of cytoplasmic pARS578 was significantly associated with decreased time to biochemical relapse (HR 2.9 (95% CI 1.0-8.2), p=0.037) and trended towards an association with decreased overall survival (p=0.076). Interestingly, dual expression of high cytoplasmic pARS81 and cytoplasmic pARS578 was associated with decreased overall survival (HR 2.1 (95% CI 1.3-3.3) p=0.001) despite neither phosphorylation site independently predicting decreased overall survival. Lastly, a study to develop a technique for isolation, propagation and characterisation of primary prostate cancer cells from TRUS biopsy specimens was undertaken. Two primary prostate cell cultures were developed which were confirmed to have a malignant luminal epithelial cell phenotype with a functional AR using flow cytometry, RT-PCR and immunofluorescence. This technique is of high translational relevance, as it provides a model with potential to identify biomarkers to predict individual patient’s response to prostate cancer therapies. Overall these results suggest that androgen receptor phosphorylated at serine 81 and serine 578 are associated with poor outcomes in prostate cancer and are potential targets for new drug therapies. Additional studies are required to validate these results in a larger multi-centre cohort of prostate cancer patients before either of these phosphorylation sites can be utilised as a biomarker in clinical practice.