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with diabetes; (ii) analyses of existing datasets from two large clinical trials; (iii) meta-analyses of published and unpublished data from large clinical trials.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.

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.

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.

In the early days of PKU, micronutrient deficiencies, undernutrition and growth failure were common features of patients with the condition. This was mainly due to the limited availability of engineered special low protein foods (SLPF) and PKU protein substitutes to use. Nowadays, SLPF foods and micronutrient-enriched PKU protein substitutes have become widely available and are free on prescription in most countries. These SLPF are high in carbohydrates and often in fat content, have a higher glycaemic index and provide more energy per weight compared with the protein-containing equivalent normal foods. Advancement in the PKU management, including dietary practices, led to nutritional problems that had never been reported before, but have become more frequent in the recent years. Overweight and obesity, rather than undernutrition, have become increasingly reported in patients with PKU, with some studies suggesting higher prevalence in females than males. Data on body composition in patients with PKU are inconsistent with some studies showing that patients with PKU have higher FM and lower FFM compared to healthy controls. This suggests that for a given body weight and height, patients with PKU might be fatter and look bigger. However, there is very little research looking at the determinants of nutrient status, body composition and obesity in patients with PKU. Hence, among the aims of this thesis was to investigate the impact of a PKU SLPF-based meal on appetite ratings, gut appetite hormones, thermic effect of feeding (TEF) and fat oxidation (Chapter 2). Twenty-three healthy adults (mean ± SD age: 24.3 ± 5.1 years; BMI: 22.4 ± 2.5 kg/m2) participated in a randomised crossover study. Each participant conducted two (PKU and Control) experimental trials which involved consumption of a PKU SLPF-based meal and protein substitute drink or an isocaloric and weight matched ordinary meal and protein-enriched milk drink. Appetite, metabolic rate, fat oxidation measurements and blood collections were conducted for the duration of 300 minutes. On completion of the measurements, an ad libitum buffet dinner was served. Responses of appetite ratings, plasma concentrations of GLP-1 and PYY (P > 0.05, trial effect, two-way ANOVA) and energy intake during ad libitum buffet dinner (P > 0.05, paired t-test) were not significantly different between the two trials. The TEF (PKU, 10.2 ±1.5%; Control, 13.2 ± 1.0%) and the total amount of fat oxidised (PKU, 18.90 ± 1.10 g; Control, 22.10 ± 1.10 g) were significantly (P < 0.05, paired t-tests) lower in the PKU than in the Control trial. The differences in TEF and fat oxidation were significant (P < 0.05, paired t-tests) for the post-meal period. Therefore, from this first study we concluded that consumption of a meal composed of SLPF has no detrimental impact on appetite and appetite hormones but produces a lower TEF and postprandial fat oxidation than an ordinary meal. We hypothesised that these metabolic alterations may contribute to the increased prevalence of obesity reported in patients with PKU on contemporary dietary management. In the second experimental chapter, we tested the hypothesis generated from the study above and measured TEF, fasting and postprandial fat and CHO oxidation in 13 patients with PKU and 10 healthy controls of similar age and BMI. Participants in the PKU group were provided an SLPF-based meal while those in the Control group were provided an isocaloric normal meal. It was found that TEF, and postprandial fat and CHO oxidation were not signifcantly different between the PKU and the Control groups. In addition, this study compared body composition characteristics (measured by Deuterium Oxide dilution technique) between PKU patients and healthy controls and revealed that differences in body composition are not significant between the two groups despite a tendency of PKU patients having higher percentage of body fatness (P=0.08). However, data generated from this study should be interpreted with caution and requires confirmation from studies with larger sample size. Micronutrient imbalance has been noted in patients with PKU despite their high provision through the PKU protein substitutes. Recent studies showed high blood levels of vitamin B12 and folate, but simultaneously deficient plasma levels of selenium and zinc in PKU patients prescribed with micronutrient-enriched PKU protein substitutes. Factors associated with micronutrient imbalance have rarely been studied in the literature. Therefore, the last chapter of this thesis aimed to evaluate the micronutrient status of children with PKU and explore factors associated with micronutrient imbalances and deficiencies. This was analysis of a large clinical dataset with serial measurements obtained from PKU children (≤16 years) attending the metabolic medicine clinic at the Glasgow Royal Hospital for Sick Children between 1990 and 2013. The study included 81 patients who provided a total of 512 blood samples for their routine annual micronutrient screening. Data on blood micronutrient measurements was available for vitamins A, B12, D, E, serum folate, and erythrocyte folate and the trace elements copper, selenium, zinc and serum ferritin as a biomarker of iron stores. Status of vitamin B12, E, and serum and erythrocyte folate measurements were above the normal range (NR) in 27%, 54%, 46% and 35% of the blood samples, respectively. However, 44% of selenium and 14% of zinc measurements were below the NR. Moreover, when we compared results with those from the UK National Dietary and Nutritional Survey, selenium and zinc deficiencies were specific to PKU condition and not a reflection of the epidemiology in the general UK population. In our PKU sample, poor metabolic control, PKU severity, and low adherence to PKU protein substitutes predicted low selenium status; while deficient zinc status was solely predicted by low adherence to PKU protein substitutes. Yet, these predictors, collectively, explained a small (5.8 – 8.8 %) variation in the status of selenium and zinc in this group of patients. Selenium and zinc deficiencies are common in PKU patients despite high levels of other nutrients including vitamin B12, E and folate. The findings of this study suggest that selenium and zinc deficiencies reported in patients with PKU may be attributed to other factors which we were unable to measure in this retrospective study, such as low bioavailability of these nutrients from the artificial PKU protein substitutes.

Introduction: Malignant Melanoma is the most lethal of the skin cancers and the UK incidence is rising faster than that of any other cancer. Breslow thickness remains the best predictor of metastasis and Sentinel Lymph Node Biopsy is the only method of detecting nodal spread in clinically node negative patients. Surgery is the only effective therapy. Angiogenesis – the growth of new vessels from pre-existing vasculature - is an absolute requirement for tumour survival and progression beyond a few hundred microns in diameter. Anti- angiogenic isoforms of VEGF have been demonstrated previously to be protective with regard to metastasis. The aims of this thesis were to determine whether VEGF expression within the tumour may allow prediction of the nodal status. Furthermore another aim was to determine whether via the “Seed and Soil” theory, by examination of angiogenic and lymphangiogenic profiles of the tumour and node can we determine that the tumour may control the microenvironment around the Sentinel Node? Finally, as a cohort of false negative patients emerged with a higher mortality rate than their true negative and true positive patient cohort counterparts, could any further patterns be established by performing the same experiments on these patients? Methods: Archived human tumour and corresponding Sentinel Node samples were used and immunohistochemistry was used to investigate the role of pro and anti angiogenic isoforms of VEGF, VEGF-C, LYVE-1 and CD31 within these patients. Results: VEGF-C expression was significantly increased in the intranodal component of positive Sentinel Lymph Nodes (p < 0.01 Bonferroni). This increased expression appeared to be independent of tumoural influences and no strong evidence for the “Seed and Soil” theory was proved. A significantly higher number of lymphatic vessel counts were identified within node negative patients (p < 0.05 ANOVA). No further significant findings were defined on examination of the false negative cohort of patients. Conclusions: This study has shown that positive Sentinel Lymph Nodes exhibit high levels of intranodal VEGF-C. This expression does not appear to be related to tumoural influences. It would therefore appear that VEGF-C expression within Sentinel Nodes warrants further investigation and may aid diagnosis of spread or represent a target to slow or even prevent the onset of metastasis.

With the current crisis of organ shortage and an increasing number of dialysis patients,studies directed at ameliorating such a substantial organ discrepancy are of considerable importance to the transplant community. The use of extended criteria donation has helped to compensate the disparity of organs however, we are still a long way from achieving satisfactory targets. Still there are many organs from older donors that are discarded primarily on the basis of chronological age. It is here that biological age may display a crucial role in allowing the transplant team to characterize donor organs with greater accuracy. Indeed both biological and chronological age are very closely related and ECD criteria are based very much on the latter, albeit with other clinical variables. However the biomarker of ageing CDKN2A which is suitably represented by Baker and Sprott’s criteria, displays closer variabilities with post-operative transplant function, at least up to one year. Telomere length known as the “Gold standard” biomarker of ageing does not display as robust a role in predicting organ function as CDKN2A. The classification of organs represented in this text from category I-IV serves merely as a guide to future studies and is yet to be validated in larger clinical trials. It is however a simple and rapid assessment tool (Gingell-Littlejohn et al PLOS One 2013). Relatively advanced cellular senescence was displayed in the mutant AS/AGU rat kidney when compared to the parent AS strain. This was exploited in a unique animal model to study the effects of ischaemia reperfusion injury on the mutant kidney, hence mimicking to a certain degree the transplant related injuries in ECD kidneys. Although ischaemic times in the model were moderate in nature, there was nonetheless a difference in the tolerance to IR injury between parent and mutant strain as evidenced by increased p16 and p21 staining in AS/AGU rats. Such a model therefore is exclusive in that interventions to improve ECD renal function post-transplantation can accurately and conveniently be represented and studied at a pre-clinical level. Anti-ischaemic compounds have been the subject of much debate over the years, however a single “Holy Grail” compound able to completely abolish the injurious effects of IR injury has never been elicited. mTOR inhibitors however, display several cellular effects and act as potent immunosuppressants. They (AZ-6) have also been shown to partially mediate the detrimental effects of IR injury on the native kidneys of AS rats in a specifically designed animal model as shown. Further studies encompassing transplanted kidneys from mutant AS/AGU rats exposed to such a promising agent would be of undoubted importance to the clinical field of transplantation, potentially leading to immeasurable economic and patient benefits.

Previous studies have identified stem cell populations in articular cartilage using colony forming assays and mesenchymal stem cell (MSC) marker expression. The specificity of classical MSC markers for isolation of stem cells within articular cartilage is insufficient, with large and highly variable quantities being reported in the literature. This study has demonstrated, for the first time, a panel of stem cell markers specific for articular cartilage-derived stem cells (ACSC). ACSCs were isolated, quantified and cultured from healthy and OA joints. Stem cells were clonally-derived cell lines that proliferated beyond 50 population doublings whilst maintaining a phenotype, and demonstrated tri-lineage potential. We discovered that OA cartilage had a two-fold increase in stem cell number, consisting of two divergent stem cell sub-populations. These divergent populations varied in proliferative capacity with only 50% of stem cells from the OA joint capable of extended proliferation in vitro. Using transcriptomic next generation sequencing of culture-expanded chondrocytes and ACSCs we successfully identified differentially expressed genes and a panel of novel markers of cartilage-specific stem cells. Novel markers were validated using qPCR and protein labelling and, were specifically expressed in ACSCs, with no expression in the culture-expanded full-depth chondrocytes. Using immunofluorescence for novel stem cell markers we found articular cartilage-derived stem cells are localised within the transitional zone in normal cartilage and the superficial zone in OA cartilage. OA cartilage was found to contain a 2-fold increase in stem cells using immunofluorescence. Subsequently, we used the panel of novel markers and fluorescent active cell sorting to isolate a sub-population from full-depth cartilage with stem cell characteristics. These cells were plastic adherent, clonogenic, with proliferative capacity greater than 50PD and displayed tri-lineage potential, therefore meeting all criteria for classification as a MSC population. The use of specific markers to isolate ACSCs will allow for further characterisation of stem cells, including a more in-depth understanding of the mechanisms of proliferation, differentiation and degeneration within articular cartilage.

Chronic lymphocytic leukaemia (CLL) is a lymphoid malignancy which despite advances in the treatment options available is still incurable. Characterised by the gradual accumulation of CD5+ B cells, the paradigm that this is due to failed apoptosis has been challenged and a significant proliferative component has been identified. However, despite the crosstalk between pathways which regulate metabolism and proliferation the metabolic characteristics of these cells are not fully understood. Furthermore, there is a renewed interest in the field of cancer cell metabolism because of the Warburg effect, a hallmark of malignancy whereby cells preferentially switch to aerobic glycolysis and rapidly consume glucose. This has led to the development of new drugs such as AZD3965 an inhibitor of monocarboxylate transporter 1 (MCT1), which along with MCT4 mediates the export of lactate, a toxic bi-product of glycolysis, out of the cell. The aim of this project was to assess whether therapeutically targeting MCT -1 and -4 would be a viable approach for CLL. Chapter 2 of this thesis examines expression of MCT -1 and -4 as well as a specific chaperone protein needed for the surface expression of these proteins, CD147. This chapter confirms the presence of both MCT -1 and -4 and CD147 in normal B cells as well as demonstrating for the first time that these transporters are expressed in CLL cells using Western blotting and qRT-PCR to assess the MCTs and flow cytometry to measure CD147. The levels of both MCTs and CD147 are demonstrated to be significantly reduced in CLL cells in comparison normal B cells likely due to the adoption of a quiescent phenotype to aid cell survival. The following chapter investigates this further by assessing whether there are any changes in expression under the influence of microenvironmental stimuli, specifically CD40 ligand (CD40L). In this chapter it is demonstrated for the first time that MCT4 is upregulated in CLL cells in response to CD40L. Analysis of gene expression using a Fluidigm Biomark™ array suggests this is due to the induction of glycolysis and that CLL cells may promote fatty acid synthesis as well as instigating changes in the metabolism of the tumour stroma possibly to provide substrates. Finally, chapter 4 evaluates the sensitivity of CLL cell lines to AZD3965 using cell death and cell viability assays. Both MEC-1 and HG3 CLL cell lines are shown to be resistant to MCT1 inhibition using AZD3965 and silencing of MCT4 using siRNA cells also has no effect on the viability of MEC-1 cells. That MCT4 can compensate for MCT1 inhibition is shown by the transient expression of MCT4 in a Raji cell line where only MCT1 is expressed. Taken together, the data presented in this study indicates that while the inhibition of MCT1 is likely to be ineffective dual inhibition of both MCT -1 and -4 may be a viable strategy for the localised inhibition of CLL in the secondary tissues. Furthermore, MCT inhibition in this disease may have the potential to negate mechanisms of resistance and protection from oxidative stress mediated by CD40L.

In this thesis I report three related studies that utilise state-of-the-art technologies to investigate germline and somatic chromosomal rearrangements in humans. Firstly, 16 patients with cytogenetically detectable deletions of 3p25-p26 were analysed with high density single nucleotide polymorphism (SNP) microarrays; Affymetrix 250K SNP microarrays (n=14) and Affymetrix SNP6.0 (n=2). Assuming complete penetrance, a critical region for congenitalheart disease (CHD) susceptibility gene was refined to approximately 200 kb and a candidate critical region for mental retardation was mapped to ~1 Mb interval containing SRGAP3. Secondly, I used SNP microarray and molecular cytogenetic studies to characterize chromosome 11p15 in 8 patients with the imprinting disorder Beckwith-Wiedemann syndrome (BWS). In addition to characterising 11p duplications in three patients, the breakpoints in two patients with balanced rearrangements were mapped to two distinct regions. Thirdly, I used high resolution SNP arrays (Affymetrix 250K Sty1 and 6.0 arrays) to identify copy number changes in renal cell carcinoma (RCC) primary tumours (n=81) and cell lines (n=23). Copy number changes most frequently involved large segments (>10Mb) and loss of 3p and gain of 5q were the most common copy number changes. A comparison of copy number changes in RCC cell lines and inherited and sporadic primary tumours was made.

Depression is one of the most common causes of periods of disability. There is evidence suggesting that the serotonin system is involved in the pathophysiology of depression. It has been suggested that synaptic serotonin levels are reduced in depressed patients, and that pharmacological blockade with antidepressants of the serotonin transporter (SERT) would result in alleviated symptoms of depression by enhancing serotonin neurotransmission. Since depression can be treated with antidepressants that target SERT, and a recently discovered 5-HTT gene-linked polymorphic region (5-HTTLPR) of the SERT gene has been shown to predispose to depression, the SERT assumes a key role in depression. Traditionally, depression severity was assessed using psychological testing of patients. However in the last 20 years, neuroimaging techniques using magnetic resonance imaging (MRI) of brain structures and molecular single photon emission computed tomography (SPECT) evolved which appear promising to better understand the pathophysiology at the tissue level. However, preclinical data on abnormalities that involve the serotonin system are limited. The studies presented in this thesis attempt to shed more light on the feasibility of using the novel MRI technique diffusion tensor imaging (DTI), and SPECT to detect disturbances of the serotonin system. Firstly, in order to elucidate the capabilities of DTI as a research tool in the detection of conceivably mild changes in white matter involving the serotonin system, a mouse model of life-long SERT deficiency was studied. Secondly, in order to validate DTI image processing methodology, a mouse model with reportedly profound myelin dysfunction was examined. Histology techniques were applied to the same mouse brains in order to explore the tissue correlate of the DTI signal changes. Thirdly, as myelin was hypothesised to interact with the serotonin system, in vitro autoradiography of SERT in mice with widespread hypomyelination was conducted in order to test this hypothesis. Lastly, in a rat model of SERT depletion, the relative abilities of a well established SPECT radioligand, [125I]βCIT (2β-carbomethoxy-3β-(4-iodophenyl)tropane), and a relatively novel SERT tracer, [123I]ADAM (2-((2-((dimethylamino)methyl)phenyl)thio)-5-iodophenylamine) were examined using micro-SPECT. The data demonstrate that DTI did not detect any changes in white matter organisation in SERT-deficient mice. Surprisingly, subtle changes in white matter microstructure were detected in mice that were haploinsufficient for SERT, i.e. heterozygous null mice, displaying a 50 % SERT reduction compared to WT as detected using DTI. On the other hand, profound hypomyelination was detected using DTI in another mouse model with white matter pathology, and correlations between DTI and histopathological markers were present, indicating that this technology provides good indications of severe pathology, while small changes, if present, may be missed. In addition, the SERT availability appeared not to be affected in mice with widespread hypomyelination. While post mortem autoradiography of SERT-depleted rats showed widespread reductions in SERT binding using dedicated specific SERT ligands, micro-SPECT using [125I]βCIT and [123I]ADAM did not show any differences. [125I]βCIT delivered good quality brain SPECT images, however analysis of [123I]ADAM scans was hampered by the poor definition of structures. Thus this thesis provides important information on the feasibility, and sensitivity of current neuroimaging modalities. In addition, methodological flaws and uncertainties in the current literature were identified, which underpins the need for improving and standardising methodological approaches, particularly in SPECT imaging.