Platelets play a crucial role in haemostasis by preventing bleeding at sites of vascular injury. Inherited or acquired platelet defects can impair haemostasis resulting in bleeding symptoms of varying severity ranging from mild to excessive which can be life threatening. Diagnosis of mild platelet-based bleeding disorders is challenging due to the absence of a gold standard technique and their variable bleeding symptoms and bleeding phenotypes observed in healthy individual as well as other haemostatic disorders. The work in this thesis built on the previous studies in the genotyping and platelet phenotyping project allowing further characterization of inherited platelet function defects in individuals with mild bleeding disorders. Platelet aggregation and secretion in samples from 206 patients were investigated during the course of this thesis and were categorised on the basis of the observed defects. Surprisingly, in over a half of these patients, an ex vivo platelet function defect was not found. The genetic investigation of selected cases using whole exome sequencing identified mutations in number of genes previously known to be critical in platelet biology. This thesis also focused on evaluation of three other platelet techniques by comparison with lumi-aggregometry to assess their overall potential in detecting platelet function defects. Further studies are still needed to further assess the potential of these techniques before they can be applied in routine clinical diagnosis.

Patients undergoing oesophagectomy are at risk of developing Acute Respiratory Distress Syndrome (ARDS), an immune mediated form of severe respiratory failure. The immunomodulatory properties of Vitamin D are increasingly recognised. We hypothesised that preoperative Vitamin D supplementation would reduce levels of perioperative alveolar oedema in patients undergoing oesophagectomy. Vitamin D deficiency is common in patients with and at risk of ARDS. High dose supplementation with cholecalciferol is a safe and effective method of increasing Vitamin D levels. Supplementation reduces perioperative increases in inflammatory alveolar oedema. Circulating levels of the active form of Vitamin D relate to long term post-operative mortality. Patients who survive at least 2 years post-op have higher preoperative circulating numbers of Natural Killer cells. We did not find any evidence of an effect of Vitamin D on Natural Killer Cells. In conclusion, preoperative Vitamin D status relates to perioperative changes in inflammatory alveolar oedema and high dose Vitamin D supplementation is a safe and effective method of improving preoperative Vitamin D status. Preoperative cholecalciferol administration should be considered in patients with and at risk of vitamin D deficiency.

The kidneys are important organs that regulate the level of water and salts in the blood, produce hormones that help to control blood pressure, and maintain homeostasis of the organism by filtering waste products. Kidney disease can either be acute or chronic, the latter progressively worsening over time to become end stage renal disease (ESRD) – a stage when kidneys are non-functional. At present, the only treatment options for ESRD are transplantation or dialysis, which both have severe drawbacks in terms of morbidity, mortality and economic cost. Moreover, the incidence of ESRD is rising annually and an alternative therapy is needed. Therefore, in order to regenerate kidney tissue or prevent worsening of the kidney condition, a new therapy should be developed. One approach is to use embryonic stem cells.

Venous thrombosis is a major health concern, with an annual incidence of ~1 per 1000 adults (Cushman 2007). This includes deep vein thrombosis (DVT) and pulmonary embolism (PE), the fatal consequence of a clot detaching and moving to the lungs, together these diseases are termed venous thromboembolism (VTE). Current treatment options for VTE are often associated with serious side effects and bleeding complications, highlighting the need for more effective prophylaxis. This study therefore aimed to identify new targets to treat DVT, which would not have the associated negative side effects. This study shows the platelet receptor CLEC-2 (C-type lectin receptor 2) plays an important role in DVT, probably through interaction with podoplanin in the IVC wall, and that lack of CLEC-2 is protective in this disease. We show other immune cells may also play a role in DVT, and demonstrate that mast cell deficiency is protective in vivo. Furthermore, we suggest that the mast cell constituent responsible for the prothrombotic phenotype is likely to be histamine. Preliminary data also suggests that T-cells may have a protective role in DVT, and that thrombin may be important for the release of neutrophil extracellular traps (NETs) from neutrophils inside a growing thrombus.

Adipose secreted cytokines are thought to contribute to pro-inflammatory state seen commonly in obese individuals, providing a potential metabolic link between obesity and osteoarthritis. The aim of this study is to further our understanding of the role of adipokines within OA by examining the serum and joint fluid adipokine expression profiles in relation to disease severity, BMI, and joint tissue turnover markers. The result of this study show that subchondral bone from overweight/obese hip OA patients exhibited reduced trabecular thickness, increased bone surface/bone volume ratio and an increase in the type I collagen α1/α2 ratio, compared to normal-weight hip OA patients. The serum concentration of resistin was higher in overweight/obese OA patients, compared to normal-weight OA patients (12740 vs 9818pg/mL respectively; p < 0.05). Stimulation of normal-weight bone explant with recombinant resistin resulted in a 2.4 fold increase in type I collagen α1/α2 ratio (1.6:1 vs 3.8:1, p < 0.01). Stimulation of primary OA osteoblasts with resistin increased Wnt signalling activation, osteoblast metabolic activity, and bone nodule formation. In addition, visfatin was elevated in the synovial fluid and in isolated synovial fibroblasts of obese hip OA patients compared to normal-weight patients. In cartilage, visfatin increased the production of 15 pro-inflammatory cytokines and chemokines, with significant increases in IL-6, CCL4, MCP-1 and in CCL20 compared to the media only control ( > 7 fold, 20-fold, 4-fold and 7-fold respectively). Visfatin significantly increased in catabolic proteases including MMP-1 (4-fold), MMP-2 (3-fold), MMP-3 (3-fold), MMP-7 (2.2-fold), MMP-8 (1.3-fold), MMP-9 (1.2-fold), MMP-10 (1.5-fold), and MMP-13 (5-fold) and localised to areas of cartilage damage. Targeted inhibition of adipokine signalling could therefore be a rewarding strategy for developing a novel therapeutic.

Hyperthyroidism is a common endocrine disorder with multiple aetiologies, manifestations and potential therapies. This thesis explores the challenges relating to the diagnosis, treatment and long-term consequences of thyrotoxicosis in real-world outpatient and inpatient settings. We performed a number of epidemiological studies analysing data from large, detailed, routinely collected data sources. We confirmed that classical manifestations of hyperthyroidism are significantly less prevalent in older patients and established that newly diagnosed thyroid dysfunction is rare in hospitalised subjects despite high volume thyroid function testing in this setting where we found a high proportion of abnormal thyroid tests in those with pre-existing thyroid dysfunction. We determined that thionamides are effective in a half of subjects treated with a prolonged course and that appropriate patient selection improves success rates. We established that treatment of hyperthyroidism with radioactive iodine results in more weight gain than antithyroid drugs and that hyperthyroidism in hospitalised patients is associated with longer hospital stays, higher frequency of admissions and increased mortality. In conclusion, this thesis provides important new insights into the diagnosis and treatment of hyperthyroidism and highlights that correct evaluation and management of patients may minimise the long-term consequences associated with this common disorder.

Mammalian skeletal muscle has a major impact on whole-body metabolic homeostasis. Hence, maintenance of a metabolically active muscle mass is key for optimal health. Notably, both muscle function and mass are profoundly negatively affected by environmental factors such as chronic smoking and physical inactivity. RNA abundance integrates genetic, epigenetic and environmental influences. Therefore, while true understanding of physiological adaptation likely require the integration between multi-level datasets, the transcriptome represents a powerful investigative tool in determining the underlying molecular mechanisms behind complex phenotypic traits. The overarching aim of this thesis was to evaluate, using omics-based systems biology approaches, the global regulation of RNAs during exogenous modulation of mammalian muscle phenotype in order to characterize local homeostatic processes as well as identify robust biomarker signatures. The first part of this thesis deals with smoke-induced peripheral muscle wasting. Initially, biological domain knowledge is used to validate a pre-clinical smoking model. Then, specific cytokines are statistically linked to limb muscle energy metabolism; a testable hypothesis supported by both animal and human data. The second part deals with the development of ‘molecular predictors’ of endurance training adaptability. Two complex clinically relevant traits are considered, namely whole-body insulin sensitivity and plasma triglyceride content. Promisingly, quantitative multi-gene predictors of response to training for both traits of interest were developed.

Patients presenting with acute upper gastrointestinal bleeding have an increased incidence of cardiovascular events, with mortality invariably related to factors other than the bleeding itself; the majority of deaths related to cardiovascular disease. Patients presenting with bleeding complicating acute coronary syndromes are at an increased risk of recurrent cardiovascular events and have higher short-term and long-term mortality when compared to patients with uncomplicated acute coronary syndromes. This study has shown the preferential use of the Blatchford score in identifying patients suitable for early discharge; using a cut-off score of 2 could avoid up to 15% of admissions. The results show significant diurnal and seasonal variation in the presentation of acute upper gastrointestinal bleeding with fewer admissions in the winter months. This study has shown an increase in levels of platelet activation and prothrombotic markers (d-dimer and vWF) in the acute upper gastrointestinal bleeding population. No such findings were seen in the acute coronary syndromes complicated by bleeding group. These novel findings may help explain the excess of cardiovascular mortality in patients with presenting upper gastrointestinal bleeding and give a biological rationale to restarting antiplatelet agents early in these patients. Further studies are required to confirm and further investigate these findings.

Background Treatment options for patients with symptomatic non-obstructive HCM are limited, driving a need to develop novel treatment strategies. Objectives To investigate the effects of biventricular pacing on symptom status and exercise capacity in patients with symptomatic non-obstructive Hypertrophic Cardiomyopathy and explore the mechanism of benefit. Methods 31 patients with symptomatic, exercise limited non-obstructive Hypertrophic Cardiomyopathy were enrolled into this double blind randomised cross over study of Biventricular vs sham pacing. The primary end point was peak oxygen consumption. Results 29 patients successfully completed the study. Biventricular pacemaker therapy increased peak Oxygen consumption (1.17ml/kg/min) and improved quality of life, without effects on systolic or diastolic mechanical dyssynchrony. A subset of patients in whom left ventricular diastolic volume fell on exercise (n = 15) achieved a greater increase in peak Oxygen consumption vs. those in whom left ventricular diastolic volume increased (1.4 vs. 0.91ml/kg/min p = 0.03) and this was associated with a normalisation of the left ventricular diastolic volume response to exercise. Conclusions Severely symptomatic patients with non-obstructive Hypertrophic Cardiomyopathy may benefit from Biventricular pacing. The mechanism of benefit from this treatment modality was a profound improvement of diastolic filling on exercise potentially due to amelioration of diastolic ventricular interaction.

The phenomenon of directed cell migration is one of many fundamental biological processes required for the existence of almost all living organisms. Efficient cell migration relies on a highly coordinated spatio-temporal system of sub-cellular processes, many of which are governed by transportation carriers known as vesicles. This thesis focuses on methods for analysing the dynamics of vesicles during directed cell migration via imaging and image analysis. Vesicle dynamics can be visualised in live cells by fluorescence microscopy. Different fluorescence microscopy techniques are better suited for studying different aspects of vesicle dynamics. First, the concept of multi-modality microscopy is explored, which combines the benefits of multiple methods into the same analyses. Thorough characterisation of vesicle dynamics requires accurately estimating thousands of trajectories from microscopy image sequences often consisting of hundreds or thousands of images. A highly efficient probabilistic multiple particle tracking frameworks is presented, which outperforms many of the state-of-the-art methods, but with a drastically reduced processing time. Finally, live cell studies show that there is a high degree of polarity of vesicle traffic of vesicles associated with the clathrin mediated endocytosis and early endosome pathways during cell migration.