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A potential benefit of the use of artificial media for the suspension of platelets as concentrates is a reduction of the morphological, functional and metabolic changes observed in platelets during storage and collectively referred to as the platelet storage lesion (PSL). A better understanding of the nature of the PSL may suggest strategies for manipulation of the storage environment to improve platelet viability and efficacy posttransfusion. In this context, two principal considerations formed the basis for the study: · The hypothesis that apoptosis is a central mechanism responsible for the changes observed in the PSL. · The investigation of this hypothesis within the applied setting of improving the storage environment of platelet concentrates. The study investigated the role on the PSL of plasma protein (in the form of albumin), acetate and glucose in leucoreduced platelet concentrates suspended in a medium with minimal plasma. A 14-day storage study on platelet concentrates in either plasma or a 70:30 ratio of a commercial additive solution (SSP+Ô) and plasma provided an overview of platelet in vitro characteristics under standard storage conditions. The work led to targeted investigations into the nature of the cell death mechanism in platelet concentrates. Results suggested that in storage media with adequate energy stores, a Bcl-2 proteinmediated mechanism of cell death was viable, though possibly storage-time dependent and limited by pre-existing levels of anti-apoptotic Bcl-2 proteins in the platelets. Further studies would be required to determine if this mechanism is akin to caspasedependent apoptosis. In media lacking glucose, a mechanism more reminiscent of necrosis was observed, associated with decreased ATP levels, accelerated mitochondrial dysfunction, elevated intracellular free calcium and culminating in platelet disruption.

Moderate physical activity and snack intake suppress the appetite of obese and lean women acutely. The associations between circulating leptin and appetite-satiety ratings suggest that there is some physiological involvement of leptin in short-term appetite regulation in response to physical activity-induced factors but only in obese women. The exercise-related factors considered in this thesis as possible mediators of leptin action were catecholamines, fatty acids, glucose and insulin. Adrenaline is unlikely to be the exercise factor responsible for the coupling between leptin and satiety since adrenaline infusion stimulated an increase in subsequent energy intake in obese women. Labetalol decreased circulating FFA and increased glucose concentrations, which confirms at least b-adrenoceptor blockade. Any conclusion with respect to the a- adrenoceptor blockade should be drawn with caution since labetalol, an a/b blocker, has greater affinity for b- than a-adrenoceptors. No differences in appetite/satiety sensations were found following exercise with adrenoceptor blockade compared to exercise alone. This indicates that the observed anorexic effect of exercise on appetite in obese women was not mediated by b-adrenoceptors. Noradrenaline is another possible exercise factor that could mediate the coupling between leptin and appetite in obese women since it is known that leptin and noradrenaline (NA) have common hypothalamic targets (e.g. NPY) and their effects are mediated by a-1 adrenoceptors. Labetalol probably was not a sufficiently strong a-adrenoceptor blocker to investigate such effects. A study of a more selective a1-adrenoceptor antagonist might be helpful in the investigation of the interaction between leptin and NA in the regulation of eating. Study 4: In endurance-trained athletes a short term detraining increases postprandial plasmin leptin, induces insulin resistance but has no effect on appetite/satiety ratings. The results of the present studies implicate leptin, insulin, insulin resistance and noradrenergic factors in the control of eating following exercise and detraining.

The findings of this study provide an original contribution to knowledge in ankylosing spondylitis (AS) and have important implications for eiThancing clinical practice. The results demonstrate the existence and significance of associations between disease and psychological status in AS, and also demonstrate the value of using longitudinal, repeated measures approach to study this long-term condition. This study is also the first to demonstrate the value of using a mixed methods approach to investigate this issue in AS. Although existing literature on prospective longitudinal cohort studies in AS is very limited (other than for studies which involve clinical trials of medications and other interventions), this project demonstrates the feasibility of sustaining such a study over an 18-month period and of recruiting large numbers of participants to both the quantitative and qualitative phases. The results are based upon a hospital-ascertained cohort of 89 adults. Both the quantitative and qualitative phases produced important new findings: 1. In this cohort, mean BASMI, BASFI and BASDAI scores remained consistent throughout the 18-month period. People with BASDAI scores higher than 4 at the beginning of the study continued to score higher than 4 throughout. 2. BASMI, BASFI and BASDAI scores correlated significantly with anxiety, depression and internality scores, but not with levels of belief in chance or powerful others, throughout the study. This demonstrates that AS disease status is closely linked to some, but not all, psychological measures. 3. There was no effect of co-existent psoriasis or iritis on either disease or psychological status, but BASMI and BASFI (but not BASDAI) scores were significantly related to age. 4. Factors which appear to influence the associations between disease and psychological status are highly complex, often differing between individuals, and usually determined by other co-morbidities and life circumstances besides AS. These results suggest that the major implication for clinical practice would be the development of a more comprehensive and integrated assessment framework for AS set within the context of a biopsychosocial model. Envisaged would be a major programme of work to critically assess and validate potential components of such a framework with the aim of determining efficacy, feasibility and acceptability of such an approach.

Neutrophils are phagocytic white blood cells which act as the first line of defence against entry of foreign microorganisms. Neutrophils are recruited to their target site through the process of spreading, extravasation and phagocytosis involving complex signal transduction within the cells, which might include the activation of the cytosolic Ca2+ activated protease, calpain-1. The work described here investigates the role of calpain-1 in regulating neutrophil functions such as spreading, trans-endothelial migration, chemotaxis, phagocytosis and Ca2+ signalling. Through the work done at European Mutant Mouse Archive (EMMA), Oxford, and by using intracellular sperm injection (ICSI) of calpain-1 deleted gene from mice generated in the USA, and with a selective genotype breeding programme, a colony of homozygous calpain-1 KO mouse has been generated in Cardiff. Homozygous calpain-1 KO neutrophils appeared to have a smaller surface spreading area and their recruitment into the peritoneal cavity of the mouse in vivo was disrupted. In vitro experiments showed significant defects in their ability to cross the ICAM-1 expressing endothelial cells in trans-endothelial migration assay. Disruption in this transmigration was only evident with ICAM-1 upregulated (TNF-treated) endothelial cells, suggesting a specific defect in the β2 integrin-ICAM-1 signalling process. Calpain-1 absence did not affect signal transduction as neutrophils were able to signal cytosolic Ca2+ in response to β2 integrin engagement (C3bi-opsonised zymosan) and also to release intracellular Ca2+ store upon IP3 uncaging. This showed that the IP3 pathway in the cells was not affected by knocking-out calpain-1 and continued to be functional. The key signalling mechanisms from β2 integrin also remained intact and this is consistent with calpain-1 activation by Ca2+ being an important event in trans-endothelial migration. In conclusion, calpain-1 absence has significantly affected the ability of neutrophils to undergo trans-endothelial migration and this effect is directed towards the event which happens downstream to the increase in cytosolic free Ca2+ concentration.

Vγ9Vδ2 T cells are a minor subset of T cells in human blood that differ from all other lymphocytes by their specific responsiveness to (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), a metabolite produced by a large range of microbial pathogens. Vγ9Vδ2 T cells can be skewed towards distinct effector functions, in analogy to, and beyond, the emerging plasticity of CD4+ T cells. Depending on the microenvironment, Vγ9Vδ2 T cells can assume features reminiscent of Th1, Th2, Th17 and Treg cells as well as professional antigen presenting cells (APCs). The main focus of this PhD was to investigate the role of the follicular B helper T (Tfh) cell derived cytokine IL-21 in enhancing the ability of human Vγ9Vδ2 T cells in providing B cell help. In order to try to mimic the physiological conditions in the GC, an in vitro system of autologous Vγ9Vδ2 T cells and B cells from tonsils or blood, the microbial metabolite HMB-PP and IL-21 was used. HMB-PP induced up-regulation of IL-21 receptor on Vγ9Vδ2 T cells. In return, IL-21 played a co-stimulatory role in the expression of the B cell-attracting chemokine CXCL13, the CXCL13 receptor CXCR5, the co-stimulatory molecules inducible co-stimulator (ICOS), OX40 and CD70 by activated Vγ9Vδ2 T cells. IL-21 also enhanced the ability of activated Vγ9Vδ2 T cells to support antibody production by B cells. Furthermore, Vγ9Vδ2 T cells not only themselves became highly activated APC marker expressing cells but also modified activation and APC marker expression on B cells. Findings presented in this thesis provide evidence that IL-21 contributes to the acquisition of B cell helper functions by human Vγ9Vδ2 T cells. In secondary lymphoid tissues, the interaction between HMB-PP-responsive Vγ9Vδ2 T cells, IL-21-producing Tfh cells and B cells is likely to impact on the generation of high affinity, class-switched antibodies in microbial infections

The biosynthesis of adrenal androgens is catalysed by steroid-modifying enzymes. Over the past decade, co-factors were explored to regulate these enzymes: P450 oxidoreductase (POR) delivers electrons to the key androgen-producing cytochrome P450 enzyme CYP17A1. In addition, sulfation of the principal androgen precursor dehydroepiandrosterone (DHEA) catalysed by the enzyme SULT2A1, supported by its co-factor 3’-phosphoadenosine-5’-phosphosulfate (PAPS) synthase 2 (PAPSS2), has been found more recently as a gatekeeper of androgen activation. Here, we have further characterised children with defects of enzymes of the androgen pathway, namely CYP17A1 and POR. We report the first human missense mutation of cytochrome b5, which supports electron transfer from POR to CYP17A1. In addition, we have explored the molecular regulation of DHEA sulfation by \(in\) \(vitro\) and \(in\) \(vivo\) studies. The results from our studies provide important information on the clinical course, the diagnostic steroid fingerprint and underlying molecular mechanisms of conditions affecting androgen generation. The \(in\) \(vitro\) studies on DHEA sulfation confirm that the PAPSS2 isoform crucially regulates SULT2A1. Our \(in\) \(vivo\) study in children with deficiencies of the steroid sulfatase (STS) enzyme, the counterpart of SULT2A1, suggests that STS does not play a major role in DHEA metabolism but is more active before puberty.

Evidence exists for the role of acute mental stress as a trigger for myocardial infarction. Despite the fact that the underlying mechanisms are not fully understood, inflammation and the vascular responses to stress are two mechanisms which have been implicated. After a critical analysis of the methods used to measure the vascular responses to acute mental stress (Chapter 2), this thesis examined the effects of acute inflammation on resting cardiac and mood measures (Chapter 3), and on the vascular responses to stress in a healthy population (Chapters 4 - 6). Two different protocols (vaccination and eccentric exercise) induced inflammation. Inflammation did not alter resting cardiac function or mood (Chapter 3). Despite no alteration in resting vascular function, acute inflammation did attenuate stress-induced vasodilation (Chapter 4 and 6). The effects of inflammation on stress-induced vasodilation were more prominent at the site of inflammation, indicating a localised impact of inflammation on stress-induced vasodilation. These findings suggest a possible interaction between inflammation and the vascular responses to mental stress, which could be a mechanism for the triggering of a myocardial infarction through mental stress. Further work is needed to identify the exact mechanisms through which this attenuation occurs, with a view to enhancing the vascular responses to stress in chronically inflamed populations.

The first chapter of this thesis comprises a review of thyroxine-binding proteins of man, with emphasis on TBG and, to a lesser extent, TBPA. The identification, isolation, physicochemical and physiological characteristics, and genetic variations of both proteins are included. The changes in protein concentration in disease are reviewed and the indications for the present study are presented. Subsequent chapters describe the purification of TBPA and partial purification of TBG. The production of monospecific antisera to TBG and TBPA, and development of immunoelectrophoretic assays for both proteins are described. TBG was measured in healthy persons, and the effects of age and sex on TBG were assessed, as was the effect of thyroid disease. TBG, TBPA and thyroid hormones were measured in patients after surgery, myocardial infarction and starvation. The inter-relationships of protein and free hormone concentrations were explored. Finally, nineteen families with inherited abnormalities of TBG concentration were studied. The typical case histories and biochemical findings of individuals with TBG abnormality were noted, especially when TBG abnormality coexisted with thyroid disease. Deficiencies of conventional thyroid function tests were found in TBG abnormality, and the use of T\(_4\) :TBG ratio as a means of assessing thyroid function was assessed retrospectively and prospectively.

Insulin causes uptake of glucose into cells and also causes increases in skeletal muscle blood flow by vasodilatation. In order for insulin to act at receptors on the skeletal muscle membrane, it must move through the capillary endothelium. In conditions associated with insulin resistance, insulin-induced glucose uptake and vasodilatation are often blunted and disordered transport across endothelium has been suggested. Firstly, studies were conducted using the hyperinsulinaemic euglycaemic (HE) clamp in rats to assess the relationship between insulin-induced muscle vasodilatation and glucose uptake into skeletal muscle. Insulin-induced vasodilatation was abolished by nitric oxide (NO) synthase inhibition in Wistar rats, implicating NO. Nutritional status and hence availability of glucose also affected vasodilatation in Wistar rats, leading to the proposal that vasodilatation was linked to glucose metabolism. Vasodilator response to insulin was significantly higher in lean versus obese Zucker rats. Nicotinic acid (NAc) did not decrease plasma FFA concentrations versus HE clamp, nevertheless, vasodilator response and glucose uptake were enhanced in lean and obese rats by some mechanism other than lowering FFA. Optimisation of the microdialysis technique allowed measurement, for the first time, of interstitial insulin concentrations in lean and obese Zucker rats and showed a significant difference under basal conditions.