Background: Patients suffering from chronic kidney disease (CKD) are at high risk of cardiovascular related premature death. In addition, traditional strategies for preventing cardiovascular disease (CVD) in patients with normal kidney function are less successful in patients with CKD. Vascular calcification (VC) and cardiac hypertrophy are common consequences. Recent evidence suggests that activation of the vitamin D receptor (VDR) is protective and is associated with significantly better survival in end stage renal disease (ESRD). Vascular smooth muscle cells (VSMCs) play a key role in the process of CKDrelated arteriopathy and are capable of phenotype transformation into chondro/osteoblast-like cells, which promotes the deleterious vascular effects, including calcification. Although the kidney is the main site of 1,25(OH)2D synthesis, some other non-renal tissues also express, VDR, 25-hydroxyvitamin D 1α-hydroxyalse (1α-OHase) an enzyme responsible for the synthesis of 1,25(OH)2D, and 1,25(OH)2D 24- hydroxylase (24-OHase), an enzyme responsible for the catabolism of 1,25(OH)2D. Extra-renal 1,25(OH)2D can affect a multitude of pathways in an autocrine/paracrine way, without the involvement of the endocrine system. Aims: This project aimed to investigate the role of autocrine vitamin D signalling in human VSMCs using both arterial tissue from ESRD and non-ESRD patients and primary cultures of human aortic smooth muscle cells (HAoSMCs). Specific aims were to characterise the expression and functionality of vitamin D system in vasculature as well as to examine the effect of classical regulators of the endocrine vitamin D system – 1,25(OH)2D3, calcium, phosphate, TNF-α and other interleukins, as well as novel factors such as FGF-23 and Klotho, on the local vitamin D metabolism in arteries. Results: We have demonstrated the presence of VDR, 1-OHase and 24-OHase in human kidney, artery and HAoSMCs. To our knowledge, we are the first to demonstrate the presence of 24-OHase in arteries. Our data suggested diminished expression of VDR mRNA and protein, with significantly decreased levels of 1α- OHase protein expression in CKD patients. Further crucial finding was the apparent increase of 24-OHase protein in CKD arteries suggested higher local vitamin D catabolism in CKD. In order to establish which factors regulate expression of 1α- OHase, 24-OHase and VDR in HAoSMCs, further studies were undertaken. Results confirmed that treatment with high calcium, phosphate, TNF-α, INF-γ, IL-6, IL-17A, FGF-23 or Klotho modulated VDR protein expression, which had further effect on expression of 1α-OHase and 24-OHase mRNA and protein. Conclusions: The results suggest that altered mineral and inflammatory environment, characteristic to CKD may favour local 1,25(OH)2D3 catabolism, potentially driving transdifferentiation of VSMCs, leading to VC. Vascular VDR activation, in particular through local metabolic activation, is crucial for vascular health, especially, under stress conditions. Local inducers and inhibitors of vascular vitamin D system have been determined, however further examination is required for potential application in future treatment of CKD related VC.

Rheumatoid Arthritis (RA) patients have high risk for cardiovascular diseases (CVD). Poor autonomic nervous system (ANS) function, (increased sympathetic and reduced parasympathetic activity) is a factor contributing to the risk for CVD in RA. The first experimental chapter includes a cross-sectional study in which the association between a measure of myocardial ischemia during an exercise tolerance test (ETT) and resting heart rate variability (HRV) was explored in 96 RA patients. Myocardial ischemia was associated with reduced HRV. The second chapter examined the parasympathetic reactivation using heart rate recovery (HRR) following ETT, and multiple factors association with HRR. Multivariate analyses revealed no factor was independently associated with HRR, but it was the overall CVD risk and disease related burden that contributed to variability in HRR. In the third chapter, the effects of a three-month exercise intervention on HRR, CVD risks, inflammation, and measures of wellbeing were investigated in 62 RA patients. Exercise reduced some CVD risk factors and improved some measures of wellbeing, however, HRR and cardiorespiratory fitness did not improve. In the last chapter, a cross-sectional study compared HRR between age-and sex-matched RA (N=43) and diabetes mellitus (N=26) patients as well as inflammatory markers, CVD risk factors, and measures of wellbeing. There was no difference in HRR or inflammation between the two groups. A sub-analysis found that cardiorespiratory fitness was an independent predictor of HRR. These findings suggest that parasympathetic activity in RA associate with several CVD risk factors, and cardiorespiratory fitness is an important factor associated with it.

Poor functional outcomes are frequent after peripheral nerve injuries despite the regenerative support of Schwann cells. Whilst motoneurons and to a lesser extent, sensory neurons survive the injuries, outgrowth of axons across the injury site is slow and the neuronal regenerative capacity is progressively reduced when neurons remain without targets and chronically denervated Schwann cells fail to support axon growth. Strategies including brief low frequency electrical stimulation that accelerates axon outgrowth and, in turn, target reinnervation and functional recovery, have excellent potential for translation to human patients. Other strategies including the insertion of cross-bridges between a donor nerve and a recipient denervated nerve stump, are effective in promoting functional outcomes after complete injuries. During muscle reinnervation the properties of the motoneurons and muscle fibers that they supply are rematched that provide some control of muscle force even when regenerating axons are misdirected to foreign targets. Axon sprouting from intact nerves is effective, although limited, in reinnervating denervated muscle fibers after incomplete injuries and in poliomyelitis. Studies in mouse models of amyotrophic lateral sclerosis however, indicate that sprouting is very limited with rapid and preferential loss of the largest and fastest contracting motor units during the asymptomatic phase of the disease.

Inherited platelet disorders vary in their associated bleeding risk. Individuals in families with known platelet gene mutations often have variable bleeding, suggesting that bleeding risk is multifactorial. Inherited platelet disorders are difficult to diagnose due to the absence of a gold standard laboratory technique and their variable bleeding phenotype, which often only manifests after haemostatic challenges. The work in this thesis furthers the previous studies in the genotyping and phenotyping of platelets project by significantly increasing the number of participants, allowing further characterisation of inherited platelet disorders in those with a normal platelet count. A bleeding assessment tool score was also recorded in all newly recruited adults. Two specific groups of patients are also studied: Those with unexplained menorrhagia, in whom the hypothesis was that some have an undiagnosed platelet defect. Those with inherited thrombocytopenia, in whom I sought to develop an assay to assess platelet function, as bleeding risk can not be predicted by platelet count alone, suggesting that qualitative defects may contribute. The genetic basis of platelet defects was investigated in many of the patients, leading to identification of mutations in genes known to be critical in platelet biology, and identification of several possible novel variants.

Optimal management of British military personnel injured in Afghanistan is best achieved in the United Kingdom; therefore aeromedical evacuation (AE) is arranged, often within the first 24 hours for the most severely injured casualties. However, early aeromedical evacuation carries its own risks, which must be considered prior to emplaning any casualty. The study aim was to investigate whether changes in tissue oxygenation occur in military casualties during aeromedical evacuation. Near infra-red spectroscopy was used in a series of studies designed to test the sensitivity of the tissue oxygen saturation monitoring technique in volunteers exposed to simulated altitude and simulated hypovolaemia. Changes in tissue oxygen saturation readings were detected in the volunteers, so an observational study was undertaken to determine whether changes in tissue oxygen saturation occur in military trauma casualties during aeromedical evacuation. Whilst the majority of casualties did not demonstrate any significant change in readings, some casualties did demonstrate reduced tissue oxygen saturation readings during their flights, although these changes were not reflected in systemic physiological monitoring techniques. Having identified that changes do occur, further work is required to investigate the cause, significance and outcome of these changes in order to fully appreciate the findings of the current study.

Cerebral autoregulation (CA) is the process by which cerebral arterial vessels constrict or dilate, in order to maintain cerebral blood flow (CBF) in the face of changes in arterial blood pressure. This thesis aimed to investigate the mechanisms that normally control CA, with specific reference to the upper limit (UL ); the point at which autoregulatory vasoconstriction is overcome. The UL is associated with an increase in CBF, which can potentially cause damaging cerebrovascular hyperperfusion. Ageing is associated with a general decrease in regulatory mechanisms, and an increased risk of stroke. Therefore we sought to assess whether changes in CA at the UL may predispose older rats to haemorrhagic stroke. We assessed CA by infusion of phenylephrine to increase ABP in an anaesthetised rat model, whilst measuring CBF to enable identification of the UL. This protocol was repeated in young (6-8 weeks) and old (12-15 months) rats in control conditions, or during removal of regulatory inputs. The UL in young, control rats was 168±4mmHg. Removal of nitric oxide-mediated dilatation and sympathetically-mediated vasoconstriction increased the UL. In old rats, the increase in UL was even greater. Thus we propose a new working hypothesis to explain the control of CA and the UL.

Background: Guidelines suggest that rehabilitation for people with stroke should adopt patient-centred goal-setting (PCGS). Methods: A literature review and two qualitative studies were done in an acute stroke-unit. Study one aimed to explore influence of PCGS within stroke rehabilitation. Patients with stroke, with ability to participate and staff caring for them were included. Data collection involved interviews, observations, document analysis and focus-groups. Analysis involved sequential and intra-case analysis methods. Study two aimed to build a resource to improve PCGS and evaluate its feasibility and appropriateness. Based on Study one and review, a resource (T-PEGS) was developed and applied in this setting. Patients with same criteria as Study one and staff who agreed to act as keyworkers were recruited. Data collection and analysis methods were similar to Study one. Findings: Study one, with thirteen patients and twelve professionals, revealed limited application of PCGS due to participants’ health beliefs, limitations in knowledge and resources. Study two involved five patients and five staff who applied T-PEGS; recording of psychosocial goals, information sharing and rapport between patients and professionals had improved. Conclusion: T-PEGS seemed to improve PCGS locally. Small study-size and single site limit generalisability. Future work should explore mechanisms and effectiveness of T-PEGS.

Previous work suggests vasodilating prostaglandins (PGs) are released during isometric handgrip exercise in an O\(_2\)-dependent manner in young men. This project investigates their contribution to the exercise hyperaemia of isometric and rhythmic handgrip contraction performed by healthy, recreationally-active young and older men. Hyperoxia (40% O\(_2\)), aspirin, and their combination equally attenuated exercise and post-exercise hyperaemia, and venous efflux of PGE\(_2\) and PGI\(_2\) in both age groups: efflux of these PGs was not attenuated with age, but their contribution to the hyperaemic response was. Further, the release of COX products evoked reflex vasoconstriction in an O\(_2\)-dependent manner. Moreover, 40% O\(_2\), aspirin, and their combination equally inhibited the exercise-evoked vasoconstriction in both age groups. However, both the exercise-evoked attenuation in perfusion of resting skeletal muscles and the contribution of COX products were attenuated with age. Additional experiments showed that adenosine contributes to the hyperaemia of electrically evoked isometric twitch contractions in an O\(_2\)-dependent manner; adenosine may contribute to the increase in the concentrations of vasodilating PGs. Importantly, unlike 60% and 100% O\(_2\), 40% O\(_2\) did not attenuate acetylcholine-evoked endothelium-dependent dilatation in either age group, supporting the argument that the effect of 40% O\(_2\) during exercise is independent of hyperoxia-related oxidative stress.

Skeletal muscle plays a central role in maintaining metabolic homeostasis in health and disease. An important metabolic component of muscle is the integrated metabolism of glucose and glucocorticoids (GCs) in the lumen of the sarcoplasmic reticulum (SR). Glucose-6-phosphate (G6P) transported into the SR by the G6P transporter (G6PT) is metabolised by hexose-6-phosphate dehydrogenase (H6PDH) to produce nicotinamide adenine dinucleotide phosphate reduced (NADPH) cofactor that is utilised by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) to reduce inactive GCs to their active forms. This thesis evaluated the SR unit as a site of integrated metabolism using a range of genetic models and screening methods. We demonstrate that perturbation of redox and metabolic balance through the distribution of H6PDH enzyme resulted in novel stress responses leading to shifts in redox and energy metabolism. Particularly, we show how the SR can elicit transcriptional changes that appear to adapt NADPH redox perturbation and also increase energy metabolism through augmented mitochondrial function. These data presented a novel sensing pathway within the SR unit that could have important relevance to metabolic and energetic control of skeletal muscle.

Age is associated with the development of multi-system function loss including the musculoskeletal, immune and cardiovascular system, as well as body composition changes. These age-related alterations lead to frailty development and disease progression, reducing quality of life. A major lifestyle change occurring in later years is reduced physical activity levels. This thesis sought to examine the associations between physical activity and multi-system function loss in a cohort of elderly individuals, and to better understand the neural mechanisms underpinning the circulatory responses to exercise. It was observed that high daily physical activity levels attenuate some but not all of the age-related changes in elderly individuals. High physical activity was associated with superior physical functioning, lower total body fat and visceral adiposity, and plasma plasminogen activator inhibitor 1 (PAI-1) concentrations. Left ventricular (LV) diastolic function was negatively associated with mean arterial pressure (MAP) and visceral adiposity, suggesting that elderly individuals with higher MAP and visceral adiposity may have inferior LV diastolic function. In terms of neural mechanisms related to circulatory responses to exercise, in models of metaboreflex over-activity whereby BP is elevated as observed in heart failure patients, left atrial systolic function is enhanced in order to maintain end-diastolic volume and SV.