Congenital thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy, usually involving ADAMTS13 gene defects. ADAMTS13 processes the multimeric plasma glycoprotein Von Willebrand factor making it less reactive to platelets. Patients differ in terms of disease severity and evidence suggests a relationship between ADAMTS13 genotype and disease phenotype. Over 140 mutations have been identified in patients but only ~30% of these has been expressed in vitro. The aim of this thesis was to study certain ADAMTS13 mutations identified in a homozygous form in congenital TTP patients to assess in vitro their effect on the secretion and activity of ADAMTS13 and to assess their contribution to disease phenotype. ADAMTS13 mutants (p.R102H, p.I143T and p.Y570C) and wild type (WT) were expressed in HEK293T cells. The p.R102H mutation partially affected the secretion of ADAMTS13 and reduced the catalytic efficiency of the mutant but not to the extent predicted based upon levels measured in patient plasma. Expressing this mutant with three ADAMTS13 polymorphisms (p.Q448E, p.P618A and p.A900V) which were also identified in the patient with this mutation further reduced the secretion and activity of ADAMTS13. When these three polymorphisms were expressed separately in WT ADAMTS13, the p.P618A polymorphism reduced the secretion and subsequently the activity of ADAMTS13 suggesting that this polymorphism in particular was responsible for the reduction observed. These results highlight the importance of ADAMTS13 polymorphisms. The p.I143T and p.Y570C mutations severely affected ADAMTS13 secretion. Immunofluorescence studies showed localisation of these mutants within the ER but less extensive localisation within the cis Golgi compared to WT ADAMTS13. The p.I143T mutant was characterised further and was shown to be degraded by the cell proteasome. Addition of a chemical chaperone (betaine) appeared to rescue the secretion defect caused by the p.I143T mutation. This may have future therapeutic implications for the treatment of some congenital TTP patients.

The optimal treatment for acute myocardial infarction (AMI) requires the rapid restoration of flow in the infarct related artery and reperfusion of the myocardium at the tissue level through angioplasty. Coronary angioplasty, in the setting of AMI, is aimed at reducing myocardial infarct size, preserving left ventricular function and improving long term outcomes. Paradoxically, the opening of an occluded vessel following a period of ischaemia can result in further myocyte death. This is called lethal reperfusion injury. Current treatments for AMI have failed to attenuate reperfusion injury and combined morbidity and mortality rates remain high. There is an urgent need for the development of novel cardioprotective therapies in the clinical arena. Remote ischaemic perconditioning describes the cardioprotective effect evoked by applying brief episodes of ischaemia and reperfusion applied to a limb with a blood pressure cuff prior to reperfusion but following the onset of ischaemia. Such strategies have been utilised in a number of proof of concept studies demonstrating the ability of remote ischaemic conditioning to attenuate reperfusion injury and reduce infarct size. As therapies for AMI improve, it has become increasingly difficult to demonstrate incremental improvements in currently used biomarkers. Cardiovascular magnetic resonance (CMR) imaging is an important imaging technique that is able to assess traditional imaging parameters with a high degree of accuracy and reproducibility. CMR is also provides supplementary information due to its unique ability to characterise the myocardium and identify the components of reperfusion injury. The identification and quantification of these adverse elements of reperfusion offer further prognostic information beyond traditional imaging biomarkers and show promise as novel endpoints in clinical cardiovascular research. Data is presented on the use of remote ischaemic perconditioning in patients undergoing primary angioplasty. CMR endpoints have been utilised to assess the efficacy of this conditioning protocol in a high-risk patient group.

Ischaemic heart disease (IHD) is a major cause of morbidity and mortality in the world. Coronary artery bypass graft (CABG) surgery is the revascularisation strategy of choice in a significant number of patients, particularly those with diabetes mellitus and complex coronary disease. During cardiac surgery, the myocardium is subjected to peri-operative myocardial injury (PMI), which has been associated with worse short and long-term clinical outcomes. Higher-risks patients are currently being operated on with subsequent higher risk of PMI and worse prognosis: therefore new strategies are required to potentiate the innate mechanisms of cardioprotection. In this regard, remote ischaemic preconditioning (RIPC) is a promising non-invasive intervention able to reduce PMI in these patients: however, not all the studies have shown significant cardioprotection with RIPC for a number of factors, amongst which the intensity of the preconditioning stimulus may play a significant role. We therefore investigated whether an enhanced RIPC stimulus, given with transient simultaneous multi-limb ischaemia/reperfusion, was able to reduce PMI and improve short-term clinical outcomes in patients undergoing elective cardiac surgery: we demonstrated that our preconditioning stimulus can significantly reduce PMI, length of intensive care unit (ICU) stay and incidence of atrial fibrillation (AF) in these patients. In addition, further retrospective analyses showed improved myocardial protection in preconditioned diabetic patients undergoing CABG surgery and in control CABG subjects receiving combined antegrade and retrograde cardioplegia compared to control CABG patients having antegrade cardioplegia or intermittent cross-clamp-fibrillation. We also conducted a multi-centre, double-blinded randomised control clinical trial, in which we investigated the effects of RIPC on clinical outcomes at 1 year in high-risk patients undergoing elective CABG surgery with or without valve surgery (the ERICCA trial). The results of this study are due to be presented in March 2015 and have the potential to significantly impact on clinical practice in cardiac surgery.

The renin-angiotensin system (RAS) is involved in maintenance of cardiovascular function and has been implicated in coronary heart disease (CHD) and type 2 diabetes (T2D). Pharmacological inhibition of the RAS improves cardiovascular disease outcomes. Angiotensin Converting Enzyme (principal component of RAS) levels are significantly associated with the ACE(I/D) polymorphism. Large studies have demonstrated a mild effect of this polymorphism on CHD risk. However the presence of RAS in diverse tissues implicated in cardiovascular diseases justify the hypothesis that genetic polymorphisms encoding proteins in this system 'modify' the risk of such diseases in the context of harmful stimuli or affect disease progression. This thesis therefore tested the hypothesis that the ACE(I/D) polymorphism 'modifies' risk of CHD and T2D, and affects progression of Hypertrophic Cardiomyopathy (HCM). Using a study of 532 myocardial infarction (MI) survivors and 574 controls (HIFMECH), an interaction between the ACE polymorphism and lipid levels on MI risk in Northern compared to Southern Europeans is demonstrated: the MI odds with high ApoB levels in D allele carriers was much greater in the North than the South. In a prospective cohort of 3052 healthy men (NPHS2), an interaction between ACE and systolic blood pressure (SBP) on CHD risk is presented where D allele carriers were protected against CHD at lower SBP, but at higher SBP were more susceptible. In the same study, ACE variants modified the risk of T2D in obese individuals, with obese D allele carriers having a substantially higher T2D risk compared to obese II homozygotes. From a retrospective study of 541 patients with HCM a possible effect of the polymorphism on left ventricular remodelling is demonstrated. Thus the work in this thesis offers strong evidence that the ACE gene modifies risk of CHD and T2D in the presence of known risk factors.

Endothelial dysfunction plays a pivotal role in the development and progression of atherosclerosis. Atherogenic risk factors harbor the potential to injure the endothelial layer by promoting apoptosis, preceded by an inflammatory reaction in the vessel wall. The maintenance of the endothelial layer is therefore crucial to ensure its integrity. More recent studies suggest an important role of endothelial progenitor cells (EPCs) in contributing towards reendothelialization after vascular injury. These EPCs home to sites of hypoxia or ischemia and take part in vascular repair. Recent studies have shown that a rare population of EPCs called endothelial colony forming cells or ECFCs can be derived from peripheral and cord blood and can be transplanted into immunodeficient mice that have demonstrated the ability to form chimeric blood vessels. Hyperoxia has been shown to be a model of mild oxidative stress, and this oxidative stress is characterized by an increase in reactive oxygen species (ROS) levels that can lead to a wide range of chemical reactions resulting in cellular necrosis and apoptosis through lipid peroxidation and DNA damage. We therefore proposed that by growing endothelial cells in vitro in hyperoxic conditions can in turn lead to cellular damage and activation of pro inflammatory markers in these cells. PGC-1α belongs to a small family of transcriptional coactivators which have been shown to regulate reactive oxygen species generation and apoptosis in mature endothelial cells. The mechanisms through which PGC-1α activates gene expression are poorly understood. This study aims at identifying a molecular signature of human endothelial progenitor cells (ECFCs) so that they can be distinguished from mature endothelial cells (HUVEC) and identifying the role of PGC-1α and its downstream targets when these endothelial progenitors and mature endothelial cells are exposed to oxidative stress. In order to achieve that goal we isolated endothelial colony forming cells and mature endothelial cells from the same donor. This was followed by their molecular profiling at mRNA level using qPCR and at protein level using immunofluorescence and FACS analysis. Cells were grown till late passages to study the growth kinetics of ECFCs and HUVEC in normoxia and hyperoxia. Telomerase activity was measured using TRAP assay and telomere length measurement was done using flow FISH. Low density array was carried out in order to distinguish between ECFCs and HUVEC in terms of gene expression analysis. The results showed that ECFCs were distinguished from early EPCs and HUVEC. PGC-1 alpha levels were up regulated in ECFCs when exposed to hyperoxia and this in turn activated several other genes that are involved in angiogenesis, oxidative phosphorylation and electron transport chain. ECFCs and HUVEC have shown to behave differently under conditions of oxidative stress and we found ECFCs to be more resistant to stress than HUVEC as shown in their growth kinetics.

Impedance cardiography (ICG) is a non-invasive technique to measure the dynamic changes in electrical impedance of the thorax. Photoplethymgraphy (PPG) is an optical- based non-invasive physiological measurement technique used to detect the blood volume pulses in the microvascular bed of tissue. These two physiological measurements have potential clinical importance to enable simple and cost-efficient ways to examine cardiovascular function and provide surrogate or additional clinical information to the measures from cardiac imaging. However, because the origins of the characteristic waveforms of the impedance and pulse are still not well understood, the clinical applications of these two techniques are limited. There were two main aims in this study: 1) to obtain a better understanding of the origins of the pulsatile impedance changes and peripheral pulse by linking their characteristic features beat-by-beat to those from simultaneous echocardiograms; 2) to validate the clinical indices from ICG and PPG with those well-established echocardiographic indices. Physiological signals, including ECGs, impedance, the first derivative impedance and finger and ear pulses, were simultaneously recorded with echocardiograms from 30 male healthy subjects at rest. The timing sequence of cardiovascular events in a single cardiac cycle was reconstructed with the feature times obtained from the physiological measurements and images. The relations of the time features from the impedance with corresponding features from images and pulses were investigated. The relations of the time features from peripheral pulses with corresponding features from images were also investigated. Furthermore, clinical time indices measured from the impedance and pulse were validated with the reference to the echocardiograms. Finally, the effects of age, heart rate and blood pressure on the image and physiological measurements were examined. According to the reconstructed timing sequence, it was evident that the systolic waves of the thoracic impedance and peripheral pulse occurred following left ventricular ejection. The impedance started to fall 26 ms and the pulse arrived at the fingertip 162 ms after the aortic valve opened. A diastolic wave was observed during the ventricular passive filling phase on the impedance and pulse. The impedance started to recover during the late ventricular ejection phase when the peripheral pulse was rising up. While the pulsatile impedance changes were mainly correlated with valve movement, the derivative impedance (velocity of impedance change) was more correlated with aortic flow (velocity of blood 2 flow). The foot of the finger pulse was significantly correlated with aortic valve open (R = 0.361, P < 0.05), while its systolic peak was strongly correlated with the aortic valve 2 closing (R = 0.579, P < 0.001). Although the pulse had similar waveform shapes to the inverted impedance waveform, the associations between the time features of these two signals were weak. During the validation of potential clinical indices from ICG, significant correlation was found between the overall duration of the derivative impedance systolic wave (359 ms) and the left ventricular ejection time (LVET) measured by aortic valve open duration from M- 2 mode images (329 ms) (R = 0.324, P < 0.001). The overall duration from the finger pulse foot to notch (348 ms) was also significantly correlated with the LVET from M-mode 2 images (R = 0.461, P < 0.001). Therefore, both ICG and PPG had the potential to provide surrogates to the LVET measurement. Age influenced the cardiovascular diastolic function more than systolic function on normal subjects. With age increasing, the reduction of the left ventricular passive filling was compensated by active filling. The ratio of the passive filling duration to the active 2 filling duration decreased with age (R = 0.143, P < 0.05). The influence of age on the diastolic wave of the impedance signals was striking. The impedance diastolic wave disappeared gradually with age. The effects of age on the peripheral pulse were mainly on the shortened pulse foot transit time (PPT) and prolonged pulse rise time. The large artery f stiffness index (SI) increased with age. Most time intervals were prolonged with heart rate slowing down. The effects of systolic blood pressure were evident on pulse transit time and pulse diastolic rising time. Driven by higher systolic blood pressure, both PPT and rising f time decreased significantly (P < 0.001). In conclusion, from the analysis based on simultaneous physiological measurements and echocardiograms, both the pulsatile impedance changes and peripheral volume pulse were initiated by left ventricular ejection. The thoracic impedance changes reflected volume changes in the central great vessels, while the first derivative impedance was associated with the velocity of blood flow. Both ICG and PPG had the potential to provide surrogates for the measures of cardiac mechanical functions from images. The PPG technique also enabled the assessment of changes in vascular function caused by age.

The aim of the work reported in this thesis was centred on the development of a fit for purpose structured education lifestyle modification programme for the self-management of Obstructive Sleep Apnoea (OSA). The programme aimed to promote compliance to Continuous Positive Airway Pressure (CPAP) therapy and increase physical activity levels. A mixed method study, the Predicting the Use of Continuous Positive Airway Pressure Therapy in Obstructive Sleep Apnoea in a UK population: The PUCOSA-UK study was carried out to explore the interplay of potential psycho-social predictors of CPAP adherence using a strong framework of psychology models in newly diagnosed and established OSA patients. The study showed that adherence to CPAP therapy was heavily dependent on the early formation of strong beliefs and preconceptions of the condition and CPAP therapy prior to trialling the therapy. The qualitative element of the study identified a number of common barriers associated with CPAP compliance including inability to recognise symptoms and link to with being diagnosed with OSA, initial negative reactions to the idea of using CPAP therapy, discomfort and inconvenience caused when wearing a CPAP mask and operating a CPAP device, lack of spousal or peer support and self-image issues. A systematic review and meta-analysis was conducted to evaluate the impact of diet, exercise and lifestyle modification interventions with or without CPAP therapy on obesity indices, OSA parameters and quality of life in adults with OSA. Intensive lifestyle intervention programmes were found to be more effective in reducing indices of obesity and in improving OSA parameters than less intensive lifestyle interventions or routine care. A structured education curriculum was written and piloted in small groups. The results of the pilot work combined with the results of the aforementioned studies informed the content of the curriculum. Overall, the patients benefited from attending the education programme and learnt useful self-management skills. Concluding, the findings from the work reported here warrant formal testing of the educational programme in a definitive randomised controlled trial.

Background: Strabismus development is a complex process involving various parts of the visual system. This complexity is increased by the presence of numerous subtypes. Although research has shown strong genetic and environmental components it is not yet clear if there are stronger associations with particular subtypes. In addition, the association between strabismus and retinal development and its limitation on visual outcome, in combination with other factors such as compliance to treatment, has not yet been explored. Methods: Strabismic subjects were recruited to four areas to assess the development and treatment of strabismic amblyopia. Genetic and environmental factors were systematically identified within the literature and through strabismic pedigrees. The influence of strabismic development on the retina was assessed using optical coherence tomography (OCT) in strabismic infants compared to healthy controls. OCT and electronic monitors were used to investigate causes of poor outcomes after amblyopia treatment. Results: Systematic reviewing of the literature revealed strong associations between strabismus and various environmental and genetic factors. By observing the inheritance of strabismus through pedigrees it was observed that accommodative forms of esotropia had a stronger association with inheritance than other subtypes. The effect of strabismus on retinal development revealed delayed or abnormal changes within particular layers of the retina. These defects were observed in both eyes of strabismic subjects and were sustained in subjects who failed to reach successful outcomes after treatment. In addition, through using electronic monitors, compliance to both glasses wearing and occlusion were significantly correlated to visual outcomes. Conclusion: This thesis continues to support growing evidence that both genetic and environmental factors play a role in strabismus development. Its development also has retrograde effects on the visual pathway in particular the retina, which is sustained unless treatment is successful. In addition, compliance to treatment has shown to be vital in obtaining a successful outcome.

Various methods of assessing peripheral vascular disease are studied by making measurements both on dogs, and patients with arterial disease. Theoretical and experimental models of blood flow in diseased arteries are reviewed. A new type of experimental inter-changeable stenosis is described; and the flow, pressure and Doppler ultrasound results from a number of dog experiments using the device are presented. The relationship between the pressure drop across a stenosis and the flow through it in-vivo is shown to differ from previously reported in-vitro values. The severity of a stenosis is shown to have little effect on peripheral resistance, and attention is drawn to the lack of concrete evidence in the literature suggesting that it does. A possible explanation for this finding is given. It is suggested that ultrasonic Doppler traces are influenced by the distal bed as well as by proximal stenoses. Experimental Doppler spectra are compared with spectra calculated from electromagnetic flow waveforms and found to be in good agreement. Measurements from over 150 vascular patients are presented, and the difficulty of determining the validity of these measurements discussed. The indirect pressure measurement method of assessing vascular disease is examined and shown to be of limited value. It is shown that an inflatable cuff can substantially alter the pressure distribution within an artery, and the possibility of worsening disease being reflected by a higher occlusive cuff pressure is introduced. It is shown that both Doppler ultrasonic velocity measurements and direct arterial pressure measurements agree with the clinical assessment in extreme cases, but that in equivocal cases the two tests often disagree with each other. The difficulty of adjudicating between tests is reaffirmed and a new method of objective evaluation suggested. Finally the problems consequent upon the dynamic nature of the disease are mentioned.

The validation of in vitro skeletal muscle models may play a pivotal role in capturing safety endpoints early within the research and development process. Therefore, the primary aim of this project was to investigate the extent of translation from an in vivo rat model to an in vitro skeletal muscle model, using a toxicogenomics approach. To this end, the mechanisms of toxicity of three novel sulfonyl isoxazoline (SI) herbicides (two triazoles and one phenyl) developed by Syngenta were investigated in vivo and in vitro. In vivo histopathology studies identified striated muscle as the target organ of SI triazole toxicity, and the stomach and liver as the target organs of SI phenyl toxicity. Mechanistic toxicogenomics was carried out on liver, heart and skeletal muscle tissues from rats treated with sub-toxic doses of the SI triazoles (177 and 197) and phenyl (907) compounds for 28 days. The biological processes perturbed by SI triazoles included mitochondrial dysfunction, oxidative stress, energy metabolism, cell death, protein regulation and cell cycle. In contrast, perturbation of cholesterol biosynthesis was identified as the SI phenyl mechanism of toxicity. Using an in vitro rat skeletal muscle cell line (L6), it was demonstrated that the SI triazoles induced mitochondrial dysfunction, mitochondrial superoxide production, cell cycle arrest, hypertrophy and apoptosis. These in vitro results were consistent with the in vivo toxicogenomics data, providing validation that these models may predict skeletal muscle toxicity. To increase detection of xenobiotic-induced mitochondrial effects in skeletal muscle, L6 cells were forced to rely on mitochondrial oxidative phosphorylation by substituting galactose for high glucose in the growth media. In galactose-grown cells, oxygen consumption was increased, glycolysis was repressed and susceptibility to mitochondrial toxicants correspondingly increased. Future work should aim to further develop the L6 model to better mimic the in vivo model using 3D and microfluidic technologies.