Study aims were: Description of health beliefs and beliefs about medicines in patients with hypertension using questionnaires based on the self-regulatory model; Examination of the relationships between these beliefs; Assessment of patient compliance and the relationship between beliefs and compliance; Evaluation of current GP practices in the management of hypertension using self-report, clinical vignettes review of prescription data and audit of an actual GP practice; Assessment of GP's cognitions in the management of hypertension in the elderly using the theory of planned behaviour; and Integration of information from both patient and doctors. A complex picture of patients' beliefs about hypertension and its treatment were found. The most important beliefs in relation to compliance were about the necessity of therapy and perception of personal control of hypertension. Emotional response to hypertension was also important. A number of demographic variables were also important, most noticeably patients' age. From this information, we suggest that compliance may be more consistently predicted by illness and medication beliefs than has been found with other factors in the past. We propose that the effects of various demographic variables may be mediated through beliefs and attitudes. We found a number of areas where current medical practice in primary care could be improved which in turn would impact hypertension control. These range from methods of measuring blood pressure, to treatment targets and choice of anti-hypertensive agents. Doctors' thought-process may be involved, but although we could show that these fit the theory of planned behaviour, relating these to actual practice was more challenging. Overall, this thesis highlights areas for further research but more importantly potential targets for interventions to improve patients' compliance and doctors' management practices. Both approaches are needed to improve the control of hypertension and reduce the burden of cardiovascular and stroke disease.

Congenital cardiovascular malformation (CVM) affects 7/1000 live births. Approximately 20% of cases are caused by chromosomal and syndromic conditions. Rare Mendelian families segregating particular forms of CVM have also been described. Among the remaining 80% of non-syndromic cases, there is a familial predisposition implicating as yet unidentified genetic factors. Since the reproductive consequences to an individual of CVM are usually severe, evolutionary considerations suggest predisposing variants are likely to be rare. The overall aim of my PhD was to use next generation sequencing (NGS) methods to identify such rare, potentially disease causing variants in CVM. First, I developed a novel approach to calculate the sensitivity and specificity of NGS data in detecting variants using publicly available population frequency data. My aim was to provide a method that would yield sound estimates of the quality of a sequencing experiment without the need for additional genotyping in the sequenced samples. I developed such a method and demonstrated that it provided comparable results to methods using microarray data as a reference. Furthermore, I evaluated different variant calling pipelines and showed that they have a large effect on sensitivity and specificity. Following this, the NovoAlign-Samtools and BWA-Dindel pipelines were used to identify single base substitution and indel variants in three pedigrees, where predisposition to a different disease appears to segregate following an autosomal dominant mode of inheritance. I identified potentially causative variants segregating with disease in all three of the pedigrees. In the pedigrees with Dilated Cardiomyopathy and Hereditary Sclerosing Poikiloderma these variants were in plausible candidate genes. Finally, NGS was used to identify rare, potentially disease causing indel variants in patients with sporadic, non-syndromic forms of CVM characterised by chamber hypoplasia. Two indel calling pipelines were used as a means to increase confidence in the identified indels. These two pipelines achieved the highest sensitivity calls using the method described above. In the 133 cases, evaluated for 403 candidate genes, indels were identified in 4 known causative genes for human cardiovascular disease, namely MYL1, NOTCH1, TNNT2, and DSC2.

This study investigated the illness and treatment representations of 214 coronary artery disease patients undergoing medication, percutaneous coronary intervention or coronary artery bypass grafting. Illness representations were investigated using the New Zealand – Illness Perceptions Questionnaire. Results revealed that with these patients, illness representations comprised the components: illness impact, duration, control and self-image. Illness identity was characterised by fatigue, breathlessness and chest-pain; and important perceived causes for their illness were, high cholesterol levels, stress or worry, and eating fatty foods. These belong to a structure of causal beliefs with the components: emotional causes, poor-lifestyle causes and external causes. Treatment beliefs were investigated through the development of a Treatment Representations Inventory which was structured around the four components: treatment value, treatment concerns, decision satisfaction and disease cure. Significant treatment group differences on the sub-scales showed a logical and explainable pattern of group differences, which reflected the treatments’ distinctive and hierarchical natures. The illness cognition scales were employed to systematically develop a path analysis model that represented the multivariate relationships between the components of illness and treatment representations. The final model proved satisfactory across a range of fit indices and elucidated significant and theoretical valid relationships within illness representations components, within treatment representations and between the two types of illness cognition. Additionally, patterns of responses to the illness cognitions were shown to form two distinct clusters of patients. One group appeared to have positive expectations for their illness and treatment, the other a more circumspect and uncertain outlook. A positive outlook led to greater positive affect, serenity, attentiveness and joviality; and less anxiety, depression, hostility and shyness than an uncertain outlook. Over time, and following revascularisation, a number of illness cognitions significantly changed. Patterns of changes differed by treatment group, reflecting the drama involved in treatments undergone and the positive orientation of patients.

The focus of my PhD has been two-­‐fold: First, to improve the understanding of the biology behind a well-­‐known cardiovascular disease (CVD) risk factor -­‐ left ventricular mass, by identifying novel genetic loci associated with this risk factor. A large-­‐scale association meta-­‐analysis in over 10,000 individuals identified four novel loci associated with electrocardiographically-­‐determined left ventricular mass. Second, to explore the application of known genetic determinants of the main blood lipid fractions, the latter being well-­‐known CVD risk factors and therapeutic targets. I assess the use of genetic variants associated with total cholesterol, low-­‐ density lipoprotein-­‐cholesterol (LDL-­‐C), high-­‐density lipoprotein-­‐cholesterol (HDL-­‐C) and triglycerides for discriminating healthy individuals from those that have a high absolute risk of CVD, those that require lipid-­‐lowering medication, and those that have a coronary event. The lipid genetic variants showed poor discriminatory ability for all three outcomes and provided no improvement over the widely-­‐used, non-­‐ genetic Framingham 10 year CVD risk score. Lipid-­‐associated genetic variants were also used to generate genetic risk score instruments for LDL-­‐C, HDL-­‐C and triglycerides, which were applied in a Mendelian randomisation analysis to determine their causal relationship with carotid-­‐intima media thickness (CIMT). CIMT has been a widely used surrogate outcome measure in clinical trials of CVD drugs. LDL-­‐C-­‐lowering drugs have shown to reduce CIMT progression and CHD risk in clinical trials. However, the extent of any causal association between HDL-­‐C or triglycerides and CIMT is unclear. The results from this MR analysis support a casual relationship with LDL-­‐C, but not with HDL-­‐C and triglycerides, which may indicate that CIMT is a less useful surrogate end point in clinical trials of primarily HDL-­‐C or triglyceride modifying therapies.

Familial Hypercholesterolaemia (FH) is a common autosomal dominant disorder of the defective plasma clearance of LDL-cholesterol. Mutations in three genes, LDLR/APOB/PCSK9, can be detected in 60-90% of definite FH patients. DNA-based testing for FH mutations has important clinical utility and is recommended by the UK and European guidelines to identify affected relatives. This thesis aimed to determine the frequency and spectrum of FH mutations in two independent cohorts of FH patients (from one Oxford lipid clinic, and of Indian background). The FH mutation spectrum was shown to be highly heterogeneous and the mutation detection rate was significantly dependent on the pre-treatment total cholesterol and triglyceride levels. This project also validated the findings that a proportion of clinically diagnosed FH patients have a polygenic cause of hypercholesterolaemia due to an accumulation of common mild LDL-C-raising alleles by analysing LDL-C gene score in 88 mutation negative and 21 mutation positive FH patients, and by replicating the results in further 231 FH patients. A high-throughput DNA sequencing method was assessed as a novel diagnostic tool for detection of FH mutations, and compared it with the currently used methods. This highlighted the need for updating the current FH mutation screening methods as well as the need for more efficient bioinformatics for the next generation sequencing data analysis. Lastly, whole exome sequencing of 125 definite FH patients with no mutations detected in known genes was performed to identify novel monogenic causes of FH. Variants in two genes, CH25H and INSIG2, were identified as potential novel FH mutations. Overall, the results of this thesis demonstrate the heterogeneous FH aetiology and help to understand the genetic architecture of the disease.

Hypertrophic cardiomyopathy (HCM) is the commonest inherited cardiac condition. Many patients have resting or provocable left ventricular outflow tract (LVOT) obstruction. Symptoms treated with drugs or surgery may improve. There is a need to improve the clinical assessment in individual patients, because of the often poor correlation between symptoms and LVOT gradient, and the association with complications such as stroke, heart failure and sudden cardiac death. In addition, in a proportion of patients with significant LVOT gradients, relief of obstruction does not adequately improve symptoms. Reduced angulation between the inter-ventricular septum and the aorta is a determinant of LVOT obstruction. However, lack of a standardised method of measurement in HCM without recourse to complex 3-D imaging limits the usefulness of this parameter in routine practice. Transthoracic echocardiography is widely available, and can be used to measure aorto-septal angulation. However, data in HCM are lacking. I validated a simple measurement of aorto-septal angulation using 2-D echocardiography and cardiac magnetic resonance imaging and determined its relation to provocable LVOT obstruction in HCM. I showed this technique to be easy, reproducible, comparable to magnetic resonance imaging, and can be quickly calculated using standard echocardiographic software. Patients have a smaller aorto-septal angle than controls, where it is associated with higher peak LVOT gradient. A reduced aorto-septal angle is highly specific for provocable LVOT obstruction and should prompt further evaluation in symptomatic patients without resting gradients. I used a non-invasive technique for measuring cardiac output to determine the relation between LVOT obstruction, cardiac output and peripheral oxygen utilisation in patients with HCM during exercise. I demonstrated that cardiac output response to exercise is impaired, caused largely by failure to appropriately augment stroke volume. LVOT obstruction is associated with greater impairment of stroke volume at peak exercise and is an independent and modifiable predictor of cardiac output reserve. However, heterogenous responses are seen between patients who otherwise appear similar using standard clinical criteria. There is therefore a strong argument for the individualisation of therapy in patients with LVOT obstruction. Invasive therapies to reduce gradients may work better in those with genuine obstruction to the outflow of blood, rather than for example myocardial ischaemia or mitral regurgitation. The non-invasive measurement of haemodynamic indices during exercise is practical, aids understanding of the complex physiological basis behind symptoms and may help to tailor therapy for HCM, and in particular LVOT obstruction.

Endothelial cells (ECs) form a dynamic surface within blood vessels, constantly monitoring and responding to their local environment by synthesising and releasing diffusible and cell surface bioactive molecules. The regulated secretion of a range of such molecules is mediated by the exocytosis of secretory granules (SGs) called Weibel-Palade bodies (WPBs). Optical data has suggested that WPBs undergo different forms of exocytosis whereby subsets of cargo molecules are released or retained based on their size. In other cell types the SG fusion pore is implicated in regulating selective release of specific molecules, however almost nothing is known about the WPB fusion pore. A highly effective method for studying fusion pore formation and expansion is carbon fibre amperometry, an electrochemical technique, allowing each stage of exocytosis to be quantified with submillisecond resolution. The primary aim of this thesis was therefore to establish carbon fibre amperometry as a technique to measure the WPB fusion pore in order to analyse its dynamics for the first time. Because WPBs did not contain endogenous oxidisable molecules suitable for amperometric detection, methods were developed to specifically load WPBs with suitable molecules. Amperometry was then used in combination with live cell imaging of WPB exocytosis to provide direct characterisation of the properties of the WPB fusion pore during Ca2+-driven WPB exocytosis. WPB fusion pore parameters were comparable to those reported for SGs in other cell types (e.g. chromaffin cells), indicating that WPBs may share similar processes controlling membrane fusion and the mobilisation and release of oxidisable species. Half of the exocytotic events demonstrated a pre-spike foot (PSF) signal prior to the current spike indicating that a restricted fusion pore had initially formed. In rare cases PSF signals showed step changes or fluctuations suggesting that during expansion the fusion pore may transition through different configurations before fully opening. Following characterisation of the WPB fusion pore under control conditions, factors which may affect the behaviour of the fusion pore were investigated, including the role of PM cholesterol. In line with previous studies, depletion of PM cholesterol increased the rate of fusion pore expansion and decreased the duration of the lifetime of the restricted fusion pore. This has been attributed to the promotion of the formation of a restricted fusion pore due to the intrinsic negative curvature of cholesterol, which is subsequently destabilised upon removal of cholesterol. Results presented here therefore support this idea. However, in contrast to results obtained from a range of cell types, which reported an inhibition of SG exocytosis following PM cholesterol depletion, WPB exocytosis remained largely unperturbed providing evidence for the insensitivity of the EC response to removal of PM cholesterol. In conclusion, the work presented in this thesis provides a detailed characterisation of the WPB fusion pore and has begun to address factors which may be important for its regulation. The extensive characterisation under control conditions now allows further elements potentially involved in the regulation of the WPB fusion pore to be investigated.

Heart Rate Variability describes the beat-to-beat variation in heart rate arising from activity of the sympathetic and parasympathetic branches of the Autonomic Nervous System (ANS). Reduced ANS tone measured by reduced heart rate variability (HRV) is a powerful predictor of adverse diagnosis in patients and is associated with increased mortality. Published research suggests that inflammation has a deleterious effect on Autonomic Nervous System tone. This study aimed to: establish if mild inflammatory conditions are associated with changes in autonomic tone as defined by heart rate variability studies in the following conditions: a. Influenza vaccination b. Reduction in oesophageal inflammation c. Reduction in weight The aim of the first study was to assess the link between inflammation resulting from the influenza vaccination and the associated changes on heart rate variability. 71 healthy volunteers opting to have a routine influenza vaccination were investigated for potential changes in cardiovascular autonomic tone associated with the temporary inflammatory effects of an Influenza vaccination. A number of temporal and frequency domain parameters of heart rate and breathing were assessed 2-5 days prior to vaccination and 1-4 days post vaccination. A sub-group of 15 volunteers who reported significant symptomatic reaction to the vaccination for at least 24 hours following vaccination displayed a statistically significant (p=<0.02) reduction in five of the six HRV parameters obtained during metronome-guided breathing. There was no evidence of significant reduction in autonomic tone following vaccination in the full sample of 71 volunteers. The aim of the second study was to establish whether inflammation resulting from erosive or non-erosive oesophagitis caused by gastro-oesophageal reflux disease had any association with changes in heart rate variability. 12 volunteers with non-erosive oesophageal reflux disease (NERD) and 8 with erosive oesophageal reflux disease (ERD) were investigated for HRV after initial diagnosis under gastroscopy. HRV assessment was repeated following 8 weeks of treatment with a proton-pump inhibitor (PPI). Initial reflux symptoms and response to PPI treatment were assessed using the GERD Impact Scale questionnaire. All participants had effective symptom response to treatment and there was no significant difference insymptoms score between NERD and ERD groups. There was a small but statistically significant increase in HRV detected following PPI treatment in the ERD group (p=0.05). The aim of the third study was to assess the link between obesity / pro-inflammatory adiposity, weight loss and the associated changes in heart rate variability. 38 clinically obese volunteers (BMI 30-39) with a family history of diabetes were reviewed for HRV prior to and following a lifestyle intervention designed to reduce body weight and BMI. Volunteers underwent repeated HRV studies after 4 months and 8 months of treatment. Volunteers on average achieved a weight loss of 11.5% (±6.0). There were statistically significant changes in HRV parameters in sub-group A (BMI ≥36) and correlation of biochemical measures with weight loss. These results further elucidate the effect of mild inflammatory triggers on autonomic tone as measured by HRV. These effects and their significance are discussed in detail in this document. The significance of the ‘cholinergic anti-inflammatory pathway’ is discussed with respect to the inflammatory conditions investigated. Suggestions for further work are proposed. In conclusion it is entirely possible to measure subtle changes in heart rate variability associated with mild inflammation and that on the evidence presented here these changes in heart rate variability are hypothesised to be reversible. My original contribution to knowledge is: 1. Changes in heart rate variability are associated with low grade inflammation resulting from the Influenza vaccination, erosive oesophagitis and increased adiposity. 2. Measurement of subtle changes in autonomic tone, associated with inflammatory challenges is possible and concurs with other published research. 3. The level of HRV attenuation does appear to be linked to those with a higher level of inflammation. In each study the most significant results came from subgroups of volunteers either demonstrating: a higher level of symptom severity, erosive oesophagitis or were in a subgroup of participants with the highest BMI / adipose tissue. 4. In the early stages of reduced heart rate variability we see that concurrent reduction in inflammation is associated with an increase in autonomic tone.

Various temporal aspects of arrhythmias containing frequent, single ventricular ectopic complexes are investi¬ gated using 24 hour tape recordings of ECGs, High-speed electronic analysis of records containing such complexes reveal characteristics difficult to reconcile with existing theories of the origin of these beats. A general hypothesis (posed by Vellani and Neilson) is investigated in order to explain these arrhythmias. The general hypothesis assumes that a ventricular parasystolic pacemaker, although protected by entrance block, is not totally impervious to sinus excitation of the surrounding ventricular myocardium. The action of sinus excitation on the ectopic focus is graded with respect to its time of arrival in the ectopic pacemaker cycle and this may cause synchronization between the otherwise separate sinus and ectopic rhythms. Divers experimental evidence and various reported arrhythmias are cited in support of the general hypothesis. Four particular hypotheses, whereby the action of sinus excitation affects the ectopic rhythm, are formulated on the basis of existing knowledge of cardiac electrophysiology. These are simulated on purpose-built electronic analogues in order to investigate their arrhythmic properties which are compared to the characteristics seen earlier in ECGs. It is concluded that two of these particular hypotheses contain the most likely explanation of the ECGs investigated, and that these hypotheses may account for many arrhythmias containing single ventricular ectopic complexes. It is pointed out that much of cardiac electrical activity, both normal and abnormal, may be viewed in terms of coupled relaxation oscillations, and that the proposed hypotheses emanate naturally from such thinking.

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