Echocardiography (echo) is a widely available method to obtain images of the heart, however, echo can suffer due to the presence of artefacts, high noise and a restricted field-of-view. One method to overcome these limitations is to use multiple images, using the "best" parts from each image to produce a higher quality "compounded" image. This thesis describes a new method to allow multiple 3D echo images to be compounded into a single better quality volume. I have proposed a definition for an "ideal" compounded image and have used this to guide the design of my compounding method, in particular designing a method to reduce the effect of image artefacts and to make use of larger numbers of images. My compounding method has been validated using phantom, volunteer and clinical images. The overall motivations for improving echo image quality are twofold: Firstly to provide clinicians with higher quality images which I hope will improve the accuracy of clinical decision making. Secondly to provide higher quality images for subsequent post-processing algorithms. A number of methods have been proposed to compound sets of ultra-sound images, all of which have reported improvements in image quality. However, previous 3D compounding methods have typically been applied to a relatively small number of images (most of them only use two images, and only one uses six images). I have investigated the effect of compounding with larger numbers of images. Results showed continued improvement in image quality up to ten images (the maximum number we deemed feasible to acquire in a clinical setting and it is approximately double of images used previously). Artefacts occur regularly within echo images, particularly shadowing artefacts (due to the highly reflecting interfaces caused by the ribs and lungs when imaging the heart). However, previous 3D compounding methods haven’t directly claimed and demonstrated the effect of artefacts. Therefore, I have proposed a 3D compounding algorithm which specifically aims to reduce the effect of echo artefacts (shadowing) as well as improving the signal-to-noise ratio, contrast, and extending the field-of-view. My method to reduce the effect of artefacts is to weight image information from different views based on a local feature coherence/consistency. I hypothesize that the presence of an artefact in an image varies greatly depending on view direction, therefore much lower consistency values will be calculated for artefact regions enabling them to be detected, and their influence on the compounded image to be greatly reduced. The accuracy of the image registration is important and errors will likely affect the final compounded images quality. In addition to registration ac-curacy my system needs to work robustly and have a large enough capture range to enable automatic registration from a suitable starting position.

TRPA1 is a member of the TRP superfamily; localised to neural and non-neuronal sites. TRPA1 is activated endogenously by products of oxidative stress, where its expression on sensory neurones leads to the release of vasoactive neuropeptides. Exogenous agonists of TRPAl, mustard oil and cinnamaldehyde, have been shown to cause concentration-dependent vasorelaxation of blood vessels via a variety of mechanisms. My PhD used TRPA1 WT and KO mice to investigate the potential for TRPA1 to alter peripheral artery tone and the implications of this on systemic blood pressure. I also studied the development of angiotensin II induced hypertension and associated pathologies. Wire myography using murine TRPA1 WT and KO mesenteric arteries showed cinnamaldehyde to cause concentration-dependent vasorelaxation comprising a TRPA1 dependent component, which was endothelial independent and mediated by CGRP and hyperpolarisaton. Basal blood pressure monitoring by both tail cuff plethysmography and telemetry showed no overall effect of TRPA1 deletion on basal hemodynamics. However, TRPAl KO mice displayed a previously unreported hyperactivity phenotype, measured by both telemetry and voluntary wheel running. 14 day infusion of angiotensin II by osmotic minipump induced similar hypertension in both TRPA1 WT and KO mice. Hypertrophy of the heart was seen in both genotypes, but of significantly increased magnitude in TRPA1 KO mice. Further analysis of associated inflammatory biomarkers by RT qPCR and MSD multiplex ELISA showed upregulation of pro-oxidative genes in hypertensive mice of both genotype. This was significantly greater in hypertensive TRPA1 KO mice than in hypertensive TRPA1 WT mice. These findings may partially explain the increase in hypertrophy in these mice. Angiotensin II infused mice of both genotypes showed increases in chemokine and cytokine expression. Striking, increases in IL6 and MCP-1 seen in hypertensive WT mice were significantly blunted in hypertensive KO mice, suggesting that TRPAl may differentially modulate inflammatory responses.

The haemoglobinopathies are the most prevalent genetically inherited disorders worldwide. Gene therapy for p-thalassaemia is particularly challenging given the requirement for high p-globin chain production in a lineage-specific manner. Advances in lentiviral vector (LV) technology, together with efforts to incorporate erythroid-specific regulatory elements into gene therapy vectors have yielded some promising results in both in vitro and in vivo studies with a number of clinical trials either underway or planned all using an ex vivo approach for the genetic correction of patient haematopoietic stem cells (HSCs). Despite this, current vectors are still sub-optimal: LV integration into the host-cell genome takes place near or within genes being actively transcribed, carrying a potential chance of causing insertional mutagenesis; long-term silencing of integrated transgenes with effect on reproducibility of expression; genetic correction in later developmental stages or adulthood that fails to avert phenotypic manifestations of the condition. This study forms the basis of a long-term programme to develop novel approaches to thalassaemia gene therapy, namely via an in utero delivery route that in principle could be readily applicable worldwide and thus have a positive global impact in the treatment of the haemoglobinopathies. We started with a comparative study with ubiquitous and tissue specific promoter/enhancer combinations in an integration defective lentiviral vector (ID-LV) configuration to assess their ability to express efficiently. Our results show that tissue specific expression of a human P-globin gene (HBE) regulated cassette from within an ID-LV can be obtained albeit at low levels of per vector copy number. The use of ID-LV for targeting HSC clearly awaits the development of versions of this vector system that are capable of replications and retention. We next performed a comparative analysis of UCOE-based lentiviral vectors in a murine neonatal intravascular delivery model system in order to find the ideal marking vector for further in utero studies, hi this comparative study UCOE-eGFP-WPRE proved to be the vector less prone to positional variegation and silencing effects, showing in reproducible levels of expression. Hence, this vector was chosen for further in vivo studies. Our main objective was to address the possibility of an in utero gene therapy approach for the haemoglobinopathies. This methodology aims to take advantage of the migration of HSCs from the foetal liver to bone marrow during development. Therefore, genetic correction of HSC in foetal liver should in principle provide a life-long cure. We initially embarked on a comparative experiment with a UCOE-eGFP-WPRE LV and its twin vector containing a luciferase reporter gene (UCOE-LUC-WPRE), to determine the optimal time of delivery in a murine in utero delivery model system. Injection via the vitelline vessel at E14 and El6 showed little difference with respect to marking of erythroid precursors. However, there appeared to be a higher level of transduced myeloid cells from injections conducted at El6. Following this, we performed vitelline vessel injection at E14 in wild-type mice with a therapeutic GLOBE-based LV containing an HBB cassette consisting of PLCR elements HS2 and HS3 linked to a mini-HBB gene, and its associated transcription termination region. Analysis of recipient mice 3 months post-injection showed vector presence (average copy number 1.347) and detectable haemoglobin levels in peripheral blood.

NADPH oxidases are a family of reactive oxygen species generating enzymes which are implicated in cardiovascular pathophysiology. The Nox2-based NADPH oxidase has been reported to be involved in the development of cardiac hypertrophy and fibrosis. However the relevant cellular source(s) involved in the above effects remains unclear. The aim of this thesis was to investigate the critical cell source of the Nox2 in a model of angiotensin II (AngII) induced left ventricular hypertrophy (LVH) and fibrosis. ROS production in resident cardiac fibroblasts was stimulated by AngII, aldosterone and TGFβ to investigate cardiac fibroblasts as a crucial cell source for Nox2. Only TGFβ elicited a Nox2 dependent ROS response thus placing Nox2 downstream of TGFβ. As AngII stimulates cardiac fibrosis and hypertrophy via downstream TGFβ, resident cardiac fibroblasts could be another critical cell source for Nox2 in the development of cardiac fibrosis and hypertrophy. The importance of Nox2 in macrophages/monocytes (peripheral circulating cell) migration was investigated by way of Dunn Chambers. Nox2 was shown to be crucial in the chemotaxis of these cells towards CSF-1 stimulation. Also the speed and persistence of their migration was significantly reduced following the loss of Nox2. Chimeric mice (WT mice with Nox2-/- marrow and Nox2-/- mice with WT marrow) were generated in-house by lethal radiation and marrow rescue with cells from mice of different genotype. Following AngII stimulation the chimeric mice demonstrated a hypertensive response. The loss of Nox2 in the circulating cells prevented the development of left ventricular hypertrophy suggesting that the peripheral circulating cells were a critical cell source for Nox2 in the development of cardiac hypertrophy. The loss of Nox2 in the resident cardiac cells prevented the development of cardiac fibrosis thus suggesting that the resident cardiac cells were the critical cell source for Nox2 in the development of cardiac fibrosis. These results indicate that Nox2 has a cell specific role in the development of cardiac fibrosis and hypertrophy following AngII stimulation.

There is growing evidence to suggest that abdominal aortic aneurysm (AAA) is a complex disease with multiple environmental and genetic risk factors. Susceptibility genes have been investigated through multiple underpowered candidate gene studies, which has led to the association of numerous genes with conflicting evidence. This study aimed to investigate the commonly cited associations between polymorphisms of the matrix metalloproteinase-9; MMP9(-1562C > T), tissue inhibitor of metalloproteinase-1; TIMP1(+434C > T) and TIMP1(rs2070584), platelet activating factor acetylhydrolase; PLA2G7(- 994G > T), estrogen receptor beta; ESR2(+1730 A-G) and Heme oxygenase 1; HMOX1(GT)n genes and AAA in a large powered study to provide definitive evidence of any association. Materials & Methods: A case-control study was performed of 1,202 patients with AAA and 1,059 screened control subjects. DNA was extracted from whole blood and genotyping was performed using polymerase chain reaction based restriction fragment length polymorphisms (PCR-RFLP). Results Two polymorphisms (ESR2 (OR 1.42, P < 0.0001) and HMOX1 (OR 1.99, P < 0.0001)) showed a potential association with AAA. One polymorphism (TIMP1 rs2070584) could not be genotyped despite using 2 different methods. Polymorphisms of the MMP9 (OR 0.99, P=0.82), PLA2G7 (OR 0.76, P=0.29) and TIMP1 (+434C > T) (OR 0.94, P=0.46) genes did not show an association with AAA. Conclusion: We have demonstrated an association between polymorphisms of the ESR2 and HMOX1 genes and AAA, although further work is essential to confirm this association. Contrary to other published data, no such association was seen in common polymorphisms of the MMP9, TIMP1, and PLA2G7 genes and AAA.

Sickle cell haemoglobin differs from normal haemoglobin by a single amino acid in its chain. This amino acid replacement, from glutamic acid to valine, causes polymerisation of proteins into defined long insoluble fibres with a typical diameter of 21.5 nm. The polymerisation is triggered by the formation of deox haemoglobin from oxyhaemoglobin in low oxygen partial pressures, which results in a conformational change in the secondary structure of the protein. Pathogenesis in sickle cell disease depends on the polymerisation and gelation of deoxygenated HbS molecules. In this work, an electrochemical method has been described to modulate the oxygen concentration in an optically transparent thin layer cell to produce deoxyhaemoglobin whilst monitoring the extent of polymerisation using turbidity measurements. The oxygen was depleted in the vicinity of the electrode and triggered the polymerisation. The dependence of protein concentration, temperature, pH and ionic strength on the nucleation and elongation of HbS polymerisation was characterised at the electrode surface and the kinetics of polymerisation was investigated using a model for fibrillogenesis describing a two-step process of nucleation followed by elongation. The rate constants, determined for a number of conditions, showed that nucleation is far slower than the growth whilst polymerisation at the surface was demonstrated to occur in three stages with an initial time delay when no structures were observed followed b growth of fibrous hair-like strands and finally gel-like aggregation. An understanding of the factors which affect polymerisation at a surface and an insight into the dynamics and mechanism of polymer aggregation and the pathophysiology of sickle cell disease has been provided. A screening method for substances that effect the fibre nucleation and/or growth that could be valuable to the pharmaceutical industry for treating sickle cell disease is also presented.

Cardiac ischaemia/reperfusion (I/R)-injury and hypertrophy are two causes of heart failure. The molecular pathways that regulate these processes include those that are stimulated by the cytokines, interferon-gamma (IFNy) and cardiotrophin-1 (CT-1). This thesis describes the investigation of components of the IFNy or CT-1 signalling pathways and their regulation of I/R-injury or hypertrophy. The transcription factor, signal transducer and activator of transcription 1 (STAT1), is the primary mediator of the IFNy signalling pathway and promotes apoptosis of cardiac cells following I/R-injury or stimulation with IFNy. It is unclear what contribution the caspase-8 and caspase-9 apoptotic pathways have in STAT1 mediated I/R-injury and IFNy induced apoptosis. Therefore, to investigate this relationship, constructs conditionally overexpressing specific caspase inhibitors were used to generate transgenic mice with inactivated caspase-8 or -9 pathways. STAT1 and the transcription factor p53 proteins are known to associate and this interaction has been demonstrated to be necessary for the maximal activation of distinct p53 target genes. STAT1 and p53 double knockout mice were generated to further investigate the role of these proteins in I/R-injury. A proteomic approach was used to identify proteins regulated by STAT1 and/or p53 in the heart where the expression of the cytokine, interleukin-6, was regulated by STAT1 and p53. The CT-1 cytokine has cardioprotective and hypertrophic effects. CT-1 expression is induced by another cardioprotective cytokine, urocortin. The mechanism by which urocortin protects the heart from I/R-injury is not fully understood, however, prevention of mitochondrial damage has been shown to be involved. In this study the protective effect of urocortin was shown to involve the mitochondrial permeability transition pore. Furthermore, the hypertrophic effect of CT-1 in cultured cardiac cells has been shown to be dependent on induction of heat shock protein 56 (hsp56). Transgenic mice overexpressing hsp56 were generated to test the cardioprotective and hypertrophic effects of hsp56 in vivo. Taken together, the data presented here demonstrate clearly that a number of key molecules regulate cardiac (patho)physiology in vivo. Targeting these signalling pathways may provide novel routes for therapeutic intervention in heart disease.

The release and clearance of von Willebrand Factor (VWF) in a group of patients with the quantitative deficiency type 1 von Willebrand Disease (VWD) was investigated. This was done by analysis of circulating VWF and VWF released from the endothelial pool on infusion of a vasopressin analogue. A variety of parameters were investigated pre-and post infusion in order to identify VWF gene linked and non-linked variables that could affect VWF clearance. Increased clearance of plasma VWF in a significant proportion of type 1 VWD patients was shown but this was not consistently associated with steady-state levels of VWF, indicating that circulating plasma VWF levels are not a consistent reflection of the VWF life-cycle in this patient group. An association between galactose exposure and reduced levels of VWF was demonstrated by the increased binding of the lectins Ricinus communis and Erythina crystagalli, this was unrelated to clearance. In addition, no significant ABO blood group effect on VWF clearance was demonstrated. The absolute level of ADAMTS-13, and the susceptibility of VWF to cleavage by ADAMTS-13 were not associated with the clearance rate of VWF in patients with type 1 VWD. Three novel candidate mutations were identified in association with significantly accelerated VWF clearance. Notably, candidate mutations were generally identified in patients with steady-state VWF levels reduced to <20 IUdL"1 and family analysis suggests absolute linkage with the VWF gene. Despite demonstrating an increased rate of clearance in the majority of patients with type 1 VWD, no single underlying common characteristic or variable was predominant within this study group.

Impairment of vascular function and tissue injury after ischaemic events is a well-known pathophysiological phenomenon. Enhanced oxidative stress by free radicals appears to be a major contributor to the ischaemic episode which can occur either by progression of a disease state (atherosclerosis or angina pectoris) or after surgical intervention (organ transplantation). As a new approach for the preservation of vascular function after ischaemia, one line of research is considering the exposure of tissues to agents or treatments that lead to the stimulation of a number of natural intracellular defense mechanisms. This phenomenon is generally known as the “stress response” and invariably involves the de-novo¬ synthesis of inducible proteins known as heat shock proteins. One such mechanism is the induction of the haem oxygenase (HO) pathway. Haem oxygenase is the mammalian enzyme which degrades haem to the antioxidant biliverdin and carbon monoxide (CO) releasing ferric (Fe3+) iron. Biliverdin is rapidly converted to another potent antioxidant, bilirubin, by the cytosolic enzyme biliverdin reductase. Of the three known isozymes, the inducible isoform (HO-1) has been proposed to act as an effective system to counteract oxidant-mediated cell injury since it is extremely sensitive to up-regulation in a variety of mammalian tissues. The aim of this research project is to investigate the role of this potentially beneficial enzyme system in ameliorating vascular dysfunction following stressful insults. In particular, 1) the way in which the HO-1 gene is modulated; 2) the role of CO and bilirubin as important effector molecules and 3) the dynamics of the HO-1/bilirubin pathway are examined.

Psychosocial factors are thought to contribute to the long term development of coronary artery disease (CAD), to the triggering of cardiac events in people with advanced disease, and to adaptation following acute coronary syndromes (ACS). My thesis presents three studies addressing different aspects of the relationship between emotional factors and CAD, using different methodologies. They focus on the role of negative emotions in vulnerability to myocardial ischaemia in daily life, the influence of acute emotional triggers of ACS on long term quality of life, and the effect of depression following ACS on a particularly important aspect of adaptation, namely return to work. The first study, called the Silent Ischaemia Study (SIS) investigated 88 out-patients with suspected CAD who underwent 24 hour ambulatory electrocardiogram (ECG) monitoring, together with saliva sampling and characterisation of daily life by a new method called the Day Reconstruction Method (DRM). The results indicated that in patients with definite CAD, depressed mood was associated with reduced high frequency and increased low frequency heart rate variability (HRV), suggestive of parasympathetic withdrawal. The Cortisol slope over the day was flatter in more depressed patients with CAD. Episodes of transient ischaemia and/or arrhythmia were also associated with increased negative affect, but their incidence was low, primarily because most patients were medicated with beta blockers. The second and third studies derive from the ACCENT (Acute Coronary Syndrome, Emotion and Triggers) study, exploring long term adaptation following ACS. Analyses showed that the likelihood of returning to work was negatively associated with depression immediately following ACS, independently of clinical and demographic factors, and that emotional triggers predicted elevated anxiety and poor mental health status at 12 and 36 months independently of covariates. In combination, these studies suggest that negative emotional status contribute both to the onset of acute cardiac events, and to adaptation following ACS.