Abdominal aortic aneurysms (AAA) account for 5,251 deaths per year in the UK (2010). Their current incidence in the NHS AAA screening programme is 1.8%, however the vast majority are small. These patients therefore undergo surveillance prior to the need for surgical intervention. Biomarkers for AAA have been extensively studied, with a meta-analysis, conducted herein, identifying a significant association between several biomarkers and AAA (upregulation of matrix metalloproteinase-2, matrix metalloproteinase-9, tissue inhibitor of matrix metalloproteinase-1, interleukin-6, interleukin-10, tumour necrosis factor-α, osteoprotegerin, osteopontin, interferon-γ, intercellular adhesion molecule-1, vascular cell adhesion protein-1, D-dimer, C-reactive protein, alpha-1 antitrypsin, fibrinogen, triglycerides, and lipoprotein(a), and downregulation of apolipoprotein-A and high density lipoprotein). However, the sensitivity and specificity of these biomarkers is poor, therefore this thesis aimed to look at the expression of microRNAs (miRNAs) in AAA, hitherto previously unstudied. MiRNAs are short, non-coding RNA sequences which are transcribed from DNA, however they are not translated into protein. They exert their effect by attaching to messenger RNA (mRNA) and causing repression of translation into protein, and deadenylation, thus causing mRNA degradation. MiRNAs are capable of interacting with over 60% of known genes. The studies within this thesis have undertaken a case control discovery and validation study into miRNAs in AAA, identifying a significant upregulation of 29 miRNAs within the discovery study, 4 of which were validated in blood (let-7e, miR-15a, miR-196b, miR-411), and miR-196b being further validated in plasma. There was however no miRNA dysregulation found in aortic tissue. In addition, these 4 miRNAs were found to have significant interactions with previously studied AAA biomarkers identified through earlier systematic review and meta-analysis. The 4 miRNAs identified within this thesis were similarly dysregulated in patients with peripheral arterial disease, therefore they may be dysregulated due to generalised atherosclerosis rather than AAA, and must be interpreted with caution.

Abdominal aortic aneurysm is an important cause of death globally, however, its impact is less today than two decades ago due to a decline in AAA mortality. Within the same timeframe changes have occurred to the way that AAA may be treated, for example an increasing use of endovascular surgical techniques. Type II endoleak is one of the most common complication of endovascular aneurysm repair. The sequela of having a type II endoleak is however unknown. My objectives within this thesis were to analyse causes of the decline in aneurysm mortality being seen in many developed countries using data derived from the World Health Organisation and investigate short/medium term outcomes of patients with type II endoleak at a single centre in the United Kingdom. Through these studies I have demonstrated a robust association between trends in established cardiovascular risk factors and mortality from AAA suggesting that a reduction in the global burden of high cholesterol (P=0.0082), hypertension (P=0.028) and smoking (P=0.017) have led to a drop in AAA mortality. Aneurysm rupture in patients with an isolated type II endoleak appears to be rare occurring in less than 1% of all literature reported type II endoleaks and no ruptures were recorded in patients with type II endoleak followed up prospectively. Patients with isolated type II endoleak demonstrate equivalent aneurysm related mortality to those without, however, there is a strong independent association between type II endoleak and 5mm of aneurysm sac expansion (P=0.0001). A conservative strategy to the treatment of type II endoleak appears to be safe and given time isolated type II endoleak appear to have a good chance of spontaneously resolving without the need for invasive intervention. For those patients with type II endoleak and 10mm of aneurysm sac expansion, further research is needed to investigate the risk versus benefit of intervention.

Coronary artery disease (CAD) is the commonest cause of death worldwide. It is a multifactorial disease caused by interplay between genes and environment. Defining genes that affect CAD risk and understanding their mechanisms may help to improve its prevention and treatment. Recent genome-wide association studies have identified several novel loci associated with CAD. In this thesis I examined two of these loci. The first was a locus on chromosome 1p13.3. I showed that this locus was also associated with LDL-cholesterol. Fine-mapping of the locus and in silico analyses suggested that SNP rs12740374, where the risk allele disrupts binding of a liver-specific transcription factor, is probably the causal variant at the locus. Finally, I showed that the lipid-lowering effect of statin is independent of this locus, suggesting that targeting the mechanism by which this locus affects LDL-cholesterol may provide additional therapeutic benefit. The second locus was on chromosome 13q34 located near the COL4A1 and COL4A2 genes which code for collagen Type 4, a key component of the arterial wall. In silico analyses identified an intronic 4-SNP haplotype within a bidirectional promoter that was likely to include the causal variant(s). A putative kidney expression QTL was identified with the CAD risk allele associated with lower expression of COL4A1 and COL4A2. Functional experiments showed DNA-protein binding to the SNPs in the haploblock. However, there were no differences in level of DNA-binding between the alleles or a differential impact on gene expression. In a clinical study, there was no difference between the 13q34 genotype groups in restenosis rates in patients undergoing percutaneous coronary angioplasty. In conclusion, the work on locus 1p13.3 shows that it is possible to identify a causal SNP from a GWA study signal, and elucidate its mechanism, but the studies on locus 13q34, indicates that this is a challenging process.

Myocardial infarction is the main cause of death in the United Kingdom. Early reperfusion of coronary artery occlusion has greatly improved mortality, though restoration of blood supply may perpetuate cell death through reperfusion injury. Preconditioning is a potent endogenous form of cardioprotection triggered through preceding brief nonperfusion of the heart’s blood supply. In remote conditioning it is triggered by intermittent tourniquet ischaemia of a limb. However a limited understanding of the mechanisms underlying transfer of a signal from the peripheries, its reception in the heart, and the impact of comorbid disease on this process hinders its application to the clinical setting of myocardial infarction. This work trials several models of reperfusion injury, and optimises a method of centrifugation of adult rat ventricular myocytes into a dense pellet to induce ischaemia, and simulate reperfusion by its dispersal. Remote preconditioning is evoked by preincubation of myocytes with serum samples taken from participants. This is used as a screening tool in order to test serum samples acquired from volunteers in control and disease states undergoing tourniquet ischaemia of a peripheral limb to reproduce the stimulus of remote preconditioning. A protective signal was seen in serum taken from healthy subjects following remote preconditioning versus baseline serum (20.5±3.3 vs 37.2±4.5 % necrosis respectively, n = 21, p < 0.001). Protection is absent in diabetes mellitus type 1 (51.5±4.6% necrosis, n = 14) and type 2 (51.3±8.2% necrosis, n = 10). The protective signal is preserved with age in healthy male participants, though appears to decline with age in a preliminary cohort of female participants. On assay of putative mechanisms of remote preconditioning, serum nitrite did not change with preconditioning in healthy volunteers, though it was found to significantly decrease in diabetes mellitus type 1. The implications for the application of this powerful yet elusive form of innate cardiac protection are considered.

Background : Nystagmus is an involuntary to and fro movement of the eyes that can be infantile or acquired and causes reduced vision. Little literature exists documenting how nystagmus affects quality of life and treatment options are largely empirical with very few randomised controlled trials. Methods : Semi-structured interviews of 21 participants were analysed using a constant comparison approach based upon grounded theory. A randomised, triple masked, controlled trial of gabapentin and memantine was performed on 66 participants with infantile nystagmus. Outcome measures were 25% improvement in visual acuity (primary outcome), 25% improvement in eye movement, absolute change in visual function and eye movement and descriptive analysis of nystagmus waveforms responding to treatment. Results : Analysis of participants’ accounts revealed six domains of living that were adversely affected by nystagmus: visual function, restriction of movement (both physical and social), standing out/not fitting in, feelings about the inner self, negativity with regards to the future and relationships. Cosmetic appearance of nystagmus was, for the first time, described as being problematic and additional to other categories was an overarching and universal distress arising from nystagmus affecting every aspect of everyday life. Gabapentin and memantine did not improve visual function as a 25% improvement or when assessing the absolute differences. In contrast null region nystagmus intensity and NAFX were significantly different for treatment as gabapentin reduced intensity and improved NAFX by 25%. For absolute differences in null region nystagmus intensity gabapentin was also significant. Pendular waveforms prevalently appeared in those participants responding to gabapentin. Baseline measurements were often a predictor for success of treatment. Both gabapentin and memantine were both reasonably well tolerated. Conclusion : Interviews revealed universally negative experiences of living with nystagmus that are previously unreported. Gabapentin, and not memantine, significantly reduces eye movement in infantile nystagmus but does not improve visual function.

Heart failure is a complex clinical syndrome that occurs at the end stage of heart disease with high costs and poor outcomes. Despite advances in therapy, improving clinical outcomes remains a challenge for physicians with 50% of patients dying within 5 years. The main aim of this study was to discover novel biomarkers in plasma that could predict treatment response in patients with heart failure using plasma proteomics. The use of two dimensional liquid chromatography coupled to electrospray ionisation tandem mass spectrometry in high definition ion mobility combined with a multiple affinity removal system column and immunoluminometric assay discovered CD180 antigen, Heat shock 70 kDa protein 4L, Leukemia inhibitory factor receptor and Neurotrimin as novel biomarkers which are able to predict treatment response in patients with heart failure. Moreover, Thyroid receptor interacting protein 11, Patatin like phospholipase domain containing protein 2 and Mannan binding lectin serine protease 2 were identified as novel biomarkers for prediction of death in patients with heart failure. Furthermore, two multiple biomarker models were developed from the findings obtained of using matrix assisted laser desorption ionisation mass spectrometry combined with C18 solid phase extraction which are able to predict treatment response in patients with heart failure. The model with seven peptide peaks showed an excellent area under the receiver operating characteristic curve (AUC) of 0.907. In particular, the model with seventeen peptide peaks achieved the maximum AUC of 1.000 (100% sensitivity and 100% specificity). The discovery of novel biomarkers in this study not only adds information to understand the pathophysiological mechanisms of heart failure better, but also may provide a more accurate prediction of treatment response to guide medical therapy. This may enable the practice of stratified medicine in the future. Moreover, novel therapeutic targets could be identified for design of new drugs to improve outcomes.

Ischaemia reperfusion injury results in metabolic disruption in cardiac cells, potentially leading to cell death and ultimately heart failure. The regulation of the cellular response to ischaemia reperfusion is not fully understood. Previously, the muscle-enriched protein MS1 was shown to be upregulated immediately after physiological and pathological muscle stimulation, possibly acting in the early phase of the stress response. The aims of this work were two-fold. Firstly, to identify transcription factors binding the UP2 site, an evolutionarily conserved putative regulatory region upstream of the MS1 transcriptional start site. In silico analyses were conducted to identify candidate transcription factors, before in vitro assays using H9c2 cardiomyoblasts were used to confirm binding of the identified factors. It was demonstrated that the Ets transcription factors Elf1 and Fli1 can bind the site in vitro, and that Fli1, in concert with GATA2, can modulate MS1 and UP2 expression. Secondly, it was shown that MS1 mRNA and protein are upregulated in response to simulated ischaemia reperfusion in vitro in a similar time-course to its upregulation in pathologically stressed rodent hearts and physiologically stressed human skeletal muscle. In addition, Elf1 knockdown ameliorated this response. It was also demonstrated that MS1 knockdown sensitised cells to oxidative stress. Overall, the findings suggest that MS1 is up-regulated in response to a simulated ischaemia reperfusion protocol in H9c2 cells, and this contributes to the protection of cells against oxidative stress. This response may be facilitated in part by the action of Ets proteins acting at the UP2 site. This thesis provides novel evidence for the involvement of MS1 in cardiomyocyte responses to reperfusion injury, possibly via UP2, and in the cellular protection against oxidant-induced cell death.

Background: Sedentary behaviour has emerged as a distinctive health behaviour paradigm, showing associations with markers of cardio-metabolic health independent of other lifestyle behaviours, such as physical activity. However, most investigations have been conducted in the general population without reference to specific risk factors and experimental research in humans is lacking. Aims: 1. Undertake narrative reviews investigating the effect of sedentary behaviour upon markers of cardio-metabolic health 2. Conduct a series of observational studies investigating the independent effects of sedentary behaviour on traditional and non-traditional markers of cardio- metabolic health. 3. Design and conduct an acute experimental study to establish whether reducing sitting time through regular bouts of non-sedentary activity improves cardio- metabolic health in women at high risk of T2DM. Key Findings: 1. Experimental studies demonstrate that regularly breaking sedentary behaviour with short bouts of walking imparts acute metabolic advantages. 2. Sedentary time was shown to have stronger associations with 2-hour glucose, triglycerides, HDL-cholesterol compared to moderate-to-vigorous physical activity (MVPA). 3. Sedentary time was associated with interleukin 6 (IL-6), heart, liver and visceral fat, independent of measured confounders, including glycaemia, adiposity and MVPA. 4. Compared to a prolonged bout of sitting, both standing and walking significantly reduced the glucose and insulin incremental area under the curve (iAUC) in women at high risk of T2DM. Conclusions: This thesis synthesizes the current evidence examining the relationship between sedentary behaviour and various health outcomes (Chapters Two and Three). Chapters Four, Five and Six investigate the independent effects of sedentary behaviour on markers of cardio-metabolic health through a series of observational studies. Chapter Seven examines the effectiveness of an experimental study and Chapter Eight discusses the implications of these findings whilst identifying areas for future research.

Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia in clinical practice. Over the past two decades, we have come to appreciate that AF has a significant heritable component. The recent advent of next-generation sequencing technology has spawned a new era of research into the genetic basis of AF. Genome-wide association studies (GWAS) have identified multiple common variants underlying AF. Further, exome sequencing has emerged as a potentially powerful technique for the identification of mutations underlying familial forms of AF. In this thesis, we sought to further elucidate the genetic basis of AF though two specific aims. Firstly, we investigated the mechanistic link between KCNN3, a potassium channel gene which was identified in a GWAS for lone AF, and arrhythmia pathogenesis. Secondly, we performed exome sequencing and classical linkage analysis in two AF pedigrees to identify novel mutations for the arrhythmia. We demonstrate that overexpression of Kcnn3 in a murine model results in an increased susceptibility to AF. Interestingly, these mice also display a high incidence of sudden death due to heart block. Exome sequencing in an AF pedigree identified a potentially causative mutation in the transcription factor gene GATA6. In a second AF pedigree, we identified a novel locus for the arrhythmia on chromosome 1. However the causative mutation at this locus remains elusive. Ultimately, the identification of the genetic substrate underlying AF is likely to uncover novel therapeutic targets as well as enhancing risk stratification for this common and morbid arrhythmia.

Background& Objective: Brain injury after cardiac surgery is a serious concern for patients and their families. Thousands of air bubbles enter the cerebral circulation during cardiac surgery, but whether these are harmful to the brain and impact adversely on cognition remains subject of speculation. The purpose of this study was to use MRI to characterise new and pre-existing cerebral ischaemic lesions in patients undergoing cardiac surgery, and to test whether the accumulation of new lesions adversely affects cognition. This study also draws upon recent advances in intra-operative bubble sizing to investigate whether high volumes of macro-bubbles have potential to result in new MRI lesions or increased risk of cognitive decline following surgery. Methods: The burden of pre-existing versus new ischaemic lesions was quantified based on analysis of 3T MR images and compared with the results of cognitive testing. Intraoperative Doppler ultrasound recordings were used to estimate the number, volume and diameters of bubbles entering the middle cerebral artery during surgery for comparison with MRI and cognitive outcome. Results: Post-operative lesions were identified in 31% of patients. Patients with pre-existing lesions were 10 times more likely to receive new lesions after surgery. Forty six percent of patients experienced postoperative cognitive decline, which was independent of whether new lesions were present. Intra-cardiac patients received over 16 times the total volume of air, 7 times as many macro-bubbles, 5 times as many emboli following aortic cross-clamp removal, and over twice as many emboli overall than CABG patients, but there were no significant differences in MRI or cognitive outcome. Conclusions: New MRI lesions and high numbers of intra-operative macro-bubbles are common during cardiac surgery, but we found no evidence of any adverse effect on cognition.