Cardiovascular disease remains a major cause of mortality and morbidity and is underpinned by Oxidative stress, within which, inactivation of nitric oxide (NO) by superoxide (SO) and other reactive oxygen species is characteristic. Two major enzyme systems are implicated within oxidative stress; NAD(P)H oxidase and endothelial nitric oxide synthase (eNOS). eNOS generates NO while at the same time, and within the same cells, NAD(P)H plays a powerful role in the generation of SO. Evidence is accumulating that polymorphisms of the genes encoding these enzyme systems may play an important role in the pathophysiology of CAD. Additionally there has been much recent interest in both biochemical markers of oxidative stress and low grade chronic inflammation as well as a non invasive vascular phenotype, pulse wave analysis. This thesis reports a series of studies (utilising the techniques described in chapter 2) which aimed to ascertain:- The reproducibility of pulse contour analysis as a non invasive intermediate cardiovascular phenotype (Chapter 3). Whether common single nucleotide polymorphisms of the p22phox gene CYBA and the endothelial nitric oxide synthase gene, NOS3, have an effect upon arterial compliance in patients with coronary artery disease (Chapters 4,5 and 6). In healthy volunteers, free of cardiovascular disease whether a relationship existed between markers of low grade inflammation and arterial stiffness (Chapter 7). Chapter 3: The reproducibility of diastolic pulse wave contour analysis and its relation to systolic pulse contour analysis. This clinical study demonstrated that both large (C1) and small artery (C2) compliance values were reproducible and that there was a significant correlation between both Augmentation Index (AIx) and C1and AIx and C2 in healthy volunteers and though there was no association between AIx and C1 in patients with coronary artery disease AIx did correlate with C2 in this population. Chapter 4: The effect of the G894T SNP of the NOS3 gene upon arterial stiffness in patients with coronary artery disease. There was no association observed between this polymorphism and blood pressure or large artery compliance however ANOVA revealed a statistically significant association for TT homozygosity and small artery compliance. The highest small artery compliance was seen in the patients homozygous for the G allele, an intermediate value observed in heterozygotes and the lowest value demonstrated in patients homozygous for the T allele. Multiple regression analysis, examining the possible contribution of confounders showed that only small artery compliance was significant when NOS3 G894T genotype was assigned as the dependent variable. Chapter 5: The C242T single nucleotide polymorphism of the CYBA gene and blood pressure and arterial compliance in patients with coronary artery disease. We sought to examine the influence of the C242T SNP of CYBA upon vascular compliance and blood pressure using the dominant allele model. The presence of the 242T allele was associated with significantly higher systolic blood pressure. Patients homozygous for the C allele had lower systolic blood pressure than heterozygotes and patients homozygous for the T allele. There was no statistically significant effect upon diastolic blood pressure but there was however a significant association observed between the 242T allele and pulse pressure. Chapter 6: Combined analysis of NOS3 G894T and CYBA C242T genotypes upon arterial stiffness. In order to contrast the arterial stiffness between the favourable versus the non-favourable genotypes patients homozygous for the NOS3 G allele and homozygous for the CYBA C allele were compared with those homozygous for the NOS3T allele and possessing the CYBA 242T allele. The former displayed higher large and small artery compliance than the latter group. Multiple regression analysis, examining the possible contribution of confounders showed that only the large and small artery compliance values contributed significantly when genotype was assigned as the dependent variable. Chapter 7 Chronic low grade inflammation and insulin resistance and arterial compliance in healthy volunteers. Within healthy volunteers multiple regression analysis showed that small artery compliance was significantly associated with IL 6, CRP and ICAM. Augmentation index showed only an association with ICAM1. There was no significant correlation between Adiponectin levels and either of the arterial stiffness parameters studied. Conclusions Diastolic pulse wave contour analysis is a reproducible assessment of arterial stiffness with the potential to represent a high fidelity non invasive vascular phenotype. Small artery compliance is correlated with Augmentation Index and although the measurements are not analogous they both represent useful means of acquiring quantitative data concerning arterial stiffness. The 242T allele of the p22phox gene, CYBA, is associated with decreased large but not small artery compliance and increased systolic and pulse pressure. Homozygosity for a common NOS3 polymorphism (894 GT) was associated with decreased small artery compliance but not with large artery compliance or blood pressure. The markers of chronic inflammation Interleukin 6, ICAM and hsCRP but not Adiponectin, a marker of Insulin resistance, predict small artery compliance in healthy individuals apparently free of vascular disease.

GATA-1 is a key haematopoietic transcription factor which plays a pivotal role in differentiation of the erythroid, megakaryocytic, eosinophilic, mast cell and dendritic cell lineages. Since its initial cloning and characterisation in 1989 a huge amount of information has been gathered on the molecular mechanisms of action of GATA-1. This knowledge has helped understanding of the processes by which cells enact differentiation programmes and suppress alternative lineage choices. GATA-1 produces at least two protein isoforms – the well characterised GATA-1 full-length (GATA-1FL) isoform and a truncated isoform – GATA-1 short (GATA-1s). GATA-1FL comprises two conserved Zinc fingers (which interact with DNA and essential co-factors), a C-terminal tail (of mostly unknown function) and an N-terminal domain (thought to confer activation properties to the molecule, but which may also be involved in transcriptional repression). GATA-1s lacks the N-terminal domain but is otherwise identical. The biological role of GATA-1s is unknown and this isoform received scant attention until the discovery that GATA-1FL mutations were linked to a rare, but highly informative, acute megakaryoblastic leukaemia seen in children with Down syndrome (constitutional trisomy 21). This discovery was particularly interesting, not only because the association between trisomy 21 and the X-linked GATA-1 mutation was extremely tight (being seen in 100% of the cases examined), but also because the GATA-1FL mutations were not randomly located, but rather clustered within the N-terminus, allowing unhindered production of the GATA-1s isoform. This finding led to interest in the pathological and physiological role of GATA-1s in haematopoiesis. Some insight has been gained into the pathological role of GATA-1s by creation of a GATA-1s knock-in transgenic mouse and by experiments looking at the ability of GATA-1s to rescue GATA-1 deficient embryonic stem (ES) cell lines. GATA-1s produces hyper-proliferation of fetal liver meg-erythroid progenitors but allows at least partial differentiation of these cells. However, a number of key questions remain. In particular what is the physiological role of GATA-1s and the reason for the tight association between trisomy 21 and GATA-1s mutations? Given this background, this thesis describes experiments designed to address the physiological role of GATA-1s, to establish whether additional GATA-1 isoforms exist, and to investigate the association between GATA-1 isoform expression and trisomy 21. Firstly a comprehensive expression analysis was performed in murine and human primary tissues and cell lines. This aimed to identify whether GATA-1s had a unique expression profile, either in particular lineages, or at distinct stages of haematological ontogeny. Reverse-transcriptase polymerase chain reaction (RT-PCR) and western blot analyses showed that the expression patterns of GATA-1s and GATA-1FL were virtually identical, with the possible exception of one human primary monocytic cell preparation which appeared to preferentially express GATA-1s. Before proceeding to further analysis of GATA-1s a search was made for additional GATA-1 isoforms using in silico analysis, RT-PCR and western blotting. This led to identification of a clone carrying a GATA-1 mutation involving the C-terminal tail, derived from a patient with chronic myeloid leukaemia. An analysis of the properties of this clone was performed, confirming its altered C-terminus and demonstrating that this conferred increased transactivation properties on the molecule as measured by luciferase assays. This observation suggests that the C-terminal tail may be an important, and previously under-recognised, functional region of the GATA-1 molecule. The discovery of this potentially hyper-functioning GATA-1 mutation led to investigation of whether GATA-1 mutations could be a widespread phenomenon in CML. However, GATA-1 mutational analysis in 21 patient samples from CML blast crisis did not reveal any additional coding mutations. To address the physiological role of GATA-1s, attempts were made to perform gene targeting in murine embryonic stem cells to produce isoform specific knock-out cells i.e. ES cells engineered so that they exclusively express the GATA-1FL isoform (a GATA-1s knock-out) or the GATA-1s isoform (a GATA-1FL knockout). These cells could then be used in in vitro haematopoietic differentiation assays and for transcriptional profiling. In this way it was hoped to establish whether GATA-1s fulfilled any unique roles in primitive or definitive haematopoiesis that could not be compensated for by the presence of the GATA-1FL isoform. Unfortunately, despite evidence of apparently successful targeting from PCR screening of ES cell clones, it was impossible to confirm the existence of endogenously targeted alleles on Southern blotting. Following exhaustive attempts at screening further clones and subclones (more than 1000 clones in total), this approach was abandoned in favour of transgenic expression of GATA-1 isoforms in cell lines. Transgenic expression studies in murine ES cells showed that whilst GATA-1FL expression led to an expansion in numbers and maturity of erythroid and non-erythroid haematopoietic colonies in vitro, GATA-1s was incapable of supporting colony formation in this assay. Studies then moved on to human cell lines. Two cell lines were identified, both capable of in vitro haematopoietic differentiation into megakaryocytic and erythroid cells, but one carrying trisomy 21 (Meg-01) and the other disomic for chromosome 21 (K562). GATA-1FL expression in these cells generally drove differentiation along the megakaryocytic or erythroid lineage as measured by DNA ploidy analysis, haemoglobinisation, upregulation of erythroid or megakaryocytic gene expression (by quantitative PCR) and suppression of alternative lineage genes (PU.1 and Ikaros) and genes associated with progenitor proliferation (cyclin D2 and c-myb). GATA-1s, in contrast, produced less evidence of differentiation with lower DNA ploidy, less up-regulation of erythroid genes and failure to repress other lineage and haematopoietic progenitor associated genes. Examination of the link with trisomy 21 confirmed that that the chromosome 21 candidate gene Erg3 was upregulated in trisomic cells and that expression of GATA-1s appeared to confer a selective advantage in the presence of trisomy 21. However, no clear mechanistic reasons for the selective advantage could be identified. Overall, these studies show widespread GATA-1s expression in haematopoietic cells, confirm the association with inadequate repression of genes associated with primitive progenitors, and suggest that the C-terminal tail of GATA-1 may be an important functional part of the molecule. Finally, these observations have generated a number of testable hypotheses which could form the basis for future work.

Problems with walking and attention are known to be prevalent in Multiple Sclerosis (MS), though no studies have reported how these two difficulties might interact. The study aimed to investigate the effects of performing a concurrent cognitive task when walking in MS and determine the effects of task demand on dual-task performance. Eighteen MS participants and 18 healthy controls took part. Participants completed walking and cognitive tasks under single and dual task conditions. MS participants, compared to healthy controls, had greater decrements in dual-task performance; including decrements in cognitive task performance, walking speed and swing time variability. Dual-task decrements were evident in titrated and fixed demand conditions. Dual-task decrements were related to fatigue, cognitive functioning and self-reported cognitive errors, but not to measures of disease severity or duration. MS participants perform differentially poorly on walking and talking dual-tasks compared to healthy controls. This may lead to difficulties in everyday life and increase the risk of falls in MS. Clinicians should independently assess dual-task walking in MS patients. The role of task demand in dual-tasking decrements remains unclear and needs further investigation. Future studies should replicate the current findings and develop practical clinical tools to assess walking and talking ability.

The Richards retrograde femoral nail (Smith and Nephew 1997) is inserted into the medullary canal of the femur via an entry point in the intercondylar fossa, just anterior the anterior cruciate ligament. This particular implant requires insertion of both proximal and distal interlocking screws for maximal axial and rotational stability. The distal screws are inserted lateral to medial in a relatively safe area of the lower thigh using an external guide. The proximal locking screws by contrast are inserted 'freehand' at the level of the lesser trochanter in an anteroposterior direction using X-Ray control. The proximal thigh contains many vital neurovascular structures and it is the proximity of the femoral nerves and vessels which is of significant concern due to the relative inaccuracy of the freehand technique (Riina et al 1998). Hypothesis This led me to my first null hypothesis that 'proximal anteroposterior locking screws of retrograde femoral nails can be inserted in the proximal end of the femur through the proximal thigh without damage to the nerves and vessels'. To test this hypothesis we proposed to: 1). Dissect the main nerves and vessels of the thigh and describe their position and relation to the lesser trochanter. 2). Place investigative drill holes in a modified anatomical procedure to imitate insertion of the proximal anteroposterior locking screws. 3). Dissect the proximal thigh to determine whether important anatomical structures are at risk of being damaged by the proximal anteroposterior locking screws. The preliminary results of this first investigation lead to a second null hypothesis:- That the nerves and vessels of the upper thigh are at risk from proximal locking screws inserted in a lateral to medial direction in the upper thigh. To test this hypothesis we propose to; 4). Plan dissections to compare lateral to medial locking with anteroposterior locking, for use with a redesigned nail. 5). Finally we proposed a modified clinical trial of the redesigned nail. This would involve attaching the redesigned nail to the side of a volunteer's thigh and taking X-Rays in theatre to determine whether the redesigned nail could be adequately screened to allow its use in practice.

Metabolic functions are one of the principal determinants of energy expenditure and are exquisitely susceptible to the effects of circulating hormones and chemical changes. Consequently, clinical experiments based on energy expenditure and metabolic functions were considered to be valid approaches to the present research. Significant abnormalities were found in the proton magnetic resonance spectroscopy of basal ganglia in CFS patients. Automatic cardiovascular responses to exercise are also impaired in a subset of CFS patients. Finally, plasma membrane injury appears to be a possible explanation for a range of observations made in this research. Subjective fatigue is a complex symptom. It is the outcome of a variable combination of physiological and neuropsychological changes induced by the primary disease process. Downstream links between brain, neuromuscular and the cardiorespiratory functions are implicated in the neural control of force output during exercises in health and disease. Higher perceived fatigue in CFS is probably caused by the central mechanisms while the sensory input to these neural regulatory mechanisms may limit endurance to maximal and submaximal exercises. Based on these findings and more indirect evidence from other studies, changes in cell membrane properties affecting neuronal signalling in the basal ganglia seem to emerge as one of the likely pathophysiological mechanisms in CFS. There is also evidence of an imbalance of the central autonomic tone in a subset of CFS patients. Surely, research in CFS has the potential to unravel the biology of central fatigue and may bridge the gap that exists between the borderland of neurology and psychiatry.

Pt. 1. Service Based Evaluation. Effective communication within and between services is important in the development of multi-disciplinary collaboration, effective service delivery and satisfactory patient treatment. The primary aim of this study was to audit assessment letter content across three therapy disciplines within Ayrshire and Arran Consulting and Clinical Psychology Services (CCPS). A secondary aim was to audit content within the three therapy disciplines. The overall aim was to ascertain service implications based on the current letter standard. A random sample of sixty letters were audited, twenty written by Clinical Psychologists, twenty by Counselling Psychologists and a further twenty by CBT Psychotherapists. The results suggested overall content inclusion was of a high standard. However, there were some inclusion gaps in specific information pertaining to the themes of case complexity, reason for seeking assistance, previous psychiatric and medical history, maintaining factors and estimated treatment length. It was concluded that training should be conducted across the service to further develop communication within these areas.

Introduction: premature ejaculation (PE) is a common sexual dysfunction. Current explanations of PE are homogenised and culture-free. These explanations do not resonate with the local Bangladeshi Muslim male population, who over-represent attendees in the local clinic. Research question: What are the factors that form Bangladeshi Muslim men’s knowledge(s) of premature ejaculation? Design: this is a first level exploratory research project using thematic analysis of semi-structured responses gained during a randomised trial comparing two biomedical interventions followed by a modified behavioural therapy. Foucault’s concept of governmentality was the theoretical foundation of the research. Results: three themes were dominant in explaining PE both in the participant narratives and the published literature. These themes were biomedical, psychological and cultural. Medicine, through disciplinary practices, positions patients in certain spaces through the use of surveillance and panopticism. Participants in the trial both occupied and rebelled against this positioning, creating tensions and contradictions between meanings of PE and clinical practice that are in marked contrast to the recommendations of biomedical literature. Motivation for seeking treatment was changes in ejaculatory recovery, and the main motivator was the man’s partner. There was little or no intimacy in Bangladeshi Muslim men that exacerbated PE. Conclusions: biomedical discourses seek to reduce sexual activity into a time-focussed goal rather than a mutually enjoyable activity. Participants in the research also reduced sexual activity to a time goal, but separated mind from body, possibly due to sexual inexperience and possibly due to cultural pressures. Sex education was minimal or absent, which explains pressures to perform, disengagement from behavioural therapy and demands for pharmacological therapies. This research has discovered new knowledge that leads to an increased understanding of PE in Bangladeshi Muslim men.

Introduction: Intensive Care Unit (ICU) medical personnel, in an ongoing process termed ventilation management, utilize patient physiology and pathology data to define ventilator apparatus settings. Aims: The aim of the research is to develop and evaluate in comparison hybrid ventilation advisor systems, that could support ventilation management process, specific to lung pathology for patients ventilated in control mode. Methodology: A questionnaire was designed and circulated to Intensivists. Patient data, as defined by the questionnaire analysis, were collected and categorized into three lung pathologies. Three ICU doctors evaluated correlation analysis of the recorded data. Evaluation results were used for identifying models basic architecture. Two custom software toolboxes were developed for developing hybrid systems; namely the EVolution Of Fuzzy INference Engines (EVOFINE) and the FUzzy Neural (FUN) toolbox. Eight hybrid systems developed with EVOFINE, FUN, ANFIS and ANN techniques were evaluated against applied clinical decisions and patient scenarios. Results: Seventeen (17) models were designed for each of the eight (8) modeling techniques. The modelled process consisted of twelve physiology variables and six ventilator settings. The number of models’ inputs ranged from single to six based on correlation and evaluation findings. Evaluation against clinical recommendations has shown that ANNs performed better; mean average error as percentage for four of the applied techniques was 0.16%, 1.29% & 0.62 for ANN empirical, 0.05%, 2.23% & 2.30% for ANFIS, 0.93%, 2.33% & 1.89% for EVOFINE and 0.73%, 2.63% & 6.56 for FUN NM, in Normal, COPD and ALI-ARDS categories respectively. Additionally evaluation against clinical disagreement SD has shown that 70.6% of the NN empirical models were performing in 90% of their suggestions within clinical SD, while the percentages were 53%, 53% and 59% for the EVOFINE, ANFIS and NN Normalized models respectively. The EVOFINE and ANFIS produced Fuzzy Systems whose architecture is transparent for the user. Visual observation of ANFIS architectures revealed possibly hazardous advices. Evaluation against clinical disagreement has shown that the NN empirical was not producing hazardous advices, while EVOFINE, ANFIS and NN Normalized were shown to produce potentially hazardous advice in 17.6%, 23% and 5.8% of the developed models.

In recent years there has been a steady increase in the number of patients being discharged within five days of coronary artery bypass graft surgery. The ability to be able to predict those patients likely to be discharged within five days of surgery is important to improve the individual patient pathway, plan resources and surgical activity, and also to achieve current policy objectives. Guided by the theory of Stress, Appraisal and Coping (Lazarus and Folkman, 1984), the aim of this observational study was to develop and validate local multivariate models from preoperative patient variables for the purpose of predicting postoperative length of stay and discharge within five days of surgery. The study also investigated the influence of previously neglected psychological variables on these outcomes. The study was conducted in two phases: Phase I A cross-sectional survey design was used for univariate and multivariate analyses of thirty one empirically or theoretically derived variables. Previously collected data was retrospectively analysed for 1043 consecutive patients undergoing first time isolated coronary artery bypass graft surgery at a single National Health Service trust during 2005. By univariate analysis twenty variables were found to be associated with postoperative length of stay as a continuous variable, and as a categorical dichotomy of either less than or equal to five days or more than five days. Multivariate analysis of these variables showed that both postoperative length of stay and discharge within five days of surgery were poorly predicted. However, the models developed were much better at predicting postoperative lengths of stay greater than five days. Phase II Another cohort of 503 patients was used to prospectively validate the models. The potential influence of perceived stress and health locus of control was also investigated. These variables were not associated with either outcome. This study identified areas for further research, including the potential of other psychosocial variables to improve the predictive ability of the models. This would increase the utility of the models in practice and contribute to improvements in both the quality of the patient journey and the business objectives of healthcare organisations.

The prevalence of asthma has increased over the last generation, in parallel with a warm indoor microclimate. Central heating, fitted carpets and tight building construction have improved standards of heating and energy efficiency in homes, at the expense of ventilation. A warm, humid environment favours the growth of the house dust mite population. Allergy to the house dust mite is the most common allergy associated with asthma in the UK. Studies of occupational asthma, seasonal asthma and at altitude infer that the environment may directly affect symptoms of asthma. Allergen avoidance has been advocated as an important aspect of asthma management, yet the evidence for its efficacy has not been clear. Large studies of conventional measures to eradicate dust mites, such as mattress covers, have not shown a benefit for symptoms of asthma. As house dust mites are sensitive to humidity, an additional strategy would be to reduce indoor air humidity by improving ventilation. A randomised, double-blind placebo-controlled study examined the effect of the installation of domestic mechanical heat recovery ventilation on asthma control in the homes of 119 adults sensitive to house dust mite allergen. The study involved collaboration between the University Departments of Architecture, Respiratory Medicine and Immunology, local General Practices, the district general hospital, the local councils and industry. 100 participants completed follow-up. At twelve months, there was a clinically significant improvement in evening peak expiratory flow in the mechanical ventilation group and fewer admissions to hospital with asthma. There was a non-significant improvement in the mechanical ventilation group in the primary outcome, morning peak expiratory flow. There was a significant reduction in the asthma control questionnaire score at 3 months, but this was not sustained to 12 months. Rhinitis visual analogue scores for sneezing, nasal discharge and nasal blockage significantly improved in the group with mechanical ventilation compared to the control group at 6 months, but not at 12 months. There was no difference in exhaled nitric oxide, a measure of airway inflammation, between the two groups at 12 months. However, these clinical improvements could not be explained by reduced allergen exposure, as although indoor air humidity was reduced during the winter months, there was no difference between the house dust mite levels between the groups, nor in the levels of specific IgE to the house dust mite. Other mechanisms, such as mould, endotoxin, viral infection and environmental tobacco smoke should be considered in future work. In the mechanical ventilation group there was a modest individual gain of 0.02 Quality-adjusted life years over 12 months. However, it may still prove a cost-effective intervention if the clinical effects are sustained. Further research is required to establish if the clinical effects are sustained for greater than one year and to investigate the mechanism of the effect of improved home ventilation on respiratory health.