The mechanical properties of the respiratory system such as resistance, elastance and mechanical work of breathing are rarely measured directly but are inferred from the effect of respiratory disease on maximal lung volumes and flows. Although such tests have proved very useful, they have shortcomings, e.g. changes in lung volumes are poor at detecting progression in interstitial lung disease and correlate only weakly with changes in functional capacity achieved post-bronchodilator in patients with airways obstruction. The direct measurement of mechanical properties is of interest as they have an obvious physical interpretation but their usefulness has as yet not been systematically tested. Resistance aside, their measurement is rarely performed as it is invasive, requiring either a sedated patient on controlled ventilation to abolish spontaneous respiratory muscle activity or measurement of oesophageal and gastric pressures. The aim of this thesis was to explore the feasibility and potential clinical value of non-invasive measurements of respiratory mechanics and work of breathing. The work is presented in three sections. Firstly, conventional methods for measuring resistance, elastance and mechanical work of breathing were reviewed and the methods for the non-invasive approaches to be used were described in detail. The results from the non-invasive methods were then validated by comparison with conventional techniques in both ventilated patients and in subjects in the pulmonary function laboratory where oesophagal and gastric manometry were performed. Finally, the non-invasive methods were evaluated in three clinical scenarios: bronchodilator reversibility testing, assessment of progression in interstitial lung disease, and monitoring recovery from exacerbation of chronic obstructive pulmonary disease.

Coronary artery disease (CAD) is the leading cause of mortality in Scotland (population 5.2 million), accounting for around 9000 deaths each year. Accurate diagnosis of the presence and extent of CAD is essential to guide management. Invasive coronary angiography (I-CA) is the gold standard diagnostic investigation but is associated with a small risk of significant vascular complications. Over the last decade multi-slice computed tomography coronary angiography (MSCT-CA) has emerged as a non-invasive imaging modality capable of visualising the coronary arteries. Incremental advances in scanner technology have greatly improved the accuracy of MSCT-CA in comparison to I-CA. Implementation of MSCT-CA in routine clinical practice in Scotland is desirable in terms of patient safety and convenience in addition to reducing pressure on cardiac catheterisation laboratory time. The latter is particularly relevant considering the recent introduction of primary percutaneous intervention for myocardial infarction. However, at the time of conducting this study the evidence for MSCT-CA accuracy was limited and only minimal guidance on appropriate use of MSCT-CA was available. Furthermore, the majority of existing evidence for MSCT-CA accuracy was derived from specialist academic centres with substantial experience in the technique and the accuracy of MSCT-CA in smaller centres with variable expertise and a more heterogeneous population was unknown. The aim of this prospective, comparative study was to determine the accuracy of MSCT-CA in comparison to I-CA for the detection of significant CAD in patients presenting to a district general hospital in Scotland and to consider the health economic implications of introducing MSCT-CA into routine clinical practice. One hundred patients with suspected CAD on the basis of symptoms and non-invasive stress testing underwent both 40-Slice MSCT-CA and I-CA. Studies were reported by independent, blinded radiologists and cardiologists and compared using the 15-Segment model of the American Heart Association. A stenosis of ≥ 50% was considered significant. The accuracy of MSCT-CA was determined in patient-based, artery-based and segment-based analyses and the impact of various patient characteristics on image quality and diagnostic accuracy was evaluated. Inter-observer agreement was determined for both MSCT-CA and I-CA and the possibility of a “learning curve effect” investigated. The cost-effectiveness of an “MSCT-CA first” strategy was considered in a health economic analysis. The primary analysis considered MSCT-CA accuracy on a per patient basis. Patient prevalence of significant CAD was 38%. Patients with MSCT-CAs deemed not fully evaluable were included and considered to have significant underlying CAD. This strategy was considered clinically relevant as in practice a patient with an unevaluable MSCT-CA would proceed to I-CA for definitive diagnosis. This work demonstrated that 40-Slice MSCT-CA has a high sensitivity (92%) and a high negative predictive value (NPV) (91%) for the detection of significant CAD on a per patient basis. Specificity and positive predictive value (PPV) were less impressive and significantly compromised by the inclusion of patients with scans considered not fully evaluable by MSCT-CA. On segment-based and artery-based analyses respectively, distal segments were more often unevaluable than proximal segments owing to their smaller size, and the right coronary and circumflex arteries were the arteries most often unevaluable, likely due to their higher mobilities. Heart rate during MSCT-CA was not optimally controlled with oral beta blockers and rate limiting calcium channel blockers. The mean number of MSCT-CA evaluable segments per patient was significantly higher in the lower heart rate group. More than half the study population had coronary artery calcification and there was a non-significant trend towards more unevaluable segments in this patient group. Coronary artery calcification had the effect of reducing NPV while increasing PPV. One third of the study patients were obese and a non-significant trend towards an increasing number of unevaluable segments with increasing body mass index was observed. Pre-test probability of significant underlying CAD was determined by the Duke Clinical Score. Fifty-nine per cent of patients had a low-intermediate pre-test probability and 41% a high pre-test probability of CAD. The prevalence of significant CAD in the high pre-test probability group and the low-intermediate pre-test probability group was 73% and 14% respectively. Correspondingly, the sensitivity and PPV of MSCT-CA were higher in the high pre-test probability group while specificity and NPV were lower. Inter-observer agreement of the MSCT-CA reporters was substantial and comparable to that of the I-CA reporters in the patient-based analysis. A small observed improvement in MSCT-CA reporter diagnostic specificity during the study was not statistically significant. This study demonstrated MSCT-CA to be cost effective in the detection of significant CAD in a patient population with low-intermediate pre-test probability and hence fairly low prevalence of disease. Savings would be increased with improved MSCT-CA specificity. A strategy of screening patients being considered for I-CA on the basis of their risk level and referring ‘low-intermediate risk’ cases for MSCT-CA could affect around 60% of patients currently referred for diagnostic I-CA in North Glasgow and subsequently avoid I-CA in at least half of these patients. To permit the development of an effective MSCT-CA service future work must focus on ensuring appropriate training for those performing and reporting MSCT-CA and on the development of local guidelines to govern patient selection for MSCT-CA. Audit of MSCT-CA referrals could determine the extent of adherence to guidelines. Further research could be observational in nature with follow-up of patients who have MSCT-CA and are then referred for I-CA and also follow-up of patients with “negative” MSCT-CA who do not have subsequent I-CA in terms of subsequent cardiac events.

Cardiovascular disease (CVD) remains the leading cause of death in the United Kingdom and is associated with a huge burden of morbidity. Within the group of cardiovascular diseases coronary artery disease (CAD) is the single largest cause of death. Death rates from CAD have been falling since the 1970s predominantly due to a reduction in the prevalence in major risk factors such as cigarette smoking. Type 2 diabetes mellitus (DM) is an important risk factor for CVD. Type 2 DM is increasing in prevalence and there is concern that this will contribute to an increase in the burden of CVD. Reducing cardiovascular risk in patients with type 2 DM has to date focussed on tight blood pressure and glycaemic control together with statin therapy to achieve tight low density lipoprotein cholesterol (LDL) targets. Recent studies such as ADVANCE and ACCORD have highlighted some of the limitations with this approach. Important vascular abnormalities underlying the development of CAD include endothelial dysfunction and increased arterial stiffness. Some of the mechanisms underlying these abnormalities are thought to include increased oxidative stress, inflammation, insulin resistance and dyslipidaemia. These processes in patients with type 2 DM are currently not fully understood. It is hoped that through increased understanding of these processes new strategies for reducing cardiovascular risk in patients with type 2 DM can be identified. This study aimed to investigate some of the processes thought to underlie CVD in patients with type 2 DM namely endothelial dysfunction, arterial stiffness, oxidative stress and dyslipidaemia. Finally this study aimed to assess the impact of two cardiovascular prevention strategies (statin therapy and increased physical activity) on these processes believed to underlie the development of CVD. One hundred and twenty six patients with CAD (36 patients with type 2 DM, 90 patients without diabetes) and 80 controls (64 healthy controls and 16 varicose vein controls) were recruited as part of the VASCAB study. In these patients in vivo and ex vivo endothelial function studies were performed. Indicators of arterial stiffness were measured using pulse wave velocity and pulse wave analysis techniques. Superoxide levels were assessed in vascular tissue, mononuclear cells and whole blood. LDL and high density lipoprotein cholesterol (HDL) subfractions were analysed in patients with CAD. To assess the impact of intensive statin therapy and tight LDL targets, endothelial function and vascular superoxide levels were compared in patients recruited as part of the VASCAB study (2007 cohort) to a group of patients recruited in 2003. Finally patients attending the cardiac rehabilitation programme following surgical revascularisation were recruited to assess the impact of increased physical activity on endothelial function and oxidative stress. Endothelial function was impaired in patients with CAD compared to controls. In patients with CAD, type 2 DM was associated with greater impairment of endothelial function compared to patients with CAD alone. Superoxide levels in the vasculature, mononuclear cells and whole blood were similar in patients with and without type 2 DM. Type 2 DM was associated with significantly lower HDL levels and a preponderance to small dense LDL compared to patients without diabetes. Arterial stiffness was increased in patients with CAD compared to controls. There was however no significant difference in arterial stiffness in patients with type 2 DM and CAD compared to patients with CAD alone. Intensive statin therapy was associated with lower LDL levels and improved endothelial function but no change in vascular superoxide levels. Following the cardiac rehabilitation programme endothelial function was improved and HDL levels increased. There were no changes in levels of oxidative stress. Endothelial dysfunction in patients with type 2 DM may partly account for the increased cardiovascular risk and worse cardiovascular outcomes seen in this group of patients. Increased oxidative stress did not explain the endothelial dysfunction associated with type 2 DM. The dyslipidaemia that was associated with type 2 DM (low HLD and small dense LDL levels) may partly explain the increased endothelial dysfunction observed. Targeting endothelial dysfunction may therefore be a strategy for reducing cardiovascular risk in patients with type 2 DM. Intensive statin therapy and increased physical activity were both associated with improvements in endothelial function. The lack of evidence for increased arterial stiffness in patients with type 2 DM may reflect deficiencies in the methods used for assessing arterial stiffness. However this study highlights the difficulties of assessing arterial stiffness clinically and raises questions regarding the impact of type 2 DM on commonly used measures of arterial stiffness. Future prospective studies assessing the impact of improving endothelial function in patients with type 2 DM on cardiovascular outcomes are required.  .

Cardiovascular disease is the leading cause of morbidity and mortality in the Western world, mainly through cerebrovascular and coronary artery related events. Cardiovascular disease is a chronic progressive disease with different stages. These stages can be assessed by a variety of biomarkers. Biomarker quantification can be used for different purposes: screening, prediction of disease recurrence, therapeutic monitoring, diagnosis and prognostication. Noninvasive, inexpensive diagnostic tests currently applied in clinical practice have a relative high rate of false positive and false negative results. Therefore further refinement of the diagnostic process could improve clinical care. Regarding prognostication the need for improvement also remains as current risk models only predict a small quantity of occurring cardiovascular events. The concept of the cardiovascular continuum postulates that cardiovascular disease consists of a chain of events, is initiated by numerous cardiovascular risk factors and subsequently progresses through pathophysiological processes, ultimately leading to end-stage heart failure. For that reason cardiovascular diseases are chronic progressive conditions and can be divided into different stages, such as early tissue dysfunction or subclinical atherosclerosis prior to development of clinically overt disease. Biomarkers suitable for prognostication and diagnosis can differ at each stage. The general aim of this thesis was therefore the investigation of a variety of biomarkers in diagnosis and prediction of cardiovascular disease at different stages of the cardiovascular continuum, as covered by three different study cohorts contributing to this thesis. This included several approaches: the comparison of central and peripheral pulse pressure in middle aged hypertensive patients in regards of their prognostic potential; the application of established circulating, functional and structural biomarkers to the diagnostic process of coronary artery disease in stable angina patients; the development/refinement of a urinary proteomic biomarker for coronary artery disease and the examination of its diagnostic potential in stable angina patients. Biomarkers successful in the diagnosis of coronary artery disease were included in multiple biomarker models. Aside from biomarker development for the general population, investigations of specific cohorts, such as patients with certain diseases and belonging to certain age groups or sharing specific biochemical features provided advances in the past. To estimate the potential of a biomarker in risk prediction association studies with surrogate biomarkers are applicable. We collected a cohort of middle-aged hypertensive patients to assess if central pulse pressure, derived from non-invasive assessment of arterial stiffness, could improve risk prediction. Central pulse pressure has been previously shown to have prognostic value in populations with end-stage renal failure, coronary artery disease and high prevalence of diabetes mellitus. Considering the prognostic information of peripheral pulse pressure in the elderly, the hypothesis that central pulse pressure could improve risk prediction is comprehensive and was investigated as part of this thesis. This was accomplished by comparing the strength of correlation between central or peripheral pulse pressure and these surrogate biomarkers. When compared to peripheral pulse pressure, central pulse pressure had stronger associations with aortic pulse wave velocity, carotid intima-media thickness, and left ventricular mass index, but equal association with the albumin:creatinine ratio. In contrast, after adjustment for age, mean arterial pressure, heart rate and hypertension status there was no significant difference between central and peripheral pulse pressure for prediction of listed surrogate biomarkers in multivariate analysis. These results suggested that central pulse pressure is unlikely to provide more prognostic information than peripheral pulse pressure in middle-aged hypertensive patients. The diagnosis of coronary artery disease is clinically relevant in symptomatic patients, either acute or stable. The diagnosis of stable flow limiting coronary artery disease is especially challenging as non-cardiac as well as other cardiac conditions can mimic symptoms. Non-invasive diagnostic tools have either moderate sensitivities or specificities, or are not widely available. Therefore new biomarkers for the diagnosis of flow limiting coronary artery disease have the potential to improve current diagnostic strategies. This could be accomplished adjacent to existing biomarkers or by replacement of such, due to cost effectiveness, better discriminatory etc. As part of this thesis, a biomarker identification and validation study was conducted into urinary proteomics of coronary artery disease. First we tried to replicate a study conducted by our research group in the past. Therein, an established coronary artery disease specific polypeptide pattern was unable to differentiate between patients with severe coronary artery disease and healthy controls despite strong cohort similarities to the original study. We therefore recalibrated the urinary polypeptide pattern using an enlarged biomarker discovery cohort and adjusted the pattern for lipid lowering and angiotensin converting enzyme inhibitor treatment effects. We calculated a score from the resulting polypeptide pattern, which identified coronary artery disease patients with a sensitivity of 79% and a specificity of 88% in a biomarker validation cohort. As the next step of biomarker development we performed a diagnostic validation study. The investigated clinical cohort consisted of stable angina patients with or without coronary artery disease. The new polypeptide pattern score was unable to differentiate between these two groups. The score however correlated strongly with coronary artery disease extent as measured by the Gensini score, implying that urinary proteomics in the diagnosis of coronary artery disease is promising, yet requires further effort before clinical employment. In addition to the urinary proteomic biomarker development second diagnostic approach was selected. As coronary artery disease is a complex chronic disease, the combination of different biomarkers should result in a better discrimination between stable angina patients with or without coronary artery disease. This approach attempts to position the individual as precisely as possible on the cardiovascular continuum including serologic, functional vascular and imaging biomarkers of subclinical atherosclerosis. Serologic markers thereby present a plasma proteomic approach covering pathophysiological processes with known correlation or causative for coronary artery disease. Functional and structural changes of the peripheral vasculature resemble the coronary artery system. We investigated circulating biomarkers and vascular biomarkers separately. A variety of circulating biomarkers differentiated patients with severe coronary artery disease from healthy control subjects. When patients with stable angina and with or without coronary artery disease as diagnosed by coronary angiography were investigated no statistically significant differences could be detected for circulating biomarkers. In the same study a microvascular biomarker, the reactive hyperaemia index, and a macrovascular biomarker, the carotide plaque score, were able to differentiated between cases and controls. Both markers either added separately or together improved the risk classification of exercise treadmill test results. This suggests that a multiple biomarker approach in the diagnosis of coronary artery disease in stable angina patients could be successful. Different aspects of the cardiovascular continuum can be applied to diagnosis and prognostication of cardiovascular disease.

Rheumatoid arthritis (RA) is an autoimmune disease that primarily affects thejoints and is characterized by bone and cartilage erosion. These patients have an increased risk of developing co-morbid cardiovascular disease (CVD). In this study the vascular adventitia from patients with coexisting RA and CVD was investigated to identify factors in the vascular environment that could affect the immune component of RA. This included the histological evaluation of aortic adventitial sections for known RA-associated autoantigens; B cells their survival factors and associated cytokines. In addition, these human studies were the impetus for the initiation of a study to investigate the effect of B cell depletion on atherosclerosis. Citrullinated proteins (CP), RA associated antigens, are an inflammation specific entity. These modified proteins are processed by (PADI), a calcium dependent enzyme expressed by inflammatory cells and some microbial species. The presence of RA specific autoantibodies (ACPA) suggests a role for citrullinated proteins in RA pathogenesis. The aim of the present study was to determine whether the inflammatory milieu in the vascular adventitia of RA patients had any association with CP. The expression of CP was confirmed in the vascular adventitia, and CVD patients with coexisting RA were found to have increased levels of CP compared to non-RA CVD patients. In addition, ACPA levels were detectable in serum samples of only RA patients. When ACPA positive RA patients were sub grouped (CP>5.5), a strong association was observed between ACPA levels and C-reactive protein (CRP) in this subgroup (CP>5.5). This is suggestive of a CP and ACPA link in vascular inflammation in patients with coexisting RA. The presence of B-lymphocytes has been shown in both murine and human vascular adventitia. In this study we demonstrated that the presence of B cell aggregates is tissue specific, since matched internal mammary artery (IMA) had no detectable B cells in the adventitia. Furthermore, BAFF and APRIL are present in the aortic adventitia and support the conclusion that a survival niche is present in the aortic adventitia; cytokine expression was demonstrated in lymphocytes, adipocytes and vascular endothelial cells. Interestingly, smoking resulted in higher levels of BAFF expression in the aortic adventitia of CVD patients. Previous studies have shown that smoking induces vasoconstriction of small vessels (vasa vasorum), and hence hypoxia of the vascular endothelial cells. We hypothesised that hypoxia would induce the increased expression of BAFF and APRIL in vascular endothelial cells. Using murine vascular endothelial cells we investigated the effect of hypoxia on BAFF and APRIL mRNA transcript levels. However, hypoxia did not have any effect on transcript levels. In addition, significant correlations were observed in RA patients only, between B cells and APRIL, B cells and CP, APRIL and CP. This part of my thesis demonstrates that the aortic adventitia of RA patients with coronary heart disease is associated with an environment that is conducive to B cell survival and harbours a known RA-associated self-antigen. Importantly, these insights provide a rationale for the perpetuation of B cell auto-reactivity outside of both lymphoid tissue and the joint in RA. Previous studies have shown that atherosclerosis is a T cell mediated disease with an imbalance in Th1 and Th2 responses. Macrophages and interleukin 18 (IL- 18, proatherogenic) cytokine have been demonstrated in aortic adventitia of humans in previous studies. However, their expression in the vascular adventitia of RA patients has not been investigated. The present study confirmed macrophage IL-18 expression in vascular adventitia, which is similar among the Subgroups (RA vs. non RA) and (smokers vs. non smokers). Furthermore, a Th2 phenotype has been proposed as atheroprotective, mediated by anti oxLDL antibody producing B cells. Interestingly, recent studies have demonstrated interleukin 33 (IL-33) a Th2 cytokine to reduce the atherosclerotic lesion in an experimental model of atherosclerosis. However, it’s expression and significance in human vessels has not been extensively investigated. In this study, using immunohistochemistry, IL-33 was localised to vascular endothelial cells of vasa vasorum in aortic adventitia and lining layer of IMA. Importantly, IL-33 was down regulated in smoker’s AA suggestive of failed protective mechanism of this Th2 cytokine. In contrast, IL-33 expression was not affected by smoking in the matched IMA tissue. In the past B-lymphocytes have been shown as an atheroprotective pathway; whereas the use of B cells depletion therapy (Rituximab) suggests a proinflammatory role of these cells in RA. Could B cell depletion therefore mediate unexpected deficit on vascular lesions in RA? My thesis revealed expression of B cells and their survival factors in the aortic adventitia. Therefore to investigate the impact of B cell depletion therapy on atherosclerosis, a mouse model (huCD20/ApoE-/-) was generated. The preliminary data are suggestive of reduction in atherosclerotic plaques with B cells depletion. These studies will be extended in future to formally test the above hypothesis. In summary I have shown for the first time the cytokine and cellular niche that exists in human aorta in RA patients. Such data will in due course inform the mechanism of accelerated vascular pathogenesis in RA.

Astrocytes are the most abundant cell within the central nervous system (CNS) and yet despite this, the true extent of their role in health and disease has not been fully elucidated. In the undamaged CNS, they are termed quiescent, where they maintain homeostasis. However, after injury or disease astrocytes become reactive where they are described as a physical and molecular barrier to regeneration. Emerging literature has suggested the existence of an additional phenotype of astrocyte, termed the activated astrocyte. These astrocytes are thought to enhance regeneration by creating a more growth-permissive environment for repair. In addition, it has also been reported that astrocytes may play a role in regulating myelination; however, it is unclear how the phenotype of the astrocytes may affect this process. Therefore, this thesis will focus on the variable phenotypic state of astrocytes and subsequently how this relates to their ability to support myelination. Using an in vitro myelinating culture, where dissociated spinal cord cells were plated on a monolayer of astrocytes, myelination can be followed over time. Since it is hypothesised that astrocytes can affect myelination we used two protocols known to affect the reactive status of the astrocyte, i) activate the astrocytes by treating with ciliary neurotrophic factor (CNTF) or ii) induce a quiescent astrocyte state by plating them on Tenascin C (TnC). It is hypothesised that CNTF changes the activation state of the astrocyte therefore making it more supportive to myelination. The addition of the astrocyte derived factor ciliary neurotrophic factor (CNTF) was shown to enhance myelination. My results demonstrate that CNTF addition does not lead to an increase in oligodendrocyte or microglia cell numbers or an increase in the diameter of the neurites, thus suggesting that this CNTF-induced increase in myelination is mediated via the astrocyte. Conversely, culturing astrocytes on the extracellular matrix molecule Tenascin-C (TnC), a method to make the astrocytes quiescent (Holley et al., 2005), resulted in a reduction in myelination. Astrocytes cultured on TnC were shown have decreased expression of nestin, which is typically a marker for reactivity. A microarray gene study comparing gene expression of the various astrocyte phenotypes identified CXCL10 to be upregulated in astrocytes on TnC. Furthermore, the addition of CXCL10 into the myelination cultures resulted in a decrease in myelination. Conversely, the addition of anti-CXCL10 to myelinating cultures on quiescent astrocytes increased myelination. Taken together, these data indicate that the astrocyte phenotype has considerable influence on myelination; where activated astrocytes support myelination whilst quiescent astrocytes do not. The identification of factors which may modify astrocyte phenotypes could lead to potential therapeutic strategies for CNS pathologies.

Cerebral autoregulation is the process by which blood ow is maintained over a changing cerebral perfusion pressure. Clinically autoregulation is an important topic because it directly effects overall patient management strategy. However accurately predicting autoregulatory state or even modelling the underlying general physiological processes is a complex task. There are a number of models published within the literature but there has been no active attempt to compare and classify these models. Starting with the hypothesis that a physiologically based model would be a better predictor of autoregulatory state than a purely statistically based one has led us to investigate approaches to model comparison. Using three different models: a new mathematical arrangement of a physiological model by Ursino, the Highest Model Frequency (HMF) model by Daley and the Pressure reactivity index (PRx) statistical model by Czosnyka, a general comparison was carried out using the Matthews correlation coecient against a known autoregulatory state. This showed that the Ursino model was approximately three times as predictive as both the HMF model and the PRx model. However, in general, all of the models predictive accuracies were relatively poor so a number of optimisation strategies were then assessed. These optimisation strategies ultimately were formed into a generalised modelling framework. This framework draws on the ideas of mathematical topology to underpin and explain any change or optimisation to a model. Within the framework different optimisations can be grouped into four categories, each of which are explored in the text of this thesis: 1) Model Comparison. This is the simplest technique to apply where the number of models under examination are reduced based on the predictive accuracy. 2) Parameter restriction. A classical form of optimisation by constraining a model parameter to cause a better predictive accuracy. In the case of both the HMF and PRx we showed between a two hundred and six hundred percent increase in predictive accuracy over the initial assessment. 3) Parameter alteration. This change allows for related parameters to be substituted into a model. Four different alterations are explored as a surrogate measure for arterial-arteriolar blood volume the most clinically applicable of which is a transcranial impedance technique. This latter technique has the potential to be a non invasive measure correlated with both mean ICP and ICP pulse amplitude. 4) Model alteration. Allows for larger changes to the underlying structure of the model. Two examples are presented: firstly a new asymmetric sigmoid curve to overcome computational issues in the Ursino model and secondly a novel use of fractal characterisation which is applied in a wavelet noise reduction technique. The framework also gives an overview of the autoregulatory research domain as a whole as a result of its abstract nature. This helps to highlight some general issues in the domain including a more standardised way to record autoregulatory status. Finally concluding with research addressing the requirement for easier access to data and the need for the research community to cohesively start to address these issues.

Chronic kidney disease (CKD) is associated with an increased risk of cardiovascular disease and end stage renal failure. Accurate identification of those with a reduced glomerular filtration rate and significant proteinuria facilitates early diagnosis and risk stratification. This thesis explores the optimal measure of proteinuria, to accurately quantify proteinuria and as a predictor of renal and patient outcomes. We examine the prevalence of CKD in a general population cohort and assess the impact of different estimated glomerular filtration rate (eGFR) formulae. We explore the prognostic role of reduced eGFR and proteinuria in patients with hypertension and present the baseline characteristics of a community cohort study of patients with predominantly early CKD. They will be followed for ten years to identify predictors of cardiovascular and renal outcome. Urine total protein:creatinine ratio (TPCR) and albumin:creatinine ratio (ACR) have largely replaced 24-hour urine collections for proteinuria quantification. The performance of these spot measures to identify significant proteinuria is compared in a cohort of 6842 patients attending a general nephrology clinic. Both tests perform well overall but TPCR is statistically significantly superior as a predictor of 24-hour total proteinuria than ACR (as measured by the area under the receiver operator characteristic (ROC) curve to predict 1g/day total proteinuria). On sub-group analysis the performance of the spot samples is poorer in women and the elderly, likely as a result of low muscle mass and low urine creatinine (the denominator in TPCR/ACR). The performance of TPCR and ACR were then compared as predictors of outcome in a similar cohort of 5586 CKD patients using a hierarchical Cox survival model. TPCR and ACR both performed well as independent predictors of death, commencement of renal replacement therapy (RRT) and doubling of serum creatinine. Notably TPCR performed well at low levels where albuminuria has been considered superior. These findings are novel. The spot samples performed as well as 24-hour collections in the sub-group with timed urine collections. The National Institute for Health and Clinical Excellence in England recommend ACR to monitor all patients with CKD; the Scottish Intercollegiate Guidelines Network recommend TPCR for non-diabetic renal disease. Therefore, we investigated the implications of these recommendations using survival modelling. The same cohort was divided into 5 groups: no proteinuria, low proteinuria (using TPCR and ACR), high proteinuria (TPCR and ACR) and two groups where TPCR and ACR were discordant (i.e. TPCR above the diagnostic threshold but ACR below it and vice versa) using the recommended thresholds (ACR 30mg/mmol/TPCR 50mg/mmol to predict 0.5g/day total proteinuria and ACR 70mg/mmol/TPCR 100mg/mmol to predict 1g/day total proteinuria). Using univariate survival analysis the discordant group had significantly poorer outcomes (using the same outcomes as previously) than those with significant proteinuria as measured by both tests. The discordant group was older with poorer renal function and some of the excess risk was abolished on multivariate analysis, however the risk did not return to the level of those without detectable proteinuria. TPCR, but not ACR, measures non-albumin proteins and these may have pathophysiological roles in progression. This requires further study. However this analysis confirmed that TPCR identifies patients at high risk of adverse outcomes. TPCR and ACR may vary as a result of muscle mass. We adjusted TPCR and ACR for estimated creatinine excretion (ECE) (calculated using the Cockcroft and Gault formula) and performed cross-sectional and longitudinal analyses. Adjusting TPCR and ACR for ECE improves prediction of significant proteinuria in sub-groups with poor baseline test performance (such as women and the elderly) using ROC curve analysis. However when adjusted and unadjusted values were compared as predictors of outcome (using a net reclassification index analysis) adjusted values were significantly inferior. Urine creatinine is an independent predictor of mortality and hence may be directly contributing to the predictive value of TPCR and ACR rather than simply correcting for urine flow rate. As such, adjusting for ECE may act to remove the effect of a second independent predictor, leading to inferior test performance. Therefore the decision to adjust TPCR and ACR for ECE depends on the test application: to predict significant proteinuria adjustment of TPCR and ACR is of benefit, but adjustment leads to inferior performance as a prognostic test. The prevalence of CKD stages 3-5 was assessed using a general population laboratory database. Overall population prevalence was 5.63% using the modification of diet in renal disease (MDRD) formula and fell to 4.94% when the CKD-Epidemiology group (CKD-EPI) formulae were applied. Those reclassified to an earlier stage of CKD were predominantly middle aged women. Prevalence over a five year period was found to be stable using the CKD-EPI formulae but rose slightly according to MDRD. Proteinuria and eGFR were assessed as predictors of outcome in a large specialist hypertension clinic cohort. On multivariate survival analysis both baseline dipstick proteinuria and an eGFR<60ml/min/1.73m2 remained strong independent predictors of cardiovascular and all-cause mortality, despite intensive specialist intervention to control blood pressure. These simple tests should be advocated for risk stratification in these patients. Lastly the baseline characteristics of a community CKD cohort are presented. We recruited 411 participants from seven general practices around Ayrshire and a detailed baseline clinical and biochemical assessment was performed. Patients were invited to participate if they were included in the primary care register of CKD stages 3-5. Over a quarter had an eGFR>60ml/min/1.73m2 on the meat-fasted study sample. Proteinuria was of notably low prevalence and the cohort had a large burden of cardiovascular disease. Complications of renal disease were uncommon. The characteristics of the cohort differ from those under hospital follow-up. Their long term outcomes should contribute to refining risk stratification in this population. Proteinuria and eGFR are key aspects of diagnosis and monitoring in CKD. Identification of the optimal measures of both is essential and findings presented here contribute to that. There is a need to refine risk stratification in CKD, to identify those who require intensive intervention, and to reassure the rest. The findings of this thesis also contribute to that. Further study is required to refine the core aspects of diagnosis and investigation of CKD.

This research programme investigates the influence of genetic variability on the clinical pharmacology of carbamazepine (CBZ) and lamotrigine (LTG). Common polymorphisms in genes that may influence the response to CBZ and LTG include CYP3A4 g.-392A>G, CYP3A5 g.6986A>G, CYP1A2 g.5734C>A, EPHX1 c.337T>C, EPHX1 c.416A>G, UGT2B7 c.802C>T, ABCB1 c.1236C>T, ABCB1 c.2677G>T/A, ABCB1 c.3435C>T and SCN2A c.56G>A. The prevalence of these common polymorphisms was evaluated in a 400-strong study population from a single research unit. Minor allele frequency ranged between 3.5% (CYP3A4 -392G) and 48.0% (ABCB1 1236T). Allele and genotype distributions were comparable to published data for other Caucasian populations. The influence of common polymorphisms in drug metabolising enzyme (DME) and sodium channel genes on the optimal dose of CBZ was assessed in a cohort of 70 patients. This study revealed that age and common polymorphisms in the EPHX1 gene (c.337T>C and c.416A>G) are potential predictors for optimal dose of CBZ. The significant effects of EPHX1 variants may be explained by their significant contribution to the inactivation of CBZ. These potential predictors explain approximately 15% of the inter-individual variation in CBZ dose requirements. Preliminary findings suggest that common polymorphisms in DME genes do not form a unique profile which increases the risk of developing intolerable CBZ adverse effects. It is unlikely that common polymorphisms in ABCB1 and SCN2A genes influence the expression and/or activity of their respective proteins to the level at which they can dictate response to LTG therapy. The influence of common polymorphisms in ABCB1 and SCN2A genes on the optimal dose of LTG was assessed in a cohort of 94 patients. Optimal dose in this study was defined as the final dose given to the patients that successfully maintained seizure freedom for at least 1 year with LTG monotherapy. Several basic clinical factors such as age and gender were also examined as potential predictors. The effect of predictors on the optimal dose of LTG was investigated using linear regression analysis. This study revealed that gender and common polymorphisms in the ABCB1 gene (c.1236C>T and c.3435C>T) are potential predictors for optimal dose of LTG. These predictors explain approximately 17% of the inter-individual variation in LTG dose requirement. These findings further highlight the potential role of P-gp in the management of epilepsy. The final study investigated the influence of ABCB1 c.1236C>T and ABCB1 c.3435C>T polymorphisms on the pharmacokinetics of LTG. A total of 156 blood samples from 50 patients receiving LTG monotherapy were available for analysis. The influence of ABCB1 variants was evaluated by population pharmacokinetics. This approach successfully estimated the oral clearance of LTG monotherapy at steady-state. However, the absorption rate constant (Ka) and volume of distribution (Vd) of LTG were poorly estimated. The inclusion of common polymorphisms in the ABCB1 gene in the pharmacokinetic model did not improve the estimation of oral clearance. This may indicate that common variants of ABCB1 do not influence clearance of LTG.

This thesis describes a series of studies which were undertaken to improve the diagnostic accuracy of the electrocardiogram (ECG), given the availability of computer assisted measurement techniques. Scalar 12 lead electrocardiograms have been recorded from over 200 cardiac patients recruited specifically for this study. Over 1,500 additional ECGs were available from normal and abnormal test populations. The ECG measurements were correlated with clinical, echocardiographic, radionuclide, coronary angiographic and contrast angiographic data of the left ventricle in order to meet the aims of the study. The history of electrocardiography was reviewed both with respect to the technique itself and the evolution of equipment through to present day computer assisted technology for recording and measurement of ECG waveforms. In addition, the development of echocardiography, nuclear cardiology and cardiac catheterization was also reviewed with particular attention being given to recent developments in technology which have allowed a reappraisal of gold standards against which the ECG can be compared. With more specific relevance to the aim of this study, the development of ECG criteria mainly with respect to post mortem examinations was reviewed so that a contrast could be drawn with present day techniques. In particular, emphasis was laid on the twin areas of left ventricular hypertrophy and ischemic heart disease. Evaluation of left ventricular function from the scalar electrocardiogram was undertaken in two ways. First of all the latest ECG scoring system of Selvester was assessed, from which left ventricular ejection fraction could be calculated. Secondly, indirect evidence of left atrial overload from the electrocardiogram was also studied. It was shown that left ventricular function could be predicted qualitatively with reasonable accuracy in the early post infarct period (third day) using the 54 criteria/32 points scoring system.