The flaws of current methods of assessing disease severity in patients with COPD and emphysema are increasingly recognised, and new methods of assessing disease activity are urgently required. Although many potential biomarkers have been suggested to fulfil this role, few have been effectively validated, and furthermore any biomarker should be based on our current understanding of the pathophysiology the disease process. This is poorly understood, however it is apparent that neutrophil proteases (particularly neutrophil elastase (NE) and proteinase 3 (Pr3)) may represent a final common pathway leading to tissue destruction. The current thesis describes the development and validation of a new marker of NE activity (Aα-Val360), and the identification of a marker of Pr3 activity, as potential biomarkers of COPD and emphysema disease activity. Methods Following in vitro validation, the performance of Aα-Val360 was assessed in a series of patient populations. Mass spectrometry was used to identify a specific marker of Pr3 activity. Results and Conclusion Aα-Val360 demonstrated acceptable in vitro and in vivo variability; related to physiological, radiological and patient reported outcomes in subjects with (or at risk of developing) COPD and emphysema (both with and without A1AT deficiency); increased during acute exacerbations; decreased in response to treatment; and partly related to disease progression in some populations. Also, a Pr3 specific cleavage product was identified which could be used to develop a new specific assay of Pr3 activity. These potential biomarkers of disease activity may be important in the assessment of patients with COPD and emphysema (or who are at risk of developing these conditions), particularly in early phase clinical trials.

Background: Decisions on which tests to use should be informed by evidence that they do more good than harm. Test-treatment RCTs are recommended as the ‘gold–standard’ approach, but have attracted criticism that question whether they are fit for purpose. Confronting this question, the thesis investigates four key challenges by finding and analysing all identifiable test-treatment RCTs (2004–2007). Methods: Capture–recapture analysis estimated the total population of trials; descriptive analysis characterised the diagnostic questions evaluated by RCT; reviews of reporting and methodological quality investigated how informative and valid trials are; analytic induction was used to develop a theoretical framework linking tests to health outcomes, from which a tool was designed. Results: Published trials were poor quality, and found to be highly complex studies that will be challenging to evaluate reliably: interventions are difficult to capture and translate into protocols; several methods traditionally used to eliminate bias are more difficult to implement; test-treatment strategies impact on patient health in numerous and highly complicated ways. Conclusion: Test-treatment trials have the potential to be very useful instruments, and though highly challenging they could be both reliable and informative. However, it must be acknowledged that trials will not be suited to all comparisons.

Parkinson's disease (PD) motor and non-motor symptoms progress relentlessly leading to frequent hospitalisations and an increase in the economic and societal burden of the disease. A major challenge in assessing treatments which can potentially reduce neurodegeneration, is the lack of tools that can be used to identify individuals with early PD with high sensitivity and specificity. There were several facets to this thesis, which were aimed at addressing these issues. Firstly a retrospective analysis of PD hospital admissions was conducted to provide background data on hospitalisation and clinical coding accuracy. A systematic review of literature for interventions to reduce hospital admissions was performed. Effect of treatments for PD motor symptoms on hospitalisation was also evaluated. Lastly, screening of potential lanthanide and 19 Fluorine based MRI probes for future use as diagnostic tools in PD was conducted. Results of these studies highlight significant underreporting ofPD hospitalisation which has a negative effect on PD resource allocation. A lack of robust evidence for measures which reduce PD admissions was demonstrated. Although the initial attempts to develop a novel MRI sensitive tool for use in PD were negative, the study refined a protocol that has potential for use in screening future probes.

The changes in the control of breathing in man at high altitude have been studied at 5,800 m (19,000 ft). The differences between 1owlanders and Sherpas were compared at 4,880 m (16,000 ft.). Ventilatory response to C0\(_2\), hypoxia and exercise were studied, and acid-base status of the blood and CSF measured. Acclimatization to altitude is characterized by a shift of the C0\(_2\) response curve to the left and an increase in its slope. The hypoxic sensitivity appears unchanged. On moderate exercise there results a progressive increase in ventilatory equivalent with increasing altitude. At maximum work rate ventilation increases more rapidly due to falling Sao\(_2\). Sherpas show no significant difference in response to C0\(_2\) but a remarkable lack of response to hypoxia. The C0\(_2\) response showed little change in slope with change of P0 \(_2\) and on exercise acutely changing PO\(_2\)had little effect on ventilation. Sherpas ventilate less on exercise and have higher maximum 0\(_2\) intakes per kg than lowlanders. The arterial pH of highlanders is normal whereas in lowlanders it remains slightly elevated after k-6 weeks at altitude. CSF pH of highlanders is about 0.04 units more acid than lowlanders at the same altitude, indicating a greater central contribution to respiratory drive and a reduced peripheral component. The role of anaerobic cerebral metabolism in respiratory acclimatization is discussed.

Neutrophils constitute the main immune defense against microbial invasion. When activated, they migrate towards the site of infection where they eliminate any foreign material in an effort to prevent wide-spread tissue damage and ultimately resolve infection. Previous work on neutrophil function in the elderly has highlighted a number of neutrophil effector functions, including phagocytosis, superoxide production and migration that exhibit decreased efficiency suggesting the potential for reduced pathogen clearance in older adults. This thesis reveals a migratory phenotype distinctive of neutrophils isolated from healthy elderly donors (> 60 years) and characterized by a maintained speed of migration (chemokinesis) but with significantly reduced directional migration (chemotaxis) and overall migratory accuracy in response to a range of chemoattractants. This migratory phenotype was shown to be associated with a constitutive basal activation of PI3Kinase in neutrophils isolated from older donors and appears to be a causative factor as treatment of neutrophils with inhibitors selective for PI3Kinase-γ and –δ, was able to restore migratory dynamics. The ‘old-migratory’ phenotype was amenable to correction by pre-incubation with 1nM Simvastatin in vitro and a two-week prescription of 80mg/day Simvastatin in vivo in healthy older adults. The ability of simvastatin to modulate migratory dynamics potentially provides a safe, cost effective intervention to reduce morbidity and mortality from infections in the elderly population.

The aim of this thesis was to explore three factors that are easily available and contribute important information for women before commencing in-vitro fertilisation (IVF) treatment: ethnicity, body-mass index (BMI) and thyroid disease. Results of the systematic review, cohort study and meta-analysis investigating ethnicity and IVF outcome showed South Asian and Black women have lower adjusted live-birth (LB) rates, after fresh cycle treatment, compared with White women. The relationship between BMI and IVF outcome was explored in a prediction model estimating chances of LB following first cycle. The model found BMI has reduced effect on IVF outcome when adjusting for other confounders such as age. The prevalence of thyroid dysfunction and thyroid peroxidase antibodies (TPOAb) was examined across the UK in >7000 women of reproductive age, and a cohort study investigating the effect of subclinical hypothyroidism (SCH) on IVF outcome was also performed. The prevalence of overt thyroid disease was 0.38% and subclinical disease 3.45%. Using an upper limit cut off for thyroid-stimulating hormone of 2.5mU/L the prevalence of SCH was 19.64%. The overall prevalence of TPOAb was 9.11%; this was 7.98% in euthyroid women. Finally, there were no significant differences in LB between euthyroid women and women with SCH.

Eukaryotic DNA is packaged into chromatin, providing a physical and structural barrier for regulatory and effector proteins to access the DNA. It has been shown that linker histone variants inhibit the double strand break (DSB) repair pathway known as non-homologous end joining (NHEJ). NHEJ, in vivo, requires the KU complex, the DNA dependent protein kinase (DNA-PK) and the DNA ligase IV/XRCC4 (LX) complex. In vitro analysis has shown that several linker histone variants are all phosphorylated by DNA-PK. Depletion of a specific linker histone variant protein levels by short interfering RNA renders cells radiosensitive and increases the levels of un-repaired DSBs following exposure to ionising radiation. This linker histone variant interacts with both DNA ligase IV and XRCC4 in vivo, along with DNA-PK, the KU complex and nucleolin. Interestingly, the interaction with nucleolin only occurs in the presence of DNA damage following exposure to ionising radiation. In vitro analysis has shown that this linker histone variant is able to form stable complexes in the presence of DNA and the LX complex, and very efficiently stimulate LX-mediated ligation of double stranded DNA. These findings establish a role for a linker histone variant in the NHEJ pathway.

Conflicting effects of sex hormones could potentially explain the increased susceptibility of females developing autoimmune diseases. In this study I found that 5\(\alpha\)-reductase expression both on the mRNA and protein was unregulated in female T cells after stimulation, which was not observed in the male T cells. This was particularly interesting as 5\(\alpha\)-reductase is responsible for the synthesis of the most potent androgen DHT, which has shown to exert anti-inflammatory effects. I did not observe any significant differences in 5\(\alpha\)-reductase expression in T cells between SLE patients and healthy controls. However, I did find a significantly higher expression of 5\(\alpha\)-reductase in B cells from SLE patients compared to healthy controls. In vitro treatment of testosterone showed that high concentrations the proportion of IL-2-producing female CD4 T cells decreased (not in the male T cells) and lower concentrations had the opposite effect. This latter observation was shown to be oestrogen-dependent as experiments using tamoxifen abolosihed the effect. Overall, sex differences are present in the expression of 5\(\alpha\)-reductase in T cells upon stimulation and regulation of 5\(\alpha\)-reductase expression is altered in SLE B cells. IL-2 production is sensitive to changes in testosterone concentration and there is an element of gender dimorphism in the T cell response to testosterone.

Cushing’s syndrome is characterised by changes in body composition and cardiovascular disease risk profiles that have similarities to the aged phenotype. 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) converts inactive glucocorticoids(GCs) to their active form (cortisone to cortisol in humans). There is growing evidence that 11β-HSD1 expression/activity increases with age in key target tissues including adipose tissue, bone, and skin. This thesis encompasses a series of novel studies investigating the role of GCs and their pre-receptor metabolism in determining the ageing phenotype, with a central focus on skeletal muscle. We show that although cure of Cushing’s disease results in rapid improvements in clinical parameters, excess mortality may persist. We show in-vitro evidence of regulation of proteolytic genes by 11β-HSD1 and that 11β-HSD1KO mice are protected from muscle weakness due to GCs and ageing. We recruited healthy subjects (n=135, 20-80 years) for in-depth phenotyping, along with muscle biopsies (analysed by gene expression array) and urine steroid metabolite analysis. Skeletal muscle 11β-HSD1 expression increased with age in women and this change may be driven by the menopause. The therapeutic potential of selective inhibitors of 11β-HSD1 in ameliorating the adverse metabolic and body composition profile associated with ageing and the menopause remains to be determined.

This thesis aims to extend the existing knowledge and enhance the methodological quality of future stepped-wedge cluster randomised trial (SW-CRTs). A systematic review of published SW-CRTs shows that pre-trial sample sizes calculations display a poor standard of reporting, with little adherence to published guidelines. The methodological rigor is often substandard, with inappropriate methods often used to determine sample size. In SW-CRTs, it is assumed that the correlation between observations is independent of the timing of them. We test the validity of this assumption by outlining a method to estimate the within-period and inter-period correlation. A case study illustrates what these correlations may look like in practice. The impact of varying cluster size in a SW-CRT is then demonstrated by comparing a design with unequal cluster size to a design with equal cluster size. A simulation study provides evidence that the SW-CRT is affected less, on average, than a P-CRT by varying cluster size. However, the potential power in a SW-CRT with unequal cluster sizes is extremely variable. A practical method for estimated power in a SW-CRT with varying cluster size is then illustrated through a Stata function.