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.

Costimulation is required for the generation of an effective T cell based immune response. The presentation of tumour associated antigens may occur in the absence of effective costimulation, inducing tolerance. Conversely effective costimulation can overcome immunosuppressive mechanisms present within the tumour. Costimulation may therefore hold significant potential for cancer immunotherapy. Using recombinant adenoviral vectors encoding the costimulatory molecules CD80 and 4-1BBL (CD137L) and the cytokine IL-12 tumour cell lines were transduced to express these molecules individually or in combination in vitro. Using PBMC from healthy donors the effect of costimulation in response to pan-T cell stimulation with the anti-CD3 antibody OKT-3 were initially studied. The combination of CD80+4-1BBL supported the proliferation of CD8+ T cells and was superior to either molecule alone. Proliferation was further enhanced by the addition of IL-12 to the combination of CD80+4-1BBL. Unexpectedly in the absence of OKT-3 costimulation with 4-1BBL or IL-12 was observed to predominantly induced the proliferation of natural killer (NK) cells. The effects of 4-1BBL on human NK cells are not clearly defined in the literature. Further experiments were therefore conducted to investigate the ability of 4-1BBL and IL-12 to stimulate NK cells. The combination of 4-1BBL+IL-12 was superior to either stimulation alone for the activation, proliferation and function of NK cells from healthy lab donors. 4-1BBL was also shown to promote the long term expansion of NK cells. Importantly renal cell carcinoma patient NK cells were shown to require a combination of 4-1BBL+IL-12 for short and long term expansion; stimulated NK cells were also shown to be functional. These data highlight the need for understanding of the pleiotropic effects of costimulatory molecules and the necessity to choose optimal combinations for the activation of not only the adaptive III but also the innate immune response. The combined intratumoural delivery of 4-1BBL and IL-12 via adenoviral vectors could potentially stimulate beneficial T cell and NK cell responses, and therefore warrant further investigation as a potential immunotherapy.

Rheumatoid arthritis (RA) results from complex interactions between genetic and environmental risk factors. Two examples of these are the genetic variant PTPN22 R620W, a disease-associated form of the protein tyrosine phosphatase (PTP) Lyp and cigarette smoking (CS). Epidemiological studies have identified interactions between R620W and CS, but the biological mechanisms behind these interactions are unclear. Lyp is expressed by all leukocytes and changes in leukocyte function are implicated in the pathogenesis of RA. Thus the aim of this study was to characterise the effects of R620W and CS on leukocyte signalling, to determine possible mechanisms by which these factors could interact to promote the development of RA. An assay to measure the specific activity of the Lyp phosphatase was developed. Healthy controls and RA patients were recruited and genotyped for the PTPN22 R620W variant. Following determination of genotype, neutrophils and CD4+ T cells were isolated and cell function assessed following cigarette smoke extract (CSE) treatment. R620W in T lymphocytes increased Lyp phosphatase activity, decreased Lyp substrate phosphorylation and increased production of the pro-inflammatory cytokines IFN-γ and TNF-α. CSE treatment decreased T cell receptor signalling which was characterised by decreased PTP activity, decreased calcium (Ca2+) flux and decreased cytokine production. R620W in neutrophils was associated with increased neutrophil activation and functions including Ca2+ flux, reactive oxygen species production and migration. Overall these data suggest that R620W may facilitate RA development and persistence by promoting the generation of inflammatory T cells and by enhancing neutrophil activation and migration. CS may promote further signalling dysfunction by oxidising the proteins controlling leukocyte signalling. These separate pathways leading to altered Lyp function may act additively or synergistically to promote the immune disturbances which underpin the development of RA.

Objectives Coeliac Disease (CD) is a common chronic autoimmune disorder characterised by sensitivity to gluten. This study investigated the relationships between CD illness representations, coping, self-efficacy, health related quality of life, wellbeing, psychological distress and dietary self-care (following a gluten free diet) among adults with coeliac disease. Design A questionnaire-based correlation design was used to investigate the hypothesised relationships between illness representations and outcomes. Methods Ninety six adults with CD, recruited through dietician clinics, completed a single booklet of questionnaires about their beliefs, experiences and behaviour in relation to their CD. In addition serology (CD antibody levels) and clinician rated levels of dietary self-care (both taken within one month of participation) were employed as additional measures of behaviour. Results Results of regression analyses showed that some aspects of illness representation, particularly estimates of consequences and emotional impact were related to some psychosocial outcomes but not levels of dietary self-care (which was generally high among the sample). Conclusions Illness representations may be useful in explaining some psychosocial outcome for CD, but more research would be needed to assess whether they are useful in explaining dietary behaviour among this population. Findings related to coping and self-efficacy are also discussed.

Chronic obstructive pulmonary disease (COPD) is an important cause of morbidity and mortality worldwide. Some studies suggest that COPD may affect work, however this relationship remains poorly understood. Various methods are used in this thesis to investigate the impact of COPD on employment and work productivity. Findings from the systematic review showed that patients with COPD had lower employment and higher sickness absence rates compared to those without COPD. From the cross-sectional analyses, breathlessness and occupational exposures to vapours, gases, dusts and fumes (VGDF) were identified as the main modifiable factors associated with unemployment and poor work productivity in COPD patients. Finally, few patients agreed to take part in the occupational intervention. Although most participating patients received a range of recommendations, these were not taken up by all. Potential benefits and suggestions for future occupational interventions were identified. This work confirms that having COPD adversely affects patients' rates of employment and work productivity. Future interventions should focus on managing breathlessness and reducing occupational exposures to VGDF to improve work ability and work productivity among patients with COPD. The development of novel interventions should draw upon lessons learned from the feasibility study reported in this thesis.

Transient Ischaemic Attack (TIA) is associated with a high early risk of stroke which can be considerably reduced by early initiation of secondary preventive drugs including antiplatelets, statins and blood pressure lowering therapy. These treatments are usually initiated by a specialist after urgent out-patient review. However, variable access to timely specialist services means that initiation of these treatments is delayed for some patients. The purpose of this thesis was to evaluate the cost-effectiveness of GP initiation of treatment following a suspected TIA compared with UK clinical practice. A Markov model was constructed to model the cost and effectiveness of urgent initiation of treatment following suspected diagnosis of TIA by GPs. In the base-case, GP initiation of treatment (followed by specialist review of treatments within a week) was compared with best practice, as stated in the National Stroke Strategy (2007). Strategies involving same-day GP initiation of treatment was found to be highly cost-effective at willingness to pay thresholds typically applied in the UK. This study illustrates the usefulness of modelling techniques to use secondary data sources to examine a policy relevant question around treatment urgency in a susceptible and identifiable group of patients where primary research is impracticable.

Femoral fracture in adolescents is a severe injury. Recent studies of intramedullary nail fixation with rigid titanium alloy helical nail viz. Expert adolescent lateral femoral nail (ALFN) have reported good results. However, there is no in vitro biomechanical data available on this nail in the literature. Experimental testing and finite element analysis (FEA) were used to establish the stiffness parameters of small composite femurs with simulated fractures stabilised using ALFN. In comparison to intact femur, construct stiffness ranged from maximum (114%) to minimum (20%) for healed fracture and segmental fracture, respectively. Simulation testing in SolidWorksTM was performed with validated FEA model to evaluate the effect of clinical and implant factors. Maximum predicted stress in the distal interlocking screw remained in an acceptable range (160.25 - 188.51 MPa) irrespective of the level of femoral shaft fracture with a relative decrease in stress values as the fracture callus healed over a 16 week period. The relative angle between the ALFN and proximal interlocking screw and implant material were two significant factors influencing stress at the interlocking screw and nail interface. In conclusion, a rigid helical titanium alloy femoral intramedullary nail can perform satisfactorily under physiological loading conditions experienced in the perioperative period.