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.

Chronic Kidney Disease (CKD) is common and often has a major impact on the health of those affected. In this thesis I have focused on areas of uncertainty that may have major implications for patient care. Firstly, I assessed the determinants of increased mortality in a multi-ethnic primary care population. Secondly, I investigated the differential progression of CKD between ethnicities. Thirdly, I assessed if tryptase, as a marker of mast cell activation, could be used to stratify risk in CKD. Finally, I investigated the impact of CKD on health related quality of life (HRQL) and the association between HRQL and clinical end-points. I found that: (i) comorbidity has a profound impact at a population level on survival in CKD; (ii) albuminuria is the principle modifiable risk factor for progression to end-stage renal disease (ESRD) in people of South Asian ethnicity; (iii) serum tryptase is an independent prognostic factor for ESRD in patients with CKD receiving treatment with an ACEi or ARB; and (iv) Low HRQL is common in CKD and reduced HRQL is associated with a higher risk for death. The findings from this thesis contribute to the understanding of CKD in ethnically diverse, high-risk populations and form the basis for further studies.

Home Treatment for acute psychiatric illness was compared to conventional hospitalisation in a design which focused on completed episodes of either exclusive alternative. The evaluation was conducted with the West Birmingham Home Treatment Team and All Saints Hospital, Birmingham. Forty cases in each group were examined with closely equivalent sociodemographic features and previous psychiatric history. The length of treatment, clinical outcome, identification and targeting of needs, readmission profile and client satisfaction were compared. The study focused on presentations involving mainly a diagnosis of severe mental illness. Home Treatment was significantly shorter and involved wider targeting of identified needs. There was no significant difference in terms of clinical outcome. Home Treatment and avoidance of admission were preferred by patients. The determinants of satisfaction with acute care in both settings was explored qualitatively. Significant design and sampling problems limit the generalisability of results. The case for and against Home Treatment is examined. The lessons learnt during the course of the study regarding the appropriate focused evaluation of Home Treatment and the place of Home Treatment as a particular model of intensive care are critically discussed.

Current methods to study enhancers in vivo involved integrating DNA sequences into the genome in a random position, in zebrafish the transposase Tol2 is utilised. However, this random genome integration can result in position effects which cause variations in reporter gene expression patterns preventing conclusions from being drawn about the enhancer being tested. To address the issue of variable position effects when using random integration techniques to study enhancers, this project aimed to establish a PhiC31 integrase system of targeted transgenesis in zebrafish. Firstly, we designed and tested a multicomponent system including a recipient vector, donor vector and PhiC31integrase mRNA. These components were co-injected into one cell stage embryos to test the integration function of PhiC31 integrase in zebrafish. The integration was detected by designing the recipient and donor vectors such that a switch from GFP to mCherry lens expression marks embryos with legitimate recombination. Over 97% of fluorescent embryos showed mCherry expression in the lens after coinjection of the three components indicating that integration of the two vectors by PhiC31 integrase is highly efficient. Legitimate recombination was shown to have occurred using PCR and sequencing techniques from total RNA extracted from embryos. A recipient transgenic line was made into which the donor vector and PhiC31 integrase RNA were co-injected. This resulted in around 70% of fluorescent embryos showing mCherry expression in the lens which suggests targeted genome integration is also efficient in zebrafish embryos. To establish whether variability of positional effects could be reduced using this system, an enhancer-promoter-reporter construct was modified to ii include donor vector sequences and injected into recipient line transgenic zebrafish embryos. Expression patterns observed in stable lines made with either Tol2 transposase or PhiC31 integrase suggest that variability in position effects is reduced when using the PhiC31 integrase system. Together, these results provide evidence that the PhiC31 integrase system I have tested is suitable for a number of uses in zebrafish, including enhancer screening.

Previously thought to be inert, nitrite is now deemed to be an important endogenous source of nitric oxide (NO), particularly during hypoxia and ischaemia. Largely animal and some limited human studies propose a role for nitrite in ‘hypoxic vasodilatation’ and ischaemia-reperfusion injury. One study explores the effect of systemic nitrite infusion on cardiac and peripheral haemodynamic parameters in healthy subjects, during normoxia and hypoxia. Despite a lack of observed effects in healthy subjects, a study performed in patients with heart failure during normoxia demonstrated favourable haemodynamic effects on cardiac output, albeit at high doses of nitrite. In patients undergoing coronary artery bypass surgery, low dose nitrite infusion afforded protection against ischaemia-reperfusion injury. Several mechanisms of nitrite reduction to NO have been described and remain to be fully elucidated. The role of one of these putative mechanisms, namely mitochondrial aldehyde dehydrogenase (ALDH2) was explored during normoxia and hypoxia. Ex vivo human vascular ring studies confirmed ALDH2 as an important nitrite reductase, in contrast to in vivo observations in the forearm vasculature, suggesting that numerous mechanisms are involved in vivo which are harder to isolate. Furthermore, this hypoxia vasodilatory role was not replicated in with GTN in the human forearm vasculature.