Rationale: Elevated asymmetric dimethylarginine (ADMA) levels contribute to the pathogenesis of hypoxia-induced pulmonary hypertension. Chronic hypoxia decreases the activity of the enzymes metabolising ADMA, dimethylarginine dimethylaminohydrolases (DDAH1 and DDAH2) but the mechanisms responsible are not understood. Objective: To elucidate the physiological significance and the mechanism of hypoxia-induced downregulation of ADMA metabolism. Methods and Results: Exposure of human pulmonary artery endothelial cells (HPAECs) to hypoxia inhibited both DDAH1 and DDAH2 gene and protein expression, reduced DDAH activity and increased ADMA levels. In contrast, in human pulmonary artery smooth muscle cells (HPASMCs) only DDAH2 was reduced while ADMA levels remained unchanged. This endothelium-specific regulation of DDAH1 and ADMA resulted from NFκB-dependent, microRNA-21 (miR-21)-mediated degradation of DDAH1 mRNA. Down regulation of DDAH1 activity contributed to hypoxia-induced endothelial barrier dysfunction and HPASMC proliferation and was prevented by overexpression of DDAH1 and miR-21 blockade. Conversely, overexpression of miR-21 mimicked the effects of hypoxia. DDAH1 overexpressing transgenic mice exposed to 2 weeks hypoxia, showed attenuated pulmonary hypertension and vascular remodelling, compared with wildtype controls. Importantly, inhibition of miR21 in vivo prevented the hypoxia-induced reduction in pulmonary DDAH1 expression and attenuated the development of pulmonary hypertension. Lung tissue samples from hypoxic mice and treatment-naïve IPAH patients also showed reduced DDAH1 expression and increased miR-21 levels, compared with controls. Conclusion: Down regulation of DDAH1 expression by miR-21 in the pulmonary vascular endothelium has a key role in the pathogenesis of hypoxia-induced pulmonary hypertension and may be of broader significance in pulmonary hypertension.

Liver transplantation (LT) is a definitive treatment for end-stage liver disease and hepatocellular cancer. As donor-recipient HLA matching is not employed, there is potential for natural killer (NK) cell-mediated alloreactivity. In practise this is not observed, and to date the recipient NK response to LT has not been characterized. This thesis describes a detailed investigation into LT recipient NK cell phenotype, function and activity. Lymphocytes were extracted from peripheral blood samples taken from LT recipients. Expression of activating receptors on NK cells was assessed using flow cytometry assays, and NK function was analysed with functional assays. Recipient NK cells exhibited a downregulated phenotype with reduced expression of activating receptors NKp30 and NKp46. There was associated hypofunctionality, with impaired NK cell cytotoxicity, degranulation and IFNγ production. These changes were found only within the differentiated CD56dim subset, and not in CD56bright cells, implying a maturation defect between the two stages of development. Similar findings were not observed in LT recipients infected with hepatitis C virus (HCV), indicating a counteracting activating effect of HCV. To explore a mechanism for the downregulation observed a microarray experiment was performed, with subsequent qPCR analysis. In LT there was downregulation of STAT-4 with associated reduction in miR-155, a microRNA target of STAT-4 and a key regulator of NK differentiation. Functional assays demonstrated impairment of the IL-12/STAT-4 pathway in LT recipients. The altered NK phenotype and gene expression were not recapitulated in vitro with immunosuppressants. The influence of HLA mismatching on LT outcome remains controversial, and this was addressed by analysing donor HLA and recipient HLA and KIR typing. No associations between HLA and HLA/KIR mismatching, NK cell activation, and clinical outcomes were found. Taken together, the data presented in this thesis indicate that LT induces recipient NK cell tolerance through altered peripheral maturation at a step prior to the acquisition of inhibitory receptors for HLA class I. Consistent with this, HLA-C matching does not influence NK alloreactivity in LT.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by loss of motor neurons in the spinal cord, brain stem and cerebral cortex. ALS is characterized by both upper and lower motor neuron symptoms and death usually occurs 3-5 years after onset. Familial histories are found in 5-10% of ALS cases while the rest are sporadic. This study is focused on analysing known and novel candidate genes in ALS and the aims of study are to characterize causal genes and risk factors for Familial ALS (FALS) and Sporadic ALS (SALS) in the Imperial Cohorts, in which genetic causes have been assigned for 64% of FALS cases. Three strategies were pursued and genes involved in proteostasis pathways were emphasized in this study. Firstly, we sequenced known candidate genes in our FALS cases excluded for known mutations. VCP and SQSTM1 genes were sequenced. We did not identify any coding changes in VCP but report a 5' hexanucleotide expansion exclusively found in ALS. Known and novel SQSTM1 mutations, P392L and E155K, were identified in FALS kindred presenting with a history of Paget's disease of bone. Secondly, we carried out association studies for two candidate genes on Chromosome 17, P4HB and NPLOC4, and showed that they were risk factors for FALS and SALS respectively. The association of P4HB SNPs with FALS survival time indicates that it is a modifier gene. Thirdly, to explore novel genes in ALS, we investigated Variable number tandem repeats (VNTR) from top candidate genes selected based on association signals from previous Genome wide association (GWA) studies and protein functions. VNTRs in NIPA1 and HSPB8 gene were associated with FALS and SALS respectively. Finally, we characterized the size of the reported hexanucleotide GGGGCC expansion in the C9orf72 gene using Southern blot analysis in our FALS cohort and interim results are presented.

A hallmark of rheumatoid arthritis (RA) is cartilage erosion by pannus - an inflamed, hyperplastic and highly invasive synovial tissue. Cartilage degradation is primarily mediated by invading synovial fibroblasts. At the cartilage invasion front of the pannus, these cells destroy the tissue, leading to the permanent loss of joint structure and function. It has been shown that membrane type 1 metalloproteinase (MT1-MMP) plays a key role in promoting RA synovial fibroblast invasion into the cartilage. However little is known about regulatory mechanisms of MT1-MMP in RA synovial fibroblasts. MT1-MMP is highly upregulated in RA synovium, but mechanisms regulating its expression are not well understood. Interestingly, several reports show high MT1-MMP levels in fibroblasts at the pannus-cartilage junction. In addition, MT1- MMP expression and activity in cultured cells can be induced by collagen. We hypothesised that cartilage, more specifically cartilage collagen, induces MT1-MMP activity in the pannus. In this study, I have confirmed that both collagen and cartilage induce MT1-MMP activity and expression in RA synovial fibroblasts. To understand mechanisms of collagen signalling I have also investigated the role of collagen receptors, namely integrins and discoidin domain receptor 2 (DDR2), in MT1-MMP activation. Knockdown of DDR2, but not collagen-binding integrins, resulted in decreased MT1-MMP activity and expression upon collagen stimulation. DDR2 knockdown also inhibited MT1-MMP-dependent collagen degradation and invasion by RA synovial fibroblasts. Analysis of DDR2 binding to intact or telopeptide-devoid collagens indicates that collagen structure might influence cell signalling. Furthermore, activation of MT1-MMP by cartilage, which is also mediated by DDR2, is enhanced by removal of proteoglycans. In summary, I have demonstrated that cartilage signalling through collagen receptor DDR2 induces MT1-MMP activity in RA synovial fibroblasts.

Medical imaging using Terahertz frequency radiation is in its infancy. Optical adjuncts to enhance the diagnostic precision of white light endoscopy are not new and almost all wavelengths of the electromagnetic spectrum have, at some point, been investigated to this end. The ultimate aim of an endoscopic technique is to identify 100% of mucosal lesions and to classify them with 100% specificity. Methods: This thesis examined the sensitivity and specificity of current technology to accurately identify colonic pathology using a diagnostic precision analysis of published data. The effect of biomaterials such as blood, mucus and faeces on Terahertz radiation was assessed using human tissue samples from health volunteers. The ability to discriminate pathological from normal colonic mucosa was assessed using terahertz radiation to interrogate excised samples of human colon. The physical nature of variation between pathological and normal colonic mucosa was assessed using histological markers in an attempt to identify the cause of terahertz radiation contrast. Results: Current technology has a sensitivity of 95% and a specificity of 78% in a non- inflamed colon. Terahertz pulsed imaging is a sensitive and specific technique for in vitro classification of colonic mucosal pathology. TPI may be a useful adjunct in the presence of inflamed mucosal tissue. Although there were too few data from the present study to quantify any potential benefit. The effects of blood, stool and mucus on TPI are similar but less pronounced than water. Immunohistochemical analysis has demonstrated an association between vascular and lymphatic density with neoplasia. This may be a mechanism for TPI discrimination of colonic pathology. TPI could in the future contribute to a minimally invasive method of in vivo diagnosis of colonic dysplasia or malignancy. Larger scale and in vivo trials of the technology are necessary to further investigate the potential clinical benefit.

The nature and resource constraints of modern health care has led to increased reliance on rigorous treatment targets and detailed policy guidelines. Whilst well intentioned, this can shift the focus away from patients themselves and places emphasis on arbitrary measures of output or performance that can relate poorly to how people experience and benefit from health care. Considerable effort and resources are spent on health care interventions, but policy makers and clinicians may be left without a clear understanding of how these treatments actually affect people in the experience of their lives. Subjective wellbeing (SWB) is a broad category of phenomena that includes people's emotional responses, domain satisfactions (e.g., health or work), and global judgements of life satisfaction. Measures of SWB offer a means to gauge the impact of changes and events in the lives of individuals. In recent years, there has been increasing interest in the use of SWB in shaping public policy (e.g. in relation to environmental and economic policies). In my thesis, I examine for the first time the ways in which measures of SWB can be used directly within a health care setting and to inform health policy decisions. In doing so, I have drawn on my understanding of clinical environments and health care systems as a practising clinician to bring a new perspective to the way that SWB is considered and used in health care and to how it can be applied in determining health policy. I explore the use of SWB at both the macro and micro levels of policy making and address the challenges faced when using measures of SWB in these ways. At the macro level, I examine the limitations and challenges of existing methods and examine where using SWB would have the most impact. I also focus on the use of SWB measures at a micro level, setting out a new model of patient experience with reference to SWB. My thesis sets out original methods for SWB data collection developed through innovative empirical work. This work into hernia surgery and on the SWB of the staff and inpatients of an acute NHS hospital has generated new data sets in clinical populations. I discuss the implications of this research and explain how, when and where SWB measures, when used in health care, can be used in health policy. All too often we lose sight of what really matters in life, and the world of heath care is no exception. Through the application of measures of SWB in health care, I offer a novel perspective that ensures a greater focus is placed on the way that patients experience health interventions when developing health policy.

Many of the more significant challenges we face in healthcare - such as reducing smoking, encouraging exercise and improving clinician adherence to evidence-based guidelines - will only be resolved if we are more successful at changing behaviours. The traditional tools used when thinking about influencing behaviour include legislation, regulation and information provision. Recently, interest has been shown in policies that 'nudge' people in particular directions; drawing on major advances in our understanding that behaviour is strongly influenced (in largely automatic ways) by the context and situation within which it is placed. Insights from across the behavioural sciences and particularly behavioural economics provide us with a powerful set of new and refined policy tools to use when trying to influence health-related behaviours. My thesis - 'Applying behavioural insights to challenges in health policy' - considers the theoretical basis for why 'nudges' might work and presents the Mindspace framework (that I co-developed with colleagues) that supports policy makers and practitioners looking to apply behavioural insights. Mindspace sets out what are considered to be the most robust effects on behaviour that operate largely, though not exclusively on automatic neurobiological and psychological systems. I explore the evidence for each of the Mindspace effects and demonstrate how they can be applied to specific areas of challenge including in enhancing the safety of medication prescribing, improving hand-hygiene compliance and reducing the trajectory of health spending in developed health systems. I do so through a mixture of qualitative and quantitative approaches, at all times grounding the work in the practicalities of my experience as a clinician. I explore the ethical and political considerations in determining whether it is appropriate to target automatic processes of judgment and make suggestions as to the direction of travel of 'nudging' in health policy going forward, particularly in relation to new information and communication technologies.

We can all use assessment and appraisal to help us improve our performance in any area of life. Healthcare researchers are no exception. For healthcare researchers a system is required to measure research performance according to an accepted global benchmark. While there are existing systems that have been created to measure research performance in general, and healthcare research performance has been appraised with several bibliometric indicators, there is a lack of evidence to prove their validity and a deficiency of indicators that embrace social behaviours such as collaboration. In this thesis we endeavoured to enhance knowledge on healthcare research performance assessment, which has the potential to be integrated into systems that specifically appraise healthcare research performance. Ultimately, these systems may promote a performance-based culture that better reflects the quality and impact of healthcare research.

Despite its great success, antiretroviral therapy (ART) fails to improve HIV-1-specific T-cell responses in treated individuals, although it does restore CMV-specific immunity. Furthermore, co-infection with CMV has been associated with increased risk of non-AIDS-related morbidities. This thesis comprehensively characterises HIV-1- and CMV-specific T-cell responses in HIV-1-infected subjects, in the context of both ART and different progression rates. Proliferative responses to CMV and HIV-1 were evaluated in HIV-1-infected and uninfected healthy controls (HC). ART led to a discordant increase in CMV-specific proliferation relative to that observed for HIV-1. High proliferative responses to both viruses in elite controllers contrasted the low CMV-specific proliferation in HC. Strong induction of the suppressive cytokine IL-10 was observed in response to CMV and HIV-1 Nef, but not to Gag, and this was higher in ART-naïve individuals compared to treated patients. Still, HC exhibited the highest levels of CMV-specific IL-10, pointing to a regulatory role for IL-10 in these responses, possibly explaining the low proliferative responses in HC. Analysis of T-cell activation, differentiation and exhaustion revealed similar frequencies of HIV-1-specific CD8 effector memory (TEM) cells between ART-naïve and treated individuals, but CMV- specific CD8 TEM cells were higher in treated individuals correlating with the increased anti-CMV T-cell function observed. Higher CD8 TEM and lower naïve CD8 T-cell frequencies were significantly associated with disease progression, whilst long-term non-progressors exhibited higher levels of CD8 terminally differentiated (TEMRA) cells. Moreover, CD8 T cells with an HLA-DR+CD38- phenotype were elevated in treated individuals compared to both HC and ART-naïve patients, and linked with cytotoxic activity in a non-progressor. Detection of replication-competent virus from HIV-1 controllers at different outgrowth rates suggests reduced replicative fitness. Overall, these results detail the complex interplay between CMV and HIV-1 co-infection, the difference between ART-mediated and spontaneous immune control and point to potential targets for immunotherapeutic interventions.

Clinical practice guidelines (CPGs) are designed to improve the care and safety of patients in hospitals. This thesis explores the promoters and barriers for CPG adherence among nurses. The research is based on a combination of a systematic literature review, qualitative research and a quantitative study. The systematic literature review included searching three data bases, namely, the British Nursing Index (BNI), Medline and Cumulative Index to Nursing and Allied Health Literature (CINAHL). The qualitative research study included one-to-one interviews and focus groups. The quantitative study consisted of a questionnaire distributed to nurses to extend and check the findings of the qualitative studies. The systematic literature review revealed that the attitude of doctors to any CPG is influenced most by the level of their agreement with the guideline and by its applicability in practice. The adherence of nurses to CPGs is influenced most by the support and feedback they receive and by team interactions. A previous framework for CPG adherence by doctors has been produced by Cabana (1999) based on a literature review. This thesis extends that framework to nurses, and adapts it on the basis of my original research findings. Three principal themes emerged from the qualitative studies; namely, nurses’ attitudes to CPGs, their knowledge of CPG and external factors that influence CPG adherence. Within these, the most prominent promoters of CPG adherence were nurses’ sense of their accountability, professional values and self-efficacy, as well as managerial monitoring and belief that a CPG would achieve the expected desirable outcome. The last of these depended to a large extent on nurses’ trust in the credibility of the guideline authors. The main barriers to CPG adherence were lack of knowledge about the guidelines caused by insufficient time to read them, poor presentation and inadequate dissemination of CPGs and the low priority given to training within a nurse’s schedule. Other barriers included lack of staff resources to apply CPGs, the exigencies of individual patient problems and wishes, the frequent movement of nurses between specialisms and a general failure to involve nurses in drafting the guidelines. All these results were confirmed by the results of a questionnaire survey. The revised framework presented here could help health care organisations, medical educators, policy makers and managers to develop better models for CPG development and awareness, especially among nurses, and to have a greater insight into the factors that promote or inhibit CPG adherence. Based on the framework, recommendations are made to help these groups of people, and nurses themselves, improve nurses’ adherence to CPGs. These are presented below, and are found as Table 7.1 in the thesis.