Systemic sclerosis (SSc) is a complex multisystem autoimmune connective tissue disease of yet unknown aetiology. It is characterised by vascular damage, autoimmunity and progressive fibrosis in the skin and internal organs, with 2 main subsets of disease: diffuse cutaneous SSc (dcSSc) and limited cutaneous SSc (lcSSc), classified mainly according to skin involvement. Although there has been tremendous progress in the understanding and treatment of autoimmune diseases in the last 20 years, SSc remains a disease with high morbidity and mortality, with no proven treatments that improve long-term outcome. There is therefore a need to improve the understanding of such a devastating disease to facilitate the development of future treatments and improve the prognosis for patients with SSc. Telomeres are nucleo-protein complexes, comprising tandem TTAGGG repeats at the ends of eukaryotic chromosomes, which protect the chromosomes from end-to-end fusion, recombination and degradation. There are a variety of telomere binding proteins which maintain telomere stability and function in sensing, signalling and repairing DNA damage. Telomere lengths have been used as a marker of biological aging for over a decade, and the technologies for measuring telomere lengths have also advanced in recent years. Quantitative real time PCR (QPCR) has now superseded Southern Blotting (SB) for telomere length measurements. Telomere erosion has been implicated in a wide range of diseases and its impact on autoimmune diseases remains unclear. As SSc is associated with particular autoantibodies which have been linked to telomeres, this thesis sought to explore telomere biology in SSc. Initial work measuring telomere lengths in SSc patients using the SB method revealed surprising results, with a lack of age-related telomere erosion in patients with lcSSc, which was in contrast to published literature at the time. The aim of this research was to measure telomere lengths of peripheral blood mononuclear cells (PBMC) in patients with SSc and related connective tissue diseases using QPCR, with the hypothesis that telomere lengths would be shorter in disease, and to explore telomere binding protein genes, with a view to gaining insight into any mechanistic differences in telomere biology in the different subsets of disease. Measurement of telomere lengths of PBMC from healthy controls and patients using QPCR showed that telomere lengths were significantly shorter in disease compared to controls, but when adjusted for age, there was no statistically significant difference in telomere lengths of patients with lcSSc compared to controls. Taking into account the complexity of telomere biology, it is possible that mediators of the inflammatory reaction at the systemic level or autoantibodies against nucleoprotein complexes directly or indirectly interfere with the homeostasis of telomere length in blood. This could occur through disrupting the proteins of the shelterin complex or associated factors relating to DNA repair. Hence, the gene expression of telomere binding proteins and other genes associated with inflammation and DNA repair were explored in patients with SSc using Taqman Low Density Arrays. The majority of these genes were under expressed in disease compared to controls, but when samples were age-matched, only 4 genes were under-expressed in patients with dcSSc: BCL2, POT1, SIRT6 and WRN, and 4 genes were under-expressed in lcSSc: ATM, BCL2, STAU1 and WRN. There was no correlation between telomere lengths and gene expression. These observations are intriguing, and the role of BCL2 and WRN merit further investigation in patients with SSc. This work has confirmed that telomeres shorten with age and disease, in keeping with the original hypothesis and published literature. However, there was no significant difference in age-related telomere erosion in patients with lcSSc. Gene expression analysis of telomere associated genes in SSc revealed lower expression in disease compared with controls. Whether this observation is a cause or effect of disease remains to be proven, but suggests that telomeres may be implicated in the pathophysiology of SSc and there are mechanistic differences between the subsets of disease.

There is a growing evidence of the potential health benefits of a diet rich in fruits and vegetables. National nutritional guidelines advise the consumption of at least five portions (400 g ) of these foods per day with the goal being a reduction in the levels of coronary heart disease (CHD) strokes and certain cancers. The beneficial properties of fruit and vegetables may be ascribed, in part to the presence of antioxidants and recent attention in this regard has focused on phenolic and polyphenolic compounds. These compounds are present in a wide variety of commonly consumed foods and beverages. Onions are a rich source of the polyphenolic flavonol quercetin-4′-glucoside. For this compound to have some health effects it must be absorbed and reach target organs in a concentration and form where they can exert an effect. To-date interest has focussed on the levels of the intact quercetin aglycone circulating in plasma and excreted in urine. However, it is now known that quercetin does not circulate in the plasma as the parent compound or the aglycone. However, at the outset of this project, the exact form(s) and concentration of metabolites circulating in plasma were unknown. The need to know what compounds are actually circulating, and at what concentration, is important if in vitro studies are to be made into the mechanisms by which quercetin could, potentially, exert a health benefit. The reasons why these issues have not been addressed are due to a number of factors. The main methodology used in studies into absorption, distribution, metabolism and excretion or (A.D.M.E) as it is know in the drug industry, is by use of chromatography coupled to various detection systems. This can range from a simple isocratic single pump linked to a single wavelength absorbance detector, to a gradient pumping system with an autoinjector linked in series to a diode array absorbance detector and mass spectrometer. The latter instruments, although initially expensive are now becoming more affordable. The original methodology used to determine the level of quercetin in plasma involved hydrolysis of the quercetin conjugates back to the aglycone. The information, which is lost by using this hydrolysis method, is vital if we are to gain a better understanding of the A.D.M.E process. There have been a large number of feeding studies carried out using onions or the flavonol contained in them. However, very little additional information was gained after the initial investigations. The objectives of the studies presented in this thesis were to develop methodology to identify and quantify the major metabolites of quercetin in man after ingestion of onions. This would initially require the use of radiolabelled [2-14C]quercetin-4´-glucoside fed to rats to facilitate the development of the method. Having successfully developed methods that would work both in rats and in man, it was of great interest to establish the fate of the complete dose of [2-14C]quercetin-4´-glucoside in rats. In Chapter 2 radiolabelled quercetin-4´-glucoside was used as a tracer to follow the metabolism of the compound as it was acted on by the digestive system of the rat. After 1 h 93% of the ingested dose was recovered in the gastrointestinal tract (GIT). Analysis using HPLC with a photodiode array (PDA) detector in series with a radioactivity monitor connected to an electrospray ion trap mass spectrometer facilitated the separation, quantification and partial identification of 18 out of 19 metabolites. The 1 h sample was part of a larger study that investigated the fate of the radiolabelled compounds up to 5 h after dosing. The latter samples formed part of another study not reported on in this thesis. Having developed the methodology, using the radiolabelled compound, it was then applied to a feed of onions to healthy human volunteers to determine if metabolite detection, identification and quantification could be carried out without the use of the radioactive tracer. In Chapter 3 plasma samples collected 1 h after a feed of onions and urine collection from 0-4 h post feed were used to test if the method could be transferred to a non labelled assay. A total of 22 metabolites plus the parent compound were identified. The metabolic profile of the plasma and urine showed marked differences, again pointing to major post absorption metabolism. The successful transfer of the method from the initial radiolabelled study to the onion feed allowed pharmacokinetic data to be obtained from all plasma samples taken over a 24 h period, along with the 0-24 h urine samples. In Chapter 4 it was seen that the metabolites are both rapidly absorbed and excreted, with plasma levels returning almost back to baseline by 6 h. The total excretion in urine accounted for 4.5% of the ingested dose. These results were controversial, as the pioneer of this field had published that the elimination half life of quercetin was of the order of 18 h. The differences between the two methods employed are discussed in Chapters 3 and 4. The fact that only 4.5% could be accounted for in this study, which was in agreement with other studies, leaves the question of what happens to the other 95.5%. It is possible that the potential health benefit attributed to this compound may have nothing to do with the parent compound but could be coming from something in the other 95.5%. Studies using patients who have undergone an ileostomy have been used to provide further information into what happens to the majority of the dose. By collecting the ileal fluid after a flavonol feed the amount of intact compound can be measured in ileal fluid (Hollman et al., 1995b; Walle et al., 2000). This work and some results from a similar trial study are discussed in Chapter 4, with regard to the process of metabolite absorption and formation. The only way to follow the parent compound throughout its passage through the body is by use of a labelled compound. In Chapter 5 a second feed of [2-14C]quercetin-4´-glucoside, which focuses on the overall fate of the compound, has samples collected for up to 72 h. The fate of the dose was monitored both in terms of the level of radioactivity excreted and found in the tissues and also the identity of the radioactive compounds detected in these samples. In Chapter 5 the results from this study and what impact they could have on quercetin’s potential ability to be the compound responsible for the health benefits are discussed.

Participation in regular physical activity among young adults (16-24 years) is suboptimal. Research has attempted to understand what determines exercise behaviour and how interventions can assist individuals in adopting and adhering to exercise. This thesis consists of a literature review, and three separate studies. In the literature review, inactivity is defined, and the current physical activity patterns of industrialised society are discussed. Models of behaviour change that enhance our understanding of the adoption and adherence process in physical activity are examined. In particular, the transtheoretical model of behaviour change [TTM] has been selected for further study. This model is unique to the study of exercise behaviour because it provides researchers with an opportunity to identify and work with an inactive population, and permits the tailoring of physical activity interventions to make them more suitable for sedentary individuals. Initially, the TTM and its core constructs are explained, followed by a critique of the model. A review of empirical research in physical activity that has been based on the TTM is included. Over fifty key studies were identified; the limitations and strengths of these studies are explored. Finally, the discussions will summarise what has been learned to date about the application of the TTM to the understanding of behaviour modification in physical activity. In study one, constructs from the transtheoretical model of behaviour change were chosen to help understand the process of exercise behaviour change of a student population. A total of 2943 respondents completed a baseline questionnaire and 1058 completed a follow-up 7 months later. A 5-item stage of change and a 40-item process of change questionnaire was used. There were significant differences in physical activity patterns from baseline to follow-up. There were also significant differences in process use across the stages. The process data was factor analysed to refine it further. A three-factor model revealed different motivational clusters underlying actual stage of behaviour change. Recommendations for intervention design suggest that adopting a positive behaviour should be treated differently to ceasing a negative behaviour. In study two, a pre-post randomised control design was used to investigate the effectiveness of a self-instructional intervention for helping a sedentary undergraduate population to become more active. The intervention was based on the transtheoretical model of behaviour change. Significantly more of the experimental group in comparison to the control group improved their stage of change from baseline. Self-efficacy and not decisional balance was found to be useful predictor of stage improvement. Discriminant analyses revealed that discrimination between stage improvement versus non-improvement was possible using the processes of change data. For stage improvers, the processes self-reevaluation and self-liberation were most frequently used, while social liberation was used significantly more by the experimental than the control group.

The first chapter of my thesis “The Gastro-oesophageal Junction” discusses the histology of the gastro-oesophageal junction and the components integral to the function of the anti-reflux barrier. It also discusses the pathology found at the gastro-oesophageal junction and describes the nitrite chemistry in this region thought to contribute to this pathology. The second chapter “Mechanisms of Gastro-oesophageal Reflux” discusses the mechanisms of gastro-oesophageal reflux. This includes factors that reduce lower oesophageal pressure, the role of the hiatal hernia and the contribution of transient lower oesophageal sphincter relaxations. The relationship between obesity and reflux disease is also discussed. The third chapter “Research into the Nature of the Acid Pocket” details previous research into the nature of the acid pocket, the primary focus of my own studies. The fourth chapter “Severe Reflux Disease is Associated with Enlarged Unbuffered Proximal Gastric Acid Pocket” details my studies comparing the postprandial acid pocket in healthy subjects and patients with severe reflux disease and my attempt to define its position relative to anatomical and manometric landmarks. 12 healthy subjects and 16 patients with severe reflux disease were studied. While fasted, a station pull-through was performed using a combined dual pH and manometry catheter. Position was confirmed by radiological visualisation of endoscopically-placed radio-opaque clips. The pullthrough study was repeated 15 minutes after a standardized fatty meal. Barium meal examination was performed before and following the meal. A region of unbuffered acid (pH≤2) immediately distal to the proximal gastric folds was more frequent in reflux patients (23/32 studies) than in healthy subjects (11/24) (p<0.05). This unbuffered acid pocket was longer in the reflux patients versus healthy subjects (median length 3cm, range 1cm to 15cm vs. 2 cm, range 1cm to 5cm; p<0.05). The acid pocket extended proximally as far as the proximal gastric folds in the patients but stopped a median of 1.1cm distal in healthy subjects (p=0.005). In healthy subjects the acid pocket occupied the distal portion of the sphincter which opened postprandially, whereas in reflux patients it corresponded to the proximal displacement of the gastric folds i.e. hiatus hernia. The fifth chapter “Paradox Of Gastric Cardia – It Becomes More Acidic Following Meals While The Rest Of Stomach Becomes Less Acidic” details stationary pH studies of the cardia in healthy subjects. The proximal cardia region of the stomach has a high incidence of inflammation, metaplasia and neoplasia. It demonstrates less acid buffering following meals than the more distal stomach. I employed novel high definition pHmetry to investigate acidity at the cardia under fasting conditions and in response to a meal. 15 healthy subjects were studied. A custom made 12 electrode pH catheter was clipped at the squamo-columnar junction with 4 electrodes recording proximal to and 8 distal to the squamo-columnar junction. The most distal pH electrode was located at the catheter tip and 9 electrodes in the region of the squamo-columnar junction were 11mm apart. The electrode situated in the cardia 5.5mm distal to the squamo-columnar junction differed from all other intragastric electrodes during fasting in recording minimal acidity (pH<4 =2.2%) while all other intragastric electrodes recorded high intragastric acidity (pH<4 =>39.%) (p<0.05). The cardia also differed from the rest of the stomach showing a marked increase in acidity in response to the meal (from 2.2% fasting to 58.4% at 60- 70min after meal; p<0.05) while the electrodes distal to the cardia all showed a marked decrease in acidity (p<0.05). These changes in acidity at the cardia following the meal caused the gastric acidity to extend 10mm closer to the squamo-columnar junction. The final discussion chapter discusses the results of our studies and my conclusions. Papers concerning the acid pocket since my own work are also discussed. My studies had full approval from the West Ethics Committee and North Glasgow Trust (COREC Reference 04/50709/26).

Cardiac hypertrophy often develops to compensate for hemodynamic overload and is associated with heart failure. Recent studies have revealed that overexpression and PKA-mediated phosphorylation of heat shock protein 20 (HSP20) at Ser16 can attenuate hypertrophic growth of cardiomyocytes and trigger cardioprotective functions following sustained β-adrenergic stimulation (Fan et al., 2004, 2005, 2006). However, the molecular mechanism of HSP20 induced cardioprotection remains to be fully elucidated. In order to gain insight into the protective mode of action of HSP20, I attempted to (1) investigate the spatiotemporal control of PKA-mediated phosphorylation of HSP20, as well as (2) identifying novel protein binding partners for HSP20 utilising cutting edge ProtoArray technology. Initially, I set up an in vitro hypertrophy model using sustained isoprenaline (ISO)-stimulated neonatal rat cardiomyocytes. Cell size, protein synthesis and fetal gene expression were assessed as parameters of hypertrophic growth. In the first section of my studies, members of the cAMP-specific PDE4 family were shown to form signalling complexes with HSP20, and that the PKA-mediated phosphorylation of HSP20 could be modulated by PDE4. Based on peptide array data, a cell-permeable peptide ‘bs906’ was developed to inhibit the interaction of PDE4 with HSP20. Interestingly, the disruption of the PDE4-HSP20 complex was shown to induce PKA-mediated phosphorylation of HSP20 and trigger cardioprotection against the hypertrophic response measured in neonatal cardiomyocytes upon chronic β-adrenergic stimulation. In the second part of my studies, protein kinase D1 (PKD1) was identified as one interacting partner that robustly associated with HSP20. This interaction was confirmed by biochemical and immunocytochemical means. Using similar approaches to those used for the PDE4-HSP20 interaction, a cell-permeable peptide ‘HJL09’ was generated to promote disruption of the PKD1-HSP20 complex. Experimentation using the peptide concluded that the disruption of the PKD1-HSP20 complex reduced HSP20 phosphorylation and attenuated the hypertrophic response in cultured cardiomyocytes as shown by reduced increases in cell size, protein content and actin reorganisation. In undertaking this work, I also defined a novel PKD phosphorylation site (Ser16) on HSP20 that conforms to the PKD phosphorylation motif of RxxS (also a PKA site). My biochemical data suggested that PKD1 may regulate the cardioprotective function of HSP20 via phosphorylation at Ser16. In situ proximity ligation assay (PLA) further revealed a role of HSP20 as ‘molecular escort’ in targeting the nuclear translocation of PKD1. This function, in part, may be responsible for the induction of fetal gene reexpression as selective disruption of PKD1-HSP20 complex using ‘HJL09’ hindered the nuclear influx of the complex, thereby attenuating hypertrophic signalling. In summary, these studies describe some exciting findings which provide further insight into novel signalling mechanism of cardiac hypertrophy in neonatal rat cardiomyocytes. I have shown that PKA and PKD1 exhibiting opposite functions despite sharing the phosphorylation site on HSP20. In this regard, HSP20 functions as a molecular nexus for the opposing actions of the PKA and PKD1 signalling pathways in hypertrophy, suggesting that crosstalk may occur between anti-hypertrophic and pro-hypertrophic pathways. The identification and characterisation of these complexes should help to build a better understanding of the hypertrophic signalling pathway, and may provide novel therapeutic strategies for the treatment of cardiac hypertrophy.

Postal questionnaires offer one of the least expensive modes of collecting patient based outcomes in health care research. Many methods of increasing response to questionnaires used in educational and market research surveys have been tested. Behavioural theories have also been applied to survey research to understand response decisions. Little attention, however, has focussed specifically on response issues to postal questionnaires used to collect data in clinical trials. This is the subject of this thesis. A systematic review of methods of improving response to postal questionnaire follow-up in health care studies was conducted. A method of improving response was then devised and its effectiveness was tested within an existing clinical trial (the Collaborative Ankle Support Trial - CAST). This method was a 'Trial Calendar'which was a prompting and reminder tool to encourage response. Qualitative data were gathered from clinical trial participants to ascertain factors influencing their response decisions. Finally, the socio-demographic characteristics of CAST participants were examined. The systematic review demonstrated that follow-up reminder systems had the most significant effect on response rates (RR 1.82, Cl 95% 1.11 to 2.99). Incorporating such reminders into a tool such as the 'Trial Calendar', however, had no effect on improving response in CAST. The qualitative study revealed aspects of behavioural theories which could be incorporated into trial information and appeals for response. Analysis of the sociodemographic characteristics of CAST participants revealed that the youngest age group (16-24 years) was less likely to respond at every followup point. It is concluded that rather than anticipating low response rates and striving to devise methods of converting non-responders into responders, efforts should be directed at preventing participants becoming non-responders in the first place. This thesis argues for the area of follow-up to postal questionnaires in clinical trials to become a theoretical research issue in its own right.

Seamless phase II/III clinical trials are attractive in development of new drugs because they accelerate the drug development process. Seamless phase II/III trials are carried out in two stages. After stage 1 (phase II stage), an interim analysis is performed and a decision is made on whether to proceed to stage 2 (phase III stage). If the decision is to continue with further testing, some dose selection procedure is used to determine the set of doses to be tested in stage 2. Methodology exists for the analysis of such trials that allows complete flexibility of the choice of doses that continue to the second stage. There is very little work, however, on optimizing the selection of the doses. This is a challenging problem as it requires incorporation of the dose-response relationship, of the observed safety profile and of the planned analysis method. In this thesis we propose a dose-selection procedure for binary outcomes in adaptive seamless phase II/III clinical trials that incorporates the dose response relationship, and explicitly incorporates both efficacy and toxicity. The choice of the doses to continue to stage 2 is made by comparing the predictive power of the potential sets of doses which might continue to stage 2.

This thesis explores the complex discipline of community palliative care. Palliative patients suffer from a range of conditions, have complex, evolving clinical problems and receive care from a wide variety of health and social care professionals. Understanding these issues is central to effective service provision and maintaining continuity of care for patients and their carers. Current community palliative care provision in the UK is the result ofthe co-evolution of the emergent specialties of palliative and primary care over the past sixty years. A critical realist and practitioner based research approach is used in a multi-method study ofthe reasons for emergency admission ofpalliative patients into hospital and a qualitative study exploring the work and experiences of health care assistants prov~ding practical supportive care for palliative patients in their homes. Examining narratives of patients, carers and health professionals enables in depth exploration ofthe fundamental elements and contexts which define the inherent complexity ofthis area. Emergency admission ofpalliative patients represents a significant breach in continuity of care often resulting in disruption for both patient and health care services. The reasons for these admissions are many and complex. Relationships of power between different health professionals and between patients and health professionals exert a major influence on community palliative care provision and the process of admission. Health care assistants are relatively powerless and employed as basic carers, yet in community palliative care they undertake emotional labour, for which they draw on their personal resources. This aspect oftheir practice is crucial for patients and their carers; however it is largely unrecognised by formal health care services. Undertaking research in the context of a community palliative care team enables the development of a community of research practice. This provides an effective model for both developing an evidence base for community palliative care and the development of appropriate local services.

The overarching aim of this thesis is to evaluate Lean implementation in the English NHS. Against a background of financial austerity measures and the ostensible widespread adoption of Lean in the UK public sector, and particularly by healthcare organisations, the objective is to understand how Lean is being implemented by NHS hospital Trusts, and whether there is any quantitative evidence that Lean implementation is improving hospital performance. Adopting Pettigrew and Whipp’s (1991) framework of strategic change, this thesis aims to present theoretically sound and practically useful research through an exploration of the context, process and content of Lean implementation by English hospital Trusts. In order to achieve this, the research employs a mixed methods research design incorporating document analysis3, quantitative analysis and case study analysis to afford an insight into the implementation of Lean from multiple viewpoints and facilitate the development of new insights relating to the phenomena of Lean implementation in English hospital Trusts. The research provides a contribution to knowledge in three key areas: firstly through the identification and validation of a typology of approaches to Lean implementation by English hospital Trusts i.e. a characterisation of the method of Lean implementation; secondly through quantitative analysis and discussion of the potential link between Lean implementation and increased performance; and thirdly a set of propositions that provide a narrative and logic to explain the influence of contextual factors upon the process of Lean implementation in English hospitals.

This thesis presents designs for a series of clinical trials where instead of designing clinical trials individually, each of the trials is designed as part of a series of trials. The framework of the design is based on a combination of classical frequentist and Bayesian approaches which is sometimes known as the hybrid approach. The unknown parameter of the treatment efficacy is assumed to be random and follows a prior distribution in the design stage but at the end of the trial a frequentist test statistic is used on the observed data to infer the parameter. The design introduced in Chapter 5 aims to determine an optimum sample size for each trial by optimizing the average power of each trial and the overall resources while fixing the conventional type I error. The design has the exibility to either run sequentially or concurrently. The design is then extended to allow interim analyses in each trial (Chapter 6). The focus of the extended design is on a series of Bayesian decision-theoretic phase II trials and one frequentist phase III trial. At each interim stage, a decision is made based on the expected utilities of subsequent actions. There are four possible actions to choose from, namely, to continue the current trial by recruiting more patients, to initiate a new phase II trial, to abandon the development plan or to proceed to a phase III trial with this treatment against a control arm. For the last action, the phase III trial is designed with the hybrid methodology as described above. Finally, the prior distributions for each treatments are assumed to be correlated and as information is gathered from the previous and current trials, the current and following prior distributions are updated (Chapter 7).