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Human embryonic stem (hES) cells have great therapeutic potential for the treatment of degenerative conditions such as Parkinson's disease, cardiac failure and type I diabetes. This potential is based on the ability of hES cells in vitro to self-renew and also differentiate to cells of all three germ layers; ectoderm, mesoderm and endoderm. Type I diabetes is due to an autoimmune disease destroying the insulin-secreting cells of the pancreas (β-cells) that regulate plasma glucose concentration. The pancreas develops from the endoderm lineage. 2. To find a cure for type I diabetes based on the use of hES, it is essential to understand the differentiation process of ES cells into the endodermal, β-cell lineage. The aim of this study was to investigate the generation of insulin-secreting cells using hES cells in vitro and to compare sue with those in the developing pancreas of the foetus.

Introduction: Type 1 diabetes is a common cause of chronic disease in young people. Over the past few decades a worldwide rise in incidence has been observed, particularly in children under the age of 5 years. life expectancy is reduced with the major causes of mortality being renal and cardiovascular disease. Good glycaeamic control has been shown to reduce the risk and progression of micro and macro vascular complications in type 1 diabetes. However, in many people, target HbA1c levels are difficult to achieve without increasing the risk of hypoglycaemia. In the past, there has been a tendency to focus on the prevention of microvascular complications, especially in young people with type 1 diabetes. However it is clear that type 1 diabetes is associated with an increased risk of cardiovascular disease and therefore a shift in emphasis towards global risk factor reduction may be indicated. This could include the management of glycaemia and cardiovascular risk factors involving lifestyle and pharmacological interventions. Aims: This thesis consists of four related studies of aspects of glycaemic and cardiovascular risk factor management in people with type 1 diabetes. The aims of these studies were: (1) Audit: To assess the achievement of glycaemic control and cardiovascular risk factor targets in people with type 1 diabetes attending a routine hospital based diabetes clinic. (2) Physical activity and glycaemic control: To clarify the relationship between aerobic fitness and glycaemic control in people with type 1 diabetes and identify the management strategies adopted by people with type 1 diabetes to maintain blood glucose control and avoid hypoglycaemia during physical activity. (3) Nurse led intervention versus routine care: To compare the effects of a nurse-led risk factor reduction clinic with routine diabetes care in achieving glycaemic and cardiovascular risk factor targets (4) Glycaemic "streaming": To investigate the stability of long term glycaemic control in a group of people with type 1 diabetes over a 5 year period, examining the possible phenomenon of glycaemic "streaming". Methods: The basic investigative methods for the 4 studies were: (1) Audit: A case note review of 218 people with type 1 diabetes. Demographic and risk factor target information (weight, HbA1Cl blood pressure, lipid profile and albumin: creatinine ratio) were collected. The same group was reassessed 3.5 years later. (2) Physical activity and glycaemic control:To assess the relationship between physical activity and glycaemic control aerobic capacity was assessed using the Chester Step Test in 141 people with type 1 diabetes. In addition self reported physical activity, frequency of hypoglycaemia and hypoglycaemic avoidance behaviour was assessed using a simple 'in-house' questionnaire in 50 of the 141 people who had completed the Chester Step Test. (3) Nurse led intervention versus routine care:To compare the effects of nurse-led intervention with routine care, 81 people with type 1 diabetes with an HbA1c ≥8.0% and at least one other risk factor for the development of cardiovascular disease were randomised to receive either routine care or nurse-led intervention. HbA1c, non-fasting lipid profile, blood pressure, weight, BMI and insulin dose were recorded at baseline, 6, 12 and 24 months. (4) Glycaemic "streaming": To examine long term glyceamic control a retrospective analysis of glycaemic control in a cohort of 181 people with type 1 diabetes (2003-2007) was conducted. Basic demographic data, sequential HbA1c" first and last HbA1c, and mean HbA1c,for the 5 year period was collected. Results: The results of each of the 4 studies are as follows: (1) Audit: Mean HbA1c was 9.7±1.9%, mean total cholesterol was 5.1±1.1 mmol/I, mean systolic blood pressure was 113±18 mmHg and mean diastolic blood pressure was 64±10 mmHg. Target HbA1c, (::; 7.5%) was achieved in only 7.9% of those audited, 54.6% had a total cholesterol above target (>4.8 mrnol/l), 13.1% had a systolic blood pressure above target (>135mmHg) and 3.8% had a diastolic blood pressure above target (>85mmHg). At re-audit mean HbA1c and total cholesterol had improved significantly and mean systolic and diastolic blood pressure had increased significantly. (2) Physical activity and glycaemic control: Initial data revealed a positive correlation between aerobic capacity and HbA1c (r=0.17, p<0.05), indicating that those with good aerobic capacity have poorer glycaemic control. Further investigation, involving 50 people with type 1 diabetes, revealed that 78% of this group took some action to prevent hypoglycaemia during physical activity despite there being no previous experience of serious hypoglycaemia associated with physical activity. (3) Nurse led intervention versus routine care: Compared with routine care, nurse-led intervention led to significant improvements in HbA1c (10.1±1.4 v 9.3±1.4%, p<0.001 total cholesterol (5.8±0.9 v 4.3±1.0 mrnol/l, p<0.001 systolic (127±22 v 115±13 mmHg, p<0.001) and diastolic blood pressure (71±13 v 65±9 mmHg, p<0.05) at 12 months. At 24 months improvements were maintained in all variables except diastolic blood pressure. In the control group only total cholesterol improved significantly after 12 months (5.9±0.9 v 5.2±1.0 mmHg, p<0.001) and this was maintained at 24 months. (4) Glycaemic "streaming":Over the 5 year study period there was a small but significant improvement in mean HbA1c in the cohort studied (181 patients) (9.0 ± 1.6 to 8.7 ± 1.5%, P = 0.003). This was accounted for by improvements in males (8.9 ± 1.6 to 8.6 ± 1.4%, P = 0.005) and those with poor control (HbA1c > 8.0%) (9.4 ± 1.4 to 9.0 ± 1.4%, P = 0.002). Females and well controlled patients did not show any improvement in mean glycaemic control. Conclusions: The basic audit data indicated that the majority of people with type 1 diabetes involved in the study had an HbA1c and lipid profile outside of the target range regardless of being managed in a clinic staffed by a multidisciplinary diabetes team experienced in the management of type 1 diabetes. Although over a 3.5 year follow- up period mean HbA1c and total cholesterol improved significantly. The majority of patients still had an HbA1c and total cholesterol above target levels. Life advice such as the encouragement of physical activity is a routine part of diabetes care on the assumption that this will have beneficial effects on glycaemic control and cardiovascular risk factors. However, the study of physical activity and glycaemic control included in this thesis, has demonstrated that increased aerobic capacity, indicating greater physical fitness, was associated with poorer glycaemic control. Further investigation revealed that this may be due to action taken by people with type 1 diabetes to avoid potential hypoglycaemia (increasing carbohydrate consumption, reducing insulin or a combination of both) which may have had a detrimental effect on glycaemic control. These findings indicate that people with type 1 diabetes require more education on the management of blood glucose during physical activity. Routine diabetes follow up reviews can be complex and time consuming with a focus not only on presenting problems but also on routine screening, risk factor reduction and lifestyle issues. The achievement and maintenance of target HbA1c levels on a long term basis can be difficult for many people with type 1 diabetes. There are several possible reasons for this. Even with modern insulin regimens it is difficult to mimic physiological insulin secretion. In addition it is possible that for some patients the drudge of the day to day self management of type 1 diabetes leads to fatigue of the efforts required. It is also plausible that concern about hypoglycaemia, leading to intentional raised blood glucose levels, may contribute to poor glycaemic control. Furthermore there may be a tendency to a "streaming" effect. In the glycaemic "streaming" study included in this thesis, a small overall improvement in HbA1c was noted over a 5 year time period. However, most patients maintained similar glycaemic control over the study period adding strength to the small body of data on the phenomenon of glycaemic "streaming". Alternatively poor glycaemic control may reflect the system of health care delivery. It may be that to achieve optimal diabetes care particular problems need to be identified and managed in a specific clinic staffed by experienced health care professionals. Traditionally diabetes specialist nurses have focused mainly on glycaemic control. However many of the lifestyle issues which impact on glycaemic control also influence blood pressure and lipid profile. In the randomised controlled study reported in this thesis nurse led intervention resulted in beneficial effects on glycaemic and cardiovascular risk targets in type 1 diabetes most of which were maintained at 24 months. These improvements may be a feature of being seen more frequently by the same person. Nurse led intervention, particularly in the first 6 months, involved identification of individual goals and regular review of these goals which may have increased motivation. Also a change in focus to global risk factor reduction, rather than just glycaemic control may have had a beneficial effect. In addition improvements in risk factors were associated with a greater use of anti-hypertensive and lipid lowering agents. The findings reported in this thesis have significant implications for practice. Targeted nurse-led global risk factor reduction may be a more effective system of achieving risk factor targets in patients with type 1 diabetes, freeing medical staff to deal with more complex cases. Adopting a global approach may encourage patients to make lifestyle changes to reduce cardiovascular risk which also have beneficial effects on glycaemic control.

Type-1 diabetes is caused by autoimmune destruction of insulin-producing β-cells in the pancreas. Today, transplantation of whole pancreas or islets is the only treatment that can restore endogenous insulin production. Within the past 20 years, pancreatic islet transplantation has become a clinical reality and an option in diabetes treatment. However, large use of this therapy is limited by shortage in organ donations and use of immunosuppressive drugs. Immunoisolation potentially allows elimination of immuosuppressive drugs and use of xenogenic tissue solving the problem of a shortage in organ donation.

Troglitazone was introduced as an anti-diabetic drug in the late 1990s, and represented the first of the thiazolidinedione (TZD) class of drugs to be marketed. Troglitazone works by activating the peroxisome proliferator-activated receptor y (PP ARy), reducing blood glucose levels by enhancing insulin sensitivity, primarily in the adipose and skeletal muscle tissues. Due to increasing concerns of troglitazone-linked hepatotoxicity, troglitazone was withdrawn from the U'K. market in 1997 and U.S.A. in 2000. A considerable effort to elucidate the mechanisms of troglitazone-induced hepatotoxicity has been made, resulting in a variety of proposed mechanisms; formation and accumulation of toxic metabolites, oxidative stress, mitochondrial dysfunction, and inhibition of bile salts excretory protein leading to cholestasis. The purpose of this study was to investigate whether a metabolomics approach can be used to further elucidate the mechanism of toxicity of troglitazone. Furthermore, metabolomic profiling was undertaken to determine if this approach can distinguish between TZDs with hepatotoxic potential (i.e. troglitazone) and those with none (i.e. rosiglitazone and pioglitazone). Conventional MTT assay revealed that troglitazone was a more potent toxin to the HuH-7 hepatoma cell line compared to rosiglitazone and pioglitazone, with an ICso value of 25.9 ± 1.8, 2: 168.9 ± 30.0, and 2: 79.4 ± 13.5 ~M, respectively. A comparison between the ICso values obtained by the MTT versus LDH assays (25.9 ± 1.8 versus 2: 42.7 ± 3.6 ~M) suggests that troglitazone affected the reductive capacity of mitochondria at lower level before causing the cells to rupture and release their intracellular contents. An untargeted and a targeted metabolomics approach revealed that troglitazone decreased the levels of intracellular glutamate, malate, energy production, GSH, and nicotinamide at concentrations that are non-toxic by the MTT assay. These observations demonstrate the discriminative ability of a metabolomics approach for the assessment of drug toxicity. The findings also suggest that troglitazone affects the tricarboxylic acid (TCA) cycle, interrupting the anaplerotic and cataplerotic balance of the TCA cycle, leading to oxidative stress and reduced energy level.

Glucagon receptor antagonism is becoming a key target area for type 2 diabetes treatment. This thesis evaluates the potential of novel peptide-based glucagon receptor analogues for type 2 diabetes therapy. Structural modifications of the well established glucagon analogue, desHis1Glu9-glucagon, was used to develop novel glucagon analogues. All peptide analogues were resistant to DPP-4 degradation and effectively antagonised glucagon-mediated cAMP production and insulin secretion when tested in vitro. desl-lis'Glu'-glucagon had a duration of biological action of 8 h and effectively antagonised glucagon-mediated glucose and insulin release in vivo. Mid-chain acylation of desl-lis'Glu/-glucagon did not hinder acute antagonistic properties and prolonged the duration of biological action to 24 h. An additional y-glutamyl Iinker in combination with acylation resulted in similar biological activity. C-terminal acylation also effectively antagonised acute glucagon-mediated glucose production in vivo. However, a C-terminal miniPEGylated version did not exhibit antagonistic properties. In general C-terminal modifications resulted in analogues with reduced acute biological activity indicating that mid-chain acylation was more effective. Pro4 substitution for Gly" without G1u9 replacement also resulted in reduced biological efficacy in relation to antagonising glucagon-mediated actions. However, Pro4 substitution did not hinder the activity of desl-lis'Glu'i-glucagon, emphasising the important role of Glu9 in biological activity. C-terminal acylation of this Pro4 analogue reduced its acute action in animals. However, chronic administration of non-acylated and mid-chain acylated forms of this Pr04 analogue improved metabolic status in high fat fed mice. Furthermore, chronic administration of the non-acylated Pro4 analogue exhibited similar beneficial effects as exendin-4 in high fat fed mice, but additive effects of combined administration were not evident. This thesis demonstrates that peptide-based glucagon antagonists exhibit prominent anti-diabetic effects in animal models of obesity-diabetes, and illustrates the necessity to further establish peptide-based glucagon receptor antagonists for type 2 diabetes therapy

Type 2 diabetes is a chronic metabolic disorder characterized by elevated blood glucose and a high risk of micro- and macrovascular complications. Prognosis depends to a substantial degree on self-management, that is, the adoption of practices to stabilize blood glucose and stave off co morbidities. This research set out to compare the narratives of people with good and poor glycaemic control (defined as 3 recent HBAIC readings <7 and >10 respectively), and of men and women. Participants (N=32) were recruited from local diabetes hospital clinics, and interviewed using a biographic-narrative method. Interviews were analysed with the use of dialogical and phenomenological concepts including voice, emotional connection to truth, and time-space elaboration of narrative. As analysis proceeded, the search for differences in the talk of participant 'groups' shifted toward a situational understanding of health practice, as participants elucidated loci of time, space, and social context which made self-management (im)possible. Health psychological notions according to which internal psychological realities play a determining role in human action were thus questioned, and placed into their historical context. Chapter 1 outlines the thesis topic, and presents an argument for a critical health psychology. Chapter 2 presents a metasynthesis of previous qualitative research on type 2 diabetes self-management. Chapter 3 explores methodology, in particular interviewing technique and analytical approach to narrative. Chapters 4,5,6, and 7 present narrative analyses of the different 'groups', with the latter being a case study to explore in detail the changing ways in which self-management intertwines with a lived life. Chapter 8 brings strands of these analyses together, in particular to explore the importance of temporal and spatial disruptions to the experience of self-management throughout the narratives. And chapter 9 offers some tentative answers to the research questions, while discussing the findings in relation to current directions in UK health policy.