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Type Two Diabetes is six times higher amongst UK Pakistanis than the remainder of the British population. This thesis examines the impact of cultural influences on diabetic health within three different generation groups of UK Pakistanis, through three qualitative studies on: I) Intergenerational aspects of food and health behaviours, 2) Attitudes toward diabetes symptoms and the seeking of medical help, and 3) The perceptions of health professionals regarding the treatment of diabetes within the UK Pakistani community. A Grounded theory approach of three studies were carried out; the first two utilised semi-structured interviews with 60 participants across three generations. The third was a focus group study with 12 health professionals. Thematic analysis of data in the first study revealed that UK Pakistanis across all three generation groups differed in their perceptions and behaviours toward 'traditional' food values and what is regarded as 'healthy'. For first and second generations, traditional values corresponded to the cultural belief that their diets were healthy, that diabetes was hereditary, and that the family was central to eating practices. This stood in contrast to the majority of the young generation, who displayed higher levels of knowledge of food, diet and diabetic health, but were also compliant to family eating practices and culture. The second study found there were differences in help-seeking behaviour for diabetes, with the majority of the first and second generation groups recognising the symptoms of diabetes as worthy of concern, and were generally willing to discuss these symptoms with others and with their GPs. However, the majority of the young generation group exhibited the tendency (often identified with 'western' ideals) to keep diabetes symptoms private, and thus to maintain a high tolerance for such symptoms. The third study found that health professionals generally held overly simplistic and ethnocentric attitudes toward the interaction of culture and diabetes within the UK Pakistani population. It also ascertained that they tended to identify foreign language, lack of education, cultural barriers to altering food choice, and culturally influenced approaches to presenting symptoms as the primary obstacles to reducing diabetes within the community. Findings were discussed in terms of an eco-cultural model, calling attention to the ways in which the concepts of ethnicity and family shape UK Pakistanis' attitudes toward food, diabetes, and the seeking of help. Inter-generational differences, society, and community all act together in the production of culture, values and practices shared in UK Pakistani families. The research extends understanding about how diabetes is defined and managed, how it is taken for granted, and how it is resisted within a small section of the UK Pakistani population.

Impaired wound healing is associated with hyperglycaemia in patients suffering from diabetes mellitus. Dicarbonyl compounds such as methylglyoxal (MG) are formed at an increased rate under hyperglycaemic conditions in diabetes. They take part in glycation of proteins and serve as precursors of advanced glycation end products (AGEs). The mechanism behind glycation and possible dysfunction of growth factors involved in wound healing is investigated in this study. Platelet derived growth factor BB (pDGF-BB) is a potent mitogenic and chemotactic factor. It plays essential roles in the recruitment and proliferation of cells involved in wound healing and tissue repair. PDGF-BB may represent a key target for sugar induced molecular modifications during hyperglycaemic conditions of diabetes. PDGF-BB was glycated using glucose-6-phosphate and MG. Matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) and sodium dodecyl polyacrylamide gel ecletrophoresis analysis were employed to confirm PDGF-BB glycation. A time and dose dependent glycation of PDGF-BB was achieved for the first time in this study. Peptide mass mapping was used following enzymatic hydrolysis and subsequent MALDI-TOF-MS analysis to localise methylglyoxal-hydroimidazolone adduct formation on arginine residues in PDGF-BB incubated with MG. The proliferation and migration of human dermal fibroblasts were significantly reduced in glycated PDGF-BB treated cells compared to native PDGF-BB treated cells. Similarly reduced wound recovery was observed in the presence of glycated PDGF-BB compared to native PDGF-BB in artificially wounded cell monolayer. These results may help to understand pathological mechanisms underlying impaired wound healing in diabetic patients. Glycation inhibitors could prevent the formation of AGEs and dysfunction ofPDGF-BB. Two new inhibitors i.e. aged garlic extract and S-allyl cysteine (SAC) were identified to inhibit formation of glucose and MG derived AGEs. Both inhibitors showed promising multi-functional and multi-stage inhibitory properties when compared to pyridoxamine and aminoguanidine, two well established glycation inhibitors. Antiglycation properties of aged garlic extract and SAC combined with their already well established antioxidant, metal chelating and carbonyl scavengmg properties may limit glycation induced complications. VI

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Background: The cardioprotection by IPC is a well-recognised phenomenon in healthy hearts, however the occurrence of IPC in diabetics is controversial. In this thesis I have investigated whether the diabetic myocardium can be preconditioned and the underlying mechanisms. Methods and results: Right atrial sections from 3 groups of patients: non-diabetics, IDDM, and NIDDM were randomised to receive one of the following protocols: aerobic perfusion, simulated ischaemia/reoxygenation, IPC, and pharmacological preconditioning with phenylephrine, adenosine, diazoxide (mitoKATP channel opener), PMA (protein kinase C agonist) or anisomycin (p38 MAPK activator). The assessment of CK leakage and MTT tissue viability at the end of the experiments demonstrated that diabetic myocardium cannot preconditioned by ischaemia or by the pharmacological activation of the signal transduction pathway upstream of mitoKATP channels but that protection can be obtained by the activation of the pathway beyond mitoKATP channels (e.g. PKC and P38 MAPK). In a second study, mitochondria isolated from the above groups of patients were used to assess for MMP after treatment with diazoxide by measuring the uptake of JC-1 dye. The partial depolarisation of MMP seen in non-diabetics was absent in the diabetic myocardium. The determination of ROS generation by the mitochondria of the non-diabetic and diabetic myocardium exposed to diazoxide showed an altered response in superoxide production in the diabetic myocardium. Finally, using specific PKC isoform inhibitors, I demonstrated that PKC a and PKC e are the two isoforms involved in IPC of human myocardium with PKCe begin upstream and PKCa being downstream of mitKATP channels. Conclusions: The failure to precondition the diabetic myocardium is caused by mitochondrial dysfunction. Since PKCa is the isoform beyond mitoKATP channels, this and P38 MAPK may represent potential clinical and therapeutic targets to protect the diabetic myocardium.

The importance of apoptosis in ischaemia-reoxygenation of the diabetic human heart is unclear. Right atrial appendages were obtained from non-diabetics, non-insulin dependent diabetics (NIDDM) and insulin dependent diabetics (IDDM) at the time of cardiac surgery. Free-hand tissue secretions were subjected to the following protocols: fresh tissue, aerobic control for 210 min, 90 min simulated ischaemia followed by 120 min reoxygenation (SI/R). Cell death by apoptosis and necrosis in non-ischaemic fresh tissue was greater in the NIDDM and the IDDM groups than in the non-diabetic group (p<0.05 in all instances). After SI/R, apoptosis and necrosis were also significantly greater in NIDDM and IDDM groups than in non-diabetics (p<0.05). The mean values of activation of Effector caspase were greater in fresh tissue in the NIDDM and IDDM groups than in the non-diabetic group (p<0.05) and values were further increased after SI/R in the NIDDM and IDDM groups as compared with non-diabetic group (p<0.05). Importantly caspase-3 inhibition reduced apoptosis by 94.0+/-1.6% in the myocardium from non-diabetics and 50.6+/-4.2% in the muscles from diabetics (p<0.05) without influencing necrosis. However, caspase -2 inhibition had no effect on either apoptosis or necrosis. Poly (ADP-ribose) Polymerase (PARP) inhibition resulted in a similar reduction in apoptosis (85.3+/-0.9% and 87.1+/-2.1%) and in necrosis (39.8+/-5.0% and 30.0+/-3.3%) in the muscles from non-diabetic and diabetics. The human diabetic myocardium is also more susceptible to ischaemia/reoxygenation injury than the non-diabetic myocardium, an effect that is mediated, at least in part, by caspase and PARP activation.

This study examined whether raised extracellular glucose concentrations (through increased oxidative stress) could induce DNA damage and hasten human vascular endothelial cell (HUVEC) senescence.;HUVEC DNA damage was measured using the comet assay. Senescence was detected by beta-Galactosidase staining at pH 6. Telomere lengths and mitochondrial genome copy numbers were obtained through Southern analysis. 8-oxoguanine excision was assessed by a nucleotide incision assay. Western blotting demonstrated Ref-1 and electron transport chain complex IV (subunit I) expression.;DNA damage peaked after four fours of high glucose (HG; 22mM) exposure. This was D-glucose specific and concentration dependent. DNA damage was attenuated by N-acetylcysteine (p < 0.01) and augmented by formamidopyrimidine DNA glycosylase (p <0.05), indicating that HG induced oxidative lesions. DNA damage was associated with premature HUVEC senescence (p = 0.011) and accelerated telomere shortening (p = 0.03). N-acetylcysteine did not prevent telomere attrition. 8-oxoguanine incision and Ref-1 expression decreased during 24-hour HG treatment, suggesting that HG could disrupt the efficiency of oxidative DNA lesion repair. No difference in mitochondrial DNA content in HG exposed HUVECs was found. However, complex IV (subunit I) expression fell in HG conditioned HUVECs ( p = 0.006), implying that HG could affect mitochondrial activity.;This study demonstrates for the first time that elevated glucose concentrations promote oxidative DNA damage in human vascular cells. This leads to accelerated telomere attrition, prompts premature human endothelial cell senescence and may profoundly disturb mitochondrial function. These observations provide a novel mechanism to implicate glucose in accelerated vascular ageing in diabetes.

Oxidative stress is thought to be elevated in diabetes as a consequence of hyperglycaemia. This thesis investigates oxidative DNA damage in diabetes, which may contribute to accelerated vascular ageing. Telomeric and mitochondrial DNA are two areas of the genome that may be more susceptible to oxidative stress and were therefore investigated.;51 patients with diabetes (aged 31-78 years) and 101 healthy controls (aged 19-75 years) were recruited. 51 of the controls were age- and sex-matched to be patient group. For both populations physiological profiles were obtained and pulse wave velocity (PWV), an index of vascular stiffness, was measured. Oxidative DNA damage was also investigated in peripheral blood using the comet assay, and in more depth by measuring terminal restriction fragment (TRF) lengths and quantifying mitochondrial DNA (mtDNA) content. PWV increased with age in both study groups (p<0.001) and was significantly higher in the patient group (8.00 +/- 2.89 versus 7.29 +/- 1.64 m/s; p=0.006), suggesting accelerated vascular ageing in diabetes. This was accompanied by elevated levels of oxidative DNA damage; 25.81 +/- 1.18 versus 21.40 +/- 0.81% Tail DNA (p=0.003) in patients and controls respectively. TRF length inversely correlated with age in both groups (p<0.05), with similar rates of attrition, and although they were shorter in the patients with diabetes, this was not significant (p=0.10). Quantification of mtDNA revealed significantly lower levels in the patients with diabetes compared to the controls (0.014 versus 0.016; p=0.020). There is accelerated vascular ageing in diabetes, which is accompanied by elevated oxidative DNA damage, and a decrease in mtDNA, but no alteration in TRF length compared to a healthy control population. The mechanisms underlying these alterations are unknown with the lack of correlations with glycaemic control suggesting it is not the sole cause but it may still contribute.

Falls are more common in older people with Diabetes Mellitus (DM) than without the condition. Falls cause significant morbidity and mortality among older adults, draining the finite resources of the National Health Service (NHS). Life expectancy of the general population is increasing and a shift in numerous social and environmental factors contribute to ever-increasing recorded cases of DM. As such, identification of risk factors for falls in the DM population is paramount. Previous research has identified intrinsic risk factors for falls in the older population however little research has been conducted specifically in people with DM with regards to primary falls prevention in this 'at risk' group. This study assessed key risk factors for falls in two patient groups; 60 older adults (over the age of 55) with DM and 60 age and gender matched control participants. Logistic regression analysis identified gait velocity, ankle dorsiflexion strength and symptoms of neuropathy (NSS) to be the key predictors of falls in the DM population. Building on the ~ identification of these risk factors, a further study (n=20, 9 males and 11 females, age range 57 - 82) investigated the effect of exercise on these risk factors, comparing the effect of an exercise class, a Nintendo Wii Fit intervention and a control group (no exercise). Interventions were provided x2 p/w for 1 hour duration. This exploratory exercise intervention (pseuso-RCT) demonstrated that individuals allocated to the exercise group achieved the most improvement in risk factors for falls in this study, with significant improvements in Neuropathy Disability Score, right step length and ankle strength for all muscle groups tested. The risk factors for falls in patients with DM are similar to those of the general population; however are more pronounced in those with DM. As such, early identification and strategic intervention such as the implementation of a structured exercise regime are critical to effective falls prevention and management, for people with DM.

Maturity-onset diabetes of the young (MODY) is caused by highly penetrant, mutations in a single gene that result in non-insulin dependent diabetes typically presenting in lean young adults. MODY accounts for approximately 1 % of diabetes and is often misdiagnosed as Type 1 diabetes or Type 2 diabetes. Over 80% of MODY patients in the UK remain unidentified. The aim of this thesis was to develop, characterise and determine the diagnostic accuracy of existing and novel biomarkers that could be used to identify patients with a monogenic form of diabetes. Blood C-peptide can aid in the diagnosis of MODY, but practical reasons limit its widespread use. In chapters 1 and 2 the measurement of C-peptide in urine is explored. We find Urinary C-peptide creatinine ratio is a stable and reproducible measure of endogenous insulin secretion, which correlates strongly with 24 hour urine C-peptide and, both fasting and stimulated, blood C-peptide in non- diabetic controls and patients with Type 2 diabetes. In chapter 3 we assess assay specific stability of C-peptide and insulin in blood on a number of analytical platforms and storage conditions. In contrast to many published reports we find C-peptide is stable in K +-EDTA whole blood for at least 24 hours at ambient room temperature. The aim of chapter 4 was to determine the prevalence of GADA and IA-2 autoantibodies in a large cohort (n=508) of patients with a confirmed genetic diagnosis of MODY. Less than 1 % of MODY patients had islet autoantibodies and a combination of GADA and IA-2A testing gave the best discrimination between Type 1 diabetes and MODY (sensitivity of 99% and specificity of 82%). A pilot study previously showed hsCRP is lower in HNF1A-MODY than in other forms of diabetes. In chapter 5 we confirm that plasma hsCRP levels are lower in HNF1A-MODY than Type 2 diabetes, Type 1 diabetes, GCK-, HNF4A- and HNF1 B-MODY. In addition, an hsCRP level of <0.75 mg/L discriminates HNF1A-MODY from Type 2 diabetes with a sensitivity of 79% and specificity of 70%. In chapter 6 we investigate the lipid profiles in HNF1A-MODY. The plasma-lipid profiles of HNF1A-MODY and the lipid constituents of HDL are similar to non- diabetic controls. HDL-cholesterol was higher in HNF1A-MODY than in Type 2 diabetes and could be used as a biomarker to aid in the identification of patients with HNF1A-MODY (sensitivity of 75% and specificity of 63%). In the final chapter we determine the diagnostic accuracy of the biomarkers studied in this thesis (C-peptide, islet antibodies, HDL-cholesterol and hsCRP) within a single test set of patients. We determine that all the biomarkers with the exception of hsCRP add discriminative value to a clinical prediction model for MODY compared to using clinical criteria alone. An overview of the major findings of each chapter, the clinical implications and areas of future research are discussed in chapter 8.