Type 2 diabetes, as a complex disease, has a range of genetic and environmental factors that underpin its aetiology. It is hoped that the emerging study of epigenetic processes will provide the necessary mechanistic insight into the genetic and environmental interactions that, to date, are poorly understood. This thesis considers the role of DNA methylation, an epigenetic modification, in the aetiology of type 2 diabetes. A range of different genome-wide and whole genome techniques are applied to a study of established type 2 diabetes and experimental models (human and animal) of fetal programming. Samples from a recent genome-wide association study of type 2 diabetes were used to identify DNA methylation patterns at areas of genetic variation associated with disease risk. Analysis of data from methylated DNA immunoprecipitation and microarray identified a genetic-epigenetic interaction in the FTO gene. At this locus, the presence or absence of a SNP created or abrogated a CpG site capable of methylation and further analyses highlighted possible functional relevance via enhancer activity. Models of fetal programming were then used to identify whether variation in DNA methylation may underlie the ‘programmed’ phenotype of diabetes and related cardiometabolic disease. Pre-existing human models of programming via maternal vitamin B12 deficiency and maternal famine exposure have been used to generate exploratory evidence of such mechanisms. Whole genome-based techniques (Medip-seq and Illumina 450k methylation array) were used to profile DNA methylation in whole blood samples from the offspring born to each of these studies. Custom bioinformatic analysis was performed to identify differences in methylation between offspring exposed versus unexposed to the in utero environmental insult. Technical replication and validation studies are ongoing to confirm or refute the presence of regions of differential methylation. Finally, this thesis considers whether a state of ‘over nutrition’ gestational diabetes, may play a role in fetal programming. This condition is of increasing prevalence across the world and is characterised by maternal hyperglycaemia and insulin resistance, often resulting in fetal overgrowth. A mouse model using an inbred strain (Lepr) of mice induced a programmed phenotype of glucose intolerance and obesity in aged offspring born to mothers with gestational diabetes. Medip-seq performed on the livers of late gestation mouse embryos identified differential methylation in cases vs. controls, located at genomic regions with potential functional relevance. A human cohort of women with gestational diabetes was collected to develop further hypothesis around the multiple environmental factors that could interact in pregnancy. Prevalent nutritional deficiencies of vitamin D, iron and one-carbon metabolites were found in women with and without gestational diabetes recruited from a local antenatal clinic. This thesis presents preliminary findings that variation in DNA methylation may be involved in the genetic and environmental risk of type 2 diabetes. The work presented highlights how future studies must incorporate integrated genetic, epigenetic and functional analysis with sufficient sample size if their results are to be translatable to diverse populations at risk of diabetes.

The molecular basis for the use of synthetic growth factors (GFs) in tissue reparation has been poorly investigated. More recently, autologous derived platelet rich growth factor has gained popularity in the field of regenerative/ reparative medicine, mostly because it fits the description of an ideal naturally existing constellation of GFs. However, its efficacy remains controversial. Hence, this study is designed to further elucidate the physiological role of PRP in treating chronic diabetic foot ulcers. Platelet -rich plasma (PRP) and Platelet -poor plasma (PPP) were prepared from blood samples taken from healthy donors and diabetic patients through the use of platelet collecting and concentrating system. The GFs released were measured through immunoassay technique. The effects of the varying concentrations of PRP/PPP in culture media was assessed through tissue culture assay (proliferation, cell migration and angiogenesis assay) on human epithelia keratinocyte, dermal fibroblast and umbilical vein endothelia cell. Furthermore, immuno-histochemistry technique was used to evaluate the differentiation, proliferation, migration and extracellular matrix alterations occurring along wound margins of patients with chronic diabetic ulcers following PRP treatment. A significant difference was observed when the expression of platelet derived growth factor-AA, epidermal growth factor, vascular endothelia growth factor, transforming growth factor and thrombospodin-I released from PRP/PPP were compared between the two groups. There was a significant proliferative, migratory and angiogenic effect of PRP over PPP in the tissue culture assay; however this effect was most prominent with 5% PRP. Overall, hyperproliferative keratin, CD44 and β1-integrin were upregulated in diabetic ulcer keratinocytes as compared with normal foot skin. The clinical study showed that 3 of the 7 diabetic foot ulcer patients treated with PRP achieved complete wound re-epithelisation. We have been able to demonstrate through in vitro studies that PRP has a positive biological effect which mimics normal physiological tissue reparation process.

Type 2 Diabetes, a metabolic disorder associated with chronic hyperglycaemia and hyperlipidaemia, is characterised by an impairment of insulin secretion and production and β-cell death. This β-cell dysfunction is determined by different factors, among which inflammatory processes, characterised by increased expression of pro-inflammatory cytokines and chemokines. Although some molecular mechanisms have been proposed to be involved in this β-cell dysfunction, they fail to explain the whole process. In this thesis, a combined approach of microarray, RNAseq, RT-qPCR and western blot will be used to elucidate the pathways affected under glucolipotoxicity, in order to discover novel molecules involved in the pathogenesis of T2D. We found that INS-1 cells exposed to 27 mM glucose, 200 μM oleic acid, 200 μM palmitic acid, show an overexpression of CD40, a TNF receptor involved in inflammation (more than 300% p<0.01), both at RNA and protein level. These data were validated in cultured human islets (p<0.05) and in islets of mice fed a high fat diet (p<0.05). We showed also that siRNA downregulation of CD40 is associated with increase in insulin secretion (p<0.05), revealing a potential new role of this receptor in β-cells. In addition, RNAseq analysis revealed a wide list of molecules differentially expressed in glucolipotoxicity, in particular molecules involved in inflammation, insulin/IGF pathway, fatty acids-cholesterol metabolism and biosynthesis. We focused our attention on potential novel targets, including the thyroid pathway, unknown microRNAs and novel genes, in order to discover new pathways involved in the impairment of insulin secretion in T2D. This work will open the way to future studies aiming to characterise these molecules and to understand their role in the insulin secretion process. Interesting candidates can then be used in the future as potential targets for the development of new and specific therapeutic strategies.

No description available.

Source: Masters Abstracts International, Volume: 47-06, page: 3553. Advisers: Michael L. Parchman; Jimmy L. Perkins. Includes bibliographical references.

The purpose of this thesis was to aid in the development of a web-based physical activity intervention for people with type 2 diabetes living in remote and rural areas. Chapter 1 introduces the research area, the design of the thesis and the key research questions. The thesis is then made up of 5 key studies. Study one, a systematic review of the literature was conducted and reported in chapter 2. This review identified the technologies that have previously been used to promote physical activity in type 2 diabetes, it identified the methodological quality of each included technology and the key components for effective change. Web based technology was the most commonly used and the most effective in increasing physical activity using components such as goal setting and physical activity trackers. These results informed study 2 (chapter 3) which explored patient and health professional's views on diabetes, physical activity and use of the internet. The need for clear information was identified with regard to diabetes as well as the call for accurate physical activity advice in relation to diabetes for both patients and health professionals. Study 3 (chapter 4) explored key information and components for an effective website. Included features were the need for a personalised approach; detailed advice on how the body responds to physical activity; a physical activity tracker and goal setting. The need for a 'virtual trainer' for support, advice and help with goal setting and interactive maps showing physical activity opportunities were all deemed important. The fourth study, chapter 5 described the design of the website and its features as well as the protocol for a six month pilot randomised controlled trial to examine the effectiveness of the development website, with and without interactive design elements. The final study in this thesis (chapter 6), describes the physical activity, physiological and biochemical results from a randomised controlled trial to test the effectiveness of the website and its features. The final chapter summarises the findings in response to the research questions and the future recommendations based on the outcomes.

Source: Masters Abstracts International, Volume: 46-01, page: 0343. Adviser: Nuria Homedes. Includes bibliographical references.