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Investigating the role of heatshock on diabetic wound healingContractor, Taha (University of Chester, 2017-05)The increasing occurrence of diabetes in the general population as a result of over nutrition and increasingly inactive lifestyle has led to an obesity epidemic which is set to grow over time. With an ever increasing obese population type 2 diabetes and cardiovascular complications are set to become the major causes of human mortality. Chronic non healing wounds are a major cause of mortality and morbidity in patients with type 2 diabetes. They are predominantly caused by macrophage dysfunction and a lack of migration of fibroblasts into the wound. This study aimed to investigate diabetic wound healing through development of an artificial scratch assay. An in vitro scratch assay developed in WS1 cells. The effect of heat shock treatments from 39°C to 45° was tested to determine if cell migration increased; however, no significant difference was seen. Mitomycin C was used to determine if wound closure occurred as a result of cell proliferation and migration or migration alone. 10μg/ml of mitomycin C inhibited cell division by 79.9% without exhibiting cytotoxicity over a 12h period. The effect of hyperglycaemia and heat shock was also tested and showed no significant difference when compared to control conditions, suggesting that fibroblast migration in vivo is hindered through other factors such as debridement or macrophage dysfunction in the wound. GLUT4 is present in insulin sensitive organs (liver, adipose and muscle) and is the major glucose transporter responsible for the clearance of glucose from the blood after a meal, thus playing a central role in glucose homeostasis. Monocytes are precursors to macrophages and can easily be isolated from whole blood. They have also been shown to express GLUT4 in response to insulin and could be used as model to assess inflammation in diabetes. A glucose uptake assay was developed in U937 cells using a fluorescent glucose analogue, 2NBDG. 2NBDG fluorescence was shown to be competitively inhibited by increasing concentrations of glucose suggesting that 2NBDG enters the cell through glucose transporters. 2NBDG uptake was also assessed at different pH and in presence of membrane fluidizers (DMSO, benzyl alcohol and phenethyl alcohol). Extremes of pH significantly reduced cell viability and only at pH 4 was 2NBDG fluorescence significantly reduced. Treatment with DMSO showed that at high concentrations (≤ 1.56%) cell viability was reduced with a concurrent reduction in 2NBDG fluorescence. The effect of benzyl alcohol and phenethyl alcohol was foundto be insignificant at the concentrations and time points tested. The presence of GLUT4 was also determined by flow cytometry and Western blotting and found to be situated in the cytoplasmic region of the cell. This study indicates that monocytes and macrophages could be a potential therapeutic target to improve diabetic wound healing as they are a source of growth factors and cytokines that can bring about resolution of inflammation and it is their dysfunction in diabetic wounds that causes poor clinical outcomes.