The incidences of obesity and type 2 diabetes and their complications are increasing globally. The presence of elevated circulating free fatty acids has been associated with the initial dysfunction of pancreatic beta cells and microvascular endothelial cells followed later by their demise. The aim of this thesis was to investigate the mechanisms by which demise occurs, and how it may be prevented. Palmitate, a saturated fatty acid, caused cell death in both INS-1 beta cells and HCMec/D3 microvascular cells, whereas the unsaturated fatty acid oleic acid did not cause cell death, and also protected against palmitate-induced toxicity. Etomoxir, the mitochondrial CPT1 inhibitor did not rescue INS-1 or HCMec/D3 cells from palmitate-induced toxicity suggesting that palmitate-induced toxicity does not occur via entry into the mitochondria. Cells were exposed to 2-bromopalmitate, a non-metabolisable fatty acid used to reduce the pool of cytoplasmic CoA, to determine whether palmitate-induced toxicity might be mediated by its ability to be activated. Pre-incubation with 2-bromopalmitate in INS-1 cells significantly prevented palmitate-induced cell death. These data suggest that the activation of palmitate with CoA might mediate cell death. Cell cycle analysis found that neither oleic acid nor palmitate caused an increase or decrease in cell proliferation in both INS-1 and HCMec/D3 cells. The data suggest that the mechanism of oleic acid-induced cytoprotection might not be via a pro-proliferative mechanism. INS-1 cells were imaged using spontaneous Raman microspectroscopy after 24-hour exposure to esterified and non-esterified fatty acids. Uni- and multi-variate analysis and spectral decomposition were carried out using a methodology optimised and validated which is presented in this thesis. The aim was to quantify changes, if any, in lipid disposition: distribution, intensity (as a measure of concentration) and composition after exogenous exposure to these fatty acids. Exposure to 0.125 mM palmitate showed a significant decrease in the percentage of lipid within the cells and a corresponding increase in the intensity of this lipid. This suggests that palmitate, alone, might be shuttled into lipid droplets. This was not observed when the cells were exposed to oleic acid, whereby an increase in the intensity of lipid was observed even though no significant change was observed in the percentage of lipid within the cells. When palmitate and oleic acid were combined, the composition of the lipid droplets changed such that the levels of palmitate decreased and the levels of oleic acid increased. These data suggest that oleic acid does not shuttle palmitate into lipid droplets. These data do not support the hypothesis that oleic acid protects against palmitate-induced cytotoxicity by shuttling palmitate into lipid droplets. The methyl esters of palmitate and oleic acid were employed to determine whether they would affect lipid disposition. No change in lipid distribution or intensity was observed when the cells were exposed to these fatty acids, validating the requirement for the free carboxyl oxygen for the covalent binding to glycerol for the formation of lipid droplets. These data also suggest that INS-1 cells cannot de-esterify esterified fatty acids.

This dissertation presents the application of MEMS-based approaches for the construction of engineered tissue substitutes. MEMS technology can offer the physical scale, resolution, and organization necessary for mimicking native tissue architecture. Micromachined nozzles and templates were explored for the fabrication of acellular, biomimetic collagenous fibrous scaffolds, microvascular tissue structures, and the combination of these structures with cell-based therapeutics. The influence of the microstructure of the tissue constructs on their macro-scale characteristics was investigated.

Microvascular permeability is a pharmacologic indicator of tumor response to therapy, and it is expected that this biomarker will evolve into a clinical surrogate endpoint and be integrated into protocols for determining patient response to antiangiogenic or antivascular therapies. The goal of this research is to develop a method by which microvascular permeability (Ktrans) and vascular volume (vp) as measured by DCE-MRI were directly compared to the same parameters measured by intravital fluorescence microscopy in an MRI-compatible window chamber model. Dynamic contrast enhanced-MRI (DCE-MRI) is a non-invasive, clinically useful imaging approach that has been used extensively to measure active changes in tumor microvascular hemodynamics. However, uncertainties exist in DCE-MRI as it does not interrogate the contrast reagent (CR) itself, but the effect of the CR on tissue water relaxivity. Thus, direct comparison of DCE-MRI with a more quantitative measure would help better define the derived parameters. The combined imaging system was able to obtain both dynamic contrast-enhanced MRI data high spatio-termporal resolution fluorescence data following injection of fluorescent and gadolinium co-labeled albumin. This approach allowed for the cross-validation of vascular permeability data, in relation tumor growth, angiogenesis and response to therapy in both imaging systems.

Diabetes mellitus is a chronic syndrome affecting carbohydrate, protein, and fat metabolism. It is characterized primarily by relative or absolute insufficiency of insulin secretion (type I diabetes or IDDM) or concomitant insensitivity / resistance to the metabolic action of insulin on target tissues (Type II diabetes or NIDDM), both resulting in hyperglycaemia. Diabetes mellitus is known to induce microvascular changes and alterations to neuronal functions. The neurovascular system comprising of unmyelinated primary afferent sensory neurones and the microvasculature innervated by these nerves play a major role in modulating inflammatory and tissue repair processes. Sensory nerve terminals respond to injury via the release of sensory neuropeptides which mediate inflammation and tissue repair. These processes are known to be altered in diabetes. This thesis is concerned with the role of diabetes in modulating microvascular blood flow directly and indirectly via modulating sensory nerve activity and the effect of these changes on repair processes in skin of 4 weeks streptozotocin (STZ)-induced diabetic rats. The following hypotheses were examined: 1. That factors implicated in long-term diabetic vascular damage play a role in altering skin microvascular function in early diabetes. 2. That preventing the deleterious effects of these factors could improve skin microvascular blood flow and skin repair processes in early diabetes. (For complete abstract open document)

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Doctor of Philosophy / Department of Anatomy and Physiology / Thomas J. Barstow / The overall aims of this dissertation are to determine the incidence and magnitude of insulin resistance (IR) in a cohort of normoglycemic college-age subjects with a family history of type 2 diabetes, and to ascertain if there is early macro- and microvascular dysfunction relative to IR. Study 1 (Chapter 2) revealed a 7-fold range in IR in healthy college subjects concomitant with measures of insulin, both fasted and during an oral glucose tolerance test, but not related with any measure of plasma glucose. These results emphasize that early in the etiology of carbohydrate dysregulation, abnormalities first occur with regard to insulin sensitivity. Using brachial artery blood flow (BABF, Doppler fluxometry) and near-infrared spectroscopy (NIRS) (Chapter 3) we extended the understanding of the use of these non-invasive tools to assess forearm resting metabolic rate and to compare the parameters of both the NIRS oxy-hemoglobin signal, as a index of perfusion in the microcirculation, and BABF, as an independent measure of microvascular reactivity during post occlusive reactive hyperemia (PORH). Resting metabolic rate ranged ~ 2 fold (2.83-5.15 [Mu]MO[subscript2]/min/100g) similar to direct measures. Amplitude, but not kinetic parameters for NIRS variables correlated with comparable parameters for BABF, providing evidence for the possible utility of NIRS in examining microvascular reactivity. In study 3 (Chapter 4), utilizing our extended understanding of hemodynamics garnered from the results of study 2, we assessed the influence of IR on macro- and microvascular reactivity. We observed that i) the magnitude of IR was significantly correlated with attenuation of endothelium-dependent vasodilation of the brachial artery (P< .01) indicating the possibility of a reduced nitric oxide bioavailability and an enhanced atherogenic milieu. Additionally we found ii) BABF at rest and during reactive hyperemia to be strongly correlated with conductance (reduced downstream resistance—an indicator of microvascular control abnormalities) independent of forearm metabolic rate, and iii) parameters of BABF (microvascular response) were also strongly correlated with brachial artery vasoreactivity (macrovascular response). In conclusion, this body of work furthers our insight into the need for earlier identification of "disease" earlier in the progression to type 2 diabetes, and provides direction for future investigations into prevention / intervention to improve microvessel functionality and to slow the atherosclerotic process in larger vessels.

Cardiovascular disease is the leading cause of death in women. Although overall mortality from coronary heart disease (CHD) has decreased, there are subsets of patients, particularly youngwomen, in whom the mortality rate has increased. Underlying sex differences in CHD may be an explanation. Women have more frequent symptoms, more ischemia, and higher mortality than men, but less obstructive coronary artery disease (CAD). Despite this, traditional risk factor assessment has been ineffective in risk stratifying women, prompting the emergence of novel markers and prediction scores to identify a population at risk. Sex differences inmanifestations and the pathophysiology of CHD also have led to differences in the selection of diagnostic testing and treatment options for women, having profound effects on outcomes. The frequent finding of nonobstructive CAD in women with ischemia suggests microvascular dysfunction as an underlying cause; therefore, coronary reactivity and endothelial function testing may add to diagnostic accuracy in female patients. In spite of evidence that women benefit from the same therapies as men, they continue to receive lessaggressive therapy, which is reflected in higher healthcare resource utilization and adverse outcomes. More sex-specific research is needed in the area of symptomatic nonobstructive CAD to define the optimal therapeutic approach.

Cardiovascular disease is the leading cause of death in women. Although overall mortality from coronary heart disease (CHD) has decreased, there are subsets of patients, particularly youngwomen, in whom the mortality rate has increased. Underlying sex differences in CHD may be an explanation. Women have more frequent symptoms, more ischemia, and higher mortality than men, but less obstructive coronary artery disease (CAD). Despite this, traditional risk factor assessment has been ineffective in risk stratifying women, prompting the emergence of novel markers and prediction scores to identify a population at risk. Sex differences inmanifestations and the pathophysiology of CHD also have led to differences in the selection of diagnostic testing and treatment options for women, having profound effects on outcomes. The frequent finding of nonobstructive CAD in women with ischemia suggests microvascular dysfunction as an underlying cause; therefore, coronary reactivity and endothelial function testing may add to diagnostic accuracy in female patients. In spite of evidence that women benefit from the same therapies as men, they continue to receive lessaggressive therapy, which is reflected in higher healthcare resource utilization and adverse outcomes. More sex-specific research is needed in the area of symptomatic nonobstructive CAD to define the optimal therapeutic approach.