Uncoupling protein-2 (UCP2) may be linked to the attenuation of reactive oxygen species (ROS), but it is unclear whether this phenomenon pertains to the pancreatic beta cell. In this study, a UCP2-deficient mouse model was used to assess the importance of UCP2 to beta cell viability. We investigated the effect of UCP2 absence in response to a beta cell cytotoxic model of diabetes induction. In vivo treatment by the cytotoxic agent streptozotocin led to overall beta cell loss, but severity was not exacerbated by UCP2 deficiency. We also examined ROS production and cell viability in islet cells exposed to various stressors associated with oxidative stress. In vitro measurements of ROS and cell death in islet cells demonstrated that the response was not influenced by UCP2 expression. In contrast with UCP2 overexpression studies showing cytoprotection, this study reveals that beta cell survival is not compromised by the absence of UCP2.

The stride interval of typical human gait is correlated over thousands of strides. This statistical persistence diminishes with age, disease, and pace-constrained walking. Considering the widespread use of treadmills in rehabilitation and research, it is important to understand the effect of this speed-constrained locomotor modality on stride interval dynamics. To this end, and given that the dynamics of children have been largely unexplored, this study investigated the impact of treadmill walking, both with and without handrail use, on paediatric stride interval dynamics. An initial stationarity analysis of stride interval time series identified both non-stationary and stationary signals during all walking conditions. Subsequent scaling analysis revealed diminished stride interval persistence during unsupported treadmill walking compared to overground walking. Finally, while the correlation between stride interval dynamics and gross energy expenditure was investigated in an effort to elucidate the clinical meaning of persistence, no simple linear correlation was found.

The innate immune system is important in both acute and chronic infection. In this thesis, I investigated the effect of H. pylori infection on 1) DCs, key orchestrators of the immune system, and 2) autophagy, recently identified as an important component of innate immunity. I determined that H. pylori activates the STAT3 pathway in DCs, increasing DC maturation and inducing production of IL-10, IL-12p40 and TNF-α, without IL-12p70. This cytokine profile may favour an immunoregulatory response, promoting persistent H. pylori infection. In addition I determined that H. pylori’s VacA toxin induced autophagy, ROS production and Parkin aggregation which has been implicated in mediating autophagy in response to mitochondrial damage. Thus H. pylori alters these key effectors of innate immunity which may play a role in promoting its chronic infection and disease.

Factor inhibiting HIF (FIH) negatively regulates hypoxia inducible factor-1 (HIF-1) transcriptional activity, selectively controlling certain HIF-1 target genes, such as vascular endothelial growth factor (VEGF) and prolyl hydroxylase domain 3 (PHD3), but not others. PHD3 and VEGF are important for placental development and function and are overexpressed in preeclampsia (PE). The purpose of this study was to examine FIH in both normal and pathological human placentae. I hypothesized that FIH regulates VEGF and PHD3 in the placenta and that this rheostat is altered in PE. Results show that FIH suppresses PHD3 and VEGF in JEG-3 cells; this effect was abrogated by FIH gene silencing. Moreover, my data indicate that seven in absentia homologue-1 (Siah-1) targets FIH for degradation in the placenta; this degradation is enhanced in PE and likely contributes to aberrant VEGF and PHD3 expression. Overall, my data suggest an important role for FIH in the pathogenesis of PE.

Androgens (such as testosterone) have been shown to increase lean body mass and reduce fat body mass in men through activation of androgen receptors (AR). While this suggests a potential clinical use for androgens, attempts at utilization of this class of hormones as a therapeutic are limited by side effects due to indiscriminate AR activation in various tissues. Thus, a greater understanding of the tissues and cells involved in promoting these changes would be beneficial. Here we show that selective overexpression of AR in muscle cells of transgenic (HSA-AR) rodents both increases lean muscle mass and significantly reduces fat mass in males. Similar effects can be induced in HSA-AR females treated with testosterone. Metabolic analyses of HSA-AR males show that these animals demonstrate increased O2 consumption and hypermetabolism. Thus, targeted activation of AR in muscle regulates body composition and metabolism, suggesting a novel target for drug development.

Ryanodine receptor 1 (RyR1) is a homotetrameric calcium channel located in the sarcoplasmic reticulum (SR) of skeletal muscle. We employed metal affinity chromatography followed by liquid chromatography mass spectrometry from HEK-293 cells to purify affinity tagged cytosolic RyR1, with interacting proteins. In total, we identified 703 proteins with high confidence (>99%). Of the putative RyR1 interacting proteins, five candidates [calcium homeostasis endoplasmic reticulum protein (CHERP), ER-golgi intermediate compartment 53kDa protein (LMAN1), T-complex protein (TCP), phosphorylase b kinase (PHBK) and four and half LIM domains protein 1 (FHL1)], were selected for interaction studies. Immunofluorescence analysis showed that CHERP co-localizes with RyR1 in the SR of rat soleus muscle. Calcium transient assays in HEK293 cells over-expressing RyR1 with siRNA suppressed CHERP or FHL1, showed reduced calcium release via RyR1. In conclusion, we have identified RyR1 interacting proteins in CHERP and FHL1 which may represent novel regulatory mechanisms involved in excitation-contraction coupling.

Dynamic nuclear polarization and dissolution of 13C-labeled metabolite allows dynamic imaging of metabolism in-vivo. However, the spatial and temporal resolutions of magnetic resonance spectroscopic imaging are limited by the duration of free-induction decay acquisitions and the T1-based, non-recoverable polarization decay. This thesis describes the implementation of a spectral-spatial radiofrequency excitation pulse with a `flyback' echo-planar readout trajectory to dynamically image [1-13C]-pyruvate and [1-13C]-lactate in an interleaved manner. This technique excites a single resonance of either [1-13C]-pyruvate or [1-13C]-lactate and generates dynamic images with 5mm in-plane resolution. Metabolite dynamics extracted from the images and the corresponding non-localized spectroscopic data reveal similar kinetic rates upon fitting to a kinetic model. This demonstrates the feasibility of probing metabolism in heterogeneous tissues in-vivo with dynamic interleaved 13C MR imaging.

Specific SNPs in intronic regions of the human TCF7L2 gene are associated with an elevated risk of T2D development and progression. Several investigations have suggested a role of TCF7L2 in pancreatic β-cells. Whether this transcription factor is indeed expressed in the pancreatic islets of rodent species, however, has been a controversial issue. Here, we found that TCF7L2 mRNA level was significantly lower in the pancreas compared to the gut or Ins-1 cell line. In addition, TCF7L2 mRNA abundance in the pancreas was decreased by insulin. Finally, both TCF7 and TCF7L1 but not LEF-1 could be detected in the mouse pancreas. mRNA abundance for these two transcription factors was also decreased by insulin, and the level of TCF7, TCF7L1, and TCF7L2 mRNAs could be down-regulated by HFD. We speculate that reduced expression of these TCF genes during hyperinsulinemia may alter the Wnt signalling pathway and therefore impair the function of β-cells.

The unfolded protein response, which is activated during ER stress, counteracts stress conditions by increasing folding capacity and by increasing the degradation of misfolded ER proteins by the ER-Associated Degradation (ERAD) system. Studies using an engineered insulinoma cell line with inducible expression of the Akita folding-deficient insulin have shown a large induction of Herp, a protein that has been implicated in the ERAD pathway. We hypothesized that Herp is an essential protein that regulates the degradation of misfolded insulin during the ER stress response. Indeed, we found that the degradation of mutant insulin is Herp-dependent and that maintaining Herp expression is vital for maintaining cell survival. We have also observed that the expression of Herp mRNA and protein is induced in various cell culture and animal models of diabetes. These results suggest that Herp is an important ER stress response protein that is induced under diabetic conditions in pancreatic β-cells.

The structure of afferent neurons is pseudounipolar. Studies suggest that they relay action potentials (APs) to both directions of the T-junctions to reach the cell body and the spinal cord. Moreover, the somata are electrically excitable and shown to be able to transmit the signals to associated satellite cells. Our study demonstrates that this transmission can go further and pass onto passive neighbouring somata, if they are in direct contact with same satellite cells. The neurons activate the satellite cells by releasing ATP. This triggers the satellite cells to exocytose acetylcholine to the neighbouring neurons. In addition, the ATP inhibits the nicotinic receptors of the neurons by activating P2Y receptors and initiating the G-protein-mediated pathway, thus reducing the signals that return to the neurons that initiated the signals. This “sandwich synapse” represents a unique pathway by the ectopic release between the somata and the satellite cells.