Rosiglitazone (RSG) is an insulin-sensitizing drug used to treat Type 2 Diabetes Mellitus (T2DM). Clinical trials show that women taking RSG experience more limb fractures than patients taking other T2DM drugs. The purpose of this study is to understand how RSG (3mg/kg/day and 10mg/kg/day) and the bisphosphonate alendronate (0.7mg/kg/week) alter bone quality in the male, female and female ovariectomized (OVX) Zucker fatty rat model over a 12 week period. Bone quality was evaluated by mechanical testing of cortical and trabecular bone. Microarchitecture, bone mineral density (BMD), cortical bone porosity, bone formation/resorption and mineralization were also measured. Female OVX RSG10mg/kg rats had significantly lower vertebral BMD and compromised trabecular architecture versus OVX controls. Increased cortical porosity and decreased mechanical properties occurred in these rats. ALN treatment prevented these negative effects in the OVX RSG model. Evidence of reduced bone formation and excess bone resorption was detected in female RSG-treated rats.

Tumours contain heterogeneous cell populations. A population enriched in tumour-initiating potential has been identified in soft-tissue sarcoma (STS) by the isolation of "side population" (SP) cells. In this study, we compared the gene expression profiles of SP and non-SP cells in STS and identified Hedgehog (Hh) and Notch pathways as potential candidates for the targeting of SP cells. Upon verification of the activation of these pathways in SP cells, using primary tumor xenografts in NOD-SCID mice as our experimental model, we used the Hh blocker Triparanol and the Notch blocker DAPT to demonstrate that the suppression of these pathways effectively depleted the abundance of SP cells, reduced tumour growth, and inhibited the tumour-initiating potential of the treated sarcoma cells upon secondary transplantation. The data provide additional evidence that SP cells act as tumour initiating cells and points to Hh and Notch pathways as enticing targets for developing potential cancer therapies.

Gastrulation is a highly complex series of cellular rearrangements that leads to the internalization of the mesoderm and endoderm. The cellular behaviors that underlie morphogenesis are dependent upon changes in cell motility and polarity. Eph receptors belong to a family of receptor tyrosine kinases that are involved in a variety of developmental processes. This study is the first to examine the role EphA4 during Xenopus gastrulation. Morpholino oligonucleotide (MO) mediated knockdown of EphA4 resulted in attenuated mesoderm involution and reduced the expression of the posterior mesoderm marker brachyury (Xbra). Expression of EphA4 in the blastocoel roof was sufficient to promote ectopic Xbra expression. I show that EphA4 can regulate Xbra expression and involution movements by signaling through PAK1. Temporal regulation of Xbra was sufficent to rescue EphA4 induced gastrulation defects. This study has uncovered a novel EphA4/PAK1 pathway which is required for mesoderm involution and Xbra expression during Xenopus gastrulation.

The objective of this thesis was to examine the role of ps20 in virus infections. We provide evidence that MHV-1 infection resulted in increased lung viral titers in ps20-/- mice. These data highlight an antiviral role for ps20 in MHV-1 infection. We also observed an increase in the percentage of GR1+ neutrophils infiltrating the BAL and in the lung draining lymph node of ps20-/- mice, on day 2 post-infection. In vitro, gene expression analysis identified an increase in expression of CXCL1 and CXCL2 in MHV-1 infected ps20-/- fibroblasts. These data suggest a role for ps20 in regulating neutrophil chemotactic factors, and migration. Next, we examined influenza A/WSN/33, and provide evidence that ps20 functions as a proviral factor. In vivo, ps20-/- mice infected with influenza A/WSN/33 exhibited decreased lung viral titers. These data suggest that ps20 functions as either a proviral or antiviral agent, dependent on the infecting virus.

Nano-FET biosensors are an emerging nanoelectronic technology capable of real-time and label-free quantification of soluble biological molecules. This technology promises to enable novel in vitro experimental approaches for investigating complex biological systems. In this study, we first explored osteocyte mechanosensitivity under different mechanical stimuli and found that osteocytes are exquisitely sensitive to different oscillatory fluid flow conditions. We therefore aimed to characterize protein-mediated intercellular communication between mechanically-stimulated osteocytes and other bone cell populations in vitro to elucidate the underlying mechanisms of load-induced bone remodeling. To this end, we devised a novel nano-manipulation based fabrication method for manufacturing nano-FET biosensors with precisely controlled device parameters, and further investigated the effect of these parameters on sensor performance.

Loss of the renal microvasculature is a common feature of almost all forms of chronic kidney disease (CKD). Here we explored the role of the angiogenic chemokine stromal cell-derived factor-1-alpha (SDF-1) and its cognate receptor CXCR4 in experimental and human CKD. CXCR4 was present on endothelial cells and podocytes, while SDF-1 was detectable on podocytes, arteriolar smooth muscle cells, interstitial fibroblasts and occasional endothelial cells. CXCR4 mRNA was elevated in the kidneys of rats with CKD and chronic antagonism of CXCR4 accelerated renal decline and capillary loss. Acute SDF-1 infusion activated glomerular endothelial nitric oxide synthase (eNOS) in vivo, while functional response to SDF-1 was impaired in glomerular endothelial cells derived from eNOS-/- mice. Finally, CXCR4 mRNA was also found to be increased in biopsies of patients with secondary focal segmental glomerulosclerosis. These observations indicate that local eNOS-dependent SDF-1/CXCR4 signaling exerts a compensatory reno-protective effect in the setting of CKD.

With the recent advent of gene therapy, anti-apoptotic therapy has been receiving spotlight as a potential modality to inhibit the deterioration of pump function in the failing heart. We hypothesized that anti-apoptotic therapy using survivin gene delivery will 1) salvage H9c2 cells exposed to doxorubicin toxicity, and 2) ameliorate the progressive decline in left ventricular function in a rat model of doxorubicin-induced cardiomyopathy. The in vitro data suggested that survivin successfully prevented cell death under doxorubicin stress by both direct and indirect/paracrine mechanisms. Doxorubicin-treated animals developed progressive left ventricular dysfunction as evident by echocardiography and invasive pressure-volume loop analysis, which was prevented by ultrasound-mediated survivin plasmid delivery, but not empty plasmid delivery. Post-mortem analysis of myocardial tissue indicated a lowered apoptotic index in survivin-treated hearts, with evidence of decrease in interstitial fibrosis. In conclusion, survivin gene therapy was shown to ameliorate doxorubicin-induced cardiomyopathy, by decreasing apoptosis and preventing adverse remodeling.

Endoglin, a co-receptor of the TGF-β superfamily, is predominantly expressed in endothelial cells and in some myeloid cells and implicated as a potential modulator of immune responses. We previously demonstrated that Endoglin heterozygous (Eng+/-) mice subjected to the dextran sulfate sodium colitis model developed persistent inflammation and epithelial ulceration, while Eng+/+ mice recovered following the acute phase of disease. Our aim was to assess potential alterations in distribution and number of immune cells, expression of inflammatory mediators and mechanisms of oxidative burst in Eng+/- mice. While the number of overall T, B and myeloid cells was unaltered between the genotypes, changes in neutrophil regulating cytokines and angiogenesis mediating factors were observed in Eng+/- mice. In addition, downregulation of phagocyte oxidative burst enzymes point to potential defects in microbial clearance in Eng+/- mice. These findings suggest a role for endoglin in regulating immune and vascular functions during inflammation.

Genomic organization of the major histocompatibility complex (MHC) can profoundly influence gene function and multigene family evolution. Situated at the interface of individual genetic variation and the adaptive immune response, MHC class I and II loci are intensively studied for disease associations and used as markers of adaptive genetic variation in evolutionary ecology research. Genomic sequence of MHC-containing cosmid clones from the red-billed gull (Larus scopulinus, Charadriiformes: shorebirds, gulls, and allies) was obtained for comparative analysis of avian MHC evolution. MHCI polymorphism was further investigated using cDNA library screening and locus-specific genotyping protocols. This first information regarding MHC organization and MHCI variation in charadriiforms suggests a complex evolutionary history to MHC architecture in birds. Duplication of MHCIIα loci in tandem MHCIIα/β pairs and their proximity to MHC-region gene COL11A2 are similar to arrangements in nonavian vertebrates, and contrast with the “minimal essential” MHC of the chicken (Gallus gallus, Galliformes: gamebirds). MHCI–TAP2 organization is shared with Galloanserae (gamebirds + waterfowl), as is a proposed major classical function for this MHCI gene. In contrast, the placement of MHCI genes adjacent to sequence from chromosomes 3, 5, and 22 of the chicken and zebra finch (Taeniopygia guttata, Passeriformes: perching birds) indicates interchromosomal rearrangements in birds and the possible genomic dispersal of nonclassical MHCI genes in the red-billed gull. Screening for avian malaria, genetic parentage tests, and field data from red-billed gulls at Kaikoura Peninsula, New Zealand were combined with MHCI genotypes to investigate relationships with disease and reproduction. Plasmodium infection was confirmed in red-billed gulls, and breeding condition was negatively associated with malarial infection and positively related to variation at the putative major MHCI locus. A low rate of extrapair paternity was identified across thirteen breeding seasons. Partners without extrapair young (EPY) had greater MHCI dissimilarity than was expected by chance, whereas lower individual MHCI variation and elevated hatching failure existed for pairs with EPY. In addition to contributing to studies of MHC evolution, sexual selection, and disease dynamics in the New Zealand avifauna, this research will facilitate studies of MHC genes in related charadriiforms, many of which are of conservation concern.

Sexual selection drives the evolution of male morphology, life history, physiology, and behaviour across taxa. Here I examine the mechanisms of sexual selection that arise at various stages in mating interactions to identify congruence or conflict between the traits selected by choice and competition. I first examine plasticity of developing male Australian redback spiders (Latrodectus hasselti) and show that male metabolic rates vary adaptively to facilitate the scramble to reach virgins. Next, I show that females cease sex pheromone production after mating and re-advertise receptivity later in their reproductive season effectively creating two windows in which males may compete. I show that females discriminate against males that do not meet a threshold courtship duration suggesting that courtship is the trait selected through choice. However, male-male competition leads to reductions in courtship effort provided to females. During the first window paternity is split equally if rival males mate in quick succession with a virgin female. However, if the second mating is delayed, there is a strong bias in the paternity of the second male. A delay in the second mating is beneficial to females as it reduces longevity costs of polyandry. However, delays in the initial mating decrease female longevity, likely because of elevated metabolic rates of virgins. My research shows that the trait favoured by female choice is in opposition to selection via male-male competition. Females’ sex pheromone production yields windows during which mating will optimize female, but not male, fitness. Studies that isolate the mechanisms of sexual selection are valuable in that they can identify the traits under selection. However, my research shows that considering these processes in isolation can lead to incorrect inferences about the net effect of sexual selection.