RhoGTPases oscillate between an inactive GDP-bound state and an active GTP-bound state. The exchange of GDP for GTP is catalyzed by guanine nucleotide exchange factors (GEFs). Different modes of GEF activation allow for localized Rho signaling in response to specific stimuli. Lfc’s activity in normal cells is tightly regulated through phosphorylation, subcellular localization and protein-protein interactions. Having identified PKA and PP2A as the potential kinase and phosphatase regulating Lfc our work has concentrated on the importance of specific sites of phosphorylation on Lfc responsible for its regulation. We have confirmed T114 and S885 as the sites of phosphorylation mediating the mutually exclusive relationship of Lfc with 14-3-3 and Tctex1, both negative regulators of Lfc activity. We have also confirmed TIP41 as a member of the Lfc-PP2A complex. Future work will focus on determining if TIP41 mediates the Lfc-PP2A interaction and the signals involved in Lfc activation by dephosphorylation.

RhoGTPases oscillate between an inactive GDP-bound state and an active GTP-bound state. The exchange of GDP for GTP is catalyzed by guanine nucleotide exchange factors (GEFs). Different modes of GEF activation allow for localized Rho signaling in response to specific stimuli. Lfc’s activity in normal cells is tightly regulated through phosphorylation, subcellular localization and protein-protein interactions. Having identified PKA and PP2A as the potential kinase and phosphatase regulating Lfc our work has concentrated on the importance of specific sites of phosphorylation on Lfc responsible for its regulation. We have confirmed T114 and S885 as the sites of phosphorylation mediating the mutually exclusive relationship of Lfc with 14-3-3 and Tctex1, both negative regulators of Lfc activity. We have also confirmed TIP41 as a member of the Lfc-PP2A complex. Future work will focus on determining if TIP41 mediates the Lfc-PP2A interaction and the signals involved in Lfc activation by dephosphorylation.

Ovarian cancer and breast cancer share many of the same risk factors. The strongest known risk factor for breast cancer is mammographic density, the radiological appearance of breast tissue on a mammogram. Even though breast and ovarian cancer share many of the same risk factors, mammographic density has never been examined in relation to ovarian cancer. The present thesis describes a pilot study that was conducted to determine the feasibility of a study looking to address the issue of mammographic density as a risk factor for ovarian cancer. It was found that a larger study was feasible and should consist of approximately 700 case-control pairs recruited from cancer centres across Ontario, with cases matched to sisters or first-degree cousins. It was also found that the use of sister controls for cases did not lead to overmatching on mammographic density, and sisters are a suitable control group.

Ovarian cancer and breast cancer share many of the same risk factors. The strongest known risk factor for breast cancer is mammographic density, the radiological appearance of breast tissue on a mammogram. Even though breast and ovarian cancer share many of the same risk factors, mammographic density has never been examined in relation to ovarian cancer. The present thesis describes a pilot study that was conducted to determine the feasibility of a study looking to address the issue of mammographic density as a risk factor for ovarian cancer. It was found that a larger study was feasible and should consist of approximately 700 case-control pairs recruited from cancer centres across Ontario, with cases matched to sisters or first-degree cousins. It was also found that the use of sister controls for cases did not lead to overmatching on mammographic density, and sisters are a suitable control group.

The stimulation of Toll-like receptors (TLRs) by pathogen-associated molecular patterns (PAMPs) activates macrophages and dendritic cells to response to pathogens. These activated cells induce many immune-related genes, including proinflammatory cytokines which are necessary to activate immune responses against infection. TLRs and their signaling components have been linked to several human diseases, including pyogenic infection and sepsis. Sepsis often occurs in cancer patients treated with chemotherapy. The first focus of this work is to understand how TLR signal transduction pathways regulate the induction of proinflammatory cytokines. TLR stimulation triggers a signaling pathway via MyD88 and IRAK-4 that is essential for proinflammatory cytokine induction. In this study, I found that MyD88-deficient macrophages had defective c-Rel activation, which has been linked to IL-12 p40 induction. In addition, the expression of C/EBPbeta and C/EBPdelta was limited in MyD88- or IRAK-4-deficient macrophages treated with LPS. Importantly, the absence of both C/EBPbeta and C/EBPdelta resulted in the impaired induction of proinflammatory cytokines stimulated by several TLR ligands. These results identify both c-Rel and C/EBPbeta/delta as important transcription factors in a MyD88-dependent pathway that regulate the induction of proinflammatory cytokines. The second focus of this work is to understand the function of TREM2 and how TREM2 regulates TLR-mediated immune responses. TREM2 and DAP12 deficiencies were found in human patients with Nasu-Hakola disease, but the biology of TREM2 remains unclear. To study the function of TREM2 in dendritic cells, TREM2-deficient mice were generated. I found that TREM2 down-regulated the expression of proinflammatory cytokines induced by TLRs. The TREM2 ligand was expressed on activated T cells, and TREM2 enhanced the expression of IFN-gamma in antigen-specific T cells. In a mouse model of autoimmune diabetes, TREM2-deficient mice were resisted to CD8+ T cell-mediated beta-cell destruction. Therefore, TREM2 can positively or negatively regulate TLR-mediated immune responses in selective conditions. Together, the results presented in this thesis provide further understanding of how c-Rel, C/EBPbeta/delta, and TREM2 control and modulate TLR-mediated responses. Understanding these processes may ultimately provide novel therapeutic strategies to modulate immune responses in patients suffered from infectious diseases and cancer.

Current applied to coils wound at the tip of an endovascular catheter can be used to remotely steer a catheter tip in a clinical magnetic resonance imaging (MRI) scanner. This study focuses on (1) derivation and experimental validation of an equation that characterizes the relationship between catheter tip deflection and a number of magnetic, mechanical, and physical factors, and (2) evaluation of resistive heating in a worst-case scenario due to application of current necessary for clinically significant deflections, and radiofrequency (RF) heating due to real-time MRI pulse sequences. The derived equation was found to accurately model the behavior of the specialized catheter tip. The equation also has implications for catheter design and device implementation, including minimization of resistive heating, which was physiologically significant (> 4°C) under certain worst-case scenario conditions. This catheter steering mechanism should improve navigational control and is a unique advantage offered by MRI-guidance of endovascular procedures.

Current applied to coils wound at the tip of an endovascular catheter can be used to remotely steer a catheter tip in a clinical magnetic resonance imaging (MRI) scanner. This study focuses on (1) derivation and experimental validation of an equation that characterizes the relationship between catheter tip deflection and a number of magnetic, mechanical, and physical factors, and (2) evaluation of resistive heating in a worst-case scenario due to application of current necessary for clinically significant deflections, and radiofrequency (RF) heating due to real-time MRI pulse sequences. The derived equation was found to accurately model the behavior of the specialized catheter tip. The equation also has implications for catheter design and device implementation, including minimization of resistive heating, which was physiologically significant (> 4°C) under certain worst-case scenario conditions. This catheter steering mechanism should improve navigational control and is a unique advantage offered by MRI-guidance of endovascular procedures.

The stimulation of Toll-like receptors (TLRs) by pathogen-associated molecular patterns (PAMPs) activates macrophages and dendritic cells to response to pathogens. These activated cells induce many immune-related genes, including proinflammatory cytokines which are necessary to activate immune responses against infection. TLRs and their signaling components have been linked to several human diseases, including pyogenic infection and sepsis. Sepsis often occurs in cancer patients treated with chemotherapy. The first focus of this work is to understand how TLR signal transduction pathways regulate the induction of proinflammatory cytokines. TLR stimulation triggers a signaling pathway via MyD88 and IRAK-4 that is essential for proinflammatory cytokine induction. In this study, I found that MyD88-deficient macrophages had defective c-Rel activation, which has been linked to IL-12 p40 induction. In addition, the expression of C/EBPbeta and C/EBPdelta was limited in MyD88- or IRAK-4-deficient macrophages treated with LPS. Importantly, the absence of both C/EBPbeta and C/EBPdelta resulted in the impaired induction of proinflammatory cytokines stimulated by several TLR ligands. These results identify both c-Rel and C/EBPbeta/delta as important transcription factors in a MyD88-dependent pathway that regulate the induction of proinflammatory cytokines. The second focus of this work is to understand the function of TREM2 and how TREM2 regulates TLR-mediated immune responses. TREM2 and DAP12 deficiencies were found in human patients with Nasu-Hakola disease, but the biology of TREM2 remains unclear. To study the function of TREM2 in dendritic cells, TREM2-deficient mice were generated. I found that TREM2 down-regulated the expression of proinflammatory cytokines induced by TLRs. The TREM2 ligand was expressed on activated T cells, and TREM2 enhanced the expression of IFN-gamma in antigen-specific T cells. In a mouse model of autoimmune diabetes, TREM2-deficient mice were resisted to CD8+ T cell-mediated beta-cell destruction. Therefore, TREM2 can positively or negatively regulate TLR-mediated immune responses in selective conditions. Together, the results presented in this thesis provide further understanding of how c-Rel, C/EBPbeta/delta, and TREM2 control and modulate TLR-mediated responses. Understanding these processes may ultimately provide novel therapeutic strategies to modulate immune responses in patients suffered from infectious diseases and cancer.

In clinical oncology and experimental therapeutics, changes in tumour growth rate or volume have been traditionally the first indication of treatment response. These changes typically occur late in the course of therapy. Currently, no routinely available imaging modality is capable of assessing tumour response to cancer treatment within hours or days after delivery of radiation treatment. Therefore, the goal of this thesis is to develop the use of ultrasound imaging and ultrasound characterization methods with frequencies of 10 to 30 MHz to assess non-invasively tumour response to radiotherapy, early, within hours to days after delivery of radiotherapy. Responses to radiotherapy were characterized initially in vitro in a well-controlled environment using cell samples. It was demonstrated experimentally that the changes in ultrasound integrated backscatter and spectral slopes were the direct consequences of cell and nuclear morphological changes associated with cell death. The research in vitro provided a basis for the in vivo research that characterized responses to radiotherapy in cancer mouse models. The results from mouse tumour models indicated that quantitative ultrasound could detect the regions in a tumour that corresponded in histology to areas of cell death. In order to understand the cellular morphological changes responsible for ultrasound scattering at these frequencies and assist in the interpretation of experimental data, numerical simulations of ultrasound scattering from four different cell lines exposed to radiotherapy were conducted and compared to experimental results. It was concluded that the increases measured in ultrasound backscatter could in part be explained by the increase in the randomization of cell nuclei resulting from the increase in the variance of cell sizes following cell death. In this thesis, it is demonstrated that ultrasound imaging and quantitative ultrasound methods were able to detect non-invasively early responses to radiotherapy in vitro and in vivo. The mechanism behind this detection was linked to changes in the acoustic properties of nuclei and changes in the spatial organization of cells and nuclei following cell death. This provides the groundwork for future investigations regarding the use of ultrasound in cancer patients to individualize treatments non-invasively based on their responses to specific interventions.

Acute and chronic hypoxia exists within the microenvironment of solid tumours and drives therapy resistance, genetic instability and metastasis. Despite its importance in solid tumour progression, very little is known regarding the functional consequences of hypoxia-mediated changes in the expression of DNA repair proteins. I studied the relationship between hypoxia and DNA repair using a prolonged chronic hypoxic gas treatment model in a variety of human tumour cell lines to mimic the dynamic state of proliferation and DNA repair in cells distant from the tumour blood vasculature. I observed decreased expression of homologous recombination (HR) and base excision repair (BER) proteins due to a novel mechanism involving decreased protein synthesis. Error-free HR was suppressed 3-fold under 0.2% O2 as measured by the DR-GFP reporter system and functional BER was impaired as assessed with a functional glycosylase assay. This decrease in protein expression and function resulted in increased sensitivity to the DNA damaging agents MMC, cisplatin, H2O2 and MMS. Additionally, chronically hypoxic cells were relatively radiosensitive (OER = 1.37) when compared to acutely hypoxic or anoxic cells (OER = 1.96 - 2.61). As HR defects are synthetically lethal with poly(ADP-ribose) polymerase 1 (PARP1) inhibition, I evaluated the sensitivity of repair-defective hypoxic cells to PARP inhibition. I observed increased clonogenic killing in HR-deficient hypoxic cells following inhibition or depletion of PARP1. PARP-inhibited hypoxic cells accumulated γH2AX foci consistent with an accumulation of collapsed replication forks. Additionally, tumour xenografts exposed to PARP1 inhibition showed increased γH2AX and cleaved caspase-3 expression in hypoxic subregions with suppressed RAD51 protein expression and decreased ex vivo clonogenic survival. I conclude that persistent down-regulation of DNA repair components by the microenvironment could result in faulty DNA repair with significant implications for therapeutic response and genetic instability in human cancers. Specifically, hypoxic cells may be sensitized to PARP inhibitors and other agents targeting repair pathways down-regulated by hypoxia as a consequence of microenvironment-mediated “contextual synthetic lethality”.