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.

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.

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.

The divalent Ca2+ metal ion acts as a universal second messenger in virtually all eukaryotic cells from fungi to plants to mammals. In mammals, Ca2+ signaling is vital to a variety of physiological processes including fertilization, cell proliferation, secretion, and muscular contraction. In electrochemically non-excitable tissues, the release of Ca2+ from intracellular stores such as the endoplasmic reticulum is tightly regulated by the inositol 1,4,5-trisphosphate receptor (IP3R). The IP3R Ca2+ release channel is activated by the binding of the small molecule inositol 1,4,5-trisphosphate (IP3) in response to extracellular stimuli such as hormones, growth factors, and neurotransmitters. The conformational changes accompanying IP3 binding were investigated using a biophysical approach. A specific focus of this work is to decipher how signals of ligand binding are transmitted from the N-terminal IP3-binding core to the C-terminal channel domain. To such end, biophysical studies of the ligand-induced conformational changes within the N-terminal domain of IP3R (a.a. 1 – 604) were performed. The results implicated the presence of two flexible linkers which join stably folded domains. This prompted the proposal of a model in which an equilibrium mixture of conformational substrates containing compact and more extended structures co-exist. Determinants within the N- and C-terminal regions of IP3R have previously been reported to be critical to channel function. Employing nuclear magnetic resonance (NMR) as well as biochemical methods, an intermolecular interaction between the S4-S5 linker, the cytoplasmic loop between the fourth and fifth transmembrane helices of IP3R, and the suppressor domain was identified. The determination of the crystal structure of the suppressor domain from isoform type 3 IP3R (IP3R3SUP) allowed us to map the residues involved in this interaction to one face of the molecule. The characterization of this interaction provides insight into the N- and C-terminal determinants essential to the IP3R channel gating mechanism.

Radiation therapy is often offered as the primary treatment for head and neck cancer(HNC). Accurate target delineation is essential for the success of radiation therapy. The current target definition technique - manual delineation using Computed Tomography(CT) - is subject to high observer variability. Functional imaging modalities such as 2-[18F]-fluoro-2-deoxy-D-glucose Positron Emission Tomography(FDG-PET) can greatly improve the visualization of tumor. FDG-PET co-registered with CT has shown potential to improve the accuracy of target localization and reduce observer variability. Unfortunately, due to the limitation of PET, the degree of improvement obtained by qualitative and simple quantitative (e.g. thresholding) use of FDG-PET is not ideal. However, both PET and CT images contain a wealth of texture information that could be used to improve the accuracy of target definition. This thesis has investigated using texture analysis techniques to automatically delineate radiation targets. Firstly, PET and CT texture features with high discrimination ability were identified and a texture analysis technique- a decision tree based K Nearest Neighbour(DTKNN) classifier – was developed. DTKNN could accurately classify head and neck tissue with an area under curve(AUC) of a Receiver Operator Characteristic(ROC) of 0.95. Subsequently, an automated target delineation technique - CO-registered Multi-modality Pattern Analysis Segmentation System(COMPASS) - was developed that can delineate tumor on a voxel-by-voxel basis. COMPASS was found to accurately delineate HNC with 84% sensitivity and 95% specificity on a voxel basis per patient. To accurately evaluate the utility of the COMPASS in radiation targeting, a validation method which can combine biased observers' contours to generate a probabilistic reference for validation was developed. The method was based on maximum likelihood analysis using a simulated annealing(SA) algorithm. The results from this thesis show that texture features of both PET and CT images can enhance the discrimination between HNC and normal tissue, and an automated delineation method of HNC using texture analysis of PET and CT images can accurately and consistently define radiation targets in head and neck. This suggests that automated segmentation of radiation targets based on texture analysis techniques may significantly reduce observer variability and improve the accuracy of radiation targeting.