This thesis explores the unequal partitioning of damaged proteins during mitosis and its implications for cell fate. Initially described in unicellular organisms, it was unclear if this method was used in vivo in multicellular organisms and had functional consequences in mammalian cells. To determine if this asymmetry was conserved in multicellular organisms, I studied three stem/progenitor populations in Drosophila: the larval neuroblast, adult female germline stem cell, and adult intestinal stem cell. Each cell type was found to asymmetrically segregate damaged proteins identified by the 2,4-hydroxynonenal (HNE) modification, which are associated with oxidative stress and age. Both the larval neuroblast and female germline stem cell were found to retain damaged proteins during division, whereas the intestinal stem cell segregated damaged proteins to differentiating progeny. I suggest that functional lifespan, and not cell type, determines the cell that receives the majority of damaged proteins during division. In each cell type, damaged proteins were associated with DE-Cadherin, a common component of the stem cell niche and removal from the niche was associated with reduced damaged protein polarization. Interestingly, when larval neuroblasts were mechanically dissociated from their niche and placed in culture, the internal polarization of damaged proteins was found to increase with progression through the cell-cycle. Therefore, I suggest that both the niche and intrinsic factors play a role in the asymmetric division of damaged proteins. To determine if an asymmetric division of damaged proteins influenced cell fate, I used a mammalian cell line with inducible expression of misfolded Huntingtin, which shares similar properties to damaged proteins. This study also revealed that the conformation of damaged proteins impacts cell fate: cells with diffuse Huntingtin displayed greater proliferation and reduced resistance to stress. Tracking cells containing an aggregate with live-imaging revealed that the cell that inherits the aggregate has a longer cell-cycle and an enhanced capacity to differentiate. Therefore, the asymmetric inheritance of damaged proteins impacts cell fate. In the final chapter of this thesis, I discuss the implications of an asymmetric division of damaged proteins on cell fate and how this information can be applied to cancer treatments.

This thesis explores the unequal partitioning of damaged proteins during mitosis and its implications for cell fate. Initially described in unicellular organisms, it was unclear if this method was used in vivo in multicellular organisms and had functional consequences in mammalian cells. To determine if this asymmetry was conserved in multicellular organisms, I studied three stem/progenitor populations in Drosophila: the larval neuroblast, adult female germline stem cell, and adult intestinal stem cell. Each cell type was found to asymmetrically segregate damaged proteins identified by the 2,4-hydroxynonenal (HNE) modification, which are associated with oxidative stress and age. Both the larval neuroblast and female germline stem cell were found to retain damaged proteins during division, whereas the intestinal stem cell segregated damaged proteins to differentiating progeny. I suggest that functional lifespan, and not cell type, determines the cell that receives the majority of damaged proteins during division. In each cell type, damaged proteins were associated with DE-Cadherin, a common component of the stem cell niche and removal from the niche was associated with reduced damaged protein polarization. Interestingly, when larval neuroblasts were mechanically dissociated from their niche and placed in culture, the internal polarization of damaged proteins was found to increase with progression through the cell-cycle. Therefore, I suggest that both the niche and intrinsic factors play a role in the asymmetric division of damaged proteins. To determine if an asymmetric division of damaged proteins influenced cell fate, I used a mammalian cell line with inducible expression of misfolded Huntingtin, which shares similar properties to damaged proteins. This study also revealed that the conformation of damaged proteins impacts cell fate: cells with diffuse Huntingtin displayed greater proliferation and reduced resistance to stress. Tracking cells containing an aggregate with live-imaging revealed that the cell that inherits the aggregate has a longer cell-cycle and an enhanced capacity to differentiate. Therefore, the asymmetric inheritance of damaged proteins impacts cell fate. In the final chapter of this thesis, I discuss the implications of an asymmetric division of damaged proteins on cell fate and how this information can be applied to cancer treatments.

Septins are a group of filament forming proteins that act as scaffolds and diffusion barriers that regulate various cellular functions, such as cytokinesis. Recently, evidence was obtained in our laboratory that septins may also regulate mammalian cell polarity through their interaction with Par3, an important metazoan polarity regulator. In this thesis I sought to examine whether septins regulated the polarization of migrating cells through their interaction with Par3 as well as how this interaction occurred. I demonstrated that the C terminus of Sept7 was capable of binding to Par3 in vitro. Sept7 was also able to colocalize with Par3, but an interaction between the two proteins could not be detected by immunoprecipitation. Migration assays used to examine the significance of Sept7 in polarization showed a small decrease in polarization after Sept7 depletion. This suggests that septins may play a non-essential role in regulating the polarization of migrating cells.

Septins are a group of filament forming proteins that act as scaffolds and diffusion barriers that regulate various cellular functions, such as cytokinesis. Recently, evidence was obtained in our laboratory that septins may also regulate mammalian cell polarity through their interaction with Par3, an important metazoan polarity regulator. In this thesis I sought to examine whether septins regulated the polarization of migrating cells through their interaction with Par3 as well as how this interaction occurred. I demonstrated that the C terminus of Sept7 was capable of binding to Par3 in vitro. Sept7 was also able to colocalize with Par3, but an interaction between the two proteins could not be detected by immunoprecipitation. Migration assays used to examine the significance of Sept7 in polarization showed a small decrease in polarization after Sept7 depletion. This suggests that septins may play a non-essential role in regulating the polarization of migrating cells.

Neutrophils, key cells of the innate immune system, are responsible for preventing bacterial infections. They are rapidly recruited to sites of infection where they eliminate bacteria through killing methods that require reactive oxygen dependent processes. It has recently been established that neutrophils are capable of rapid and complex changes in gene expression during inflammatory responses. The concept that neutrophils only directly kill bacteria has been replaced by the concept that activated neutrophils can influence the immune response through the secretion of a variety of cytokines and by acting as antigen-presenting cell (APC) expressing MHC Class II, allowing for activation of T cells. Recent advances in neutrophil biology demonstrated that neutrophils also have an active regulatory role in angiogenesis and tumoral fate. It has been noted that a number of diseases including arthritis, periodontitis and acute respiratory distress syndrome (ARDS) are associated with neutrophil hyperactivity that results in significant tissue damage. Our group has previously shown that for some periodontal diseases, neutrophil hyperactivity is a key determinant of disease progression and severity. However, it remains unclear what factors are responsible for a patient developing a hyperactive neutrophil mediated disease. I hypothesize that local gene expression changes in neutrophils are responsible for the hyperactive behaviour of these cells during an inflammatory response. In order to assess this, I characterized the neutrophil gene expression profile in various compartments (bone marrow, blood and peritoneum in mice and blood and oral cavity in humans) and then characterized this genetic and phenotypic profile during an inflammatory response. I hypothesize that the neutrophil has a characteristic set of genes that are normally activated when it enters a site of inflammation from the circulation and that neutrophils can be polarized into a different functional subset under certain conditions that result in inflammation mediated diseases. To identify changes in neutrophil gene expression in the circulation and inflamed tissue I used recent advances in neutrophil isolation, RNA amplification, and microarray technologies to characterize the specific transcriptome associated with neutrophil site-specific responses.

Recently, we demonstrated that the anti-bacterial agent tigecycline preferentially induces death in leukemia cells through the inhibition of mitochondrial protein synthesis. To better understand the mechanisms of sensitivity and resistance to mitochondrial translation inhibition, we treated TEX leukemia cells with increasing concentrations of tigecycline over 4 months, and selected a population of cells resistant to tigecycline (RTEX+TIG). Compared to their wild type counterparts, the resistant RTEX+TIG cells had an altered metabolic profile with diminished oxidative phosphorylation and a greater reliance on glycolysis. Upon removal of tigecycline from RTEX+TIG cells, the cells re-established aerobic metabolism and oxidative phosphorylation to wild type levels. At the molecular level, these cells had increased levels of HIF1a. Strikingly, tigecycline-resistant cells had decreased expression of CD34 and CD117, clonogenic growth potential and engraftment capabilities in vivo. Thus, chronic inhibition of mitochondrial translation leads to the establishment of rho-zero-like metabolic phenotype, and is associated with differentiation of leukemia cells.

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections worldwide and it has been linked to increased risks of cervical cancer. Our study aims to understand the mechanisms by which chlamydia infection contributes to higher risks of tumorigenesis. We discovered in over 90% of infected human epithelial cells that chlamydia inclusions localized to the host cell centre during mitosis and prevented the completion of cleavage furrow ingression. Infected cells that failed to divide into two daughter cells re-entered interphase as tetraploid multinuclear cells, which are known initiators of tumorigenesis. In order to address whether chlamydia actively sought the host cell centre during mitosis, we created vacuoles using internalized latex beads and our findings indicated that chlamydia inclusions localized to the host cell centre much more frequently than similarly sized vacuoles. In addition, we demonstrated that metabolically inactive chlamydia inclusions localized to the cell centre less frequently than inclusions under normal conditions. Together, these results suggested that chlamydia actively localized to the host cell centre to efficiently block host cell mitosis. We took advantage of the microtubule-displacing capability of the C. trachomatis inclusions and demonstrated that astral microtubules could promote furrow initiation independent of the central spindle microtubules. Microdomains on chlamydia inclusions play important roles in inclusion localization and chlamydia replication. We discovered that these structures were surprisingly resistant to bacterial protein synthesis and host Src family kinase inhibitors. Finally, we demonstrated that multinuclear cells that resulted from unsuccessful mitosis contained significantly higher Golgi content, an important nutrient source for chlamydia. Our results indicated that C. trachomatis in multinuclear cells intercepted Golgi-derived lipids faster than in mononuclear cells. Together, my results reveal that C. trachomatis inclusions robustly localize to the cell centre to block host cell mitosis in order to acquire host Golgi nutrients more quickly in multinucleated cells.

The Corticotropin Releasing Factor (CRF) peptides are an evolutionarily conserved peptide family integral to the stress response. In 2002, an investigation was undertaken into possible homologues of CRF, which led to the discovery of the Teneurin C-terminal Associated Peptide (TCAP) family located on the C-terminal end of the teneurin proteins. This peptide family is involved in neuroprotection against stress. TCAP possesses a number of biological actions independent of teneurin, however, evidence of how TCAP is processed from its proprotein had not been determined. I hypothesize that TCAP is transcribed independently and has independent functionality in promoting filopodia elongation and outgrowth. Here, I show that TCAP-1 is independently expressed as an mRNA separate from the teneurin gene. Moreover, a key step in the TCAP-1 mediated molecular mechanism of filopodia outgrowth may require an interaction between Elongation Factor-1α (EF-1α) and the actin cytoskeleton, where this mechanism is not modulated by CRF.

The Corticotropin Releasing Factor (CRF) peptides are an evolutionarily conserved peptide family integral to the stress response. In 2002, an investigation was undertaken into possible homologues of CRF, which led to the discovery of the Teneurin C-terminal Associated Peptide (TCAP) family located on the C-terminal end of the teneurin proteins. This peptide family is involved in neuroprotection against stress. TCAP possesses a number of biological actions independent of teneurin, however, evidence of how TCAP is processed from its proprotein had not been determined. I hypothesize that TCAP is transcribed independently and has independent functionality in promoting filopodia elongation and outgrowth. Here, I show that TCAP-1 is independently expressed as an mRNA separate from the teneurin gene. Moreover, a key step in the TCAP-1 mediated molecular mechanism of filopodia outgrowth may require an interaction between Elongation Factor-1α (EF-1α) and the actin cytoskeleton, where this mechanism is not modulated by CRF.

Colorectal cancer (CRC) is the third leading cause of death from cancer in North America. The KRAS gene is mutated in approximately 40-50% of all CRC, and this mutation precludes treatment with promising targeted therapeutics. c-MET is a receptor tyrosine kinase that is overexpressed in ~70% of CRCs, and expression is correlated with disease progression. We hypothesized that high c-MET plus mutant KRAS would result poor survival of CRC patients, by activating unique signalling pathways that may be targeted for therapeutic purposes. To this end, we used phosphoproteomics in a KRAS mutant cell line, and identified proteins phosphorylated on tyrosine in response to HGF stimulation, including a subset of those that contain SRC family kinase consensus motifs. Small molecule inhibitors of either SRC or c-MET reduced tyrosine phosphorylation of both proteins, indicating reciprocal signalling. We chose the c-MET target p190RhoGAP for future study, as it is often ubiquitously bound to p120RasGAP via phosphorylated tyrosine. We found that RasGAP expression is mediated in part by KRAS signalling, and that expression of RasGAP could partly rescue tumourigenicity of a CRC cell line where the mutant KRAS allele has been inactivated, indicating the requirement of both mutant KRAS and RasGAP expression in this model. We then conclude by looking at CRC patient samples to determine the role of KRAS mutation in the progression and survival of CRC. We found that both KRAS and c-MET copy number are correlated to KRAS mutation status, and that c-MET polysomy plus KRAS mutation leads to worse overall survival than KRAS mutation alone. Overall, we identified novel targets of c-MET and KRAS oncogenic signaling, and identify a population which may derive the most benefit from treatments targeting both of these lesions.