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.

Hsp104 is a protein remodeling factor that is crucially important for induced thermotolerance and prion propagation in yeast. Recent work demonstrates that Hsp104 is able to directly recognize and interact with synthetic polypeptide substrates, and that this interaction is dependent on the amino acid composition or sequence (Lum et al., 2008). Here this concept is applied to the in vivo substrate Sup35. Sup35, a translation termination factor, also forms the yeast prion [PSI+]. The maintenance of the prion is critically dependent on the expression levels of Hsp104. Over-expression of Hsp104 leads to the loss of prions, as does inhibition of this protein remodeling factor. As part of this thesis, an in vitro assay was established in which spontaneous nucleation, the event preceding of fiber formation, was suppressed. Fibrilization itself then becomes strictly dependent on the chaperones Hsp104, huHsp70p and Ydj1. In line with in vivo observations, Hsp104 mutants that fail to propagate [PSI+] also fail to overcome nucleation inhibition in this assay. Following this, the next part of this work established that the middle (M) domain of Sup35 inhibited this process, while not affecting spontaneous fibrilization under non-inhibitory conditions. This finding was reproduced in vivo, as middle domain over-expression also led to curing of weak [PSI+]. This suggested that the M-domain contains an Hsp104 binding site. This hypothesis is supported by data presented in this thesis which show that a small segment 129-148 within the Middle domain has enhanced Hsp104 binding properties. Deletion of this 20-mer peptide also reduced the Hsp104 ability to interact with this prion substrate; it also results in the destabilization of the prion and enhanced curing by the prion curing agent guandidinium hydrochloride. This represents the first ever Hsp104 binding site identified within a natural substrate.

Planar cell polarity (PCP) provides positional information to a field of cells, coordinating the orientation of polarized structures or the direction of polarized cell movements. An evolutionarily conserved signalling pathway regulates PCP, however, the cue that establishes PCP is unknown. There is a strong precedent for Wnt signalling to act as the cue to establish PCP. Here I perform in vivo assays of cell polarity to examine the role of non-canonical Wnt signalling in regulating PCP, using zebrafish neural progenitor cells and asymmetric membrane localization of GFP-Prickle (a PCP cytoplasmic effector molecule) as a model system. My preliminary evidence suggests Wnt4a provides positional information to cells in the neural tube. In addition, using a membrane-yeast-two-hybrid approach to discover novel regulators of PCP, I identified Ring Finger 41 as a new binding partner to Van-gogh-like-2 (an essential PCP signalling molecule) and a novel regulator of vertebrate PCP.

In a previous study, three determinants of fitness were identified as mutant alleles (each designated "e") that arose in yeast populations propagated in divergent environments. In a low-glucose environment, MDS3e and MKT1e interacted positively to confer a fitness advantage. PMA1e from a high-salt environment interacted negatively with MKT1e in low glucose, indicating a mechanism of reproductive isolation. In this thesis, I demonstrated that the negative interaction between PMA1e and MKT1e is mediated by alteration in intracellular pH and likely by a delay of the cell division cycle, while the positive interaction between MDS3e and MKT1e is mediated by changes in gene expression affecting glucose transporter genes. I also confirmed the evolutionary significance of the positive interaction by showing that an MDS3e genetic background is required for the recapitulation of the MKT1e mutation. Collectively, these results illustrate how epistasis can play a central role in both adaptation and speciation.

Recent advances in the field of bionanotechnology have enabled researchers to design a variety of tools to detect, image and monitor biological process in cells. Despite this progress, the limited understanding of nanomaterial-cellular interactions has hindered the widespread use of these nanomaterials in biological systems. In this thesis, we examined the potential effects of metallic nanoparticle geometry on important cellular processes such as membrane trafficking, intracellular transport and subcellular signalling. We found that the size of nanoparticles plays an important role on their ability to interact with the cell surface receptors thus dictating their subsequent ability to activate intracellular signalling cascades. Interestingly, trafficking of these nanoparticles was dependent on their size due to biochemical and thermodynamical constraints. These findings suggest that nanomaterials actively interact with biological systems, thus, directly modulating vital cellular processes. In addition, by utilizing various physical and chemical properties of nanomaterials, we developed a novel class of hybrid nanoscaled carrier systems capable of delivering semiconductor quantum dots (QDs) into live cells without inducing membrane damage. Using biodegradable polymeric nanoparticles, bioconjugated QDs were encapsulated and delivered into trafficking vesicles of live cells. The environmentally sensitive surface charge of the polymeric nanoparticles exhibited positive zeta potential inside acidic endo-lysosomes, thus enabling their escape from the vesicular sequestration into the cytosol. Hydrolytic-induced degradation then releases the bioconjugate QDs for active labelling of subcellular structures for real-time studies. Unlike previously described intracellular QD delivery methods, the proposed system offers an efficient way to non-invasively deliver bioconjugated QDs without inducing cell damage, enabling researchers to accurately monitor cellular processes in real-time. The understanding of both physical and chemical properties of nanomaterials is crucial to the design of biocompatible nanosystems to study fundamental processes in biological systems. Here, we demonstrated that both the size and surface chemistry of nanoparticles can be modified to obtain desired biological responses. Future experimental efforts to study other physical and chemical properties could allow the development of more sophisticated and effective platforms for biological applications.

Epithelial sheets line the surfaces of the body, forming a barrier between the external environment and internal tissues. During development, regulation of epithelial architechture can drive morphogenesis and build the three-dimensional structures of the body. Epithelial form and function derive from the polarized morphology of epithelial cells, which have apical surfaces that face the external environment, lateral surfaces containing cell-cell junctions and basal surfaces that connect to the underlying tissue. A network of polarity proteins establishes the apico-basolateral axis, while a system of polarized membrane traffic ensures delivery of specialized cargo to distinct membrane surfaces. How these systems of polarity and trafficking are integrated is still poorly understood. The focus of my study was to investigate how the apical polarity proteins Cdc42, Par6, Bazooka and aPKC (the “Par complex”) regulate polarity and adherens junction (AJ) integrity during Drosophila development. Upon perturbation of Cdc42/Par activity during embryogenesis, apical membrane proteins accumulate in sorting endosomes. This trafficking defect occurs throughout the ectoderm, but in the ventral neuroectoderm (VNE) is accompanied by a concomitant depletion of the apical proteins from the plasma membrane (PM) and a loss of AJ integrity. I have demonstrated that the VNE phenotype is a consequence of the relatively high morphogenetic activity of this tissue. Furthermore, I have shown that the AJ defects are likely a downstream consequence of the depletion of important apical polarity factors, such as Crumbs, from the PM. To further characterize the mechanism of apical trafficking, I searched for interactors of Cdc42/Par in the membrane trafficking machinery. I describe interactions between several trafficking genes and Cdc42/Par and provide evidence that Vps26, a component of the retromer complex that retrieves proteins from endosomal membrane and delivers them to the Golgi for re-secretion, is phosphorylated by aPKC and acts as an aPKC effector in the recycling of apical membrane proteins. I propose that Cdc42/Par regulate the retromer to promote the PM localization of apical proteins, which is important to maintain AJ integrity in morphogenetically active tissues.

The tyrosine kinase receptor Tie-2 is expressed on the surface of endothelial cells and is necessary for angiogenesis and vascular stability. To date, the best characterized ligands for Tie-2 are Angiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2). Ang-1 has been identified as the main activating ligand for Tie-2 while the role of Ang-2 has been controversial since its discovery; some studies reported Ang-2 as a Tie-2 antagonist while others described Ang-2 as a Tie-2 agonist. The purpose of this thesis was to understand: (1) how the receptor Tie-2 is regulated by Ang-1 and Ang-2 in endothelial cells, (2) to compare the effects of Ang-1 and Ang-2, and (3) to determine the arrangement and distribution of Tie-2 in endothelial cells. The research presented in this thesis indicates that Tie-2 is arranged in variably sized clusters on the endothelial cell surface. Clusters of Tie-2 were expressed on all surfaces of cells: on the apical plasma membrane, on the tips of microvilli, and on the basolateral plasma membrane. When endothelial cells were stimulated with Ang-1, Tie-2 was rapidly internalized and degraded. Upon Ang-1 stimulation, Tie-2 localized to clathrin-coated pits on all surfaces of endothelial cells indicating that one pathway mediating Tie-2 internalization is through clathrin-coated pits. After activation of Tie-2, Ang-1 dissociates from the endothelial cell surface and accumulates in the surrounding medium. When experiments were repeated with Ang-2, it was discovered that Ang-2 induced all of the same effects on Tie-2 as Ang-1 but at a much reduced level and rate, indicating that Ang-2 likely functions as a partial agonist for Tie-2 in endothelial cells.

Osteoclasts are multinucleated, bone resorbing cells that carry out their function using specialized actin-based structures called actin rings and podosomes. Rho GTPases function as molecular switches that regulate the actin cytoskeleton in osteoclasts and many other cell types. Filamin A (FLNa) and SBDS are two proteins that have the potential to interact with both F-actin and Rho GTPases, and thus regulate osteoclast formation, differentiation, or function. We found that in FLNa-null pre-osteoclasts, activation of RhoA, Rac1, and Cdc42 was perturbed, leading to defective pre-osteoclast migration prior to fusion. Ablation of SBDS resulted in the blockage of osteoclast differentiation downstream of RANK and defective RANKL-mediated upregulation of Rac2 that is required for pre-osteoclast migration. Therefore, both FLNa and SBDS are required to coordinate Rho GTPase activation during osteoclastogenesis, in addition to a role for SBDS in osteoclast differentiation downstream of RANK.

Planar cell polarity (PCP) provides positional information to a field of cells, coordinating the orientation of polarized structures or the direction of polarized cell movements. An evolutionarily conserved signalling pathway regulates PCP, however, the cue that establishes PCP is unknown. There is a strong precedent for Wnt signalling to act as the cue to establish PCP. Here I perform in vivo assays of cell polarity to examine the role of non-canonical Wnt signalling in regulating PCP, using zebrafish neural progenitor cells and asymmetric membrane localization of GFP-Prickle (a PCP cytoplasmic effector molecule) as a model system. My preliminary evidence suggests Wnt4a provides positional information to cells in the neural tube. In addition, using a membrane-yeast-two-hybrid approach to discover novel regulators of PCP, I identified Ring Finger 41 as a new binding partner to Van-gogh-like-2 (an essential PCP signalling molecule) and a novel regulator of vertebrate PCP.

In a previous study, three determinants of fitness were identified as mutant alleles (each designated "e") that arose in yeast populations propagated in divergent environments. In a low-glucose environment, MDS3e and MKT1e interacted positively to confer a fitness advantage. PMA1e from a high-salt environment interacted negatively with MKT1e in low glucose, indicating a mechanism of reproductive isolation. In this thesis, I demonstrated that the negative interaction between PMA1e and MKT1e is mediated by alteration in intracellular pH and likely by a delay of the cell division cycle, while the positive interaction between MDS3e and MKT1e is mediated by changes in gene expression affecting glucose transporter genes. I also confirmed the evolutionary significance of the positive interaction by showing that an MDS3e genetic background is required for the recapitulation of the MKT1e mutation. Collectively, these results illustrate how epistasis can play a central role in both adaptation and speciation.