The Slit family (Slit1-3) of secreted glycoproteins and their cognate Roundabout family (Robo1-4) of transmembrane receptors provide important repulsive signals to guide cell migration during development and postnatal life. The dynamic regulation of Slit/Robo signaling is poorly understood in vertebrates. In this study, we identified a novel role for endocytosis in regulating Slit2 /Robo1 expression. Using heterologous expression systems, Slit2 was found be endocytosed in a Robo1-dependent manner and subsequently degraded in the lysosome, while Robo1 was found to be primarily recycled. An AP-2 consensus binding site, which mediates clathrin-dependent endocytosis, was identified in the Robo1 cytoplasmic tail and found to be required for Slit2 down-regulation and Slit2-induced endocytosis of Robo1. Preliminary data suggests that Slit2-induced endocytosis of Robo1 may be required for downstream signaling. These findings have important implications for how Slit/Robo signaling may be dynamically regulated during cell migration.

The aggressive nature of pancreatic cancer, characterized by invasiveness, resistance to treatment, rapid progression, and its high prevalence in the population urges the need for developing more effective treatments. Many studies have attributed resistance to therapeutics of pancreatic cancer to activity of the transcription factor nuclear factor kappa B (NF-kB). NF-kB is regulated by the serine/threonine kinase glycogen synthase kinase-3 (GSK-3). GSK-3 is a key mediator of pathways such as insulin, wnt, and PI3K/Akt and has roles in proliferation, glucose metabolism, apoptosis, motility and neuroprotection. Depending on the cellular context, GSK-3 activity can promote or inhibit cell survival. GSK-3 inhibition was recently reported to have anti-cancer effects against pancreatic cancer cells. This effect was in part attributed to suppression of NF-kB. In this thesis, I showed that while blocking GSK-3 disrupts NF-kB, and has anti-survival effects on pancreatic cancer cells, it does not sensitize to the chemotherapeutic drug gemcitabine. NF-kB inhibition by curcumin also resulted in similar effects. These results questions previous reports that NF-kB activation plays a major role in chemo-resistance of pancreatic cancer. The inhibition of NF-kB by genetic disruption of GSK-3 was previously reported to sensitize mouse embryonic fibroblasts and hepatocytes to TNF-alpha cytotoxicity. I therefore tested whether GSK-3 inhibition could sensitize pancreatic cancer cells to apoptosis induced by the clinically applicable member of the TNF-alpha family, TNF-alpha related apoptosis inducing ligand (TRAIL). In contrast to the results obtained with gemcitabine, the combination of genetic or pharmacological inhibition of GSK-3 and TRAIL was found to be highly synergistic in apoptosis induction. Analysis of the apoptotic mechanisms, point towards effects of GSK-3 inhibition on caspase-8 activation, consistent with inhibition of the death receptor signalling pathway. It was found that not only caspase-8 but also mitochondrial anti-apoptotic proteins such as Bcl-XL and Mcl-1 were mediating the TRAIL sensitization. Furthermore, for the first time the in vivo effects of GSK-3 inhibition in combination with TRAIL treatment was investigated. The results indicate a significant enhancement of apoptosis in pancreatic cancer xenografts with minimal toxic effects. Together, these studies provide a rationale for developing combination treatments based on GSK3 inhibition and TRAIL death receptor activation to treat pancreatic cancer.

Spinal cord injury (SCI) pathophysiology occurs as a primary traumatic event followed by secondary injury, resulting in the loss of neurons, oligodendrocytes and demyelination of residual axons. Unfortunately, endogenous spontaneous regeneration of oligodendrocytes is minimal. Previously, a method to generate multi-potential stem cells (MPSC) from umbilical cord blood (UCB) has been reported using lineage negative cells (Linneg) grown in fibroblast growth factor 4 (FGF4), stem cell factor (SCF) and fms-like tyrosine kinase receptor-3 ligand (Flt-3l) supplemented serum free medium. These MPSC have the ability to differentiate into bone, muscle and endothelial cells. In this thesis, the ability of MPSC to differentiate into oligodendrocytes was investigated as a potential treatment for SCI. Culturing MPSC under conditions that mimic normal timing of oligodendrocyte differentiation resulted in cells that expressed oligodendrocyte markers in vitro and were morphologically similar to them. I next investigated the ability of MPSC to improve functional recovery in a SCI compression injury model. Although the cells did not differentiate into oligodendrocytes in vivo as we initially hypothesised, a modest but significant improvement in hindlimb function was observed. A cytokine assay revealed that MPSC secrete elevated levels of anti-inflammatory, angiogenic and neurotrophic factors, possibly contributing to indirect mechanisms of repair by reducing secondary injury. Shiverer mouse neonates were next used as an alternative non-injury model to investigate the differentiation potential of MPSC. We hypothesised that transplanting MPSC into a host with an immature immune system and an actively myelinating environment would lead to engraftment and differentiation into oligodendrocytes. However no MPSC that differentiated into oligodendrocytes could be detected. Altogether, our in vitro data adds support for the reprogramming of cells, with further studies needed to test the functionality of resulting oligodendrocyte-like cells. Although MPSC failed to differentiate in both in vivo models, several potential therapeutic targets to treat SCI were found.

In 1962, Gross and Lapiere described collagenolytic activity in the degradation of tadpole tails during amphibian metamorphosis. This activity was later attributed to a collagenase enzyme belonging to the matrix metalloproteinase family. Over the past 49 years, steady growth in the field of metalloproteinase biology has uncovered that degradation of extracellular matrix components represents only a fraction of the functions performed by these enzymes. The regulatory roles of these enzymes in numerous aspects of mammalian biology remains poorly understood. This thesis investigates the metalloproteinase ADAM17 and its natural inhibitor TIMP3 in acute and chronic inflammation. My work describes the generation of new murine experimental systems of compartmentalized ADAM17 or TIMP3 deficiency and their applications in acute liver inflammation (i.e. fulminant hepatitis and T-cell mediated autoimmune hepatitis) and atopic dermatitis. Loss of Timp3 protected mice against fulminant hepatic failure caused by activation of the death receptor Fas. We determined that TIMP3 simultaneously promotes pro-apoptotic signaling through TNFR1 while suppressing anti-apoptotic EGFR activation in the liver. Mechanistically, we identified that ADAM17 is critical in shedding TNFR1 and EGFR ligands (e.g. Amphiregulin, HB-EGF, TGF) and extended this finding to clinically relevant drug-induced hepatitis. Adult TIMP3 deficient mice also exhibited spontaneous accumulation of CD4+ T cells in the liver. Consequently, polyclonal T cell activation with the lectin Concanavalin A (con A) in a model of autoimmune hepatitis resulted in accelerated liver injury. We identified that this immunopathology relied on TNF bioavailability as mice lacking both Timp3 and Tnf were resistant to con A. Using bone marrow chimeras we established that non-hematopoietic tissues were the physiologically relevant source of TIMP3 in vivo, thereby highlighting an immunosuppressive role for this stromal metalloproteinase inhibitor in cellular immunity. Finally, we investigated epithelial:immune crosstalk in the epidermis by generating tissue-specific ADAM17 deficiency in basal keratinocytes. These mice developed spontaneous inflammatory skin disease that was physiologically consistent with atopic dermatitis. Focused investigation of keratinocyte-specific signaling deregulated by ADAM17 deficiency revealed its requirement for tonic Notch activation, which in turn antagonized transcriptional activity of AP-1 transcription factors on the promoters of epithelial cytokines TSLP and G-CSF. In summary, these works identify cellular mechanisms governing cytokine-mediated communication between epithelial and immune cells to modulate inflammation. The findings that TIMP3 and ADAM17 act as regulators of key inflammatory, proliferative and developmental pathways provide impetus to expand our understanding of this important family of enzymes in mammalian signal transduction.

Autophagy is an evolutionarily conserved degradative eukaryotic cell pathway that plays a role in multiple cellular processes. One important function is as a key component of the cellular immune response to invading microbes. Autophagy has been found to directly target and degrade multiple intracellular bacterial species. In this thesis, I identify and characterize two distinct regulatory mechanisms for this pathway involving the second messengers: diacylglycerol and cyclic adenosine monophosphate (cAMP). Salmonella enteric serovar Typhimurium (S. Typhimurium) is a Gram-negative bacterial species that has been shown to be intracellularly targeted for degradation by autophagy. While targeting of this species has been previously shown to involve ubiquitination, this pathway accounts for only half of targeted bacteria. Here I show that ubiquitin-independent autophagy of S. Typhimurium requires the lipid second messenger diacylglycerol. Diacylglycerol localization to the bacteria precedes autophagy and functions as a signal to recruit the delta isoform of protein kinase C (PKC) in order to promote the specific autophagy of tagged bacteria. Furthermore, I have found that the role of diacylglycerol and PKC delta is not limited to antibacterial autophagy but also functions in rapamycin-induced autophagy indicating a general role for these components in this process. Multiple bacterial species have been found to be targeted by autophagy and while some have developed strategies that allow them to avoid targeting, no bacterial factor has yet been identified that is able to inhibit the initiation of this process. Here I show that two bacterial species, Bacillus anthracis and Vibrio cholera inhibit autophagy through the elevation of intracellular cAMP and activation of protein kinase A. Using two different bacterial cAMP-elevating toxins, I show that multiple types of autophagy are inhibited in the presence of these toxins. This is indicative of a general inhibitory function for these toxins and identifies a novel bacterial defence strategy. This work characterizes both a novel regulatory signal for the induction of autophagy and identifies a novel bacterial tactic to inhibit this process. Together the data presented in this thesis provide novel insight into the regulation of autophagy and offer potential targets for modulation of this process.

SPARC is a highly conserved trimodular Ca2+- and Collagen-binding matricellular protein with diverse functions during development, wound healing and cancer metastasis. Our lab previously generated an embryonic lethal Drosophila SPARC null mutant, Df(3R)nm136, analysis of which revealed that SPARC was required for the deposition of Collagen IV into basal laminae and normal nervous system development during embryogenesis. In contrast to these previous studies, my data revealed that SPARC is not required for the deposition of Collagen IV into embryonic basal laminae or embryonic nervous system development. Further analysis showed that the Df(3R)nm136 chromosome carried a second-site mutation in the Neuralized locus which caused the nervous system defects and embryonic lethality previously associated with a loss of SPARC. Removal of this second site mutation and reanalysis of the SPARC mutant phenotype revealed that SPARC is required for larval development where it appears to play a role in the regulation fat body remodelling. SPARC mutant fat bodies showed changes in cell shape and basal lamina remodelling which resemble the fat body remodelling process that normally occurs during pre-pupal stages via up-regulation of MMP2 in response to the steroid hormone ecdysone. The effects of loss of SPARC on fat body cells were shown to be cell autonomous. Structure-function analysis of SPARC showed that secretion of SPARC is required for its function, whereas Domain1 is dispensable. Together, my studies indicate that SPARC has essential intra and extracellular roles during Drosophila larval fat body development.

It has been hypothesized that tumours are caricatures of normal tissue organization, where a minority cell population, the ‘stem cell’ of cancer, holds the exclusive ability for tumour propagation. These cancer stem cells (CSCs), or tumour-initiating cells, possess extensive self-renewal ability, through which they ensure maintenance of the tumourigenic clone. Such cells have been identified in various cancers, including colorectal, and have been proposed to be the source of tumour re-initiation following therapy. An important and currently unanswered question in solid tumours is whether all CSCs are equal or whether there is a gradient of potency within the CSC compartment. Using primary human colon tumour cells and sensitive in vivo functional assays, we have determined that colon CSCs are not uniform; rather, they vary with respect to their proliferative capacity, which is also linked to their response to chemotherapy. These findings hold therapeutic implications for colorectal cancer treatment since all types of CSCs must be eradicated to remove the risk of tumour relapse. While the CSC model may provide attractive answers to some challenging questions, it remains controversial. Ascertaining the importance of CSCs will come from targeted CSC therapies. Here we demonstrate that human colorectal CSC function is dependent on the self-renewal regulator BMI-1. Down-regulation of BMI-1 inhibits the ability of colorectal tumour-initiating cells to self-renew resulting in the abrogation of their tumourigenic potential. Treatment of primary colorectal cancer xenografts with small molecule BMI-1 inhibitors resulted in colorectal tumour-initiating cell loss with long-term and irreversible impairment of tumour growth. Targeting the BMI-1 related self-renewal machinery provides the basis for a new therapeutic approach in the treatment of colorectal cancer. Collectively, we have advanced the CSC field in two areas of importance. We show for the first time that the CSC pool encompasses a gradient of proliferative potential linked to chemotherapeutic response. Second, we provide critical proof for the clinical relevance of CSCs by inhibiting tumour growth through targeting of the self-renewal machinery. This body of work significantly advances our understanding of colorectal tumour-initiating cells.

DNA damage represents a major challenge to the faithful replication and transmission of genetic information from one generation to the next. Cells utilize a highly integrated network of pathways to detect and accurately repair DNA damage. Mutations arise when DNA damage persists undetected, unrepaired, or repaired improperly. Mutations are a driving force of carcinogenesis and therefore many of the DNA damage surveillance and repair mechanisms guard against the transformation of normal cells into cancer cells. Central to the detection and repair of DNA damage is the relocalization of DNA damage surveillance proteins to DNA damage where they assemble into subnuclear foci and are capable to producing a signal that the cell interprets to induce cellular modifications such as cycle arrest and DNA repair which are important DNA damage tolerance. In this work, I describe my quest to understand the mechanisms underlying the assembly, maintenance, and disassembly of these DNA damage-induced foci and how they affect DNA damage signaling in Saccharomyces cerevisiae. First, I describe phenotypic characterization of a novel mutation that impairs assembly of the 9-1-1 checkpoint clamp complex into foci. Second, I describe my work to further understand the roles of the histone phosphatase Pph3 and phosphorylated histone H2A in modulating DNA damage signaling. Third, I include my work to uncover the possible mechanism by which the helicase Srs2 works to enable termination of DNA damage signaling. In summary, this thesis documents my efforts to understand the cellular and molecular nature of DNA damage signaling and how signaling is turned off in coordination with DNA damage repair.

DNA double-strand breaks (DSBs) are a threat to cell survival and genome integrity. In addition to canonical DNA repair systems, DSBs can be converted to telomeres by telomerase. This process, herein termed telomere healing, endangers genome stability since it usually results in chromosome arm loss. Therefore, cells possess mechanisms that prevent the untimely action of telomerase on DSBs. In this work, I reported the completion of a transposon mutagenesis screen in budding yeast and the identification of five novel genes (RRD1, CIK1, CTF18, RTS1, and IRC6) critical for telomere healing. The characterization of Rrd1 led to the surprising finding that Rrd1 facilitates telomere healing at DSBs with little or no TG-rich sequences but not at DSBs with long tracts of telomeric sequences. Pph3, a PP4 phosphatase, acts in conjunction with Rrd1 to promote telomere healing. Conversely, Mec1, the ATR ortholog, phosphorylates Cdc13 on its S306 residue to suppress its accumulation at DSBs. Rrd1 and Pph3 oppose Cdc13 S306 phosphorylation and are necessary for the efficient accumulation of Cdc13 at DSBs. Next, I found that Cik1 and its kinesin partner Kar3 are both important for telomere healing. Importantly, Kar3 contributes to telomere healing through its motor function. In contrast to Rrd1, Kar3 contributes to telomere healing regardless of telomeric sequence lengths adjacent to the break. Finally, Cik1 and Kar3 have a general role in DNA repair and physically associate with DSBs, which is dependent on the process of anchoring DSBs to nuclear periphery. In conclusion, I identified a mechanism by which the ATR family of kinases enforces genome integrity, a phosphoregulatory loop that underscores the contribution of Cdc13 to the fate of DNA ends, and a kinesin complex critical for the spatial organization of DNA repair.

Dictyostelium is a well established model for the study of differentiation and morphogenesis. It has previously been shown that Ca2+ and its primary sensor calmodulin (CaM) have roles in cell differentiation and morphogenesis in Dictyostelium and higher eukaryotes. Here I further elucidated the role of Ca2+ and CaM in cell differentiation in Dictyostelium. No previous work existed on the regulation of CaM-binding proteins (CaMBPs) or their binding partners by developmental morphogens. First, I gained insight into the developmental role of nucleomorphin (NumA1), a novel CaMBP, as well as its binding partners Ca2+-binding protein 4a (CBP4a) and puromycin-sensitive aminopeptidase A (PsaA). I showed that NumA1 and CBP4a expression is co-regulated by differentiation-inducing factor-1 (DIF-1), a stalk cell morphogen. Both proteins likely have a role in prestalk-O cell differentiation. On the other hand, I showed that PsaA expression is regulated by cAMP and PsaA regulates spore cell differentiation. Thus, NumA1 likely differentially regulates stalk and spore cell differentiation by interacting with CBP4a and PsaA, respectively. I also used Dictyostelium as a model to gain insight into the mechanism of action of colchicine, a microtubule disrupting agent that has been shown to affect differentiation and morphogenesis in many organisms. I identified that colchicine affects cell motility, disrupts morphogenesis, inhibits spore cell differentiation and induces stalk cell differentiation through a Ca2+ and CaM-dependent signal transduction pathway. It specifically induced differentiation of ecmB expressing stalk cells, independent of DIF-1 production. Lastly, I analyzed for the first time the role of Ca2+ and CaM in ecmB expression in vivo. I showed that Ca2+ and CaM regulate ecmB expression in intact and regenerating slugs and that Ca2+ and CaM also regulate cell differentiation, motility and slug shape. In conclusion, Ca2+ and CaM play integral roles in cell motility, cell differentiation and morphogenesis in Dictyostelium.