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.

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.

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.

Tie-2 expressing monocytes (TEMs) were identified to be a subset of monocytes that was potent inducer of tumor angiogenesis in various types of cancer. On the other hand, monocytes and macrophages are crucially involved in the innate immune response, but the function of TEMs in this process is not well understood. Therefore, we studied the role of Tie-2 in regulating the inflammatory response initiated by lipopolysaccharide in murine macrophages. First, we observed that a small percentage of bone marrow-derived macrophages express Tie-2. Furthermore, we observed that Tie-2 ligands were able to induce phosphorylation of signalling proteins in macrophages. However, we found that Tie-2 ligands were not able to regulate pro-inflammatory cytokine secretion, nor were the ligands able to prevent apoptosis induced by lipopolysaccharide in macrophages. These results suggest that contrary to current evidence in the literature, Tie-2 ligands may not play a role in modulating the pro-inflammatory response in TEMs.

eneurin C-terminal associated peptides (TCAPs) are a novel family of peptides encoded on the last exon of the teneurin genes. The predicted peptide sequences are highly conserved across metazoans and possess the structural hallmarks of a cleavable bioactive peptide that are 40 or 41 amino acid residues. One of the peptides in the family, TCAP-1, is a potent regulator of neurite outgrowth and dendritic spine density in the hippocampus and inhibits corticotropin-releasing factor (CRF)-associated stress-induced and cocaine-seeking behaviours. The effects of TCAP-1 are long lasting, suggesting that TCAP-1 plays a significant role in the regulation of cell-to-cell communication and cellular plasticity. Moreover, TCAP-1 regulates cellular energy, metabolism and cell survival and may, therefore, possess functional attributes outside of the CNS. However, the molecular mechanisms associated with TCAP-1-mediated trophic effects are not known. My research was aimed to 1) determine whether TCAP-1 exerts its effects as part of a direct teneurin-1 function, whereby TCAP-1 represents a functional region of the large teneurin-1 protein, or if it has an independent role, either as a splice variant or post-translational proteolytic cleavage product of teneurin-1; 2) map the distribution of TCAP-1-immunoreactivity and TCAP-1 binding sites in mouse; 3) elucidate the molecular mechanism by which TCAP-1 regulates cytoskeletal dynamics; and 4) investigate a role for TCAP-1 in male reproduction. My research establishes that the C-terminal region of teneurin-1, corresponding to TCAP-1, can be both structurally and functionally independent from teneurin-1 in both the brain and testis of the adult mouse. Furthermore, I provide novel evidence that functionally links the teneurin-TCAP-1 system with the dystroglycan complex and provide new insight into the molecular and signaling mechanisms by which TCAP-1 regulates cytoskeletal dynamics. These studies implicate the teneurins in a broader range of neuroendocrine and trophic functions than previously thought and furthers our understanding of the mechanisms associated with TCAP-1-mediated function in the body.

Tie-2 expressing monocytes (TEMs) were identified to be a subset of monocytes that was potent inducer of tumor angiogenesis in various types of cancer. On the other hand, monocytes and macrophages are crucially involved in the innate immune response, but the function of TEMs in this process is not well understood. Therefore, we studied the role of Tie-2 in regulating the inflammatory response initiated by lipopolysaccharide in murine macrophages. First, we observed that a small percentage of bone marrow-derived macrophages express Tie-2. Furthermore, we observed that Tie-2 ligands were able to induce phosphorylation of signalling proteins in macrophages. However, we found that Tie-2 ligands were not able to regulate pro-inflammatory cytokine secretion, nor were the ligands able to prevent apoptosis induced by lipopolysaccharide in macrophages. These results suggest that contrary to current evidence in the literature, Tie-2 ligands may not play a role in modulating the pro-inflammatory response in TEMs.

eneurin C-terminal associated peptides (TCAPs) are a novel family of peptides encoded on the last exon of the teneurin genes. The predicted peptide sequences are highly conserved across metazoans and possess the structural hallmarks of a cleavable bioactive peptide that are 40 or 41 amino acid residues. One of the peptides in the family, TCAP-1, is a potent regulator of neurite outgrowth and dendritic spine density in the hippocampus and inhibits corticotropin-releasing factor (CRF)-associated stress-induced and cocaine-seeking behaviours. The effects of TCAP-1 are long lasting, suggesting that TCAP-1 plays a significant role in the regulation of cell-to-cell communication and cellular plasticity. Moreover, TCAP-1 regulates cellular energy, metabolism and cell survival and may, therefore, possess functional attributes outside of the CNS. However, the molecular mechanisms associated with TCAP-1-mediated trophic effects are not known. My research was aimed to 1) determine whether TCAP-1 exerts its effects as part of a direct teneurin-1 function, whereby TCAP-1 represents a functional region of the large teneurin-1 protein, or if it has an independent role, either as a splice variant or post-translational proteolytic cleavage product of teneurin-1; 2) map the distribution of TCAP-1-immunoreactivity and TCAP-1 binding sites in mouse; 3) elucidate the molecular mechanism by which TCAP-1 regulates cytoskeletal dynamics; and 4) investigate a role for TCAP-1 in male reproduction. My research establishes that the C-terminal region of teneurin-1, corresponding to TCAP-1, can be both structurally and functionally independent from teneurin-1 in both the brain and testis of the adult mouse. Furthermore, I provide novel evidence that functionally links the teneurin-TCAP-1 system with the dystroglycan complex and provide new insight into the molecular and signaling mechanisms by which TCAP-1 regulates cytoskeletal dynamics. These studies implicate the teneurins in a broader range of neuroendocrine and trophic functions than previously thought and furthers our understanding of the mechanisms associated with TCAP-1-mediated function in the body.

The renal capsule is a flattened layer of cells which surround the kidney. Expression of the transcription factor Foxd1 is required for normal development of the capsule. Furthermore, current evidence suggests that during development the capsule progenitors are in a state of active hedgehog signaling. We hypothesize that hedgehog plays a role in modulating capsule morphogenesis in the embryonic kidney. To test the hypothesis hedgehog signaling was inhibited in the capsule via Foxd1Cre mediated deletion of Smoothened (Smo), the activator of the pathway. Mutant kidneys were approximately 48% smaller in volume and had a 42% decrease in nephron number. Furthermore, mutants displayed abnormal patterning of the capsule where regions on the surface of the kidney had no capsule cells. The discontinuous capsule phenotype was observed only after E13.5. Additionally, capsule cells progressively lost expression of known markers Foxd1 and Raldh2 and their proliferative capacity was decreased by 54% at E13.5.

The renal capsule is a flattened layer of cells which surround the kidney. Expression of the transcription factor Foxd1 is required for normal development of the capsule. Furthermore, current evidence suggests that during development the capsule progenitors are in a state of active hedgehog signaling. We hypothesize that hedgehog plays a role in modulating capsule morphogenesis in the embryonic kidney. To test the hypothesis hedgehog signaling was inhibited in the capsule via Foxd1Cre mediated deletion of Smoothened (Smo), the activator of the pathway. Mutant kidneys were approximately 48% smaller in volume and had a 42% decrease in nephron number. Furthermore, mutants displayed abnormal patterning of the capsule where regions on the surface of the kidney had no capsule cells. The discontinuous capsule phenotype was observed only after E13.5. Additionally, capsule cells progressively lost expression of known markers Foxd1 and Raldh2 and their proliferative capacity was decreased by 54% at E13.5.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a human intestinal pathogen, which can cause severe disease. EHEC O157:H7 is responsible for outbreaks of diarrhea and hemorrhagic colitis. EHEC produces a potent cytotoxin known as Vero (Shiga-like) cytotoxin, which causes diarrhea-associated hemolytic uremic syndrome (HUS), the most common cause of acute renal failure in children. Current treatment remains predominantly supportive in nature because antibiotics and non-steroidal anti-inflammatory drugs exacerbate the condition. Therefore, alternative therapeutic approaches that will prevent the EHEC colonization without the release of toxins need to be delineated. Understanding the pathobiology of disease is likely to yield novel approaches to interrupt the infectious process. My hypothesis was that pathogen-derived effectors associate with lipid rafts and, thereby, promote the recruitment of host signal transduction proteins to lipid rafts in response to EHEC O157:H7 infection. In this thesis, specific host signalling pathways hijacked by EHEC O157:H7, through lipid raft signalling platforms, to elicit pathogenic effects are studied using complementary approaches, including epithelial model cell lines and an animal model of infection (Citrobacter rodentium challenge of mice). A lack of osteopontin resulted in decreased attaching effacing lesions and reduced colonic epithelial cell hyperplasia in response to C. rodentium infection. These findings suggest that C. rodentium, mimicking EHEC O157:H7 infection, is capable of utilizing host cell components to elicit its pathogenic effects. In vitro data showed that EHEC O157:H7 effector proteins manipulate cell signalling through lipid rafts employed as platforms to recruit and activate host second messengers. PKC and PI3K activation led to attaching and effacing lesions, disruption of tight junctions, and the initiation of both innate and adaptive host immune responses. The results pointed towards a role for atypical PKC in EHEC-induced attaching and effacing lesion formation. The role of lipid rafts in EHEC O157:H7 pathogenesis was also studied using Citrobacter rodentium-infected Niemann-pick type C (NPC) mice. Infection of NPC mice, which lack lipid rafts, with C. rodentium resulted in delayed colonization and delayed onset of attaching-effacing lesion formation, compared with infected wild type mice. C. rodentium-infected NPC mice also demonstrated reduced colonic epithelial hyperplasia and decreased secretion of the pro-inflammatory cytokine, interferon-γ. Taken together, the findings presented in this thesis highlight the importance of host cell signal transduction cascades in EHEC O157:H7 disease pathogenesis, and demonstrate a role for lipid rafts and OPN in mediating host cell signaling responses to non-invasive enteric microbial pathogens.