The mammalian kidney and reproductive systems both derive from a common embryological origin, the intermediate mesoderm. Abnormal intermediate mesoderm development can result in congenital abnormalities of the urogenital system, yet the molecular mechanisms that govern intermediate mesoderm development are incompletely defined. The spatial and temporal expression of the proteins BMP2 and 4 and their receptor ALK3, in urogenital tissue, suggests a function for BMP-ALK3 signaling in the intermediate mesoderm. It was found that Alk3IM null kidneys display renal hypoplasia, associated with a decrease in kidney size and nephron number. The phenotype of renal hypoplasia in Alk3IM nulls was associated with early decreased number of developing nephron structures and secondary defects in branching morphogenesis. While neither apoptosis nor cell proliferation differed in metanephric mesenchyme cells in Alk3IM nulls, markers of renal progenitor cells were decreased in mutant animals. It was observed that Alk3 expression in the intermediate mesoderm also controls mesonephric tubule number. Alk3IM nulls had fewer mesonephric tubules and fewer derivative Leydig cells. The reduction in Leydig cells resulted in decreased levels in serum testosterone and defects in seminal vesicle formation and fertility. Alk3 expression was also required for normal development of the corpus epididymis. The morphological defects in nephrogenesis were associated with decreased phospho-p38 MAPK expression and in the testis with decreased Phospho-SMAD1/5/8. These results elucidated a requirement for Alk3 signaling in controlling progenitor cells derived from the intermediate mesoderm.

Objective: Glucagon-like peptide-1 receptor (GLP-1R) agonists have been shown to reduce atherosclerosis in non-diabetic mice. We hypothesized that treatment with GLP-1R agonists would reduce the development of atherosclerosis in diabetic Apoe-/- mice. Results: Exendin-4 treatment (10 nmol/kg/day) of high-fat diet-induced glucose-intolerant mice for 22 weeks did not significantly reduce oral glucose tolerance (P=0.62) or HbA1c (P=0.85), and did not reduce plaque size at the aortic sinus (P = 0.35). Taspoglutide treatment for 12 weeks (0.4-mg tablet/month) of diabetic mice reduced body weight (P<0.05), food intake (P<0.05), oral glucose tolerance (P<0.05), intrahepatic triglycerides (P<0.05) and cholesterol (P<0.001), and plasma IL-6 levels (P<0.01); increased insulin:glucose (P<0.05); and unaltered oral lipid tolerance (P=0.21), plasma triglycerides (P=0.45) or cholesterol (P=0.92). Nonetheless, taspoglutide unaltered aortic atherosclerosis (P=0.18, sinus; P=0.19, descending aorta) or macrophage infiltration (P=0.45, sinus; P=0.26, arch). Conclusions: GLP-1R activation in either glucose-intolerant or diabetic mice does not significantly modify the development of atherosclerosis.

Pathogenic bacteria employ a number of mechanisms to induce infection and survive in host tissues, including toxin secretion and the formation of protective multicellular structures called biofilms. Type IV Pili (T4P) are highly conserved organelles essential for both the establishment of infection and biofilm maturation. The goal of this research is to gain a molecular level understanding of the function of the highly dynamic T4P of Pseudomonas aeruginosa. The pilMNOPQ operon encodes 5 members of a transmembrane complex that facilitates pilus function. While PilQ is the putative outer membrane secretin through which the pilus exits the cell, the roles of the PilM/N/O/P proteins are less well defined. Using both in vivo and in vitro techniques our characterization of PilP has provided significant insight into organization of the apparatus. PilP is an inner membrane lipoprotein essential for T4P function, but lipidation is dispensable, suggesting that its interactions with other T4P components are sufficient for PilP function. We showed that PilN/O/P form a stable heterotrimer when expressed in E. coli, and we suggest that they form a similar subcomplex in P. aeruginosa. Additionally we were able to show that PilP is also able to interact with a periplasmic fragment of the outer membrane pore protein PilQ. Structural and bioinformatics studies suggest that the organization of PilN/O/P/Q complex is similar to that of the transenvelope complex of another important Gram-negative virulence factor – the Type II Secretion System (T2SS). Our structural and functional characterization of PilP, the PilN/O/P complex and the striking similarities between the T4P and T2S systems, as well as important differences that make each molecular machine unique, will be presented.

Pathogenic bacteria employ a number of mechanisms to induce infection and survive in host tissues, including toxin secretion and the formation of protective multicellular structures called biofilms. Type IV Pili (T4P) are highly conserved organelles essential for both the establishment of infection and biofilm maturation. The goal of this research is to gain a molecular level understanding of the function of the highly dynamic T4P of Pseudomonas aeruginosa. The pilMNOPQ operon encodes 5 members of a transmembrane complex that facilitates pilus function. While PilQ is the putative outer membrane secretin through which the pilus exits the cell, the roles of the PilM/N/O/P proteins are less well defined. Using both in vivo and in vitro techniques our characterization of PilP has provided significant insight into organization of the apparatus. PilP is an inner membrane lipoprotein essential for T4P function, but lipidation is dispensable, suggesting that its interactions with other T4P components are sufficient for PilP function. We showed that PilN/O/P form a stable heterotrimer when expressed in E. coli, and we suggest that they form a similar subcomplex in P. aeruginosa. Additionally we were able to show that PilP is also able to interact with a periplasmic fragment of the outer membrane pore protein PilQ. Structural and bioinformatics studies suggest that the organization of PilN/O/P/Q complex is similar to that of the transenvelope complex of another important Gram-negative virulence factor – the Type II Secretion System (T2SS). Our structural and functional characterization of PilP, the PilN/O/P complex and the striking similarities between the T4P and T2S systems, as well as important differences that make each molecular machine unique, will be presented.

Objective: Glucagon-like peptide-1 receptor (GLP-1R) agonists have been shown to reduce atherosclerosis in non-diabetic mice. We hypothesized that treatment with GLP-1R agonists would reduce the development of atherosclerosis in diabetic Apoe-/- mice. Results: Exendin-4 treatment (10 nmol/kg/day) of high-fat diet-induced glucose-intolerant mice for 22 weeks did not significantly reduce oral glucose tolerance (P=0.62) or HbA1c (P=0.85), and did not reduce plaque size at the aortic sinus (P = 0.35). Taspoglutide treatment for 12 weeks (0.4-mg tablet/month) of diabetic mice reduced body weight (P<0.05), food intake (P<0.05), oral glucose tolerance (P<0.05), intrahepatic triglycerides (P<0.05) and cholesterol (P<0.001), and plasma IL-6 levels (P<0.01); increased insulin:glucose (P<0.05); and unaltered oral lipid tolerance (P=0.21), plasma triglycerides (P=0.45) or cholesterol (P=0.92). Nonetheless, taspoglutide unaltered aortic atherosclerosis (P=0.18, sinus; P=0.19, descending aorta) or macrophage infiltration (P=0.45, sinus; P=0.26, arch). Conclusions: GLP-1R activation in either glucose-intolerant or diabetic mice does not significantly modify the development of atherosclerosis.

Mutations in the SLC4A1 gene result in misfolding and trafficking defects of the human erythroid (AE1) and kidney (kAE1) forms of the anion exchanger 1 glycoprotein. This affects the amount of functional protein at the cell surface, resulting in hematological and renal diseases. In this thesis, the role of the quality control system of molecular chaperones (cytosolic and ER) was examined during the biosynthesis of wild type and mutant AE1 in different cellular models. The hypothesis to be tested is that molecular chaperones are responsible for the intracellular retention of AE1 mutants. Chaperones were found to interact with AE1 and kAE1 in vitro and in vivo (HEK-293, K562, MDCK cells). Disruption of the calnexin-AE1 interaction in K562 cells did not affect the cell surface levels of wild type or mutant erythroid AE1. AE1 also trafficked to the cell surface in mouse embryonic fibroblasts completely deficient in calnexin or calreticulin. In contrast, in MDCK cells, disruption of the calnexin-kAE1 interaction allowed functional dominant (R589H, R901stop), but not misfolded kAE1 mutants (kSAO, G701D), to escape the ER and traffic to the cell surface. Calnexin is therefore not required for the cell surface expression of erythroid AE1, but can be responsible for the intracellular retention of certain kAE1 mutants in cells with the complete complement of molecular chaperones. Components involved in membrane glycoprotein folding and quality control (calnexin, ERp57, Hsc70, Hsp70), were lost at later stages during the differentiation of CD34+ erythroid progenitor cells. This suggests that the loss of molecular chaperones may facilitate the massive production of red cell glycoproteins, allowing erythroid AE1 mutants to escape quality control, traffic to the plasma membrane, and be present in mature red blood cells. These studies demonstrate that the role chaperones play varies, depending on cellular context. By understanding the cellular context and factors involved, therapeutic strategies may be tailored to deal with protein misfolding diseases, and in the case of kAE1, rescue the cell surface trafficking of misfolded, but functional, transport protein using pharmacological modulators.

Mutations in the SLC4A1 gene result in misfolding and trafficking defects of the human erythroid (AE1) and kidney (kAE1) forms of the anion exchanger 1 glycoprotein. This affects the amount of functional protein at the cell surface, resulting in hematological and renal diseases. In this thesis, the role of the quality control system of molecular chaperones (cytosolic and ER) was examined during the biosynthesis of wild type and mutant AE1 in different cellular models. The hypothesis to be tested is that molecular chaperones are responsible for the intracellular retention of AE1 mutants. Chaperones were found to interact with AE1 and kAE1 in vitro and in vivo (HEK-293, K562, MDCK cells). Disruption of the calnexin-AE1 interaction in K562 cells did not affect the cell surface levels of wild type or mutant erythroid AE1. AE1 also trafficked to the cell surface in mouse embryonic fibroblasts completely deficient in calnexin or calreticulin. In contrast, in MDCK cells, disruption of the calnexin-kAE1 interaction allowed functional dominant (R589H, R901stop), but not misfolded kAE1 mutants (kSAO, G701D), to escape the ER and traffic to the cell surface. Calnexin is therefore not required for the cell surface expression of erythroid AE1, but can be responsible for the intracellular retention of certain kAE1 mutants in cells with the complete complement of molecular chaperones. Components involved in membrane glycoprotein folding and quality control (calnexin, ERp57, Hsc70, Hsp70), were lost at later stages during the differentiation of CD34+ erythroid progenitor cells. This suggests that the loss of molecular chaperones may facilitate the massive production of red cell glycoproteins, allowing erythroid AE1 mutants to escape quality control, traffic to the plasma membrane, and be present in mature red blood cells. These studies demonstrate that the role chaperones play varies, depending on cellular context. By understanding the cellular context and factors involved, therapeutic strategies may be tailored to deal with protein misfolding diseases, and in the case of kAE1, rescue the cell surface trafficking of misfolded, but functional, transport protein using pharmacological modulators.

Dictyostelium discoideum is a social amoebozoan that is used a model system for studying a variety of cell and developmental processes, especially cell motility and chemotaxis. Genome analyses suggest that this model organism possesses a higher percentage of Epidermal Growth Factor (EGF)-like (EGFL) repeats than any other sequenced eukaryote, including humans. EGFL repeats share strong sequence similarity with EGF. In mammals, EGF binds to an EGF receptor (EGFR) to initiate intracellular signalling that regulates a diversity of cellular processes including cell motility and chemotaxis. Some EGFL repeats, like EGF, have also been shown to increase the rate of cell motility by binding to the EGFR and activating EGFR-dependent signalling. Despite their abundance in Dictyostelium, a function for EGFL repeats in this model eukaryote had not previously been studied. This thesis presents a collection of studies that investigated the function of a specific EGFL repeat from the extracellular, cysteine-rich, calmodulin (CaM)-binding protein CyrA. A synthetic peptide (DdEGFL1), equivalent in sequence to the first 18 amino acids of the first EGFL repeat (EGFL1) of CyrA, was shown to increase random cell motility and cAMP-mediated chemotaxis via a novel signalling pathway that did not require either of the two cAMP receptors that are active during early development of Dictyostelium. Several intracellular signalling components were identified and then incorporated into a model detailing the signal transduction regulating EGFL repeat-enhanced cell movement in Dictyostelium. Finally the expression, secretion, and localization of CyrA are presented to couple the findings from studies on DdEGFL1 function with those for the full-length protein. In mammals, a protein that localizes to the extracellular matrix (ECM) and modulates cellular processes by binding to a cell surface receptor and initiating intracellular signalling is termed a ‘matricellular’ protein. The research presented in this thesis suggests that CyrA is the first matricellular protein identified in Dictyostelium.

The flyingfishes (Teleostei: Exocoetidae) are a family of 53 epipelagic marine species distributed throughout tropical and subtropical surface waters. They form a key mid-trophic link between zooplankton and predators, and have evolved special adaptations to survive in the open ocean. However, little is known about their basic evolutionary history and ecology. Here, I apply a multidisciplinary approach to better understand the evolution and ecology of flyingfishes. I propose the first species-level phylogenetic hypothesis for the group, based on nuclear and mtDNA sequences, and show that the most speciose genus (Cheilopogon) is paraphyletic. Gliding evolved progressively from two- to four-wing strategies, and habitat preference is correlated with species range size. I also analyzed patterns of genetic diversity within the most abundant genus, Exocoetus, and found no evidence of cryptic species. Instead, I found that this genus likely consists of three genetically distinct species (in contrast to the five currently recognized) and two indistinct species that diverged very recently. Population genetic analysis of Exocoetus volitans (266 samples from 97 localities) indicates a single, circum-tropical population that is well connected; yet the Isthmus of Panama and an Equatorial barrier limit gene flow. Finally, I investigated species abundance, richness, diversity, and distributions within the eastern tropical Pacific Ocean (11,125 specimens). My results provide critical updates on species distributions and habitat preferences. Predictive modeling indicates that sea surface temperature is important for defining flyingfish habitat. This thesis addresses central issues concerning both evolution and ecology in the epipelagic zone, and highlights the need for better understanding remote marine regions and organisms.

Dictyostelium discoideum is a social amoebozoan that is used a model system for studying a variety of cell and developmental processes, especially cell motility and chemotaxis. Genome analyses suggest that this model organism possesses a higher percentage of Epidermal Growth Factor (EGF)-like (EGFL) repeats than any other sequenced eukaryote, including humans. EGFL repeats share strong sequence similarity with EGF. In mammals, EGF binds to an EGF receptor (EGFR) to initiate intracellular signalling that regulates a diversity of cellular processes including cell motility and chemotaxis. Some EGFL repeats, like EGF, have also been shown to increase the rate of cell motility by binding to the EGFR and activating EGFR-dependent signalling. Despite their abundance in Dictyostelium, a function for EGFL repeats in this model eukaryote had not previously been studied. This thesis presents a collection of studies that investigated the function of a specific EGFL repeat from the extracellular, cysteine-rich, calmodulin (CaM)-binding protein CyrA. A synthetic peptide (DdEGFL1), equivalent in sequence to the first 18 amino acids of the first EGFL repeat (EGFL1) of CyrA, was shown to increase random cell motility and cAMP-mediated chemotaxis via a novel signalling pathway that did not require either of the two cAMP receptors that are active during early development of Dictyostelium. Several intracellular signalling components were identified and then incorporated into a model detailing the signal transduction regulating EGFL repeat-enhanced cell movement in Dictyostelium. Finally the expression, secretion, and localization of CyrA are presented to couple the findings from studies on DdEGFL1 function with those for the full-length protein. In mammals, a protein that localizes to the extracellular matrix (ECM) and modulates cellular processes by binding to a cell surface receptor and initiating intracellular signalling is termed a ‘matricellular’ protein. The research presented in this thesis suggests that CyrA is the first matricellular protein identified in Dictyostelium.