Prior research has suggested that life style factors, such as diet and physical activity, influence prostate cancer (PCa) progression. Metformin intake has been shown to be associated with decreased cancer risk in type II diabetic patients. We hypothesize that a low carbohydrate diet, prolonged aerobic exercise and metformin treatment can all independently slow prostate tumor development and a combination regimen will have an additive benefit. We used LNCaP xenografts to test this hypothesis. Results revealed that a diet low in carbohydrate reduced food consumption and a combination with exercise significantly reduced animal body weights. Ten weeks of metformin did not significantly alter tumor growth rate compared to control animals. Ten weeks of exercise significantly inhibited tumor growth. Out results suggest that dietary carbohydrate alteration or the administration of metformin alone cannot significantly influence prostate tumor progression. A suitable sustained exercise regimen may offer a more protective effect against PCa progression.

For many species, vision contributes to a number of fitness-related tasks, including mating and the detection of prey and predators. Selection on the visual system should therefore be strong, especially when ecological or genomic changes open new avenues for evolutionary changes. Visual system proteins are thus attractive systems for molecular evolutionary analyses. This thesis presents a collection of evolutionary studies on two gene families, opsins and crystallins. Opsin proteins determine the wavelengths of light detected by the retina, while crystallin proteins contribute to lens transparency and refractory power. My studies focus on teleost fishes, because teleost visual ecology is exceptionally diverse and because gene duplication is common in this group. In Chapter One, I outline the relevance of protein variation to organismal evolution and describe the analytical methods employed throughout this thesis. Chapter Two considers the long-wavelength sensitive (LWS) opsins of the guppy (Poecilia reticulata). The guppy is shown to possess multiple LWS opsins that have accumulated differences at functionally important amino acid sites since duplicating. Chapter Three focuses on the guppy’s main predator, the pike cichlid Crenicichla frenata, which is shown to have a greater capacity for short-wavelength vision than previously believed. However, this cichlid possesses three fewer opsins than closely-related African cichlids, a difference partly due to duplication of a green-sensitive (RH2) opsin in African cichlids. In Chapter Four, this RH2 duplication event is studied in greater depth; variation in selective constraint is documented following gene duplication and between species from different lakes. Some of the analytical methods employed in Chapter Four were newly developed, as detailed in Chapter Five, where a test for functional divergence among clades is evaluated and then improved upon through the presentation of a new null model that better accommodates among-site variation in selection. In Chapter Six, phylogenetic relationships within the βγ lens crystallin superfamily are clarified, and the functionally distinct γN family is shown to have evolved conservatively compared to other crystallin families. The thesis concludes with suggestions for future directions for evolutionary research on opsins and crystallins, and summarizes recent work that has built on these studies.

Autotransporters represent a large superfamily of known and putative virulence factors produced by Gram-negative bacteria. They consist of an N-terminal “passenger domain” responsible for the specific effector functions of the molecule and a C-terminal “β domain” responsible for translocation of the passenger across the bacterial outer membrane. The serine protease autotransporters of Enterobacteriaceae (SPATEs) represent those autotransporters produced by Enterobacteriaceae where, as the name suggests, the passenger domain functions as a serine protease. Members of this family of autotransporters include among others the extracellular serine protease EspP produced by enterohemorrhagic Escherichia coli (EHEC) O157:H7. EHEC, especially those of serotype O157:H7, have been implicated as causative agents of hemorrhagic colitis and hemolytic-uremic syndrome, both of which include disruption of the normal processes in human blood responsible for maintaining good health. EspP has previously been shown to cleave human coagulation factors V and VIII and has been hypothesized to possibly contribute to the mucosal hemorrhage in patients infected with EHEC. This thesis aims to better understand the functional significance of EspP in EHEC pathogenesis by analyzing the crystallographic structure of the mature passenger domain of EspP and by investigating, in vitro, its effects on the coagulation and fibrinolytic processes in human blood. Like the previously determined autotransporter passenger domains, the EspP passenger domain is found to contain an extended right-handed parallel β-helix preceded by an N-terminal globular domain housing the catalytic function of the protease. Of note, however, is the absence of a second globular domain protruding from this β-helix. Furthermore, EspP is found to alter hemostasis in vitro by drastically decreasing the activities of human blood coagulation factors V, VII, VIII and XII, by enhancing platelet-fibrin clot formation, and by accelerating fibrinolysis. These results provide compelling evidence for a pathogenic role played by EspP during EHEC infection.

Autotransporters represent a large superfamily of known and putative virulence factors produced by Gram-negative bacteria. They consist of an N-terminal “passenger domain” responsible for the specific effector functions of the molecule and a C-terminal “β domain” responsible for translocation of the passenger across the bacterial outer membrane. The serine protease autotransporters of Enterobacteriaceae (SPATEs) represent those autotransporters produced by Enterobacteriaceae where, as the name suggests, the passenger domain functions as a serine protease. Members of this family of autotransporters include among others the extracellular serine protease EspP produced by enterohemorrhagic Escherichia coli (EHEC) O157:H7. EHEC, especially those of serotype O157:H7, have been implicated as causative agents of hemorrhagic colitis and hemolytic-uremic syndrome, both of which include disruption of the normal processes in human blood responsible for maintaining good health. EspP has previously been shown to cleave human coagulation factors V and VIII and has been hypothesized to possibly contribute to the mucosal hemorrhage in patients infected with EHEC. This thesis aims to better understand the functional significance of EspP in EHEC pathogenesis by analyzing the crystallographic structure of the mature passenger domain of EspP and by investigating, in vitro, its effects on the coagulation and fibrinolytic processes in human blood. Like the previously determined autotransporter passenger domains, the EspP passenger domain is found to contain an extended right-handed parallel β-helix preceded by an N-terminal globular domain housing the catalytic function of the protease. Of note, however, is the absence of a second globular domain protruding from this β-helix. Furthermore, EspP is found to alter hemostasis in vitro by drastically decreasing the activities of human blood coagulation factors V, VII, VIII and XII, by enhancing platelet-fibrin clot formation, and by accelerating fibrinolysis. These results provide compelling evidence for a pathogenic role played by EspP during EHEC infection.

Given a shared toolbox of pathogenicity-related genes among a set of species, why is one species a biotroph and specialist while another is a necrotroph and generalist? Is it the result of selection on primary sequence, or on proteins, or alternatively, differences in the timing and magnitude of gene expression? The Sclerotiniaceae (Ascomycota, Leotiomycetes, Helotiales) is a relatively recently evolved family of fungi whose members include host generalists and host specialists, and the spectrum of trophic types. Based on a phylogeny inferred from three, presumably evolutionarily conserved housekeeping genes, the common ancestor of the Sclerotiniaceae was necrotrophic, with at least two shifts from necrotrophy to biotrophy. Phylogenies inferred from eight pathogenicity-related genes, involved in cell wall degradation and the oxalic acid pathway, were incongruent with the presumably neutral phylogeny. Site-specific likelihood analyses, which estimate the rate of nonsynonymous to synonymous substitutions (dN/dS), showed evidence for purifying selection acting on all pathogenicity-related genes, and positive selection on sites within five of eight genes. Rate-specific likelihood analyses showed no differences in dN/dS rates between necrotrophs and biotrophs, and between host generalists and host specialists, indicating that selection acting on the genes does not drive divergence toward changes in trophic type or host association. In vitro screens for oxalic acid production demonstrated that all necrotrophic generalists produce oxalic acid by 72 hours, while production was either absent or delayed among biotrophs and host specialists. This pattern was also observed during the course of Arabidopsis thaliana infection, in which large spikes of expression were seen in the oxalic acid pathway-related gene, oah, within eight hours of inoculation among necrotrophic generalists only. Results suggest that necrotrophic generalists can be distinguished from biotrophs and host specialists in the Sclerotiniaceae by the ability to produce abundant amounts of oxalic acid early in infection and to cause large proliferating lesions on A. thaliana.

Identifying the genetic basis for phenotypic variation is a central question in evolutionary biology and can be studied in detail using model organisms. Fecundity variation in different isolates of C. briggsae has been observed previously, but the genetic causes of this variation are unclear. Crosses between C. briggsae advanced-intercross recombinant inbred lines (AI-RILs) and parental strains yield near isogenic line (NIL) strains, which I created to provide a powerful genetic resource to fine-map the basis for fecundity and other trait differences. Phenotypic analysis of the NILs shows the complexities of possible epistatic interactions on phenotypic expression. These NIL strains contribute a valuable genetic resource toward the long-term goal of identifying the genes responsible for differences in fecundity in this species. The elucidation of the basis for this trait variation will also contribute further into the mechanisms for how genotype and phenotype and environment all interact.

Given a shared toolbox of pathogenicity-related genes among a set of species, why is one species a biotroph and specialist while another is a necrotroph and generalist? Is it the result of selection on primary sequence, or on proteins, or alternatively, differences in the timing and magnitude of gene expression? The Sclerotiniaceae (Ascomycota, Leotiomycetes, Helotiales) is a relatively recently evolved family of fungi whose members include host generalists and host specialists, and the spectrum of trophic types. Based on a phylogeny inferred from three, presumably evolutionarily conserved housekeeping genes, the common ancestor of the Sclerotiniaceae was necrotrophic, with at least two shifts from necrotrophy to biotrophy. Phylogenies inferred from eight pathogenicity-related genes, involved in cell wall degradation and the oxalic acid pathway, were incongruent with the presumably neutral phylogeny. Site-specific likelihood analyses, which estimate the rate of nonsynonymous to synonymous substitutions (dN/dS), showed evidence for purifying selection acting on all pathogenicity-related genes, and positive selection on sites within five of eight genes. Rate-specific likelihood analyses showed no differences in dN/dS rates between necrotrophs and biotrophs, and between host generalists and host specialists, indicating that selection acting on the genes does not drive divergence toward changes in trophic type or host association. In vitro screens for oxalic acid production demonstrated that all necrotrophic generalists produce oxalic acid by 72 hours, while production was either absent or delayed among biotrophs and host specialists. This pattern was also observed during the course of Arabidopsis thaliana infection, in which large spikes of expression were seen in the oxalic acid pathway-related gene, oah, within eight hours of inoculation among necrotrophic generalists only. Results suggest that necrotrophic generalists can be distinguished from biotrophs and host specialists in the Sclerotiniaceae by the ability to produce abundant amounts of oxalic acid early in infection and to cause large proliferating lesions on A. thaliana.

Identifying the genetic basis for phenotypic variation is a central question in evolutionary biology and can be studied in detail using model organisms. Fecundity variation in different isolates of C. briggsae has been observed previously, but the genetic causes of this variation are unclear. Crosses between C. briggsae advanced-intercross recombinant inbred lines (AI-RILs) and parental strains yield near isogenic line (NIL) strains, which I created to provide a powerful genetic resource to fine-map the basis for fecundity and other trait differences. Phenotypic analysis of the NILs shows the complexities of possible epistatic interactions on phenotypic expression. These NIL strains contribute a valuable genetic resource toward the long-term goal of identifying the genes responsible for differences in fecundity in this species. The elucidation of the basis for this trait variation will also contribute further into the mechanisms for how genotype and phenotype and environment all interact.

Master of Science / Department of Biomedical Sciences / Sanjeev K. Narayanan / Fusobacterium necrophorum is an anaerobic Gram-negative non spore forming rod shaped bacteria that is a normal inhabitant of the alimentary tract of humans and animals. Two subspecies of F. necrophorum have been recognized- subspecies necrophorum and subspecies funduliforme. Subspecies necrophorum is an opportunistic pathogen in animals causing diseases such as bovine hepatic abscesses and sheep foot rot while as subspecies funduliforme is linked with human oral and hepatic infections such as sore throats, Lemierre’s syndrome and hepatic abscesses. The pathogenic mechanisms of F. necrophorum are complex and are not well understood or defined. Several virulence factors such as leukotoxin, haemolysin, haemagglutinin and adhesin have been described. One of the most important factors in F. necrophorum bacterial pathogenesis is the adhesion of the bacteria to the host cell. The adhesion of the bacteria to the host cell helps it colonize the host tissue and this is followed by intracellular multiplication with dissemination to other tissues, which could ultimately lead to septicemia and death. Bacteria use adhesins which are proteins found in the outer membrane which help them bind with host receptors and this helps with the adhesion of the bacteria to the host cell. Not much is known about F. necrophorum adhesins. Here, we describe and characterize a novel adhesin.

Master of Science / Department of Biology / A. Lorena Passarelli / Cydia pomonella granulovirus (CpGV) is a member of the Baculoviridae family of viruses. The CpGV open reading frame 46 (CpGV-ORF46) predicts a 545 amino acid protein that shares homology with matrix metalloproteases (MMPs), a family of zinc-dependent endopeptidases that degrade extracellular matrix proteins. In silico analyses revealed the presence of putative mmp genes in all species from the Betabaculovirus genus, while no mmps were identified in members of the Alphabaculovirus, Gammabaculovirus or Deltabaculovirus genera. Unlike most cellular MMPs, baculovirus MMPs do not have a propeptide domain, a domain involved in regulating MMP activation, or a hemopexin-like domain, which is necessary for substrate binding and specificity in many MMPs. However, Betabaculovirus MMPs do contain a predicted conserved zinc-binding motif (HEXGHXXGXXHS/T) within their catalytic domain. The function of CpGV-MMP and its effects on baculovirus replication in cultured cells and insect larvae were investigated. CpGV-MMP was expressed in and purified from Escherichia coli, and activity was measured using a generic MMP substrate in vitro. CpGV-MMP had in vitro activity and its activity was specifically inhibited by MMP inhibitors. To study the effects of CpGV-MMP on virus replication and dissemination, CpGV-MMP was expressed from Autographa californica nucleopolyhedrovirus (AcMNPV) under the control of a strong and constitutive promoter, the Drosophila heat shock 70 protein promoter. Expression of CpGV-MMP did not affect virus replication in cultured cells. The effects of expressing CpGV-MMP from AcMNPV during larval infection were evaluated in the presence or absence of the AcMNPV chitinase and cathepsin genes. Insect bioassays showed that the absence of cathepsin resulted in a significant delay in larval time of death; however, this delay was compensated by expression of CpGV-MMP. In addition, larval time of death was accelerated when cathepsin, chitinase, and CpGV-MMP were all expressed. Finally, we determined the effects of CpGV-MMP on larvae melanization and liquefaction. CpGV-MMP was able to promote larvae melanization in the absence of cathepsin. CpGV-MMP, in the absence of cathepsin, was not able to promote larvae liquefaction. When chitinase was engineered to be secreted from cells, CpGV-MMP rescued liquefaction in the absence of cathepsin. In conclusion, CpGV-MMP is a functional MMP which can enhance larvae mortality with the presence of cathepsin. In addition, CpGV-MMP can promote larvae melanization; however, it can only promote liquefaction when chitinase is engineered to be secreted from cells.