Doctor of Philosophy / Division of Biology / Jeroen Roelofs / The ubiquitin-proteasome pathway is the major pathway of selective protein degradation in the cell. Disruption of this pathway affects cellular protein homeostasis and contributes to diseases like cancer, and neurodegeneration. The end point of this pathway is the proteasome, a complex protease formed by 66 polypeptides. Structurally, it can be subdivided into the Core Particle (CP) and the Regulatory Particle (RP). The CP harbors the proteolytic sites, whereas, the RP contains six orthologous AAA-ATPases, the Rpt proteins. These Rpt’s are essential for proteasome function and are at the interface between RP and CP. The work in this thesis focuses on the Rpt subunit Rpt5 from yeast. The C-terminal tail of Rpt5 has been shown to contribute to the binding with the CP. However, our study showed it is also essential for the interaction with Nas2, one of nine proteasome-specific chaperones. Thus, Nas2 might function as a regulator of the Rpt5-CP interaction. Further analyses suggested that Nas2 has an additional function in assembly, and that mutating the tail of Rpt5 results in increased binding of the proteasome-associated protein Ecm29 to the proteasome. We showed that Ecm29 binds Rpt5 directly, thereby inducing a closed conformation of the CP substrate entry channel, and inhibiting proteasomal ATPase activity. Consistent with these activities, several proteasome mutant strains showed Ecm29-dependent accumulation of unstable substrates. Thus, Ecm29 is an inhibitor of the proteasome in vivo and in vitro. Interestingly, besides the Rpt5 mutants, several other proteasome mutants show increased levels of Ecm29, suggesting Ecm29 has a role in quality control. Consistent with this, we observed that Ecm29 associates preferably with specific mutants and nucleotide-depleted proteasomes. Based on our data we propose a model, where early in assembly Nas2 binds to the Rpt5 tail inhibiting the Rpt5-CP interaction directly. Later in assembly Ecm29 performs a quality control function, where it recognizes and remains bound to defective proteasomes. By inhibiting these proteasomes Ecm29 prevents the aberrant degradation of proteins.

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.

Master of Science / Department of Entomology / Jeremy L. Marshall / Males providing females with gifts in order to mate is not a novel occurrence. Indeed, depending on the taxonomic system, males may provide gifts ranging from dead insects, to nutritious ejaculates to even subjecting themselves to cannibalistic feeding. Interestingly, while the burden of the costs of these gifts is primarily carried by the male, net fitness of providing or receiving such gifts must be positive for both sexes, if these gifts are to be maintained in a population. If this is not the case, and sexual conflict has exerted a cost-benefit imbalance between the sexes, then the expectation is that the system will evolve towards the reduction of nuptial gift giving. Here, utilizing the Allonemobius socius complex of crickets where females benefit greatly from cannibalizing male blood as a nuptial gift, we explore the possibility that sexual conflict is acting on gift size. To do this, we assess the gift-size distribution, and their associated fitness functions, for twelve populations that span the phylogeny and geographic distribution of this complex. We find that gift-size distributions are shifted towards small or no gifts in the majority of populations. Moreover, fitness data suggest that males providing a small or even no gift are equally successful to their large gift-giving counterparts. Taken together, the population profiles indicate that at least half of these populations are evolving towards the near or complete loss of these cannibalistic gifts – a predicted, but previously undocumented, outcome of sexual conflict. We also assess the speed at which sexual conflict is acting to alter gift size distributions in populations by comparing gift sizes between different time points in populations and across phylogenetic history. The implications of these results are discussed relative to antagonistic coevolution of behavioral traits and sexual conflict theory in general.

Doctor of Philosophy / Department of Biology / Stephen Keith Chapes / Ehrlichia chaffeensis is an obligate, intracellular bacterium that causes human monocytic ehrlichiosis (HME). The bacteria are vectored by the Lone Star tick (Amblyomma americanum), which is found primarily in the Midwestern and Southeastern United States E. chaffeensis was first reported in 1986 and HME was designated a nationally reportable disease by the United States Centers for Disease Control in 1999. Ehrlichia grows in several mammalian cell lines, but NO consensus model for pathogenesis exists for arthropods or vertebrates. Moreover, the host genes required for intracellular growth of this bacteria are unknown. We first established that the bacteria could infect and replicate both in vitro and in vivo in Drosophila melanogaster S2 cells and adult flies, respectively. We performed microarrays on S2 cells, comparing host gene expression between permissive or non-permissive conditions for E. chaffeensis growth. A total of 210 permissive, exclusive and 83 non-permissive, exclusive genes were up-regulated greater than 1.5-fold above uninfected cells. We screened flies mutant for genes identified in our microarrays for their ability to support Ehrlichia replication. Five mutant stocks were resistant to infection with Ehrlichia (genes CG6479, separation anxiety, CG3044, CG6364, and CG6543). qRT-PCR confirmed that bacterial load was decreased in mutant flies compared to wild-type controls. In particular, gene CG6364 is predicted to have uridine kinase activity. Thus, the in vivo mutation of this gene putatively disrupts the nucleotide salvage pathway, causing a decrease in bacterial replication. To further test the function of gene CG6364 in bacterial replication, we obtained cyclopentenyl cytosine (CPEC) from the National Cancer Institute. CPEC is a cytidine triphosphate (CTP) inhibitor known to deplete CTP pools in various cancers and to exhibit antiviral activity. Consequently, it inhibits de novo nucleotide synthesis, but doesn’t affect the nucleotide salvage pathway. When S2 cells were treated with CPEC and infected with Ehrlichia, an increase in bacterial replication was confirmed by qRT-PCR. Furthermore, addition of cytosine to S2 cells also resulted in increased bacterial replication. Therefore the nucleotide salvage pathway through cytidine appears necessary for bacterial replication. Our approach has successfully identified host genes that contribute to the pathogenicity of E. chaffeensis in Drosophila.

Master of Science / Department of Biology / S. K. Chapes / Ehrlichia chaffeensis is a Gram-negative, obligatorily intracytoplasmic bacterium and the causative agent of a tick-borne disease, human monocytic ehrlichiosis. In vertebrates, E. chaffeensis exhibits tropism for monocytes /macrophages. However, no clear requirements for cell tropism have been defined in ticks. Previously, our group identified two host genes that control E. chaffeensis replication in vivo in Drosophila. We used these two genes, CG6364 and separation anxiety (san) to test the hypothesis that E. chaffeensis replicates in arthropod hemocytes. Using the UAS/GAL4 RNAi system, we generated F1 flies (RNAi flies) and confirmed ubiquitous-or tissue-specific reduction in the transcript levels of the targeted genes. When RNAi flies were screened for Ehrlichia infections, we found that when either CG6364 or san were specifically suppressed in the hemocytes or in the fat body E. chaffeensis failed to replicate or cause infection. Deletion of these genes in the eyes, wings or the salivary glands did not impact fly susceptibility or bacterial replication within these organs. Our data demonstrate that in Drosophila, E. chaffeensis replicates within the hemocytes, the insect homolog of mammalian macrophages, and in the fat body, the liver homolog of mammals. This study provides insights about replication sites of E. chaffeensis in arthropods.

The Hedgehog (Hh) signaling pathway has received attention regarding its important role in embryonic development, however the mechanism by which pathway regulators, such as Suppressor of fused (Sufu), Kinesin family member 7 (Kif7), and primary cilium, mediate Hh signaling transduction is not entirely understood. The work presented here examines the roles of Sufu and Kif7 in regulating Hh signaling in growth plate chondrocytes, as well as how they mediate parathyroid hormone-like hormone (Pthlh) signaling during chondrocyte development. I show here that Sufu and Kif7 are essential regulators of Indian hedgehog (Ihh) signaling. While Sufu negatively regulates Gli transcription factors, Kif7 functions both positively and negatively in chondrocytes. Kif7 plays a role in Sufu protein degradation and the exclusion of Sufu-Gli complexes from the primary cilium. Importantly, halving the dosage of Sufu restores normal Hh pathway activity and chondrocyte development in Kif7-null mice, demonstrating that the positive role of Kif7 is to restrict the inhibitory function of Sufu. Furthermore, Kif7 exerts inhibitory function on Gli transcriptional activity in chondrocytes when Sufu function is absent. Therefore, Kif7 regulates the activity of Gli transcription factors through both Sufu-dependent and Sufu-independent mechanisms. I show that Sufu is crucial for mediating the negative effect of Pthlh on Gli transcriptional activity and chondrocyte hypertrophic differentiation, whereas Kif7 and primary cilium are dispensable in this process. Although primary cilium is required for Hh ligand-mediated activation of Gli transcription, Pthlh negatively controls Gli transcriptional activity in a cilia-independent manner. The results of this work provide insight into how Hh signaling is regulated by Sufu and Kif7 in the context of primary cilium, but also suggest Sufu serves as an important link between Ihh and Pthlh signaling during growth plate chondrocyte development.

Sperm play a pivotal role in determining the reproductive success of individuals whose sperm must compete directly with that of others. I used sperm precedence assays and experimental evolution to examine the role of sperm traits in the reproductive success of hermaphrodites and males in the androdioecious nematode Caenorhabditis elegans. First, sperm size and the rate of reproduction were analyzed, in the context of male-male sperm competition, for evidence of natural heritable variation. Sperm size proved to be a strong indicator of second-male sperm precedence in the genotypes examined. Second, I tested the theoretically predicted effect of larval development time on the number of self-sperm produced by hermaphrodites. I demonstrated that a short larval development period favored the evolution of fewer sperm, inline with theoretical predictions. These results provide important insights into C. elegans reproductive biology and more generally to our understanding of the evolution of reproductive systems.

Carboxyl esterases, particularly arylesterases, were predicted from 16 microbial genomes, and then expressed in E. coli. Of the more than 175 cloned genes, 86 were expressed in soluble form. These were screened for activity using a range of both commercial and natural substrates. Forty-eight proteins were active on pNP-acetate at pH 8 whereas 38 proteins did not exhibit any activity towards any substrates. Among the 48 active proteins, 20 proteins showed arylesterase activity. To date, 8 bacterial esterases and 2 archaeal arylesterases were characterized in terms of pH stability and optima, thermal inactivation, solvent stability, and kinetics. To our knowledge there is only one other published report of arylesterases from archaea. The synthetic capability of arylesterases can transform phenolic acids to value-added chemicals. Accordingly, this project provides an arsenal of industrially significant activities that can extend the antioxidant properties of lignin-derived molecules in a broader range of renewable products.

Food and nutrition are important for energy balance, reproduction and maintenance of health in all species. Drosophila melanogaster feed on yeast and sugar and food availability affects reproduction. In this thesis, I show that mating frequency and fertility are affected by the composition of food in two D. melanogaster wild-type strains, Canton-S and Oregon-R. Canton-S flies mate multiple times in the presence of yeast and sugar, while Oregon-R only remate in the presence of yeast. However, Oregon-R flies have higher fertility counts on all food types compared to Canton-S. These effects of food do not appear to depend on smell or taste, because both chemosensory mutants and artificial sweeteners tested fail to block the effects of food on reproduction. Moreover, Canton-S, but not Oregon-R flies show an interaction between food and group size. I conclude that genetic differences, social context and nutrition interact to regulate reproduction in flies.

Apolipoprotein B (ApoB) synthesis is partially regulated at the translational level; however, the molecular mechanisms that govern translational control of apoB mRNA remains largely unknown. We imaged intracellular apoB mRNA traffic and determined whether insulin silences apoB mRNA translation by trafficking into translationally-silenced cytoplasmic RNA granules called Processing Bodies (PBS). ApoB mRNA was visualized by using a strong interaction between the bacteriophage MS2 protein and a specific phage RNA sequence that binds MS2 protein. We observed a statistically significant increase in the localization of apoB mRNA into PBs, 4h, 8h, and 16h after insulin treatment. Conversely, acute insulin treatment (1h) did not show any significant effect. Insulin was also found to reduce polysomal association of apoB mRNA 4h and 16h post treatment in HepG2 cells. Overall, our data suggest that chronic insulin treatment silences apoB translation in HepG2 cells by localizing apoB mRNA into PBs and reducing translationally-competent mRNA pools.