Viral sequence data has great potential for answering questions about the epidemiological dynamics and evolution of viruses. Classical approaches have sought amino acid changes that alter pathogenesis or transmissibility by influencing a virus's ability to enter or replicate within cells. However, this approach rarely recognises the fundamental impact of heterogeneous host contact structures and existing immunological responses on viral transmission. This thesis draws heavily on ecological and immunological concepts to explore the epidemiological dynamics, diversity and evolution of viruses using molecular sequence data. A number of different research approaches and study systems are used in this thesis. I begin by describing a novel polyomavirus in a European badger, and apply phylogenetic techniques to analyze the evolutionary history of the Polyomaviridae. I subsequently describe a large metaviromic study in a population of wild mute swans, for which host demographic data are available. I describe nine new viral species and test whether age and season are associated with differences in abundance and prevalence of different viral taxonomic groups. The study highlights the potential of metaviromics for investigating viral epidemiological dynamics in natural populations. Influenza A viruses of avian origin (AIV) threaten human and animal health. Using phylogeographic methods, I reconstruct the spatial spread of an H5N8 virus at a regional scale, and investigate how bird density and migration shaped this dispersal. Despite the importance of acquisition of humoral immunity to different strains throughout the lifespan of wild birds for epidemiological dynamics, this topic is poorly understood. I assess the accumulation of immune responses to AIV with age in mute swans. I consider how ecological factors, including age-structured immunity, might have affected the epidemiology of an H5N8 outbreak in the population.

Endometriosis is one of the most common gynecological diseases as it is thought to affect up to 15% of the female population. Characterized by the growth and proliferation of endometrial tissue outside of the uterine cavity, it is a complex condition with varying degrees of severity and can affect multiple regions of the body with symptoms ranging from a total lack of symptoms to debilitating pain and infertility. The most accepted theory of how endometriosis initiates is that of retrograde menstruation; however, approximately 90% of women with unobstructed fallopian tubes are thought to have some menstrual debris in the peritoneal cavity. Therefore, this theory does not explain in full why endometriosis occurs in some but not all women who experience retrograde bleeding. Genetic factors are thought to play a major role in the pathogenesis of endometriosis as women with a family history are 5 to 10 times more likely to develop the disease. The goal of this study was to determine if common chromosomal aberrations in the form of additions, deletions, or regions of loss of heterozygosity that may contribute to the establishment or progression of the disease are present in a population of endometriosis patients. DNA was isolated from the peripheral blood of endometriosis patients and endometriosis tissue biopsies, and it was analyzed using oligonucleotide based array comparative genomic hybridization. The results suggest that an addition on chromosome 17p13.3 may play a role in the biological mechanisms involved in endometriosis as it was identified in 75% of the DNA samples obtained from the peripheral blood and 100% of the DNA samples obtained from the tissue biopsies. This chromosomal imbalance is of particular interest as it is located in a region that harbors the tumor suppressor gene, hypermethylated in cancer-1 (HIC-1), whose aberrant expression has been reported in multiple cancers. Endometriosis has long been thought of as a benign disease despite its malignant characteristics, and individuals with endometriosis have been demonstrated to have an increased chance of developing ovarian cancer. This was the first study to examine the DNA from endometriosis patients using oligonucleotide based array comparative genomic hybridization to investigate genetic abnormalities in endometriosis. The findings may provide a novel target for future therapeutic options as well as indicate a link between endometriosis and cancer that has not been previously reported.

The objective of this study was to identify genes that are differentially regulated during diapause when compared with nondiapausing pupae in Sarcophaga crassipalpis. The results of a Suppression Subtractive Hybridization procedure was used to indentify genes that are differentially regulated in both diapause and nondiapausing states while suppressing genes that are common to both states. Randomly picked colonies from both subtractive libraries were isolated and the inserts sequenced. The sequences were analyzed using the bioinformatics tools NCBI, BlastX, Clustal W, etc. Out of 384 clones, 59 genes were found to be upregulated during diapause and 37 genes were found to be upregulated during a nondiapause pupal stage, no genes were found to be expressed commonly in both the diapause and nondiapause constructed libraries.

The evolution of phenotypic plasticity is currently a topic of paramount interest in a diverse field of sub-disciplines. Salience is placed by all fields in describing the interaction of selection and phenotypic plasticity and the consequence of this interaction more broadly on evolution. Lacking in the discussion is substantial empirical description of genotype/phenotype interactions that by definition constitute the plastic response to novel and stressful environments. Here, I present empirical observations that bring the interaction of genotype and phenotype into focus. Drosophila melanogaster populations subjected to selection for tolerance to low food or high alcohol conditions each exhibited an enhancement of adaptive plasticity consistent with predictions associated broadly with the Baldwin Effect. Furthermore, each appears to have followed different courses of regulatory modification to achieve these ends. Broadly implicit in the results is the observation that previous exposure of the population to the conditions of induction may dictate the course of subsequent evolution of the phenotype.

The soil bacterium Rhodococcus is a member of the phylum Actinobacteria and is related to Streptomyces, which is known for its production of many secondary metabolites. Recent genomic investigation of Rhodococcus has uncovered many silent gene clusters that appear to code for nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKS) of unknown function. Previous work, showed that Rhodococcus species strain KCHXC3 produces an inhibitory compound in agar culture extracts that displays prominent activity against several Gram positive and Gram negative species including the pathogens Rhodococcus equi, Shigella dysenteriae and Pseudomonas aeruginosa. Using the engineered Rhodococcus transposon vector, pTNR, the goal of this investigation is to screen random mutants of KCHXC3 for strains that no longer produce the inhibitory molecule. A library of 1825 random insertion mutants was produced via electroporation then screened for production of the inhibitory molecule by a disk diffusion assay against Shigella dysenteriae. From this screening, 7 mutants which no longer produce the compound of interest were identified.

Genetically modified (GM) foods technology is a novel idea for improving food and crop production, but the supposed health risk of GM foods, such as possible negative long-term health effects to humans, animals and the environment, have provoked the European Union to create assessment protocols to monitor and regulate the safety of GM foods and crops. This research investigates the perception and attitudes of unlabeled GM foods of the WKU faculty and staff. A survey was administered via WKU Qualtrics, and chi-square tests were performed to see how the benefits and disadvantages of GM foods may affect the purchasing decisions of the educated consumer, and to see if the WKU faculty and staff wants GM foods to be labeled or not. The research confirms that the benefits and disadvantages of GM foods do affect the purchasing decisions of the educated consumer. The survey revealed that about 60% of the WKU faculty and staff buys GM foods, and 40% do not buy GM foods, and approximately 92% of the WKU faculty and staff wants GM foods to have proper labeling and information. The research provides information about how the educated consumer of Kentucky may feel about unlabeled GM foods. The research also recommends some trade-off benefits of GM foods, including that approximately 35% of the WKU faculty and staff reported that they would buy GM foods if it helps to lower cholesterol and fight diabetes, and 20% say they would buy GM foods if it is cheaper than other foods. Some of the disadvantages of GM foods are that the pesticide chemicals used in the production methods of GM foods are toxic to humans, animals, and the environment. Approximately 54% of the participants say they would not buy GM foods because they are concerned about how it may affect their long-term health, and about 35% reported that they don’t buy GM foods because of improper labeling and information.

Amphibians face an unknown future in a time of rapid environmental change due to global climate perturbations. Since amphibians are perceived to be indicators of ecosystem health, understanding the causes of their declines can improve our perception of threats to other species. Molecular techniques have allowed us to explore how environmental change affects genetic variation and to predict evolutionary adaptive potential of amphibian populations. The identification of populations with the greatest potential to respond to changing environmental variables may be an important conservation strategy to aid in future management efforts. I utilized targeted exon capture sequencing to identify adaptive variation in California tiger salamanders (CTS; Ambystoma californiense), a species threatened by land use change and hybridization with barred tiger salamanders (A. mavortium). I identified 17 and 26 outlier loci for balancing selection in historic and recent samples of CTS respectively. The outlier loci corresponded to genes of various functions, though none of the outliers associated significantly with the change in several tested environmental variables. Despite the lack of environmental correlations detected, it must also be considered that the outlier loci could be involved in epistatic interactions where many genes with small effects influence a single phenotype with fitness benefits. Additional hypotheses to explain the observed changes in allele frequencies and outliers may be the effects of UV-B radiation, pesticide use, or indirect effects of climate change.

CP1 is a well-conserved cathepsin L-like protease essential for proper growth and development in Drosophila melanogaster. Previous research has demonstrated that CP1 has the ability to break down the extracellular matrix. Using the UAS-GAL4 system, immunohistochemistry, and antibody-staining, this research attempts to characterize the role of CP1 and its effects on basement membrane degradation and signaling. These effects include actions at the cellular level and on a known signaling pathway. The genes involved in this pathway are known to be required for proper development of the wing disc into the adult wing. We have demonstrated the collagenase activity of CP1 as well as a possible mechanism via TIMP. We have shown that cp1 is part of the wingless signaling pathway and potentially acts as an upstream regulator on wingless and nubbin. Finally, we have successfully inserted the cDNA of a potential inhibitor of CP1, titled crammer, into the vector pUAST to create transgenic flies. Understanding how CP1 affects Drosophila development through cellular and gene activity is important because cathepsins are highly conserved between flies, humans, and have been implicated in several diseases, including cancer. Discovering the mechanisms by which CP1 functions allows for discoveries to be made in connection with disease processes.

The Annual Killifish, Austrofundulus limnaeus, survives the seasonal drying of their pond habitat in the form of embryos entering diapause midway through development. The diapause trajectory is one of two developmental phenotypes. Alternatively, individuals can "escape" entry into diapause and develop continuously until hatching. The alternative phenotypes of A. limnaeus are a form of developmental plasticity that provides this species with a physiological adaption for surviving stressful environments. The developmental trajectory of an embryo is not distinguishable morphologically upon fertilization and phenotype is believed to be influenced by maternal provisioning within the egg based on observations of offspring phenotype production. However, incubation temperature may override any such maternal pattern suggesting an environmental influence on the regulation of developmental trajectory. We hypothesize that maternally packaged gene products coordinate the cellular events prior to the maternal-to-zygotic transition (MTZ) that determine developmental trajectory in embryos of A. limnaeus. In addition, we propose that environmentally responsive gene expression after the MTZ can sustain or override any such maternal provisioning. Using high-throughput RNA-sequencing, we have generated transcriptomic profiles of protein-coding messenger RNA and noncoding RNA during development in A. limnaeus. Embryos destined for either the diapause or escape phenotypes display unique expression profiles immediately upon fertilization that support hormone synthesis, well before the stage when phenotypes are morphologically distinct. At stages when the trajectories diverge from one another, differential expression of the vitamin D receptor signaling pathway suggests that vitamin D signaling may be a key regulator of developmental phenotype in this species. These data provide a critical link between maternal and environmental influences on the genetic regulation of phenotypic plasticity. These results will not only impact our understanding of the genetic mechanisms that regulate entrance into diapause, but also provide insight into the epigenetic regulation of gene expression and development. Uncovering genetic mechanisms in a system exhibiting alternative developmental trajectories will elucidate the role of maternal packaging in regulating developmental decisions, and in sustaining metabolic depression during diapause.

Globally, habitat fragmentation has had a major impact on the conservation and management of many species and is one of the primary causes of species extinction. Habitat fragmentation is loosely defined as a process in which a continuous habitat is reduced to smaller, disconnected patches as the result of habitat loss, restriction of migration or the construction of barriers to movement. Aquatic systems are particularly vulnerable to habitat fragmentation, and today an estimated 48% of rivers are fragmented worldwide. My dissertation evaluates how habitat fragmentation has influenced the populations of four different species of fish in the Lake Champlain basin. In chapter 1 I summarize the current state of habitat fragmentation research, I broadly describe habitat fragmentation, review how habitat fragmentation pertains to population genetics, and describe the legacy of habitat fragmentation in the Lake Champlain basin. In chapters 2, 3 and 4 I evaluate and discuss the impact of nine lake causeways on the population structure of slimy sculpin (Cottus cognatus), rainbow smelt (Osmerus mordax), and lake whitefish (Coregonus clupeaformis). The genetic effects of causeways are limited. However, causeways appear to have had a significant influence on rainbow smelt demographics, and the genetic structure observed in lake whitefish may be a product of reduced effective population size resulted from commercial harvest in the late 1800s. In chapter 5 I evaluate how the basin-wide population of tessellated darters (Etheostoma olmstedi) is naturally structured throughout Lake Champlain and three different major tributaries and evaluates the effect that different types of habitat fragmentation (dams, causeways, and natural fall lines) have on tessellated darter populations. Tessellated darters appear to be highly structured by river drainage but not by dams, causeways or fall lines. My dissertation highlights how comparative population genetic studies can be used to identify patterns of isolation within large populations. My results stress the value of reporting both the presence and absence of barrier induced population sub-structuring.