Climate change has caused an increase in extreme rain events and flooding in certain regions across the globe. During rain events, water flows over impervious surfaces structures such as roads and sidewalks, picking up contaminants such as metals, fertilizers and other nutrients, and various organics that which may impact organisms in such as streams, river, and lakes. Previous work has found significant differences in survival of organisms that were exposed to contaminated stormwater runoff. This study investigated stormwater chemistry parameters at collection sites on the East Tennessee State University campus. Sites were selected based on the extent of human interaction and traffic in the areas. Additionally, acute toxicity of stormwater samples was investigated through 48-h bioassays with the cladoceran, Daphnia magna. In September and November 2022, water chemistry and toxicity analyses were conducted across multiple rain events and over a six-hour time course of an individual rain event. For each of the events and the time course, chlorophyll levels, specific conductivity, pH, temperature, and dissolved oxygen were measured. No statistical difference between the water chemistry parameters between sampling sites or between rain events were observed. Additionally, no significant differences in 48-h survival of D. magna were detected between sampling locations or during the single event time course study. These data suggest that there were no pollutant surges at the collection sites and that D. manga survival was not affected by the contaminants.

Weddell seals (Leptonychotes weddellii) are among the deepest diving pinnipeds (i.e., seals, sea lions, and walrus) and one of the best studied marine mammals in the world; as such, these seals are considered a model species for the study of diving physiology and behavior. Adult Weddell seal dive physiology is rather comprehensively understood, yet previous research has excluded an examination of pups’ initial independent diving attempts, beginning instead with the diving capabilities of near-weaning individuals at four to five weeks of age. This is beyond the point many pups have attempted their first independent dives; pups begin to enter the water at 8-10 days after birth, with some observed in the water earlier. The aim of this study was to investigate the development of diving capabilities and fine-scale behaviors of Weddell seal pups beginning at one week of age throughout their dependence period. Pups were sampled longitudinally at 1, 3, 5, and 7 weeks of age. Total body oxygen stores (TBO2, mL O2) were calculated as the sum of blood, muscle, and lung oxygen stores for each seal at all time points. Blood samples were collected under sedation, muscle oxygen parameters were interpolated, and lung oxygen content was extrapolated from adult values. Flipper-mounted time-depth recorders were used to collect concurrent dive behavior data. In chapter 1, I hypothesized that diving capability (TBO2) would be more strongly correlated with dive experience than calendar age; to examine this, age, mass, and diving parameters were correlated with oxygen stores. I instead found mass and age were most significantly correlated with individual tissue oxygen stores and TBO2. I predicted diving experience would be an important driver of oxygen storage development due to hypoxia exposure, but pups spent the majority of their time in the water at the surface and had little to no exposure to hypoxia during dependence. Increases in mass may enable early advances in diving ability, and with increased diving capabilities, pups will be able to become successful independent foragers. Later exposure to hypoxia may be the key to the subsequent increases in TBO2 observed in yearlings and juveniles. In chapter 2, I used TDR data to predict when pups would be in the water based on developmental, temporal, and environmental factors including age, weaning status, time of day, and weather parameters. Pups spent the most time in the water and made their deepest, longest, and most frequent dives during the late night and early morning hours. These data indicate pups are following the diving patterns of their mothers, which follow the diurnal vertical migration of their prey. The data also suggest Weddell seal pups most likely prioritize learning to swim and navigate as opposed to practicing foraging while still dependent. It is critical for pups to develop their swimming, navigational, and diving abilities while they are still with their moms to ensure their survival. This study is the first to describe the complete trajectory of the development of diving physiology and behavior in Weddell seal pups throughout dependence. It is important to understand how the internal diving physiology of Weddell seal pups develops because this directly determines their diving capabilities and their ability to forage successfully, which in turn directly correlates with their survival. Pup survival is an indicator of population growth rates, so the development of diving physiology in pups can lend insights into larger population-level trends.

Amphibians suffer from large-scale population declines globally, and emerging infectious diseases contribute heavily to these declines. Amphibian Perkinsea (hereafter Pr) is a worldwide anuran pathogen associated with mortality events, yet little is known about its overall impact on amphibian populations and species. Thus, we sought to determine the epidemiological patterns of Pr across individual hosts, populations, communities, and the overall landscape. To accomplish this, we conducted two field-based and one experimentally-based study to identify specific factors that contribute to pathogen prevalence, infection intensity, and overall disease outcomes for Pr in individuals and populations. We collected 1973 total anurans across the two field chapters of this dissertation with 32% of individuals infected with Pr in more "natural" habitats while 8.8% of individuals were infected with Pr in the urban Xenopus tropicalis invasion range. We identified that co-variates including co-infection status, host species, host life stage, sampling month, and sampling site explained much of the variability of infection status within individuals and sites. In the experimental infection studies, we identified that host susceptibility to Pr varied significantly by species, but sub-lethal impacts may still occur in tolerant species. This study is the first to establish epidemiological patterns of Pr across space, time, and host species and fills knowledge gaps in our understanding of how invasive species alter pathogen dynamics. Ultimately, this work highlights the need for ongoing monitoring, experimental studies, and mitigation efforts to address the challenges pathogens pose to amphibian biodiversity.

Oligonucleotides enable many biotechnological applications; however they are easily degraded by nucleases. Many nucleotides modified at the 2’ position are degraded at decreased rates which improves oligonucleotide utility. Most applications of oligonucleotides rely on enzymatic synthesis. Unfortunately, native DNA polymerases do not recognize most useful modified nucleotide substrates. Directed evolution has been used to identify mutants of Taq DNA polymerase I (Taq) that recognize substrates with 2’ modifications. While mutant enzymes capable of modified nucleotide addition have been identified, to date, all of these enzymes are limited by their inability to synthesize full length modified DNA. Despite considerable efforts to evolve new activity there has been little work done to quantitatively characterize these evolved enzymes. This thesis work presents efforts to synthesize modified primers that will help comparatively and quantitatively characterize three enzymes previously evolved to recognize 2’ modified substrates. Using the methods developed in this thesis project, our lab will be able to characterize the relationship between the number of modified nucleotides in the primer terminus and the rate of modified and unmodified nucleotide addition. Future work will identify key enzymatic steps that limit extension in these enzymes with implications for the future design of Taq mutants capable of synthesizing long 2’ modified oligonucleotides.

Approximately 1 in 11 births in the United States are preterm (gestation). Within the United States, there are huge racial disparities for risk of preterm birth, an issue understudied and rarely addressed by research in the field. There is a wealth of biological knowledge surrounding pregnancy and labor, but causes for preterm birth are poorly understood. A genetic factor that has been shown to play a key role in many biological processes crucial to a healthy pregnancy and timely labor is microRNA (miRNA). MiRNA have an active role in the regulation of various tissues, especially developing tissues like those found in the placenta and uterus. Additionally, oxidative stress has been shown essential to placental development and the initiation of labor. Here, a study is proposed that aims to address the effect of oxidative stress on myometrial miRNA expression, specifically the miR-200 family and miR-199/214 cluster. This work also underscores the importance of addressing racial disparities with regards to preterm labor during research, while bringing up ethical considerations for conducting such research. The thesis will conclude with an outline of the many considerations vital for discussing the research and analysis of preterm birth disparities using a feminist, antiracist, queer self-reflexive analysis.

Fluvastatin, the HMG-CoA reductase inhibitor known for its role in the treatment of hypercholesterolemia and cardiovascular disease, has more recently been shown to play a role in the immune response. Given the critical role that mast cells play in allergy and inflammatory diseases such as asthma, which effects one third of America’s population, we assessed the effect of fluvastatin on mast cell and basophils function. We demonstrate that fluvastatin downregulated IgE-mediated cytokine production. Additionally, in vivo studies showed that fluvastatin suppressed IgE-mediated anaphylaxis. Interestingly, the effects of fluvastatin showed dependence on genetic background, as C57BL/6 mast cells were sensitive, while 129/Sv mast cells were resistant to fluvastatin. Characterizing the role of fluvastatin on mast cells may prove to be therapeutically important.

The field of epigenetics is gaining popularity and speed, due in part to its capability to answer lingering questions about the root cause of certain diseases. Epigenetics plays a crucial role in regulation of the cell and cell survival, particularly by cytosine methylation. It remains controversial if DNMT’s which facilitate methylation are present in mammalian mitochondria and what the functional significance they may have on modification of mitochondrial DNA. CRISPR-Cas9 technology enabled genome editing to remove the MTS (mitochondrial targeting sequence) from DNMT1 of HCT116 cells, purposefully minimizing effects on nuclear cytosine methylation, while exclusively impacting mitochondrial modification. Removal of the DNMT1 MTS did not completely prevent the localization of this enzyme to the mitochondria according to immunoblot analysis. As well, deletion of the MTS in DNMT1 revealed only a small decline in transcription; not until removal of DNMT3B did we see a two-fold decrease in transcription from mitochondrial protein coding genes. No significant decline in transcription occurred when a DNMT3B knockout also lost the MTS of DNMT1; this study is evidencing that DNMT3B is possibly the more significant methyltransferase in the mitochondria. Our aim from this study and future research is to clearly characterize which enzymes in the mitochondria are controlling cytosine modifications and to understand the mechanistic complexities that accompany cause and consequence of epigenetic modifications.

In the yeast Saccharomyces cerevisiae, the IME4 methyltransferase, interacts genetically with methyl binding protein, Pho92, to affect the expression of PHO regulon target genes. Cells mutant in IME4 or PHO92 show increases in the RNA abundance of PHO regulon target genes. The increase in the RNA abundance of the PHO regulon target genes is not additive in the cells double mutant in IME4 and PHO92. Hence, Ime4 and Pho92 interact in a single pathway in PHO regulon. Surprisingly, cells overexpressing IME4 and MUM2 shows increase in some PHO regulon target genes, indicating that IME4 affects the PHO regulon target genes through multiple mechanisms in different conditions. A promoter swap experiment revealed that one of the PHO regulon mRNAs that codes for phosphatase, PHO5, is a direct target of Ime4. Further experiments are required to examine whether the same is true for all PHO regulon mRNAs.

Snakes represent an impressive evolutionary radiation of over 3,500 widely-distributed species, categorized into 515 genera, encompassing a diverse range of morphologies and ecologies. This diversity is likely attributable to their distinctive morphology, which has allowed them to populate a wide range of habitat types within most major ecosystems. In my first chapter, I provide the largest-yet estimate of the snake tree of life using maximum likelihood on a supermatrix of 1745 taxa (1652 snake species + 7 outgroup taxa) and 9,523 base pairs from 10 loci (5 nuclear, 5 mitochondrial), including previously unsequenced genera (2) and species (61). I then use this phylogeny to test hypotheses regarding heterogeneity in diversification rates and how this shaped overall patterns of snake diversity in Chapter 2. I also used the species-level phylogeny to test the evolution of habitat use in snakes, morphological variation, and whether distantly-related species exhibit morphological convergence in Chapter 3. Finally, in Chapter 4 I investigate how prehensile tails effect striking performance in arboreal snakes.

Thermotolerance limit in aquatic organism is set by the ability to sustain aerobic scope to sudden temperature shifts. This study tested the genetic and plastic differences in thermotolerance of Daphnia that can be explained by the differences in the ability to retain mitochondrial integrity at high temperatures. Five genotypes with different biogeographic origins were acclimated to 18ᵒC and 25ᵒC. We developed a rhodamine 123 in-vivo assay to measure mitochondrial membrane potential and observed higher fluorescent in heat damaged tissues as the disruption of the mitochondrial membrane potential. Significant effects on temperature tolerance were observed with CCCP and DNP but not with NaN3. Effects of toxins were significant in temperature sensitive genotype and high concentration of lactate was observed in 18ᵒC acclimated genotype only. We conclude that genetic and physiological differences are intricately linked to the ability of sustaining aerobic respiration at high temperatures which sets limit to the thermotolerance.