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Title: Exploring Cis Elements and Trans-Acting Factors Involved in the Human Inactive X Chromosome Organization and Compaction.
Creator: Sun, Zhuo, Chadwick, Brian P., Gunjan, Akash, Dennis, Jonathan Hancock, Yu, Hong-Guo, Deng, Wu-Min, Florida State University, College of Arts and Sciences, Department of...
Show moreSun, Zhuo, Chadwick, Brian P., Gunjan, Akash, Dennis, Jonathan Hancock, Yu, Hong-Guo, Deng, Wu-Min, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: In this dissertation, I have explored cis and trans factors involved in the organization and compaction of the human inactive X chromosome (Xi). I describe here three trans factors (SMCHD1, LRIF1 and SETDB1) that were found to have important roles in Xi chromatin compaction, as demonstrated by a doubling of the Xi volume in their absence. I also report a novel enhancer element on the Xi that is reactivated in SETDB1 mutants and is in part responsible for the Xi decompaction phenotype, and...
Show moreIn this dissertation, I have explored cis and trans factors involved in the organization and compaction of the human inactive X chromosome (Xi). I describe here three trans factors (SMCHD1, LRIF1 and SETDB1) that were found to have important roles in Xi chromatin compaction, as demonstrated by a doubling of the Xi volume in their absence. I also report a novel enhancer element on the Xi that is reactivated in SETDB1 mutants and is in part responsible for the Xi decompaction phenotype, and displays complex cis and trans communication between the active X chromosome and Xi. We have generated SMCHD1 and LRIF1 mutants using both TALENs and CRISPR-Cas9 genome engineering platforms. Loss of either protein results in Xi decompaction and reactivation of some Xi genes. Using the X-linked choroideremia locus (CHM) as an example of a reactivated gene, we show that reactivation is coupled with a reduction in the repressive heterochromatin markers histone H3 trimethylated at lysine 9 (H3K9me3) and 27 (H3K27me3) and an increase in the euchromatin marker histone H3 trimethylated at lysine 4 (H3K4me3) in the promoter region. Alongside these chromatin changes, we observed movement of the CHM locus away from the H3K9me3 territory towards the H3K27me3 territory. Previous data from our lab showed that loss of the macrosatellite repeats DXZ4 from the Xi resulted in large-scale changes in cis to the three-dimensional organization of the Xi, including fragmentation of the chromosome territory as observed by light microscopy. Intriguingly, deletion of SMCHD1 in DXZ4 Xi mutants results in a more pronounced Xi decompaction phenotype than that of SMCHD1 loss alone, suggesting that both perform complementary roles to compact the Xi. In the effort to determine which histone lysine methyl-transferase is responsible for H3K9me3 at the Xi, we isolated SETDB1 TALEN mutant clones and discovered that like SMCHD1 and LRIF1, loss of SETDB1 leads to decompaction of the Xi territory. Furthermore, in the SETDB1 mutants, we observed drastic chromatin changes within the 3’ third of the 1.4 megabase Interleukin 1 Receptor Accessory Protein-Like 1 (IL1RAPL1) gene. In this genomic interval, there is localized loss of repressive chromatin defined by H3K9me3 coupled with a gain of the active makers defined by histone H3 di-methylated at lysine-4 (H3K4me2) and acetylated at lysine 27 (H3K27Ac). The DNA underlying the major peak of H3K27Ac possesses very powerful enhancer activity in vitro and is located immediately adjacent to the long terminal repeat of an endogenous retrovirus element ERVL-MaLR that is reactivated from the Xi in the SETDB1 mutants. Reactivation of the ERVL-MaLR results in a significant increase in the transcription of novel bi-directional transcripts originating from the 3’ region, coupled with a significant reduction in full-length IL1RAPL1 transcripts originating from the endogenous 5’ promoter. To determine if this enhancer element contributes to decompaction of the Xi, clones were isolated in which it had been deleted from either the Xa or Xi using CRISPR-Cas9 system. We found that deletion of the enhancer from the Xi increased detection of full-length IL1RAPL1 transcript in trans, but did not result in Xi decompaction. In contrast, deletion of the enhancer from the Xa decompacted the Xi territory and resulted in a total loss of transcript originating from the 5’ promoter on the Xa. These data revealed complex cis and trans effects that affects IL1RAPL1 gene expression and Xi chromatin compaction. Importantly, this same interval is centrally located in a known fragile site on the X chromosome that is frequently lost in patients with intellectual disability. Portions of this dissertation have been published or are being prepared for publication. Parts of Chapter 1 has been published as a book chapter in Epigenetics: Current Research and Emerging Trends, Caister Academic Press (Chadwick, 2015). The data presented in Chapter 3 has been published in Epigenetics & Chromatin (Sun and Chadwick, 2018). Data presented in Chapter 2 are in the process of being prepared as manuscripts for publication.
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Date Issued: 2019
Identifier: 2019_Spring_Sun_fsu_0071E_15036
Format: Thesis

Title: Spawning Patterns, Trophic Ecology, and Toxicology: Conservation Related Research of an Iconic Reef Fish, the Atlantic Goliath Grouper.
Creator: Malinowski, Christopher Robert, Coleman, Felicia C., Huettel, Markus, Burgess, Scott C., Grubbs, R. Dean, Miller, Thomas E., Travis, Joseph, Florida State University, College of...
Show moreMalinowski, Christopher Robert, Coleman, Felicia C., Huettel, Markus, Burgess, Scott C., Grubbs, R. Dean, Miller, Thomas E., Travis, Joseph, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: The Atlantic Goliath Grouper, a large, long-lived reef fish in the southeastern United States, was fished to near extinction decades ago. It is now showing signs of recovery in Florida waters due to protection from fishing in state and federal waters that started in 1990. While providing some optimism for their future, recent discovery of high mercury levels in this species is cause for concern. Indeed, I contend that such levels could not only impede their full recovery but present a human...
Show moreThe Atlantic Goliath Grouper, a large, long-lived reef fish in the southeastern United States, was fished to near extinction decades ago. It is now showing signs of recovery in Florida waters due to protection from fishing in state and federal waters that started in 1990. While providing some optimism for their future, recent discovery of high mercury levels in this species is cause for concern. Indeed, I contend that such levels could not only impede their full recovery but present a human health problem if the fishery were re-opened. For my dissertation, I investigated their spawning patterns and trophic ecology, the latter of which included investigations of the organisms in their diet that lead to mercury accumulation in their tissues. Mercury presents a problem because of its neurotoxic effects and its ability to bioaccumulate in some forms. My research demonstrates that mercury levels in Goliath Grouper are not only much higher than the Environmental Protection Agency’s suggested safe levels for human consumption, but that they are likely impacting the overall health and reproductive potential of this iconic species.
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Date Issued: 2019
Identifier: 2019_Spring_MALINOWSKI_fsu_0071E_15181
Format: Thesis

Title: Proximate Mechanisms Influencing Individual Variation in Cooperative Behavior.
Creator: Cusick, Jessica Ashley, DuVal, Emily H., Hull, Elaine M., Travis, Joseph, Miller, Thomas E., Burgess, Scott C., Florida State University, College of Arts and Sciences,...
Show moreCusick, Jessica Ashley, DuVal, Emily H., Hull, Elaine M., Travis, Joseph, Miller, Thomas E., Burgess, Scott C., Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Cooperation is a complex behavior in which individuals act in ways that increase the fitness of others, at some cost to themselves. In cooperatively breeding vertebrates, helpers capable of reproducing forgo their own reproduction to assist raising offspring produced by breeders. Cooperative breeding in many species is facultative. In such cases, breeders within a single population differ in whether they are assisted by helpers, and potential helpers differ in whether they join a group and...
Show moreCooperation is a complex behavior in which individuals act in ways that increase the fitness of others, at some cost to themselves. In cooperatively breeding vertebrates, helpers capable of reproducing forgo their own reproduction to assist raising offspring produced by breeders. Cooperative breeding in many species is facultative. In such cases, breeders within a single population differ in whether they are assisted by helpers, and potential helpers differ in whether they join a group and provide alloparental care. A major challenge in the study of cooperative breeding behavior is understanding why individuals differ in their cooperative tendency and their contributions to cooperative activities. The ultimate causes of variation in cooperative breeding behavior are increasingly well understood. Our understanding of the underlying physiological mechanisms and behaviors associated with individual differences in cooperative tendency remains poorly studied. In order to understand why cooperative behavior varies within a population, it is necessary to consider the proximate mechanisms associated with variation in cooperative breeding behavior from both the breeders’ and potential helpers’ perspectives. I investigated proximate mechanisms underlying individual variation in cooperative breeding behavior in a wild, color-marked population of facultative, cooperatively breeding brown-headed nuthatches (Sitta pusilla). The study population was located in north Florida at Tall Timbers Research Station. In this population, cooperation varies among breeders and helpers. Approximately 30% of breeding pairs are assisted by at least one second-year, male helper. Second year males also vary in their cooperative tendency, some males become helpers, others attempt to breed independently, and some do not associate with a social group. From 2013-2018, I used observations and experiments to investigate (1) how helpers contribute to breeders’ reproductive effort and how breeders alter their own investment when assisted by a helper, (2) how breeders’ prior nesting success and cooperative status affect subsequent helper recruitment, (3) how potential helpers’ early life physiological mechanisms influence individual variation in cooperation, and (4) how variation in aggressive behavior among breeders influences variation in cooperative behavior. Breeders assisted by helpers did not reduce their investment in offspring production or care, and as a result, nestlings raised by cooperative groups received more food, weighed more, and were more likely to fledge compared to nestlings raised by just the breeding pair. Variation in cooperation among breeders was not explained by differences in breeders’ prior nesting success or cooperative status. Eight-three percent of breeders that recruited helpers had fledged offspring the previous breeding season, yet 56% of unassisted breeders had also fledged young the previous year. These data suggest fledging young is neither necessary nor sufficient in explaining variation in cooperative behavior among breeders. Furthermore, variation in aggressive behavior was unrelated to variation in helper recruitment among breeders. Breeders’ aggressive behavior in response to a heterospecific competitor model was unrelated to breeders’ current cooperative status, and did not predict future recruitment of helpers. These data suggest that, while we thought that aggression and cooperation would represent a behavioral conflict, they do not. Variation in cooperation behavior among potential helpers was related to variation in hormone concentrations, and not variation in relatedness among group members. For potential helpers, variation in nestling corticosterone (the primary stress hormone in birds), not relatedness, predicted which individuals became helpers. Nestlings with lower levels of stress-induced corticosterone were more likely to become helpers, but were not significantly more related to the breeding male compared to their non-helping siblings. My dissertation research investigated proximate mechanisms that influence individual variation in the decision to cooperate, a relatively unexplored aspect of cooperative breeding behavior. This study is one of the first to document a link between glucocorticoids and future helping, and demonstrates that variation in the expression of cooperative behavior may be due to individual differences in underlying physiological mechanisms and behaviors, not relatedness alone. This research contributes to our understanding of variation in cooperative behavior and cooperation among unrelated individuals.
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Date Issued: 2019
Identifier: 2019_Spring_Cusick_fsu_0071E_15096
Format: Thesis

Title: Identification and Characterization of Linker of Nucleoskeleton and Cytoskeleton (Linc) Complex Components in Maize (Zea mays L.) Meiosis and Development.
Creator: Gumber, Hardeep Kaur, Bass, Hank W., Gunjan, Akash, McGinnis, Karen M., Yu, Hong-Guo, Florida State University, College of Arts and Sciences, Department of Biological Science
Abstract/Description: The LINC (Linker of Nucleoskeleton to Cytoskeleton) complex is an essential multi-protein structure spanning the nuclear envelope in all eukaryotic organisms. The core of the LINC complex is comprised of SUN (Sad1/Unc-84) domain proteins in the inner nuclear membrane, physically interacting in the periplasmic space, with the KASH (Klarsicht/ANC-1/Syne homology) domain proteins of the outer nuclear membrane. The N-terminus of SUN domain proteins interact with chromatin and lamin or lamin-like...
Show moreThe LINC (Linker of Nucleoskeleton to Cytoskeleton) complex is an essential multi-protein structure spanning the nuclear envelope in all eukaryotic organisms. The core of the LINC complex is comprised of SUN (Sad1/Unc-84) domain proteins in the inner nuclear membrane, physically interacting in the periplasmic space, with the KASH (Klarsicht/ANC-1/Syne homology) domain proteins of the outer nuclear membrane. The N-terminus of SUN domain proteins interact with chromatin and lamin or lamin-like proteins at the nuclear periphery, whereas, the N-terminus of KASH domain proteins interact with the cytoskeletal components in the cytoplasm, making a bridge across the nuclear envelope. LINC complex is essential for maintaining the shape and size of the nucleus; nuclear migration; and chromosome movements during meiosis. Despite of being an essential component of the cell, the knowledge of LINC complex is limited in plants, especially in monocots which include grasses, the most important agronomic crops. To fill this knowledge gap, using bioinformatic and biochemical approaches, we identified 17 new maize candidate genes coding for core LINC or associated proteins. These include 10 Maize LINC KASH (MLK) proteins, 3 inner nuclear membrane Nuclear envelope Associated Proteins (NEAP), 2 lamin functional homologs NMCP/CRWN homolog (NCH) proteins and 2 NCH-interacting KAKU proteins. Several of these candidates have been verified for their nuclear envelope localization and SUN-interaction by FRAP and co-immunoprecipitation assays in heterologous expression system. In previous studies from our lab, 5 SUN proteins were identified in maize and SUN2 was characterized for its meiotic functions in tethering telomeres to the nuclear envelope, while the KASH protein remained unknown. In second part of the dissertation, I have reported the functional characterization of Maize LINC KASH AtSINE-like2, MLKS2, which encodes a SINE-group KASH protein with characteristic armadillo repeats (ARM) at its N-terminus. Genetic analysis of transposon-insertion mutations, mlks2-1 and mlks2-2, showed defects in multiple aspects of meiosis. 3D cytology of mutant meiocytes show defects in nuclear positioning, bouquet formation and aneuploidy resulting in inviable grains. The mutants show defects in perinuclear actin accumulation. An ARM-dependent MLKS2-actin co-localization was observed in heterologous expression system. Together, these studies support a working model in which the nucleus is connected to F-actin cytoskeleton through a possible VELCRO-type mechanism provided by the MLKS2 ARM-domain on the nuclear surface. This places MLKS2 in the meiotic chromosome segregation pathway, likely mediated by a chain connecting chromosomes to actin through SUN-MLKS2 LINC complex.
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Date Issued: 2019
Identifier: 2019_Summer_Gumber_fsu_0071E_15267
Format: Thesis

Title: Investigating the Interaction of Sleep and Alcohol.
Creator: Noakes, Eric J. (Eric Joseph), Lyons, Lisa C., Arbeitman, Michelle N. (Michelle Nina), Houpt, Thomas A., Deng, Wu-Min, Florida State University, College of Arts and Sciences,...
Show moreNoakes, Eric J. (Eric Joseph), Lyons, Lisa C., Arbeitman, Michelle N. (Michelle Nina), Houpt, Thomas A., Deng, Wu-Min, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Alcohol abuse is more prevalent in populations in which sleep deprivation is more common including shift workers and older individuals. While much research has investigated the impact of alcohol use and abuse on sleep quality, little is known about the role of sleep in alcohol sensitivity and toxicity. Using Drosophila melanogaster, a well-established model for sleep and alcohol studies, I investigated the modulating effect of sleep on alcohol sensitivity, toxicity, and tolerance. Mechanical...
Show moreAlcohol abuse is more prevalent in populations in which sleep deprivation is more common including shift workers and older individuals. While much research has investigated the impact of alcohol use and abuse on sleep quality, little is known about the role of sleep in alcohol sensitivity and toxicity. Using Drosophila melanogaster, a well-established model for sleep and alcohol studies, I investigated the modulating effect of sleep on alcohol sensitivity, toxicity, and tolerance. Mechanical sleep deprivation increased alcohol sensitivity and alcohol-induced mortality following an acute binge-like exposure to alcohol. Genetically sleep deficient flies exhibited increased alcohol-induced mortality which accumulated with age, but not increased alcohol sensitivity. Conversely, pharmacologically increasing sleep protects against the toxic effects of alcohol. Sleep deprivation blunted long-term but not short-term functional alcohol tolerance. Finally, sleep deprivation, alcohol exposure, and the combination of sleep deprivation and alcohol exposure induce both distinct and overlapping changes in gene expression. Taken together, the results of this study suggest that sleep modulates alcohol toxicity and impacts functional alcohol tolerance. This study lays the foundation for a future in-depth investigation of potential mechanisms governing the interaction of sleep and alcohol.
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Date Issued: 2019
Identifier: 2019_Summer_Noakes_fsu_0071N_15449
Format: Thesis

Title: Assessing Variation in Dispersal Decisions in a Cooperatively Breeding Passerine.
Creator: Dietz, Samantha Lauren, DuVal, Emily H., Underwood, Nora C., Burgess, Scott C., Florida State University, College of Arts and Sciences, Department of Biological Science
Abstract/Description: Natal dispersal, the period where an organism moves from its birthplace to the area where it settles and attempts to breed, may have significant consequences for individual fitness. Individuals vary in both the decision to initiate dispersal and the decision to settle and attempt reproduction. In cooperative species, some individuals delay their departure from the natal territory and forego reproduction for one or more breeding seasons, while others disperse much sooner. The timing of when...
Show moreNatal dispersal, the period where an organism moves from its birthplace to the area where it settles and attempts to breed, may have significant consequences for individual fitness. Individuals vary in both the decision to initiate dispersal and the decision to settle and attempt reproduction. In cooperative species, some individuals delay their departure from the natal territory and forego reproduction for one or more breeding seasons, while others disperse much sooner. The timing of when individuals depart their natal site can affect their ability to locate and establish a breeding territory. Availability of local breeding sites, competition among the natal group, and an individual's development are hypothesized to influence dispersal initiation. Once an individual departs the natal territory, they also must choose a settlement area that will affect their access to potential mates, resources, and exposure to predators. Understanding how a juvenile's experience prior to dispersal influences their timing and settlement decisions may help explain variation in fitness among individuals within a population. Despite the importance of settlement site, individuals often appear to settle in low-quality habitat when high-quality habitat is available. The Natal Habitat Preference Induction hypothesis posits that individuals may choose breeding habitat that is similar to their natal habitat, rather than habitat of the highest quality. I investigated factors that influenced variation in dispersal behavior in a population of cooperatively breeding Brown-headed Nuthatches (Sitta pusilla) by addressing two questions: (1) What factors influence whether and when individuals depart from the natal territory? and (2) How do individuals make settlement decisions? I found that males dispersed earlier when they experienced more competition within the natal group, and females dispersed earlier when they were smaller in size relative to their siblings, and when local breeding opportunities were constrained. I found no evidence that individuals were choosing settlement sites based on habitat cues as predicted by the Natal Habitat Preference Induction hypothesis. My thesis broadens understanding of how multiple aspects of an individual's experience might affect dispersal decisions, and assesses one hypothesis that potentially explains how dispersers make settlement decisions.
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Date Issued: 2019
Identifier: 2019_Summer_Dietz_fsu_0071N_15341
Format: Thesis

Title: Characterization of Linc Complex Assembly in Budding Yeast.
Creator: Fan, Jinbo, Yu, Hong-Guo, Wang, Yanchang, Bass, Hank W., Chadwick, Brian P., McGinnis, Karen M., Florida State University, College of Arts and Sciences, Department of Biological...
Show moreFan, Jinbo, Yu, Hong-Guo, Wang, Yanchang, Bass, Hank W., Chadwick, Brian P., McGinnis, Karen M., Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: The linker of the nucleoskeleton and cytoskeleton (LINC) protein complex bridges the inner and outer nuclear membranes and regulates a range of nuclear activities that include telomere tethering and chromosome movement. The canonical LINC complex is composed of a pair of transmembrane-domain proteins, with the KASH protein localized to the outer nuclear membrane, and the SUN protein to the inner nuclear membrane. In budding yeast, Csm4, which is specific to meiosis, and Mps2 are two KASH-like...
Show moreThe linker of the nucleoskeleton and cytoskeleton (LINC) protein complex bridges the inner and outer nuclear membranes and regulates a range of nuclear activities that include telomere tethering and chromosome movement. The canonical LINC complex is composed of a pair of transmembrane-domain proteins, with the KASH protein localized to the outer nuclear membrane, and the SUN protein to the inner nuclear membrane. In budding yeast, Csm4, which is specific to meiosis, and Mps2 are two KASH-like proteins, whereas Mps3 is the sole SUN protein. The current notion posits that Mps3 pairs with either Csm4 or Mps2 to form separate LINC complexes at the telomere and the centrosome, respectively. Here we show that Mps2 mediates the interaction between Csm4 and Mps3 to form a functional heterotrimeric composition of LINC complex that regulates telomere attachment and meiotic recombination. Csm4 binds to Mps2, and both localize to telomeres. Csm4's localization depends on Mps2 and Mps3, but Mps2's association with the telomere depends on Mps3 but not Csm4. The Mps2-mediated heterotrimeric LINC complex controls nuclear shape, telomere bouquet formation, recombination, and homolog pairing in prophase I. These findings reveal the heterotrimeric composition of the yeast LINC complex and have implications for understanding LINC variants in higher eukaryotes. To further characterize the heterotrimeric LINC complex, we have reconstructed the meiotic LINC complex in vegetative yeast cells by ectopically expressing Csm4. In the wild-type cells, both Mps2 and Mps3 are concentrated at the centrosome. In the presence of Csm4, Mps2 and Mps3 form "mitotic patches" at the leading edge of the budding daughter cell during mitosis. Importantly, the presence of Mps3 patch depends on Mps2, while the presence of Mps2 patch does not depend on Mps3, demonstrating that Mps3's interaction with Csm4 requires Mps2. Furthermore, we show that the Mps2/Mps3 patch is absent in yeast cells treated with the actin depolymerizing drug latrunculin B, indicating that ectopic t-LINC formation in vegetative cells depends on actin. These findings support our meiotic model in which the yeast telomere-associated LINC complex is composed of Mps3, Mps2, and Csm4. Finally, we have revealed that Csm4 is a short-lived protein, whose degradation appears to regulate meiotic telomere-associated LINC complex disassembly. The protein level of Csm4 peaks during prophase I but is barely detectable by Western blotting after metaphase I. We hypothesize that the disassembly of the yeast telomere-associated LINC complex is regulated by the degradation of Csm4. To test this hypothesis, a targeted genetic screen was performed and two CSM4 interacting factors were identified, UBC7 and DOA10, which encode the E2 ubiquitin conjugating enzyme and the E3 ligase of the endoplasmic-reticulum-associated protein degradation (ERAD) pathway, respectively. These findings therefore provide a clue to how the yeast telomere-associated LINC complex is downregulated during the cell cycle. In summary, we show that a heterotrimeric LINC complex is assembled at the telomere in budding yeast meiosis, and Mps2 is the linker between Mps3 and Csm4. Our work not only clarifies the composition and function of the telomere-associated LINC complex in budding yeast, but also provides implications for LINC variant formation in other organisms. In addition, we show that the KASH-like protein Csm4 is likely subject to ERAD pathway regulation for protein turnover, which may provide a mechanism for LINC complex disassembly during the cell cycle.
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Date Issued: 2019
Identifier: 2019_Summer_Fan_fsu_0071E_15268
Format: Thesis

Title: Socially Mediated Plasticity and Polymorphism: Integrating Theory and Experiment to Predict Alternative Phenotypes.
Creator: Lange, Elizabeth C., Hughes, Kimberly A., Hull, Elaine M., Travis, Joseph, DuVal, Emily H., Levitan, Don R., Florida State University, College of Arts and Sciences, Department...
Show moreLange, Elizabeth C., Hughes, Kimberly A., Hull, Elaine M., Travis, Joseph, DuVal, Emily H., Levitan, Don R., Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Understanding the maintenance of phenotypic variation within populations has long been a puzzle in evolutionary biology. Many models ignore that fact that animals are not living alone; instead social factors have the potential to alter the development and fitness consequences of alternative phenotypes to promote variation in many systems. Furthermore, while there is empirical evidence that individuals alter phenotypes in response to social cues, it is unclear under what conditions socially...
Show moreUnderstanding the maintenance of phenotypic variation within populations has long been a puzzle in evolutionary biology. Many models ignore that fact that animals are not living alone; instead social factors have the potential to alter the development and fitness consequences of alternative phenotypes to promote variation in many systems. Furthermore, while there is empirical evidence that individuals alter phenotypes in response to social cues, it is unclear under what conditions socially-cued plasticity will evolve and be adaptive. My dissertation research combines theory with developmental experiments in sailfin mollies (Poecilia latipinna) to understand if and how individuals alter phenotypes in response to social cues. The first chapter of my dissertation uses an individual-based modelling approach to determine if and when individuals should evolve a strategy that uses social cues during development to alter the expression of alternative phenotypes. We found that socially-cued plasticity evolves under limited conditions where selection acts on survival differences between alternative phenotypes and the expression of socially-cued plasticity is costly. Socially-cued plasticity was not adaptive when selection acted on fecundity. Because costs facilitated the evolution of adaptive socially-cued plasticity, our results suggest that socially-cued plasticity is a special case of plasticity where general models do not hold. Furthermore, we found that socially-cued plasticity is a self-limiting strategy; using social cues to alter phenotypes in adulthood was most likely to evolve when the majority of the population was not using socially-cued plasticity; this scenario allowed social cues to be reliable predictors of environmental conditions. In the second and third chapter of my dissertation, I used sailfin mollies to determine how species with alternative reproductive phenotypes (ARPs) alter their life history and mating behavior in response to social cues during development. Sailfin mollies are a livebearing fish that exhibit extensive variation in body size and correlated traits including age at maturity, morphology and mating behavior, both within and between populations. Together these traits make up a male's ARP. Smalls males mature quickly (50-60 days) and use their disproportionally longer intromittent organ in sneaking behavior. Large males take longer to mature (130-150 days) and use their disproportionally larger dorsal fins in courtship displays to entice female cooperation in mating. Intermediate-sized males, which are intermediate in morphology and time to maturity, switch between courting and thrusting depending on the social context. Previous studies have examined the role of abiotic environmental factors on male ARP in mollies, but found that these factors cannot account for the observed inter- and intra-population variation. Since mollies are gregarious and social environment has been shown to influence adult male behavior, we hypothesize that variability in social conditions can influence the relationships between genotype and phenotype to produce ARP variation. My second chapter describes an experimental study where we examined the relationship between genotype and phenotype by determining how the variation in social environment during development influenced sex-specific differences in life history phenotypes. We found that both variation in the social environment influences life history development in both males and females, but there were sex-specific differences in how social environment modulated the genotype-phenotype relationship. These results suggest that social environment is an important driver of life history differences in sailfin mollies. My final experiment tested the hypothesis that social environment during development affects male alternative mating behaviors. We found that courtship and sneaking behaviors were affected by variation in the social environment, but these effects manifested in complex interactions between experimental treatments. For example, the relationship between body size and courtship displays was affected by a genotype by social environment interaction, and there was a three-way interaction between genotype, developmental stage, and the male’s own body size. In addition,. These results implicated alternative reproductive morph, social environment during development, stage, and body size as non-independent factors in the expression of male ARPs. Results from my dissertation demonstrate that conditions for adaptive evolution of socially-cued plasticity are limited, but despite this, variation in social cues elicited substantial variation in life history and behavior, in ways not accounted for by current life history or sexual selection theory. These seemingly paradoxical results may be resolved by considering the natural history of mollies. Sailfin molly males of different ARPs have differences in survival and therefore results from the modelling chapter suggest mollies may be a system where socially-cued plasticity would evolve. To determine if the patterns observed in this dissertation are adaptive, or are accounted for by gene flow, by exposing animals to social environments they would not typically encounter in nature, or by other non-adaptive processes, future studies should assess mortality in different social environments and reproductive success to determine how social environment affects fitness. Taken together, my dissertation provides a better understanding of how phenotypic plasticity evolves and how social environment affects life history and mating behaviors.
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Date Issued: 2019
Identifier: 2019_Fall_Lange_fsu_0071E_15531
Format: Thesis

Title: Modeling Zika Virus Infection and Pathogenesis in Neuroglia and 3D Cerebral Organoids.
Creator: Ogden, Sarah Catherine, Tang, Hengli, Megraw, Timothy L., Dennis, Jonathan Hancock, Li, Yan, Zhu, Fanxiu, Florida State University, College of Arts and Sciences, Department of...
Show moreOgden, Sarah Catherine, Tang, Hengli, Megraw, Timothy L., Dennis, Jonathan Hancock, Li, Yan, Zhu, Fanxiu, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Zika virus is an arbovirus that has reemerged in recent years as a global health concern. A member of the Flavivirus genus, Zika virus is closely related to other major human pathogens such as dengue virus and West Nile virus, but each of these viruses exhibit varied clinical pathologies. While Zika virus was first discovered in 1947, it was not until the outbreaks across the Pacific and Americas from 2013 onward that Zika virus’ unique neuropathies were revealed. Of particular concern was...
Show moreZika virus is an arbovirus that has reemerged in recent years as a global health concern. A member of the Flavivirus genus, Zika virus is closely related to other major human pathogens such as dengue virus and West Nile virus, but each of these viruses exhibit varied clinical pathologies. While Zika virus was first discovered in 1947, it was not until the outbreaks across the Pacific and Americas from 2013 onward that Zika virus’ unique neuropathies were revealed. Of particular concern was the potential for vertical transmission and teratogenic effects in infected pregnant women, prompting the World Health Organization to declare Zika virus a public health crisis of global concern in 2016. Even with a growing knowledge of Zika virus’ adverse fetal outcomes and the characterization of congenital Zika syndrome, much research is still needed to understand the cellular and molecular mechanisms underlying these potentially devastating infection consequences. Given this need, we therefore established a cerebral organoid system to model Zika virus exposure throughout fetal brain development. When exposed to Zika virus, organoids, which recapitulated features of brain development in the first trimester, exhibited microcephaly-like restrictions in maturation. This included an overall reduction of organoid growth, enlargement of the ventricle-like structures, and increased cell death in organoids exposed to both the prototypical and modern epidemic strains of Zika virus. In older organoids resembling the fetal brain structure of the second trimester, Zika virus preferentially infected neural progenitor cells as compared to neurons, and also infected several other early brain cell types, demonstrating the potential cellular affects underlying Zika virus-induced microcephaly. We next utilized a neuroglia-derived cell line highly permissive to Zika virus infection to find Zika virus host factors in neural cells. We identified the host protein, AXL, as an important factor for Zika virus infection, but observed a difference in the requirement of AXL for the infection of Zika and dengue virus. Chimeras of Zika and dengue virus revealed that the structural proteins displayed on the virion surface were a major determinant of AXL-dependent viral infection. Lastly, we investigated the molecular mechanisms contributing to AXL-dependent infection and found that suppression of the IFN response as mediated by AXL was minimally involved in AXL-dependent Zika virus infection, in contrast to dengue virus, suggesting that different aspects of AXL signaling may facilitate Zika and dengue infection in neuroglia. Our data provide a model system for evaluating Zika virus exposure during fetal brain development. We show that Zika virus infects both neural progenitor cells and neuroglia, cell types which are present at different stages of cerebral cortex development. Our work highlights the importance of AXL in Zika virus infection of neuroglia and the differential requirement of AXL for Zika and dengue virus infection. Collectively, these findings contribute toward dissecting the mechanism by which Zika virus utilizes AXL for viral entry and infection, which may be important for understanding Zika virus pathogenesis as AXL is expressed on many of the cell types infected by the virus in vivo.
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Date Issued: 2019
Identifier: 2019_Fall_Ogden_fsu_0071E_15517
Format: Thesis

Title: Cytogenetic Analysis of Male Meiosis in Humulus Lupulus L. (Hop): An Investigation of a Highly Structurally Variable Genome.
Creator: Easterling, Katherine Aline, Bass, Hank W., Levenson, Cathy W., Mast, Austin R., McGinnis, Karen M., Miller, Brian G., Florida State University, College of Arts and Sciences,...
Show moreEasterling, Katherine Aline, Bass, Hank W., Levenson, Cathy W., Mast, Austin R., McGinnis, Karen M., Miller, Brian G., Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Hop (Humulus lupulus L.) is an important crop worldwide, known as the main flavoring ingredient in beer. The diversifying brewing industry demands variation in flavors, superior process properties, and sustainable agronomics, which are the focus of advanced molecular breeding efforts in hops. Hop breeders have been limited in their ability to create strains with desirable traits, however, because of the unusual and unpredictable inheritance patterns and associated non-Mendelian genetic marker...
Show moreHop (Humulus lupulus L.) is an important crop worldwide, known as the main flavoring ingredient in beer. The diversifying brewing industry demands variation in flavors, superior process properties, and sustainable agronomics, which are the focus of advanced molecular breeding efforts in hops. Hop breeders have been limited in their ability to create strains with desirable traits, however, because of the unusual and unpredictable inheritance patterns and associated non-Mendelian genetic marker segregation. To better understand the transmission genetics of hop we genotyped 4,512 worldwide accessions of hop, including cultivars, landraces, and over 100 wild accessions, using a genotyping-by-sequencing (GBS) approach. From the resulting ~1.2M single nucleotide polymorphisms, pre-qualified GBS markers were validated by inferences in population structures and phylogeny. Analysis of pseudo-testcross mapping data from F1 families revealed mixed patterns of Mendelian and non-Mendelian segregation. We used genome-wide association studies and FST analysis to demonstrate selection mapping of genetic loci for key traits, including sex, bitter acids, and drought tolerance. Among the possible mechanisms underlying the observed segregation distortion from the genomic data analysis, the cytogenetic analysis points to meiotic chromosome behavior as one of the contributing factors. Cytogenetic analysis of meiotic chromosome behavior has also revealed conspicuous and prevalent occurrences of multiple, atypical, non-disomic chromosome complexes, including those involving autosomes in late prophase. To explore the role of meiosis in segregation distortion, we undertook 3D cytogenetic analysis of hop pollen mother cells stained with DAPI. Our initial DAPI survey of late meiotic prophase nuclei in two cultivars and two wild hop revealed conspicuous and prevalent occurrences of multiple, atypical, non-disomic chromosome complexes, including autosomes. We then systematically examined all stages of meiosis for meiotic irregularities utilizing telomere and 5S rDNA fluorescent in-situ hybridization (FISH). We used telomere FISH to demonstrate that hop exhibits a normal telomere clustering bouquet. Highly variable 5S rDNA FISH patterns within and between plants, together with the detection of anaphase chromosome bridges, reflect extensive departures from normal disomic signal composition and distribution. Subsequent FACS analysis revealed variable DNA content in a cultivated pedigree. To date, linkage groups in hop have not been assigned to physical chromosomes, rendering genome assemblies and karyotype development particularly challenging, given its genomic structural variability. Consequently, understanding genome evolution in Humulus represents a considerable challenge, requiring additional resources, including integrated genome maps. In order to facilitate cytogenetic investigations into the transmission genetics of hop, we report here the identification and characterization of 18 new and distinct tandem repeat sequence families. A tandem repeat discovery pipeline was developed using k-mer and dot plot analysis of PacBio long-read sequences from the hop cultivar, Apollo. We produced oligonucleotide FISH probes for some of these families and demonstrated their utility via 3D FISH to stain meiotic chromosomes from wild hop, var. neomexicanus. Probes derived from the tandem repeat sequence families, HSR0, HuluTR120 and HuluTR235, were shown to be particularly useful for detection of meiotic abnormalities in wild and cultivated hop. Collectively, these tandem repeat sequence families represent unique and valuable new reagents with the capacity to inform genome assembly efforts and support comparative genomic analyses. The findings shed light on long-standing questions on the unusual transmission genetics and phenotypic variation in hop, with major implications for breeding, cultivation, and the natural history of Humulus. The data in this dissertation have been or are being considered for publication. The data presented in Chapter 2 has been published in The Plant Genome (Zhang et al., 2017). The data presented in Chapter 2 has been published in Frontiers in Plant Science (Easterling et al., 2018). The data in Chapter 4 is submitted to Frontiers in Plant Science.
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Date Issued: 2019
Identifier: 2019_Fall_Easterling_fsu_0071E_15522
Format: Thesis

Title: Constructing a Single-Cell Transcriptomic Atlas of Cells Regulating Drosophila Oogenesis and Ovulation and Understanding the Role of Notch Signaling during Development and Tumorigenesis.
Creator: Jevitt, Allison M., Deng, Wu-Min, Jones, Kathryn M., Megraw, Timothy L., Hughes, Kimberly A., Lenhert, Steven John, Florida State University, College of Arts and Sciences,...
Show moreJevitt, Allison M., Deng, Wu-Min, Jones, Kathryn M., Megraw, Timothy L., Hughes, Kimberly A., Lenhert, Steven John, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: The Drosophila ovary is an important model system for studying oogenesis and has provided insight into broader, biological topics such as stem cell niche, differentiation, migration, morphogenesis, signaling, cell-size regulation, and tumorigenesis. The purpose of this dissertation was to build a comprehensive single-cell transcriptomic atlas of cells regulating oogenesis and ovulation and to use the ovarian follicle cells, as a model system to interrogate the role of ectopic Notch expression...
Show moreThe Drosophila ovary is an important model system for studying oogenesis and has provided insight into broader, biological topics such as stem cell niche, differentiation, migration, morphogenesis, signaling, cell-size regulation, and tumorigenesis. The purpose of this dissertation was to build a comprehensive single-cell transcriptomic atlas of cells regulating oogenesis and ovulation and to use the ovarian follicle cells, as a model system to interrogate the role of ectopic Notch expression in development and during tumorigenesis. Single-cell RNA sequencing analysis revealed transcriptional signatures for each of the 28 expected ovarian cell types and developmental stages. Each stage of oogenesis was then separately analyzed with a special focus on the most diverse cell type, the follicle cells. This identified key cell-type specific expression patterns governing processes like differentiation, mitotic-to-endocycle switch, migration, morphogenesis, phagocytic removal of nurse cells, eggshell formation, and a newly identified shift of the pre-corpus lutuem cell expression from oogenesis-to-ovulation. Additional characterization of the interconnected tissues in the dataset identified novel cellular heterogeneity of the oviduct and a population of hemocytes associating closely with it. We further describe how this association occurs in a non-mating-dependent manner as early as the pupal stage (during oviduct development). Additional characterization of the hemocyte cluster reveals expression of many phagocytic genes including the newly identified enzymatic marker, Cp1. Additional validation of cell-type markers classifies these macrophage-like cells as plasmatocytes, one of the most common types of hemocytes in flies. Armed with a rich dataset describing proper signaling during development we turn to the follicle cells and switch focus to study dysregulated signaling of the Notch pathway and its involvement in tumor formation. First, we expressed ectopic Notch (NICD) in follicle cells and discovered an uneven pattern of nuclear NICD retention in a cell-cycle dependent manner. We find that the cell-cycle regulator, string (cdc25), can strongly impact the localization of NICD regardless of the cell-cycle status of the cell. We also identify that Vps proteins involved in forming the ESCRT complex can similarly regulate the nuclear NICD pattern. Next, we examined the role of ectopic Notch signaling in tumorigenesis and found that while not sufficient for tumor formation, Notch functions as a tumor promoter and leads to a more dysplastic tumor phenotype. Tumor cells with ectopic NICD gain a survival advantage which may be due to a bypass of the DNA damage sensing checkpoint. RNA sequencing revealed unique expression of these tumor cells with ectopic NICD in both pre- and post-tumor conditions. Specifically, we identified DNA stability related genes, RecQ4 and Xpd, which are upregulated in NICD-overexpressing pre-tumor cells.
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Date Issued: 2019
Identifier: 2019_Fall_Jevitt_fsu_0071E_15554
Format: Thesis

Title: Stress Response and Coloration as Mediators of Behavioral and Physiological Variation.
Creator: Humphrey, Eve, Travis, Joseph, Eckel, Lisa A., Hughes, Kimberly A., DuVal, Emily H., Burgess, Scott C., Florida State University, College of Arts and Sciences, Department of...
Show moreHumphrey, Eve, Travis, Joseph, Eckel, Lisa A., Hughes, Kimberly A., DuVal, Emily H., Burgess, Scott C., Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Differences in behavior and physiology can arise and change because of variation in life history strategies or differences in discrete polymorphisms. These differences can result from the indirect effects of predation, or responses to erratic, repeated exposures to predators in which the prey individual is not consumed (non-consumptive predation). Behavioral responses in the presence of predators, like changes in mating or antipredator behavior may differ because of life history variation or...
Show moreDifferences in behavior and physiology can arise and change because of variation in life history strategies or differences in discrete polymorphisms. These differences can result from the indirect effects of predation, or responses to erratic, repeated exposures to predators in which the prey individual is not consumed (non-consumptive predation). Behavioral responses in the presence of predators, like changes in mating or antipredator behavior may differ because of life history variation or discrete morphs within populations. There are also physiological responses that occur with behavioral responses. These physiological responses occur through the animals’ stress response. A stress response is an accumulation of endocrine and neuroendocrine responses that return the body back to its normal metabolic and hormonal levels. The least killifish, Heterandria formosa is an excellent system to understand how life history variation influences responses to predation, while, melanic and silver morphs in the Eastern mosquitofish, gambusia holbrooki, are ideal for understanding how variation within populations influence responses to predation. The overarching goals of this dissertation were to evaluate variation in behavior and physiology from predation risk as a result of differences between populations of the least killifish and within a population of the Eastern mosquitofish as well as to review pleiotropic effects that stem from the central melanocortin system and their potential influence on behavior and stress within and between populations. To answer our first questions in the least killifish we created control and predator treatments and measured both populations’ differences in male mating behavior when exposed to a predator. We also analyzed female cortisol levels between the two populations to see if their stress responses varied. We exposed male and female least killifish from both populations to predator and control treatments over 30 days to measure the effect of treatment and population on reproductive output. Results showed that males from the high-predation population were more active overall than males from the low predation population in the control treatment but that males from both populations responded to the presence of predators. Females from the low predator population had higher cortisol levels in predator treatments however cortisol levels from females in high predator population were similar in control and predator treatments. Our results reflect conclusions of another study in which predator presence did not influence reproductive output in another population of H.formosa. In the Eastern mosquitofish we exposed silver and melanic males to predator and control treatments and followed behavior assays with cortisol analyses. We found that melanic males were more active than silver males in their mating behavior and this result is consistent with other studies. All males decreased their mating behaviors in the presence of predators and increased antipredator freezing behavior in predator treatments. Melanic males had higher cortisol levels on average than silver males in control treatments; melanic male cortisol levels were only half as high in the predator treatments as in the control treatments. Differences in morph behaviors and physiology may be mediated by the pleiotropic effects correlated with black coloration and also may help rare eastern mosquitofish morphs persist. Finally, we reviewed the melanocortin system and its pleiotropic effects determined the frequencies of melanic morphs within natural populations and reflected on other pathways that influence black coloration. We found that melanic morphs are rare in polymorphic populations. We also found that populations with discrete polymorphisms mostly consisted of melanic morphs that occurred 33% or less of the time in the population with melanic morphs frequencies higher than 75%. In conclusion, this work is the first to review the frequencies of melanic morphs in populations and assess behavior and stress response in the least killifish and Eastern mosquitofish. We highlight the importance of assessing short-term behavioral responses and also measuring long-term responses through predation exposure in H.formosa. We also provide evidence for the use of short and long-term measures and not solely relying on short-term responses as a means to predict fitness. We also demonstrate how variation within populations, specifically melanic coloration may influence differences in morph behaviors, the propensity to be approached or attacked by predators, and additional behavioral and physiological responses that may arise because of pleiotropy in the melanocortin system.
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Date Issued: 2019
Identifier: 2019_Fall_Humphrey_fsu_0071E_15472
Format: Thesis

Title: Evaporative Edge Lithography: A New Method for Assaying the Effect of Lipophilic Drugs on Migration and Outgrowth of Cells over Patterned Surfaces.
Creator: Vafai, Nicholas Rahim, Lenhert, Steven John, Guan, Jingjiao, Jones, Kathryn M., Keller, Thomas C. S., Dennis, Jonathan Hancock, Florida State University, College of Arts and...
Show moreVafai, Nicholas Rahim, Lenhert, Steven John, Guan, Jingjiao, Jones, Kathryn M., Keller, Thomas C. S., Dennis, Jonathan Hancock, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Cells sense and respond to topographical cues in their microenvironment that influence growth, development, and migration. Cell migration and outgrowth assays have been used to study cellular movement or changes in cellular morphology and topography. Such assays are promising tools in drug discovery, especially when implemented with high-throughput and high-content imaging systems. These techniques have also been useful for screening and analyzing the effect of different compounds on neurite...
Show moreCells sense and respond to topographical cues in their microenvironment that influence growth, development, and migration. Cell migration and outgrowth assays have been used to study cellular movement or changes in cellular morphology and topography. Such assays are promising tools in drug discovery, especially when implemented with high-throughput and high-content imaging systems. These techniques have also been useful for screening and analyzing the effect of different compounds on neurite outgrowth and topography which in turn may lead to the discovery of beneficial targets for regeneration of nervous tissue. Typically, high-throughput screening of large chemical libraries is employed during the early stages of discovering new drug entities. However, these screening assays do not utilize different topographical surfaces. Many common techniques such as the scratch wound assay are limited in their compatibility with patterned surfaces. Therefore, there is a need to develop novel technologies capable of identifying potentially therapeutic compounds in early stage of drug discovery processes that can regulate cell behaviors and are not limited in their throughput and compatibility with patterned surfaces. A potentially scalable approach is the “fence” assay in which cells are cultured on topographical surfaces which are partially covered by a removable barrier. Upon removal of the barrier, cells are free to spread and migrate on the freshly uncovered topographies. In this thesis, a novel technique called evaporative edge lithography (EEL) is demonstrated as an approach to miniaturize the fence assay and can be used for high-throughput screening (HTS) in early stages of drug discovery. Furthermore, EEL is a new method to fabricate lipid-based drug delivery microarrays. Lipid multilayer micro-patterns offer a promising approach to applications such as drug screening and biosensing that require well defined patterns and fluidity. It is shown in this thesis that the factors that govern stability and instability of lipid multilayer nanostructures upon immersion using fluorescence microscopy and observed the following four mechanisms of lipid multilayer instability and strategies are derived to control immersion stability based on these findings: (1) Dissolution by the air/water interface; (2) Disruption by shearing from flowing solution; (3) Spreading at the solid-liquid interface; (4) Diffusion into solution. Based on these studies, a lipid multilayer microarray was developed that is suitable for cell-based assays without detectible cross-contamination by culturing cells on lipid patterns. It is shown in this thesis that this assay is compatible with poorly soluble lipophilic drug compounds that pose a challenge for HTS microarray assays. EEL was demonstrated for topographically patterned surfaces for screening compounds on adherent cells. Lipophilic compounds including docetaxel and BFA were screened using EEL with cultured HeLa cells to test if migration is affected and can be quantified with this approach. These results indicate that docetaxel and BFA were delivered locally into cells locally from surface supported lipid films and significantly inhibited cellular migration. Subsequently, EEL was used to screen docetaxel on cultured primary olfactory bulb neuronal cells to test the effect on neurite outgrowth. EEL is a novel approach that allows delivery and subsequent study of the effects of poorly water-soluble drugs on cell migration as well as in vitro screening of different drugs for their effects on cell structures and functions. In addition, this migration assay is a scalable and promising approach for high throughput drug screening microarrays since multiple drug compounds at different dosages can be screened simultaneously on the same surface. This work will advance future studies in developing a portable assay capable of screening lipophilic cancer and neurotropic compounds for topographically-driven cell outgrowth and migration.
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Date Issued: 2018
Identifier: 2018_Sp_Vafai_fsu_0071E_13835
Format: Thesis

Title: Genetic Dissection of Cis-Elements in Spatio-temporal Control of DNA Replication.
Creator: Sima, Jiao, Gilbert, David M., Gunjan, Akash, Dennis, Jonathan Hancock, Zhu, Fanxiu, Fadool, Debra Ann, Florida State University, College of Arts and Sciences, Department of...
Show moreSima, Jiao, Gilbert, David M., Gunjan, Akash, Dennis, Jonathan Hancock, Zhu, Fanxiu, Fadool, Debra Ann, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: DNA replication in all Eukaryotes follows a defined temporal order termed replication-timing program (RT), which is coupled with the spatial separation of chromatin distribution inside the nucleus. Early or late replicating chromatin self-organizes in 3D into sub-nuclear compartments at the nucleus interior or proximity to nuclear lamina respectively. RT is also highly correlated with multiple other features of the genome including transcriptional activity, chromatin composition, and...
Show moreDNA replication in all Eukaryotes follows a defined temporal order termed replication-timing program (RT), which is coupled with the spatial separation of chromatin distribution inside the nucleus. Early or late replicating chromatin self-organizes in 3D into sub-nuclear compartments at the nucleus interior or proximity to nuclear lamina respectively. RT is also highly correlated with multiple other features of the genome including transcriptional activity, chromatin composition, and mutational landscape. The molecular mechanisms regulating RT and linking these events are unclear. To investigate the role of DNA sequences in RT regulation, I adopted two parallel approaches to test the sufficiency and necessity of specific DNA segments in these processes. In the first approach, I developed an extra-chromosomal vector system (E-BAC) to show that determinants for RT and A/B compartmentalization are genetically encoded in ~200kb DNA sequences. In the second approach involving CRISPR (clustered regularly interspaced short palindromic repeats) mediated genome-editing, I identified three “early replication control elements” (ERCEs) internal of the domain that act redundantly and interdependently to give rise to both early replication and A/B compartmentalization of a pluripotency associated domain in mouse embryonic stem cells. The three ERCEs and other ERCE-like elements form the strongest CTCF-independent interactions among each other, which could drive the formation of A/B compartments inside the nucleus. The ERCEs also display a combination of active chromatin features resembling promoters and/or enhancers. They are implicated in gene regulation possibly by mediating the formation of transcription factories. These findings underscore the genetic influence on controlling multiple cellular processes, and highlight the complexity of cis regulation from the linear genome. The discovery of cis regulatory elements offers mechanistic insight linking highly correlated genomic features/activities, and provides opportunities to further dissect their relationship from a 3D perspective. Deeper understanding of genome regulation will hopefully enable the manipulation of these processes in cell function and disease.
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Date Issued: 2018
Identifier: 2018_Sp_Sima_fsu_0071E_14411
Format: Thesis

Title: How Males Shape Up: The Evolution of Male Body Morphology in Poeciliid Fishes.
Creator: Landy, Joseph Alexander, Travis, Joseph, Slice, Dennis E., DuVal, Emily H., Erickson, Gregory M., Hughes, Kimberly A., Florida State University, College of Arts and Sciences,...
Show moreLandy, Joseph Alexander, Travis, Joseph, Slice, Dennis E., DuVal, Emily H., Erickson, Gregory M., Hughes, Kimberly A., Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Understanding how environmental forces, genetic variation, and developmental process combine to mold adaptations remains a core challenge in evolutionary biology. Our research is answering this challenge. More specifically we focused on how these three drivers of adaptation have shaped the evolution of body morphology in poeciliid fish. The body morphology of poeciliid fish has been shown to be under strong ecological selection and sexual selection. In the poecilid fish Poecilia reticulata,...
Show moreUnderstanding how environmental forces, genetic variation, and developmental process combine to mold adaptations remains a core challenge in evolutionary biology. Our research is answering this challenge. More specifically we focused on how these three drivers of adaptation have shaped the evolution of body morphology in poeciliid fish. The body morphology of poeciliid fish has been shown to be under strong ecological selection and sexual selection. In the poecilid fish Poecilia reticulata, body morphology can evolve rapidly, over a span of four to five years. These attributes makes it an ideal system to study the processes leading to adaptive evolution. Here we studied this adaptive evolution in poeciliid fish at two scales: among populations of a single species and within populations of a single species. At the broadest scale, we investigated local adaptation among populations of a single poeciliid species, Heterandria formosa. We quantified patterns in morphological variation among populations and tested for associations between this variation and ecological data, which are derived from long term population censuses. Results from this study illustrate the complicated construction of multivariate phenotypic variation and suggest that different agents of selection have acted on different components of body morphology. These patterns in inter-population phenotypic variation can be evidence of local adaptation; however, they can also be reflective of patterns in phenotypic plasticity induced by environmental or maternal effects. The role of maternal effects are especially relevant in H. formosa as females are live bearing and provide nutrients to developing embryos via a placenta. We used a common garden experiment and a large factorial breeding experiment to explicitly test for genetically based differences among populations in their responses to environmental variation (norms of reaction). This laboratory work allowed a definitive diagnosis of which features actually represented local adaptations among populations of Heterandria formosa. Results showed that male body morphology has a significant genetic component and signs of population specific response to both the environment during post-parturition development and in response to the maternal environment during embyronic development. The narrowest scope of our work focuses on the evolution of body morphology within an experimental population of the Trinidadian guppy. Interestingly, the strength and direction of selection on phenotypic variation is not the same among all individuals within a population. In particular, genetic correlations between the sexes can produce intralocus sexual conflicts (ISC) when selection favors different trait values in the genders. This form of sexual conflict can inhibit the evolution of males and females so that neither sex can reach its optimal trait value. Theory suggests that ISC will have a minimal effect in populations off there adaptive peak such as those in a novel or variable environment. However, ISC is likely to have an inhibitory effect in populations in a stable environment near their adaptive, thereby limiting adaptive evolution. Here we used an experimental population of Trinidadian guppies in a novel environment to unravel the emergence of sexual conflict as populations adapt to novel environments. Guppies from a high predation environment were translocated to a drainage with minimal predation. The phenotypic evolution of the population was tracked monthly. This work incorporates mark-recapture methods, complete pedigree reconstruction using high throughput sequencing, and geometric morphometric shape analysis. Results indicate that there are fitness differences among individuals and different optimal values in males and females but the role of sexual conflict within this population is currently negligible. To conclude, our work investigated the environmental and genetic factors influencing the evolution of body morphology in poeciliid fish. We demonstrate the complicated nature of selection with different selective agents acting on different aspects of body morphology. We found that populations of fish have unique environmental maternal effects which may ultimately be adaptive. We also conclude that while there are optimal trait value differences in males and female guppies but being off the adaptive peak limits the influence of ISC on morphological evolution.
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Date Issued: 2018
Identifier: 2018_Su_Landy_fsu_0071E_14692
Format: Thesis

Title: Zika Virus Infection Induces DNA Damage Response and S-Phase Arrest in Human Cortical Neural Progenitors.
Creator: Hammack, Christy, Tang, Hengli, Megraw, Timothy L., Chadwick, Brian P., Gilbert, David M., Li, Yan, Zhu, Fanxiu, Florida State University, College of Arts and Sciences,...
Show moreHammack, Christy, Tang, Hengli, Megraw, Timothy L., Chadwick, Brian P., Gilbert, David M., Li, Yan, Zhu, Fanxiu, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus of significant public health concern closely related to other highly pathogenic flaviviruses, such as dengue virus (DENV) and West Nile virus (WNV). With the rise of ZIKV in Brazil in 2015, its potential link to microcephaly and other severe neurological birth defects prompted the World Health Organization to declare ZIKV a Public Health Emergency of International Concern. Since this time, numerous studies have provided ample...
Show moreZika virus (ZIKV) is a re-emerging mosquito-borne flavivirus of significant public health concern closely related to other highly pathogenic flaviviruses, such as dengue virus (DENV) and West Nile virus (WNV). With the rise of ZIKV in Brazil in 2015, its potential link to microcephaly and other severe neurological birth defects prompted the World Health Organization to declare ZIKV a Public Health Emergency of International Concern. Since this time, numerous studies have provided ample evidence to establish ZIKV as the causative agent of microcephaly, yet the molecular mechanisms underlying these neurodevelopmental defects are not well understood. We therefore establish a tractable experimental model system to investigate the impact of ZIKV on human neural development. We demonstrate that ZIKV efficiently infects human cortical neural progenitor cells (hNPCs) derived from induced pluripotent stem cells, but less efficiently infects other cells along the neural differentiation pathway, including immature cortical neurons. Infected hNPCs further release infectious ZIKV particles. Importantly, ZIKV infection disrupts cell cycle progression and induces cell death in hNPCs contributing to their attenuated growth. Global transcriptome analyses of ZIKV-infected hNPCs reveal transcriptional dysregulation, notably a downregulation of cell-cycle-related genes, highlighting the potential involvement of cell cycle pathways in ZIKV biology. We then study the molecular mechanisms by which ZIKV manipulates the cell cycle in hNPCs and the functional consequences of cell-cycle perturbation on the replication of ZIKV and related flaviviruses. We demonstrate that host cell-cycle disruption is unique to ZIKV among the flaviviruses tested, including DENV and WNV, however similar among the two strains of ZIKV tested, including the prototype Uganda strain and a Puerto Rican strain. ZIKV, but not DENV, infection induces DNA double-strand breaks, triggering the DNA damage response through the ATM/Chk2 signaling pathway, while suppressing activation of the ATR/Chk1 signaling pathway in hNPCs. Furthermore, ZIKV infection impedes the progression of cells through S phase thereby preventing the completion of host DNA replication. Recapitulating the S-phase arrest state with S-phase inhibitors leads to an increase in ZIKV replication, but not of WNV or DENV replication. Together, our results identify hNPCs as a direct target of ZIKV and the damaging impact of ZIKV on the growth of hNPCs. Importantly, our data demonstrate ZIKV’s ability to induce host DNA damage and arrest cell cycle progression, which results in a cellular environment favorable for its replication. As hNPCs generate the cortical neurons during early fetal brain development, the ZIKV-mediated growth retardation likely contributes to the neurodevelopmental defects of the congenital Zika syndrome.
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Date Issued: 2018
Identifier: 2018_Sp_Hammack_fsu_0071E_14286
Format: Thesis

Title: Elucidating a Mechanism for Hepatitis C Virus Induced Steatosis and Identification of Anti-Viral Compounds for Treating Zika Virus Infection.
Creator: Lee, Emily M. (Emily Michelle), Tang, Hengli, Ren, Yi, Keller, Thomas C. S., Zhu, Fanxiu, Meckes, David G., Florida State University, College of Arts and Sciences, Department of...
Show moreLee, Emily M. (Emily Michelle), Tang, Hengli, Ren, Yi, Keller, Thomas C. S., Zhu, Fanxiu, Meckes, David G., Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: During the past 30 years, there have been several Flaviviridae threats. Among them, Hepatatis C virus (HCV) emerged in the Western hemisphere as the previously unidentified etiological agent of non-A non-B hepatitis in transfusion patients. Less than twenty-five years after the 1989 discovery of HCV, several high-efficacy direct acting antivirals (DAAs) boasting a >95% cure rate were approved for treatment of HCV infected patients. While the advent of these DAAs has revolutionized the...
Show moreDuring the past 30 years, there have been several Flaviviridae threats. Among them, Hepatatis C virus (HCV) emerged in the Western hemisphere as the previously unidentified etiological agent of non-A non-B hepatitis in transfusion patients. Less than twenty-five years after the 1989 discovery of HCV, several high-efficacy direct acting antivirals (DAAs) boasting a >95% cure rate were approved for treatment of HCV infected patients. While the advent of these DAAs has revolutionized the prognosis for chronically infected HCV patients, a high level of HCV-induced disease burden remains due to low rate of diagnosis (% here) and the high cost of therapy. Without treatment, 80% of chronically infected hepatitis C individuals exhibit hepatic intracellular lipid accumulation, termed steatosis. This liver damage can eventually contribute to cirrhosis (20% of individuals) and hepatocellular carcinoma (5% of individuals), necessitating a liver transplant for patient survival. Hepatitis C virus (HCV) infection perturbs host lipid metabolism through both cellular and viral-induced mechanisms, with the viral core protein playing an important role in steatosis development. In the first three years of my Ph.D., I sought to identify mechanisms contributing to HCV-induced steatosis. This work was published and Spotlighted in Journal of Virology. In this study, we identified a liver protein, the cell death-inducing DFFA-like effector B (CIDEB), as a HCV entry host dependence factor that is downregulated by HCV infection in a cell culture model. We then further investigated the biological significance and molecular mechanism of this downregulation. Importantly, we validated our in vitro finding with an in vivo model system, and saw that HCV infection in a live mouse model downregulated CIDEB in the liver tissue. We also found that CIDEB gene knockout in a hepatoma cell line resulted in multiple aspects of lipid dysregulation that can contribute to hepatic steatosis, including reduced triglyceride secretion, lower lipidation of very low density lipoproteins, and increased lipid droplet (LD) stability. The potential link between CIDEB downregulation and steatosis was further supported by the requirement of HCV core and its LD localization for CIDEB downregulation, which utilized a proteolytic cleavage event that is independent of the cellular proteasomal degradation of CIDEB. In late 2015, the global scientific community became aware of the emergence and threat of another Flaviviridae virus called Zika virus (ZIKV) in Brazil. The previously obscure ZIKV, which had laid relatively dormant for the previous 70 years, began spreading rapidly through the Western hemisphere, thus prompting the World Health Organization (WHO) to declare a public health emergency in February 2016. In response to the global health emergency posed by the ZIKV outbreak and its link to microcephaly and other neurological conditions, we established a collaboration with Dr. Wei Zheng at the National Center for Advancing Translational Sciences (NCATS) at this time, with whom we together performed a drug repurposing screen of ~6,000 compounds that included approved drugs, clinical trial drug candidates and pharmacologically active compounds. We reported these results in Nature Medicine in August 2016. From this initial research, we identified 37 lead compounds that either inhibit ZIKV infection or suppress infection-induced caspase-3 activity in different neural cells. We found that emricasan, a pan-caspase inhibitor, inhibited ZIKV-induced increases in caspase-3 activity and protected human cortical neural progenitors in both monolayer and three-dimensional organoid cultures. Ten structurally unrelated inhibitors of cyclin-dependent kinases inhibited ZIKV replication. Niclosamide, a category B anthelmintic drug approved by the US Food and Drug Administration, also inhibited ZIKV replication. Finally, combination treatments using one compound from each category (neuroprotective and antiviral) further increased protection of human neural progenitors and astrocytes from ZIKV-induced cell death. We then continued our work to identify additional compounds by refining our high-throughput assay. We developed a high-throughput ZIKV-NS1 based FRET detection assay to rescreen all 6,000 compounds, and then validated 256 hits by a semi-automated viral titer assay we developed in our lab in collaboration with NCATS. From this combinatorial approach, we identified an additional 117 compounds for use in further antiviral development. Among these, we found a conserved role of proteasome inhibitors in inhibiting ZIKV infection, and identified additional lead compounds including emetine, an anti-protozoal small molecule compound. In collaboration with NCATS and Dr. Anil Mathew Tharappel who completed mouse studies, we found that emetine is effective in a live animal model at reducing Zika viral load and likely inhibits viral replication via a direct block on the ZIKV NS5 RNA dependent RNA polymerase.
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Date Issued: 2018
Identifier: 2018_Sp_Lee_fsu_0071E_14503
Format: Thesis

Title: Physiological Ecology of Elasmobranchs in the Gulf of Mexico and Northwestern Atlantic.
Creator: Prohaska, Bianca Karoli, Grubbs, R. Dean, Eckel, Lisa A., Travis, Joseph, DuVal, Emily H., Burgess, Scott C, Gelsleichter, James J., Florida State University, College of Arts...
Show moreProhaska, Bianca Karoli, Grubbs, R. Dean, Eckel, Lisa A., Travis, Joseph, DuVal, Emily H., Burgess, Scott C, Gelsleichter, James J., Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: A thorough understanding of the physiology of elasmobranchs (sharks, skates, and rays) is important from an applied aspect as most species are captured either directly or indirectly in commercial and recreational fisheries, or affected by other anthropogenic stressors such as habitat loss. Since we know very little about this group's physiology, it is unknown how they respond to these stressors, and how they survive stressful events. This is particularly important for endangered and...
Show moreA thorough understanding of the physiology of elasmobranchs (sharks, skates, and rays) is important from an applied aspect as most species are captured either directly or indirectly in commercial and recreational fisheries, or affected by other anthropogenic stressors such as habitat loss. Since we know very little about this group's physiology, it is unknown how they respond to these stressors, and how they survive stressful events. This is particularly important for endangered and threatened species such as the smalltooth sawfish Pristis pectinata, but also for those species that are data deficient like many of the deep-sea sharks in the Gulf of Mexico. Through this dissertation I aimed to investigate the physiology of a variety of elasmobranchs, and investigate ecological, evolutionary, and applied research questions. Similar to other elasmobranchs, the smalltooth sawfish Pristis pectinata is slow-growing, matures late in life, and produces relatively few young, all factors which have contributed to its sensitivity to dramatic population declines from overfishing and habitat loss. Currently, the physiological stress response of these fish to capture or to other physiological challenges such as habitat loss, climatic changes, or pollution is unknown. We examined basic stress physiology over ontogeny and as a function of capture using different fishing gears. We also examined stress parameters to test whether degraded habitat and water quality from altered habitats may have resulted in chronic stress in juveniles. Results suggested that the stress response to capture by all methods was low, particularly for blood lactate, compared to other elasmobranchs examined to date. Metabolic stress was found to change over ontogeny, with young of the year (YOY) eliciting the highest responses. Glucose, pCO2, bicarbonate, potassium, and hematocrit indicated gillnet capture induced greater stress responses than longline capture. Significantly higher metabolic stress was observed in YOY and juveniles captured in the two nurseries most influenced by anthropogenic activities, the Peace and Caloosahatchee rivers, than in the two relatively pristine nurseries in Everglades National Park. Prior to the Deep-Water Horizon (DwH) oil spill, little research effort was focused on studying the physiology of deep-sea sharks inhabiting the Gulf of Mexico. While the biology of these fishes remains virtually unknown, they are routinely captured in commercial fisheries as bycatch. Also unknown is what potential detrimental effects the DwH oil spill, which occurred at 1,500 m deep, has had and will continue to have on these organisms. The basic physiological post-capture stress response was examined and compared among seven deep-sea shark species including Mustelus sinusmexicanus, Mustelus canis, Squalus cubensis, Squalus clarkae, Centrophorus uyato, Centrophorus granulosus, and Hexanchus griseus and as functions of depth and proximity to the oil spill. Results suggested there may be taxonomic similarity in the stress response, but the responses we observed were more likely driven by habitat as two congeners that inhabit two distinct habitats displayed very different responses, and much closer to the other sharks in their respective habitats. We found a greater relative stress response in shallower inhabiting sharks as well as smaller bodied sharks. With increasing body temperature metabolic rate increases, as does the capture stress response; however, the core temperatures of the larger bodied deeper dwelling species were not altered as drastically as the smaller bodied sharks, indicating that the stress response of these sharks in this study may have been delayed. No increase in the stress response was detected with proximity of capture to the DwH oil spill site. The scalloped hammerhead Sphyrna lewini, and the great hammerhead Sphyrna mokarran are large, coastal to semi-oceanic shark species common to waters of the U.S. east coast and are regularly taken in commercial and recreational fisheries, particularly the bottom longline fishery, in this region. High rates of hooking mortality and low rates of population growth are believed to have caused severe declines in the U.S. Atlantic populations of these species. The objective was to determine the physiological stress induced by bottom longline capture in both S. lewini and S. mokarran, and to assess the post-release survivorship of S. lewini using survivorship pop-off archival satellite tags (PSATLIFE). Nine PSATLIFE tags were deployed and the results suggested 89% survival post release, with hook durations ranging from 6-136 minutes, and release conditions of excellent, good and fair. The one observed mortality experienced a hooking duration of 54 min and was released in fair condition. The physiological stress parameters lactate, pCO2, and pH were found to scale negatively with time since hooking and condition factor in both species. These data will be useful for providing at boat mortality estimates of S. mokarran and the physiological stress response to longline capture in both species to the Atlantic bottom longline fishery.
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Date Issued: 2018
Identifier: 2018_Fall_Prohaska_fsu_0071E_14867
Format: Thesis

Title: Neuromodulation of the Kv1.3 Ion Channel by Satiety and Metabolic Hormones.
Creator: Bell, Genevieve A., Fadool, Debra Ann, Schatschneider, Christopher, Bhide, Pradeep, Johnson, Frank, Keller, Laura R., Florida State University, College of Arts and Sciences,...
Show moreBell, Genevieve A., Fadool, Debra Ann, Schatschneider, Christopher, Bhide, Pradeep, Johnson, Frank, Keller, Laura R., Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Have you ever noticed when you are hungry and looking for something to eat, you can smell the freshly salted fries of McDonalds before seeing the golden arches? That is because our sense of smell, olfaction, is intimately linked with metabolic state and feeding behavior. In fact, many of the circulating hormones and gut peptides that regulate feeding behaviors have receptors located in the olfactory bulb (OB), and thus, have been reported to influence olfactory functioning; increasing...
Show moreHave you ever noticed when you are hungry and looking for something to eat, you can smell the freshly salted fries of McDonalds before seeing the golden arches? That is because our sense of smell, olfaction, is intimately linked with metabolic state and feeding behavior. In fact, many of the circulating hormones and gut peptides that regulate feeding behaviors have receptors located in the olfactory bulb (OB), and thus, have been reported to influence olfactory functioning; increasing sensitivity while hungry and decreasing odor perception once satiated. If metabolic state becomes disrupted, as observed in metabolic disorders such as obesity and Type 2 Diabetes, this can negatively impact one’s sense of smell, which is why many obese and/or diabetic patients also display symptoms of anosmia, or loss of smell. My laboratory has pinpointed the OB as an internal metabolic sensor, in particular, the mitral cells (MCs) within the OB, which are the major output neurons of the OB and are responsible for sending signals to higher-order processing regions in the brain. Gut peptides and important metabolic factors such as insulin and glucose can increase MC firing frequency via modulation of the voltage-gated potassium channel, Kv1.3. Kv1.3 channels are widely distributed across the body, but are highly expressed in the OB and predominantly located on MCs; Kv1.3 channel carries 60 – 80% of the outward current of these cells. The central theme of my dissertation involves understanding how excess energy substrates can perturb the operation of the brain to effect behavior of the organism. My research involves specifically targeting Kv1.3, that is modulated by circulating neuropeptides and hormones. Our laboratory has demonstrated gut peptides and important metabolic factors such as insulin, glucose, and glucagon-like peptide -1 (GLP-1) can increase MC firing frequency via modulation of Kv1.3, and ultimately modify olfactory and metabolic behaviors. This dissertation examines two aspects in which energy substrates modulate Kv1.3 activity, behaviorally and physiologically. Results from these experiments revealed that 1. Long-term intranasal delivery of insulin does not affect olfactory or metabolic behaviors, but changes meal bout and dampens Kv1.3 phosphorylation. 2. Intranasal delivery of the specific Kv1.3 inhibitor, stichodactyla (ShK) 186 toxin, partially improves metabolism in obese animals. 3. GLP-1 modulates Kv1.3 activity via serine phosphorylation. This work will further expand upon how feeding-related hormones can shape olfactory ability and bring forth new insight into using sensory systems to control food intake and overconsumption, a leading cause of obesity in America.
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Date Issued: 2018
Identifier: 2018_Su_Bell_fsu_0071E_14684
Format: Thesis

Title: Shifting Distributions of Marine Sponges and the Ecology of an Endemic Species.
Creator: Kaiser, Kathleen, Wulff, Janie L., Levitan, Don R., McCoy, Sophie J., Florida State University, College of Arts and Sciences, Department of Biological Science
Abstract/Description: The Florida Gulf of Mexico coast extends over both tropical and subtropical zones resulting in an intermingling of fauna typical to both zones. Cold winter water temperatures historically limited the distribution of many tropical species and allowed sub-tropical species to thrive. In the past 60 years, average winter sea surface temperatures have increased 2-3°C in the Northern Gulf of Mexico (NOAA OSPS), potentially allowing range expansion of tropical species and local extinction of...
Show moreThe Florida Gulf of Mexico coast extends over both tropical and subtropical zones resulting in an intermingling of fauna typical to both zones. Cold winter water temperatures historically limited the distribution of many tropical species and allowed sub-tropical species to thrive. In the past 60 years, average winter sea surface temperatures have increased 2-3°C in the Northern Gulf of Mexico (NOAA OSPS), potentially allowing range expansion of tropical species and local extinction of subtropical species. While poleward range expansion is possible for species along the Atlantic coast, species in the Gulf of Mexico face land barriers that prevent northward movement. Distribution patterns of sponges in the Northeastern Gulf of Mexico have changed since previous studies in the 1950’s and 1960’s. A little over half of the common species (56%) are still widespread throughout the Northeastern Gulf of Mexico. Eleven species (44%) previously reported as widespread are either missing entirely or are no longer widespread throughout the region. Two species are newly reported in the Northeastern Gulf of Mexico (Niphates erecta and Haliclona curacaoensis) and eight other species are more widespread than before. Caribbean species make up most of the common species composition of the Northeastern Gulf of Mexico (de Laubenfels 1953, Little 1963, Storr 1976, current study). Due to the limited latitudinal range of the Northeastern Gulf of Mexico it is necessary to look at a wider scale to determine species’ northern range limits. When compared to the wide-scale Caribbean fauna, it appears that only a handful of Caribbean species occure in the Gulf of Mexico and the North West Atlantic. Of 90 common Caribbean species only 27% (24 species) were found in the Northeastern Gulf of Mexico and 38% (34 species) at Gray’s Reef in Georgia U.S.A. while 53% (48 species) were not found in either the Northeastern Gulf of Mexico or Gray’s Reef (Hopkinson et al. 1983, Freeman et al. 2007). While the species composition of Caribbean sponges at Gray’s Reef and in the Northeastern Gulf of Mexico is similar, each contain unique species that are not found at the other location. This may be due to a combination of biotic and abiotic factors that differ between the Atlantic and Gulf coasts of Florida, aiding the migration of some sponge species while hindering others. One species commonly found in the Northeastern Gulf of Mexico is endemic to the region. Halichondria corrugata Diaz, van Soest & Pomponi, 1993 is the most abundant sponge in shallow seagrass beds and on dockside fouling communities in the Northeastern Gulf of Mexico. The variation in abiotic conditions between these habitats and H. corrugata’s ubiquity make it an interesting system to test what aspects of ecology allow it to be so successful. H. corrugata utilizes different ecological strategies in seagrass and dock habitats. H. corrugata is flexible in its reproductive strategy, utilizes predator avoidance techniques when needed, and seems to be resilient to light variation despite being reliant on photosynthetic symbionts for supplemental nutrition.
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Date Issued: 2018
Identifier: 2018_Su_Kaiser_fsu_0071N_14788
Format: Thesis

Title: The Biomechanical Evolution of Mammalian Prismatic Enamel with Potential Application to Biomimetic Ceramic Development.
Creator: Kuhn-Hendricks, Stephen Michael, Erickson, Gregory M., Oates, William, Inouye, Brian D., Parker, William C, Steppan, Scott J., Florida State University, College of Arts and...
Show moreKuhn-Hendricks, Stephen Michael, Erickson, Gregory M., Oates, William, Inouye, Brian D., Parker, William C, Steppan, Scott J., Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Biological hard materials are a remarkable class of materials combining large volumes of mineral with minute organic components into often complex, hierarchical microstructural arrangements. These intricate microstructures offer ideal systems from which form-function relationships can be dissected due to their limited functional demands. They are also of increasing interest to the materials science community due to their high combinations of stiffness and toughness unexpected of ceramic-like...
Show moreBiological hard materials are a remarkable class of materials combining large volumes of mineral with minute organic components into often complex, hierarchical microstructural arrangements. These intricate microstructures offer ideal systems from which form-function relationships can be dissected due to their limited functional demands. They are also of increasing interest to the materials science community due to their high combinations of stiffness and toughness unexpected of ceramic-like materials. Individually, each approach for understanding these materials has suffered from a lack of insight from the other field: the biological perspective has suffered from a lack of analytical rigor while the engineering perspective has been ignorant to the intricacies of evolution as needed to accurately infer the original and current function of these structures. Here I present and execute a unified framework for examining biological hard materials. In order to identify the mechanical import of microstructural changes, this framework tests changes in biologically relevant material properties by measuring mechanical response across the transformation series of microstructures observed in conjunction with ecological shifts. In order to apply this framework, I use mammalian dental enamel as a model system. Dental enamel is the most mineralized tissue in the vertebrate body and is non-repairable and irreplaceable if damaged. Arguably, it has only two functions: transfer masticatory loads to ingesta and resist its own degradation. In mammals, the evolution of a critical tissue constituent--the enamel prism--has resulted in a multitude of enamel microstructural arrangements, some of which have independently evolved consistently in ecologically similar contexts. I sought to characterize changes in the mechanical response of enamel microstructures by providing a survey of elastic modulus and fracture toughness for a diversity of mammals showing a broad array of microstructural forms. Considering the mechanics of damage to mammalian enamel as they pertain to documented microstructural changes within lineages, I then identified three critical functional transitions in enamel microstructures. These functional transitions include: (1) the evolution of the enamel prism, (2) the adaptation to a high wear diet, and (3) the adaptation to a high fracture diet. I investigated potential changes in material response across these transitions. Methodologically, I measured elastic modulus using instrumented nanoindentation across a series of reptilian and mammalian enamels to examine differences in resistance to elastic deformation. I then verified and executed a new method for determining the intrinsic fracture toughness of enamel, crack tip opening displacement, and identified changes in small scale resistance to fracture. I used Vickers microindentation to evaluate differences in resistance to plastic deformation. Lastly, I developed a novel method for quantifying fracture orientation, called Crack Analysis of Propagation Orientation (CAPO). CAPO identifies directions of preferred cracking and provides a proxy of resistance to large-scale fracture effects. These data provide consistent evidence that mammalian enamel microstructures are remarkably consistent in elastic modulus, intrinsic fracture toughness, and hardness. This consistency and their correspondence to values reported in the literature suggests that selection has acted to make enamel microstructures as stiff, hard, and intrinsically tough as possible given the inherent developmental constraints of amelogenesis and material constraints of hydroxyapatite. However, they display marked quantitative and qualitative differences in their resistance to large-scale fracture. Contact with hard particulates in the environment such as plant phytoliths or exogenous grit are expected to result in local indentation damage and the removal of enamel through microcrack growth. Grazing taxa have enamels which include modified radial enamel, a microstructure that channels indentation crack growth into a single direction and suppresses subsurface lateral crack growth. Together, these mechanisms would reduce the removal of enamel pieces by inhibiting microcrack coalescence and offer increased resistance to severe wear. Conversely, contact with large objects such as bone are expected to result in fractures which propagate across the tooth surface. Carnivoran Hunter-Schreger bands qualitatively suppress fracture across bands; this behavior could provide resistance to fatigue crack growth. These results provide evidence that mammalian enamel microstructures are consistent in many of the commonly reported material properties but differ primarily in their large-scale fracture behavior. They further offer avenues for biomimetic ceramic composites with consistent hardness and moduli but with potential damage and fatigue tolerance specific to the loading scenario.
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Date Issued: 2018
Identifier: 2018_Su_KuhnHendricks_fsu_0071E_14758
Format: Thesis

Title: Discovering Change Using Herbarium Specimens: Plant Phenology, Distributions, and Biological Outliers.
Creator: Pearson, Katelin D. (Katelin Delight), Mast, Austin R., Burgess, Scott C, Nelson, Gil, Riccardi, Gregory A, Florida State University, College of Arts and Sciences, Department of...
Show morePearson, Katelin D. (Katelin Delight), Mast, Austin R., Burgess, Scott C, Nelson, Gil, Riccardi, Gregory A, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Herbarium specimens and the professionals who collect them can be powerful resources for understanding significant biological change, and many opportunities remain to improve specimen data analysis, collection, and exploration to maximize this impact. In this thesis, I develop and apply novel approaches in each of these aspects of specimen data use. First, I use a new method of assessing specimen phenology to investigate differing phenological sensitivities of asteraceous plant species in the...
Show moreHerbarium specimens and the professionals who collect them can be powerful resources for understanding significant biological change, and many opportunities remain to improve specimen data analysis, collection, and exploration to maximize this impact. In this thesis, I develop and apply novel approaches in each of these aspects of specimen data use. First, I use a new method of assessing specimen phenology to investigate differing phenological sensitivities of asteraceous plant species in the U.S. Southeastern Coastal Plain—an under-studied region in the field of phenology. These analyses reveal contrasting phenological responses of spring- and fall-flowering species to warming climate in this region that could have significant ecological and evolutionary effects on, e.g., pollinator and herbivore interactions. Second, I propose two avenues by which the collecting community can contribute in an even greater capacity to studying biotic change: (1) by documenting and reporting specimen outliers, which could be indicators of change, and (2) by more consistently noting taxa associated with the specimens they collect, which could enable augmentation of existing occurrence data by up to 18% according to an analysis of over 84,000 specimen records. These contributions represent advances in the burgeoning field of biodiversity informatics, which has the potential to vastly improve our understanding of life on Earth and the changes it is undergoing.
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Date Issued: 2018
Identifier: 2018_Su_Pearson_fsu_0071N_14634
Format: Thesis

Title: Effects of Sperm Environment on the Evolution of Gamete Traits in Ciona Robusta.
Creator: Kosman, Ellen T., Levitan, Don R., Beerli, Peter, Winn, Alice A., Houle, David, Travis, Joseph, Florida State University, College of Arts and Sciences, Department of Biological...
Show moreKosman, Ellen T., Levitan, Don R., Beerli, Peter, Winn, Alice A., Houle, David, Travis, Joseph, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Fertilization is a complex process, and gamete traits can affect the rate at which sperm and egg collide and fuse, making them prime targets for selection. This is particularly true for broadcast spawners, whose fertilization success mainly depends on interactions between gametes. Gamete traits can modify collision and fusion rates affecting fertilization success, but sperm availability can affect the rate at which gametes can interact. Because of this the strength and direction of selection...
Show moreFertilization is a complex process, and gamete traits can affect the rate at which sperm and egg collide and fuse, making them prime targets for selection. This is particularly true for broadcast spawners, whose fertilization success mainly depends on interactions between gametes. Gamete traits can modify collision and fusion rates affecting fertilization success, but sperm availability can affect the rate at which gametes can interact. Because of this the strength and direction of selection on gamete traits to optimize fertilization success is dependent upon sperm availability. While many studies have examined differences in selection pressures due to sperm availability on individual traits, rarely has the effect of interactions between traits been examined. Yet, interactions between traits may have an important impact on selection by modifying the focal trait's effect on fertilization success, and that modification can be contingent on the sperm environment. For instance, eggs that increase collisions and are quicker to fuse with sperm may be more prone to polyspermy (reproductive failure due to multiple sperm fusions) at lower sperm availabilities than eggs that increase collisions but are slower to fuse with sperm. Therefore, determining how interactions between traits can affect fertilization success, and how that effect can change across different sperm environments, is important for understanding the selective pressures a particular trait may face for a given sperm environment. Additionally, while many studies have postulated that interactions between different male and female gamete recognition protein (GRP) variants can affect fertilization success by altering fusion rates, it has yet to be examined. In this dissertation, I present the results of manipulative and observational experiments designed to determine how interactions between a suite of gamete traits may affect fertilization success. I conducted a series of no-choice fertilization assays over a range of sperm availabilities, in order to determine whether collision rates, genetic variability in GRPs (which mediate compatibility), or interactions between the two were the most important in determining fertilization success. I also attempted to determine if there was a difference in compatibility between different male and female GRP variants by examining whether individuals garnered a greater share of paternity based on their respective genotypes. This was accomplished by conducting fertilization assays, in which the eggs were offered a mix of two males' sperm. I also examined whether there might be a functional link between genetic variation in GRPs and chemoattractant-mediated differences in sperm behavior. This was accomplished by examining whether there was a difference in sperm chemotaxis or chemokinesis based on the respective GRP genotypes of the eggs and sperm using video analysis and dichotomous chambers. Finally, I looked at whether the trends seen in the laboratory were found in a natural population. I correlated changes in settler density (as a proxy for sperm availability) with changes in collision trait values, as well as examining for increases in assortative mating based on GRP identity with increasing settler density. I found that interactions between traits tended to explain most of the variance in fertilization success for most sperm environments. While I was unable to determine differences in compatibility, my results suggest that sperm that have the same female GRP genotype as the eggs they were exposed to tended to garner a higher share of the paternity. Additionally, my results also suggest that sperm will aggregate around eggs when they both share the same GRP genotype at the receptor locus, offering a mechanism by which assortative mating between gametes based on GRP genotype could occur. I then found that assortative mating based on the female receptor genotype does occur in a natural population, as more homozygous settlers were produced than expected under random mating as settler density increased. Additionally, while I found that collision rate traits changed in the directions predicted from previous studies based on increasing settler density, that relationship could be modified by GRP genotype. This suggests that assortative mating based on multiple trait values can occur, most likely due to the fact that different combinations of traits can maximize fertilization success. Overall, interactions of gamete traits with compatibility played a large role in fertilization success, particularly as sperm density increased. These results highlight the importance of examining the interplay of multiple traits under differing spawning conditions, in order to truly understand how they can affect fitness, and shape trait evolution.
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Date Issued: 2018
Identifier: 2018_Fall_Kosman_fsu_0071E_14911
Format: Thesis

Title: Notch-Induced Neoplastic Tumorigenesis in a Drosophila Transition Zone Model.
Creator: Yang, Sheng-An, Deng, Wu-Min, Megraw, Timothy L., Arbeitman, Michelle N., Cui, Hongchang, McGinnis, Karen M., Florida State University, College of Arts and Sciences, Department...
Show moreYang, Sheng-An, Deng, Wu-Min, Megraw, Timothy L., Arbeitman, Michelle N., Cui, Hongchang, McGinnis, Karen M., Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Transition zones are regions in the animal body where two types of epithelial tissue meet. Many transition zones are known high-risk sites for tumorigenesis. However, little is known on why transition zones are more susceptible to tumor formation, mainly due to the lack of a suitable study model. In this dissertation, I report that the Drosophila salivary gland imaginal ring can be used as a model to study tumorigenesis in transition zones. Drosophila melanogaster imaginal rings are larval...
Show moreTransition zones are regions in the animal body where two types of epithelial tissue meet. Many transition zones are known high-risk sites for tumorigenesis. However, little is known on why transition zones are more susceptible to tumor formation, mainly due to the lack of a suitable study model. In this dissertation, I report that the Drosophila salivary gland imaginal ring can be used as a model to study tumorigenesis in transition zones. Drosophila melanogaster imaginal rings are larval tissues composed of progenitor cells that are essential for the formation of three adult tubular structures, including the salivary gland, foregut, and hindgut. In the first part of this dissertation (Chapter 2), I show that during the larval stage, Notch signaling is activated in all three imaginal rings and canonical Notch signaling positively controls cell proliferation in these imaginal tissues. In addition, Serrate (Ser) is the ligand provided from neighboring imaginal ring cells that trans-activates Notch signaling, whereas both Ser and Delta could cis-inhibit Notch activity when the ligand and the receptor are in the same cell. In the second part of this dissertation (Chapter 3 and 4), I demonstrate that constitutive activation of Notch signaling in the imaginal ring during the third larval instar stage is sufficient to induce neoplastic tumorigenesis in the tumor hotspot at the posterior end of salivary gland imaginal rings, which is also a transition zone between diploid salivary gland imaginal ring cells and polyploid salivary gland cells. In this region, local endogenous JAK-STAT and JNK activation creates a tissue microenvironment that is susceptible to oncogenic Notch induced tumorigenesis. JNK activates a matrix metalloprotease, MMP1, to determine where the neoplasms form. Moreover, ectopic MMP1 can transform the anterior area of the salivary gland imaginal ring, which is normally refractory to oncogenic Notch-induced tumorigenesis, into a tumor "hotspot". In the third part of this dissertation, I further report that the cells in tumor hotspot of salivary gland imaginal ring adopt an endoreplicative cell fate after the second instar larval stage. These endoreplicating cells are normally lost during the pupal stage. However, overexpression of Notch induces re-mitosis in these polyploid tumor hotspot cells, which results in aneuploidy contributing to advanced tumor development. Loss of endoreplication or re-mitosis activity is sufficient to rescue the malignancy of Notch-induced tumors. Taken together, these findings reveal how endogenous signaling creates tumor-favoring microenvironments, and post-endoreplication mitosis promotes neoplastic tumorigenesis at the Drosophila transitional zone model, and ultimately establish the salivary gland imaginal ring as an in vivo model for studies of site-specific tumorigenesis.
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Date Issued: 2018
Identifier: 2018_Fall_Yang_fsu_0071E_14869
Format: Thesis

Title: Insects in Variable Plant Patches.
Creator: Merwin, Andrew Charles, Underwood, Nora C., Inouye, Brian D., Cogan, Nicholas G., Winn, Alice A., Burgess, Scott C, Travis, Joseph, Florida State University, College of Arts and...
Show moreMerwin, Andrew Charles, Underwood, Nora C., Inouye, Brian D., Cogan, Nicholas G., Winn, Alice A., Burgess, Scott C, Travis, Joseph, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Animals move through landscapes where their resources are unevenly and often patchily distributed. When animals move and choose among their scattered resources in predictable ways, ecologists may be able to anticipate the spatial distribution of their populations and the relative strength of their trophic interactions (e.g. predation, or facilitation). Likewise, an understanding of animal movements can inspire the design and preservation of habitat for conservation or the promotion of...
Show moreAnimals move through landscapes where their resources are unevenly and often patchily distributed. When animals move and choose among their scattered resources in predictable ways, ecologists may be able to anticipate the spatial distribution of their populations and the relative strength of their trophic interactions (e.g. predation, or facilitation). Likewise, an understanding of animal movements can inspire the design and preservation of habitat for conservation or the promotion of ecosystem services. However, movement-based predictions of animal populations—and the human interventions these predictions inspire—are only as reliable as our understanding of the determinants of animal movement. The research presented here addresses three basic aspects of animals' environments and experiences that have the potential to influence our understanding of animal movement, population distributions, and ecological interactions: (1) animals' conspecific density (2) prior experience with resources, and (3) the composition and geometry of habitat patches. These topics are well-studied, but rarely in the context of spatially heterogeneous landscapes, and many prior studies have confounded important explanatory variables. For the first study, I performed a lab experiment using the bean beetle, Callosobruchus maculatus, and two of its host beans, Vigna unguiculata and V. radiata, to explore how consumer density influences resource choice and the relative distribution of damage among resources in a patch. My results demonstrated that the damage a focal resource type receives can depend on the frequency of neighboring resource types, and that this frequency dependence decreases with regional consumer density. These findings illustrate the importance of consumer density in mediating indirect effects among resources, and suggest that accounting for consumer density may improve our use of mixed‐crop pest management strategies. For the second study, I used a field experiment to test whether prior hostplant experience influences the distribution of offspring on and damage to hostplants among and within plant patches that varied in hostplant density and composition. Specifically, I reared diamondback moths, Plutella xylostella, on either collard or mustard plants (Brassica oleracea or B. juncea, respectively) and recorded the number of offspring on and damage to plants in three patch types within large field cages: two collards, four collards, and mixed patches of two collards and two mustards. I found that in cages with collard-reared moths, there were more offspring and damage per plant in four-collard patches than in two-collard patches, while mustard-reared moths did not respond to collard density. In contrast, I found no effect of natal hostplant experience on hostplant choice within mixed patches, and no influence of mustard plants on attacks on collards in mixed patches versus two-collard patches (i.e. there were no associational effects). These findings suggest that accounting for prior hostplant experience may improve our understanding of how some herbivores and their damage are distributed in patchy environments through time. For my final dissertation study, I used a correlated random walk model to make predictions for animal density in patches that vary in area and perimeter-to-area ratio. I then tested predictions from this model by manipulating the area and perimeter-to-area ratios of plant patches and observing the densities of two predaceous beetles: a relative habitat specialist, Calosoma sayi, and a relative habitat transient, Tetracha carolina carolina. The model predicted that as habitat specialists spend more time in patches relative to non-habitat, patches with lower perimeter-to-area ratios should have higher animal density. However, for relatively transient species, defined as spending more time within non-habitat than in the habitat patches, the model predicted slightly higher densities in higher perimeter-to-area patches or no difference between patch types. Area per se, in contrast, did not influence mean density. Contrary to the model, I found that in the field experiment patch area and perimeter-to-area ratio interacted to influence the movement and density of the relative habitat specialist, C. sayi. Their density increased significantly with patch size in high perimeter-to-area patches, but patch size had no influence on C. sayi's density within low edge-to-area patches. By contrast, densities of T. carolina were slightly higher in high perimeter-to-area ratio patches once the influence of plant height was accounted for, which was consistent with the model. These results underscore the importance of considering both patch area and perimeter-to-area ratio as well as species-specific movement behaviors for the management of habitat for ecosystem services. Together these projects highlight exciting new areas of consideration for the study of plant-insect interactions in heterogeneous habitats, which could improve our ability to predict insect distributions and interactions in natural and managed populations.
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Date Issued: 2018
Identifier: 2018_Fall_Merwin_fsu_0071E_14835
Format: Thesis

Title: The Role of Evolution in Maintaining Coexistence of Competitors.
Creator: Pastore, Abigail I. (Abigail Ilona), Miller, Thomas E., Bertram, R. (Richard), Inouye, Brian D., Steppan, Scott J., Winn, Alice A., Florida State University, College of Arts and...
Show morePastore, Abigail I. (Abigail Ilona), Miller, Thomas E., Bertram, R. (Richard), Inouye, Brian D., Steppan, Scott J., Winn, Alice A., Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Species interactions can regulate a population’s density and therefore can act as a selective force on that population. Such evolutionary responses have the potential to feedback and change ecological interactions between species. For species that compete for resources, the interaction between ecological and evolutionary dynamics will regulate the stability of the species interactions, determining whether competing species can coexist. The outcome of competition between species is determined...
Show moreSpecies interactions can regulate a population’s density and therefore can act as a selective force on that population. Such evolutionary responses have the potential to feedback and change ecological interactions between species. For species that compete for resources, the interaction between ecological and evolutionary dynamics will regulate the stability of the species interactions, determining whether competing species can coexist. The outcome of competition between species is determined by two factors: (1) niche overlap, or the similarity in how species use resources and are affected by their environment, and (2) fitness differences, or differences in how efficiently each species uses resources in their environment. Decreasing niche overlap will decrease competitive interactions, thereby stabilizing coexistence. Decreasing fitness differences makes species more equal in their competitive abilities, facilitating coexistence. In the absence of evolutionary constraints, both niche overlap and fitness differences among species are subject to change as a consequence of evolution among competitors, and thus ecological dynamics between two species will also change. In this dissertation, I develop a broader understanding of (1) how niche overlap and fitness differences between species change after evolution in response to competition, (2) how changes in niche overlap and fitness differences are mediated through changes in resource use of protists, and (3) what role evolutionary history plays in shaping ecological and evolutionary dynamics. I address these goals with a suite of approaches including theoretical models, an experimental lab system, and comparative methods. I constructed a quantitative genetic model of trait evolution, where the trait of a species determined its resource use, and found that species are prone to change in their niche overlap as well as their fitness differences as a result of trait evolution. However, the magnitude of changes in niche overlap and fitness differences were determined by the resource availability within the environments. When resources were broadly available, species changed more in their niche overlap, whereas when resources were narrowly available, species changed more in their fitness difference. To test these predictions, I developed a system in the laboratory where protists competed for a bacterial resource. Species were allowed to evolve in either monoculture or a two-species mixture; the effects of evolution on competition, niche overlap and fitness differences were quantified using parameterized models. In general I found that species tended to converge in their niche as a result of evolution, however, changes in fitness differences between species were larger and more influential on coexistence than changes in niche differences. Both increases in niche overlap, and increases in fitness differences decreased coexistence among species pairs. By describing the bacterial communities associated with these protists before and after selection I determined that protists tended to converge or not change in which bacteria they were consuming as a result of selection. Additionally, for eleven protist species, I determined whether traits or relatedness predicted competitive ability by placing species on a molecular phylogeny and conducting pairwise competition experiments for all pairs. I found no correlations, suggesting neither traits, nor evolutionary history was informative for explaining current ecological and evolutionary interactions in this deeply divergent clade. There are two major conclusions from this dissertation: (1) when species evolve in response to competition, changes in fitness differences may often be more important than changes in niche overlap, (2) evolution can, and may be likely to, decrease the ability of species to coexist through increases in niche overlap and increases in fitness differences. This work suggests that one must simultaneously consider the role of evolutionary and ecological processes to understand community processes. Specifically, when researchers are attempting to explain mechanisms of coexistence between species, they must consider how evolutionary dynamics may change the ecological interactions within communities of competitors.
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Date Issued: 2017
Identifier: FSU_FALL2017_Pastore_fsu_0071E_14135
Format: Thesis

Title: Composition and Stability of Single-Stranded DNA Viral Populations in Wastewater Treatment Plants.
Creator: Pearson, Victoria M., Rokyta, Darin, Beerli, Peter, Dennis, Jonathan Hancock, Hughes, Kimberly A., Tang, Hengli, Florida State University, College of Arts and Sciences,...
Show morePearson, Victoria M., Rokyta, Darin, Beerli, Peter, Dennis, Jonathan Hancock, Hughes, Kimberly A., Tang, Hengli, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Regular emergence and re-emergence of viral pathogens emphasizes the importance of understanding viral biogeography and migration. Single-stranded DNA (ssDNA) viruses are among the least understood groups of microbial pathogens, yet the group contains known agricultural pathogens, which infect both livestock and crops (Circoviridae and Geminiviridae), and model organisms (Microviridae). Wastewater treatment plants (WWTPs) receive water from multiple sources, becoming reservoirs for the...
Show moreRegular emergence and re-emergence of viral pathogens emphasizes the importance of understanding viral biogeography and migration. Single-stranded DNA (ssDNA) viruses are among the least understood groups of microbial pathogens, yet the group contains known agricultural pathogens, which infect both livestock and crops (Circoviridae and Geminiviridae), and model organisms (Microviridae). Wastewater treatment plants (WWTPs) receive water from multiple sources, becoming reservoirs for the collection of many viral families that infect a large range of hosts. Investigations utilizing high-throughput sequencing have determined that local viral diversity is extremely high but does not scale to produce an exponentially higher global diversity. It follows that similar genotypes can be found great distances apart, although they may not be permanent constituents of any single population. Transient genotypes have been observed in temporal surveys of closed systems, where genotypes migrate between individual populations. This study focused on the geographic and temporal population stability of single-stranded DNA (ssDNA) viruses in open systems. Sampling from WWTPs in three neighboring cities in Northwest Florida, which receive constant inflow and potentially receive the same viruses from the local environment, was conducted across a nine-month time span. A combination of polyethylene glycol (PEG) precipitation and filter concentration was used to isolate whole viral particles from the complex wastewater samples. The ssDNA viruses were isolated from larger viruses using a sucrose gradient for size selection and rolling circle amplification was performed to both bias the sample towards ssDNA and prepare the samples for high-throughput sequencing. Amplified genomes were sequenced using Illumina platforms and de novo assembled. Given the increased potential for migration, we expected the populations would be mostly homogenous with relatively few viruses that are unique to individual WWTPs. Viral genotypes with genetic similarity to Circoviridae, Geminiviridae, and Microviridae were recovered from all three WWTPs, however <25% of recovered genes match genotypes (>80% amino acid identity) recovered from neighboring sample sites. We determined that <10% of the genotypes were present in all three plants and the majority of genotypes were specific to one WWTP. Unexpectedly, the WWTPs that were closest to each other geographically were the least similar, and the plants geographically distant from each other had the most observed genetic overlap. These results highlight the high level of diversity within each population, while the high observed heterogeneity indicates localized genetic success and limited migration opportunities between the WWTPs. Throughout time the communities experienced a large degree of genetic turnover. Only 30% of the genotypes were present in more than one time point, 5% were recovered in three of more samplings and <1% were present in all five time points. This thesis concludes that viral genomes are continually moving through the environment and their presence in any given area may be temporary. Therefore, viruses are a continual selective force on their host species through the sheer volume of genetic potential in an area at any given time.
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Date Issued: 2017
Identifier: FSU_FALL2017_Pearson_fsu_0071E_14170
Format: Thesis

Title: Evolution of the Material Properties of Mineralized Dental Tissues.
Creator: Kay, David Ian, Erickson, Gregory M., Lenhert, Steven John, Oates, William, Steppan, Scott J., Florida State University, College of Arts and Sciences, Department of Biological...
Show moreKay, David Ian, Erickson, Gregory M., Lenhert, Steven John, Oates, William, Steppan, Scott J., Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: During their 473 million-year diversification, gnathostomes came to exploit an unprecedented variety of trophic niches. Modifications to dental form and mineralized tissue constituents (i.e. enamel, dentines and in a few taxa, cementum) facilitated their exploitation of novel prey and/or plant matter. In general, it has been assumed that the intra-tissue level biomechanics of these constituents had little bearing on whole-tooth functionality, aside from enamel in mammals showing dental...
Show moreDuring their 473 million-year diversification, gnathostomes came to exploit an unprecedented variety of trophic niches. Modifications to dental form and mineralized tissue constituents (i.e. enamel, dentines and in a few taxa, cementum) facilitated their exploitation of novel prey and/or plant matter. In general, it has been assumed that the intra-tissue level biomechanics of these constituents had little bearing on whole-tooth functionality, aside from enamel in mammals showing dental occlusion. Specifically, many mammals possess teeth that self-wear to functionality and show a diversity of derived dental tissues (e.g. prismatic enamel fabrics, coronal cementum) – some which have been shown to possess unique biomechanical attributes to resist wear and fracture. Here I formally test the hypothesis that the primitive gnathostome hard tissue material properties remained static prior to the cladogenesis of Mammalia. An ancillary goal is to glean initial insights on how the material properties of these dental tissues in non-mammalian and mammalian taxa may contribute to whole-tooth form, function, performance and diets. Properties were tested and examined using two standardized material science techniques, microindentation and nanoindentation, as well as a novel technique for quantifying fracture propagation from cracks formed during microindentation. The results from this investigation suggest these material properties are highly variable among gnathostome dentitions. Aside from hardness, there is not a significant relationship between most material properties and diet aside from enamel hardness. There are also complex fracture patterns seen in the enamels of mammals and chondrichthyans, showing that gnathostome lineages independently evolved properties to control fracture and damage done to tooth enamel. Overall, this study suggests that in the case of enamel hardness, natural selection operated at the tissue level to bring about shifts in tooth functionality throughout the gnathostome radiation. More material properties (i.e. fracture toughness) need to be investigated to uncover the true functional import of material properties in dental tissues and establish how the tissue complexes contributed to whole tooth function.
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Date Issued: 2017
Identifier: FSU_FALL2017_Kay_fsu_0071N_14152
Format: Thesis

Title: Why Dominant Individuals Cooperate — Fitness Consequences of Cooperative Courtship in a System with Variable Cooperative Display Coalitions.
Creator: Jones, Megan Anlis, DuVal, Emily H., Mesterton-Gibbons, Mike, Hughes, Kimberly A., Houle, David C., Steppan, Scott J., Florida State University, College of Arts and Sciences,...
Show moreJones, Megan Anlis, DuVal, Emily H., Mesterton-Gibbons, Mike, Hughes, Kimberly A., Houle, David C., Steppan, Scott J., Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Understanding the evolution of cooperative behaviors is a major goal of evolutionary biology, but the majority of research in this field has focused on why helpers assist others. Helpers’ reproductive costs introduce a clear paradox to our understanding of natural selection as helpers in cooperative systems apparently sacrifice reproductive opportunities to increase others’ fitness. This puzzle in cooperative behaviors has led to significant advances in our understanding of indirect and...
Show moreUnderstanding the evolution of cooperative behaviors is a major goal of evolutionary biology, but the majority of research in this field has focused on why helpers assist others. Helpers’ reproductive costs introduce a clear paradox to our understanding of natural selection as helpers in cooperative systems apparently sacrifice reproductive opportunities to increase others’ fitness. This puzzle in cooperative behaviors has led to significant advances in our understanding of indirect and delayed fitness benefits for helpers. However, as cooperation results from the interaction of individuals that may have very different incentives for participation it is equally important to understand whether and how cooperation benefits the dominant recipients of this help. There has been relatively little attention paid to why the recipient of the apparent help participates in the cooperative relationship, in part because the advantage to the dominant individual seems apparent in many systems. Existing work reveals a variety of potential benefits for dominant individuals and that the benefits for dominants may be less obvious than assumed. To date investigations into costs and benefits of cooperation to dominant individuals have been largely limited to cooperative breeding behavior. My dissertation research investigates the fitness consequences of cooperative courtship display for dominant individuals, in the White-ruffed Manakin, Corapipo altera. Manakins (Aves: Pipridae) are small, primarily lekking passerines, and, in some species, males cooperate in their courtship displays. Previous work on manakin cooperative display behavior has focused on benefits to subordinate males. The fitness consequences of cooperation for dominant individuals has not yet addressed in a system with variation in cooperative strategies. I found strong evidence of cooperation among male C. altera. I also found that, within a single population of C. altera on the Atlantic slope of Costa Rica, some males participate in coordinated display with other males (45.4±20% were classified as cooperative in any given year), while other males appear to only display singly. My dissertation research investigated the causes and consequences of cooperation by dominant C. altera males by quantifying aspects of the males' fitness including how inclusive fitness benefits may facilitate the maintenance of cooperative display coalitions and the consequences of cooperative display coalitions for males’ annual reproductive success, survival, and social status — important parts of lifetime fitness for long-lived, iteroparous species including C. altera. I found that cooperative males were not more closely related than expected at random from the population. Males that cooperated did not have higher annual reproductive success than males that displayed solo nor was there a significant difference in the frequency of copulations after a solo courtship display and a courtship display by multiple males. In a survival analysis, cooperation did not significantly affect the survival of dominant males. There was no consistent pattern of cooperation (or non-cooperation) among males across their tenure as dominant male: some were always cooperative, some always non-cooperative, but many males with multi-year tenures switched between cooperative and non-cooperative statuses. However, more males than expected employed strictly solo strategies across their tenure as dominant individuals, given the population-wide rates of survival and cooperation. The degree to which males cooperated, defined as the proportion of tenure classified as cooperative, was unrelated to variation in lifespan or length of tenure as a dominant male. Additionally, the proportion of total tenure classified as cooperative did not explain the patterns of lifetime reproductive success. Together, these results reject the hypotheses that dominant males in cooperative partnerships gain indirect or direct fitness benefits from their associations with subordinate males. Seeking to understand processes underlying patterns of fitness consequences from cooperative behaviors, I conducted three experiments to determine if males at sites where the dominant male was cooperative were faster or more intense in their response to an experimental stimulus. Cooperative males were not faster to respond to a female at the display site nor were they faster to respond to the vocalization of an unknown male conspecific at the display site. Cooperative males were not significantly more likely to respond to a predator model, however, they were significantly more likely to spend time near the snake and lizard models. There could be benefit of sociality in the detection of terrestrial predators. This research addresses previously unexplored aspects of cooperative courtship display, and therefore represents a significant contribution to the more general understanding of the costs and benefits of cooperation. The variation in the amount of cooperation expressed by different individuals of this species offers a unique opportunity to separate the fitness consequences of cooperation by comparing differences in success not only among individuals, but also those among displays in different cooperative contexts by the same individual.
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Date Issued: 2017
Identifier: FSU_SUMMER2017_Jones_fsu_0071E_13625
Format: Thesis

Title: Reproductive Dynamics of Gulf Black Sea Bass in the Northeastern Gulf of Mexico.
Creator: Mckenzie, Ryan Wilson, Coleman, Felicia C., DuVal, Emily H., Travis, Joseph, Florida State University, College of Arts and Sciences, Department of Biological Science
Abstract/Description: Our knowledge of the reproductive dynamics of many economically important marine fish species is remarkably poor. This limits our ability to assess and manage the effects of exploitation on their reproductive potential. The Gulf Black Sea Bass Centropristis striata melana is a temperate serranid that contributes to both recreational and commercial fisheries in the state of Florida, however, the reproductive dynamics of this species is not well understood. To fill this gap, I conducted a...
Show moreOur knowledge of the reproductive dynamics of many economically important marine fish species is remarkably poor. This limits our ability to assess and manage the effects of exploitation on their reproductive potential. The Gulf Black Sea Bass Centropristis striata melana is a temperate serranid that contributes to both recreational and commercial fisheries in the state of Florida, however, the reproductive dynamics of this species is not well understood. To fill this gap, I conducted a fisheries-independent survey to explore the spatial and demographic scales of spawning populations in the northeastern Gulf of Mexico. To ensure effective and non-biased sampling, I assessed gear type and fish behavior sampling biases for the Gulf Black Sea Bass. Baited fish traps and hook-and-line were equally selective for fish size, however, hook-and-line had a higher catch efficiency. Body size was strongly correlated to social dominance in the Gulf Black Sea Bass, however, larger individuals in the population were not more susceptible to hook-and-line gears. These results indicated that hook-and-line was the optimal sampling method with relatively high efficiency and low sampling bias. Using hook-an-line fishery-independent surveys, I assessed the spatial and temporal dynamics of the Gulf Black Sea Bass spawning populations to test whether spawning populations were consistent across spawning habitats and describe demographic trends in spawning. Spawning populations were not consistent across available spawning habitat and displayed a high degree of spatial variability over scales of no more than 10 kilometers. These patterns were likely influenced by juvenile recruitment rates. Demography was a clear factor in the timing of reproduction as the proportion and average size of females and males significantly changed over the course of the spawning season. Larger females began spawning earlier in the spawning season and larger males were present on spawning habitats for longer periods. Overall, the findings of this study highlighted the important roles of spatial and demographic variation in the reproduction of the Gulf Black Sea Bass, and warrant future investigation due to their implications into the conservation and management of this economically important fishery.
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Date Issued: 2017
Identifier: FSU_SUMMER2017_Mckenzie_fsu_0071N_14086
Format: Thesis

Title: The Role of Seasonal and Geographic Temperature Variation in the Life Cycle of the Clonal Sea Anemone Diadumene Lineata (Verrill).
Creator: Ryan, Wilbur Helcat, Miller, Thomas E. (Professor of Biological Science), Huettel, Markus, Hughes, Kimberly A., Levitan, Donald R., Wulff, Janie L., Florida State University,...
Show moreRyan, Wilbur Helcat, Miller, Thomas E. (Professor of Biological Science), Huettel, Markus, Hughes, Kimberly A., Levitan, Donald R., Wulff, Janie L., Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Clonality is the general term that encompasses all manner of pinching, splitting, budding, and fragmenting behaviors by which organisms divide their somatic body tissues into more or less independent units. It can be as straight forward as fragments of a sponge surviving after being rent apart by a hurricane or as convoluted as the telescoping generations of parthenogenic aphids. Clonality is a widespread feature of animal life cycles and the degree of clonal investment is expected to affect...
Show moreClonality is the general term that encompasses all manner of pinching, splitting, budding, and fragmenting behaviors by which organisms divide their somatic body tissues into more or less independent units. It can be as straight forward as fragments of a sponge surviving after being rent apart by a hurricane or as convoluted as the telescoping generations of parthenogenic aphids. Clonality is a widespread feature of animal life cycles and the degree of clonal investment is expected to affect everything from spatial genotypic and genetic structure to evolutionary dynamics and ecology interactions. Yet, the shear diversity and complexity of clonal behavior has hampered efforts to develop a general understanding of how and why clonality evolves as the adaptive benefits of these behaviors may be as idiosyncratic as the mechanisms by which cloning occurs. Contrary to some past formulations of the problem, the production of clonal progeny is not typically an alternative to sexual reproduction, as most clonal organisms also reproduce sexually. While there is often an immediate tradeoff where a unit of energy can either be invested in gametes or clonal progeny at any given time, there is not inherently a tradeoff between asexual and sexual reproduction over the span of a lifetime. Dividing somatic tissue in to separate units can be a way of increasing total lifetime fecundity by increasing total biomass, more efficiently colonizing open space or promoting longevity by spreading the risk of mortality over spatially-separated somatic units. With this perspective, understanding the adaptive value of clonality becomes a matter of analyzing the holistic suite of fitness effects that arise from variation in allocating energy among unitary growth, clonal propagation and gametogenesis. The amount of energy available and the fitness value of a particular investment strategy are governed in large part by the environment and so understanding the environmental context is key to understanding the forces shaping life cycle evolution. Temperature, in particular, affects the metabolic cost of maintaining body tissues and is key in determining the energetically optimal body size for a unitary animal. Where temperatures fluctuate seasonally or where clonal replicates may spread across a heterogeneous landscape, the reaction norm of fission rate, body size or traits associated with gamete production may be an important target of selection, influencing which life cycle patterns can evolve. In this dissertation I examine the influence of seasonal and geographic temperature variation on fission rate, body size and gamete production of the clonal sea anemone, Diadumene lineata (Verrill 1869), to better understand the constraints and tradeoffs that govern the evolution of resource allocation strategy; and ultimately, the factors that drive the evolution of clonality in this species. Through a combination of laboratory experiments, field observations, optimality modeling and genetic tools I demonstrate that (1) fission rates are strongly temperature dependent, resulting in seasonal and geographic variation in clonal behavior, (2) the production of gametes is closely tied to body size and shows an inverse latitudinal pattern with fission rate, (3) the observed reaction norm of fission rate with temperature is consistent with selection to maximize gamete production across the locally experienced range of temperatures, as opposed to selection for maximum clonal proliferation, per se, and (4) there is a latitudinal decrease in genotypic richness and diversity that corresponds with changes in fission rate, suggesting that variation in fission rate leads to changes in the spatial structure of genetic variation among sites. Together, these results are consistent with the hypothesis that clonality is adaptive under conditions where individual body size is constrained by the environment. Under these conditions more gametes may be produced over a lifetime by genets dividing somatic tissue into multiple small units rather than remaining a single large unit. In this species, there is an immediate cost to dividing a large body into two pieces as the number of gametes produced by two small individuals sums to less than those produced by a large individual, yet, the lost reproductive potential may be able to be compensated for over time by an increased growth rate at a smaller body size. Additional costs and benefits imposed by changes in mortality rate, competitive ability or mate choice as fission rate changes remain to be investigated and may be equally important in understanding and predicting the evolution of clonal behavior in this and other species.
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Date Issued: 2017
Identifier: FSU_SUMMER2017_Ryan_fsu_0071E_13999
Format: Thesis

Title: Assortative Mating in the Tropical Sea Urchin Lytechinus Variegatus.
Creator: Nunez, Jose Alberto Moscoso, Levitan, Donald R., Hughes, Kimberly A., Burgess, Scott C., Florida State University, College of Arts and Sciences, Department of Biological Science
Abstract/Description: Non-random mating is presumed to be an important mechanism that allows for the maintenance of genetic variation. Assortative mating has been studied extensively in organisms that possess defined ways in which sperm is transferred to eggs (e.g. via copulation, courtship or vector assisted pollination in plants), but rarely in broadcast spawners. Broadcast spawning is perceived as a mating event that allows for mixing of gametes and promotes random mating. However, there are multiple pathways...
Show moreNon-random mating is presumed to be an important mechanism that allows for the maintenance of genetic variation. Assortative mating has been studied extensively in organisms that possess defined ways in which sperm is transferred to eggs (e.g. via copulation, courtship or vector assisted pollination in plants), but rarely in broadcast spawners. Broadcast spawning is perceived as a mating event that allows for mixing of gametes and promotes random mating. However, there are multiple pathways in which spawning adults can affect fertilization of gametes in non-random ways. For example, positive assortative mating can occur in broadcast spawners if similar phenotypes spawn closer together in space or time, or possess similar gamete recognition proteins that expedite fertilization. Here, I propose to examine assortative fertilization, patterns of aggregation and gamete recognition protein genotype of the sperm bindin gene as a function of spine color in the sea urchin Lytechinus variegatus as well as evaluating deviations from Hardy-Weinberg Equilibrium (HWE) based on color. Results indicate that laboratory crosses of urchins within color morphs yielded higher fertilization success than crosses between color morphs. Field surveys determined that these sea urchins are aggregating by color at times of their reproductive season when they are more likely to spawn. Tests for HWE using field data of urchin phenotypes suggest strong deviations from HWE. However, DNA sequences of regions of the sperm bindin gene for sea urchins of different color do not show evidence of genetic structure of the population. Paternal success in broadcast spawners is largely determined by the proximity of males to spawning females and the compatibility between them at the time they release their gametes. Selection is predicted to favor traits and behaviors that increase the likelihood of spawning near a more compatible neighbor. These results provide strong evidence for assortative mating and an explanation for the maintenance of color variation in this species.
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Date Issued: 2017
Identifier: FSU_SUMMER2017_Moscoso_fsu_0071N_14093
Format: Thesis

Title: Characterizing the Functional Role and Epigenetic Regulation of Large Tandem Repeats on the Inactive X Chromosome.
Creator: Darrow, Emily M. (Emily Michelle), Chadwick, Brian P., Hurt, Myra M., McGinnis, Karen M., Deng, Wu-Min, Bass, Hank W., Florida State University, College of Arts and Sciences,...
Show moreDarrow, Emily M. (Emily Michelle), Chadwick, Brian P., Hurt, Myra M., McGinnis, Karen M., Deng, Wu-Min, Bass, Hank W., Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: X chromosome inactivation (XCI), the mammalian form of dosage compensation, is a canonical example of epigenetic regulation and involves the transcriptional repression of nearly an entire chromosome (Xi) while preserving the transcriptional activity of its homologue (Xa) in females. Since the initial report describing a dense nuclear cytological feature in female feline neurons and Mary Lyon’s subsequent hypothesis of random X chromosome inactivation as a means of compensating for the lack of...
Show moreX chromosome inactivation (XCI), the mammalian form of dosage compensation, is a canonical example of epigenetic regulation and involves the transcriptional repression of nearly an entire chromosome (Xi) while preserving the transcriptional activity of its homologue (Xa) in females. Since the initial report describing a dense nuclear cytological feature in female feline neurons and Mary Lyon’s subsequent hypothesis of random X chromosome inactivation as a means of compensating for the lack of two X chromosomes in males, XCI has yielded decades of insights into the mechanisms of epigenetic regulation. This dissertation focuses on the three-dimensional organization of the Xi and the functional potential of large tandem repeats. The large X-linked tandem repeat, DXZ4, adopts a euchromatic conformation on the Xi in contrast to the largely heterochromatic chromosome and is able to form CTCF-dependent interactions with other euchromatic repeats exclusively on inactive X chromosome in females. We demonstrate here that DXZ4 has a critical role in maintaining the three-dimensional organization of the Xi as well as the separation of multi-megabase domains containing different types of heterochromatin. While characterizing the genomic interval of DXZ4, we uncovered transcriptional activity corresponding to two novel, long non-coding RNAs (lncRNAs) which originate on opposite sides of the DXZ4 and are transcribed antisense to one another. Both of these lncRNAs traverse the array in human embryonic stem cells (hESCs). Developmentally associated transcription suggests a potential connection between their transcription activity and maintenance of a heterochromatic DXZ4 on the Xa and male X prior to differentiation. Mouse and human genomes largely share the same gene content in accordance with Ohno’s law; however, the mouse genome has undergone rearrangements involving large syntenic blocks and has acquired several multi-megabase, lineage-specific regions of repetitive DNA that are absent in human. To further our understanding of the mouse inactive X chromosome and highlight another difference between human and mouse XCI, we characterized an approximately 20-Mb repeat that, similar to DXZ4, displays marks of euchromatin on the Xi and conversely displays marks of heterochromatin on the Xa. Overall, this work gives new insight into the function and epigenetic regulation of macrosatellites as well as the relationship between higher-order chromatin organization and heterochromatin maintenance of the Xi.
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Date Issued: 2017
Identifier: 2018_Sp_Darrow_fsu_0071E_14252
Format: Thesis

Title: Dissecting the Roles of Signaling Pathways and microRNAs in Proliferation and Tumorigenesis.
Creator: Shu, Zhiqiang, Deng, Wu-Min, Ma, Teng, Arbeitman, Michelle N., Cui, Hongchang (Professor of Biological Science), McGinnis, Karen M., Florida State University, College of Arts...
Show moreShu, Zhiqiang, Deng, Wu-Min, Ma, Teng, Arbeitman, Michelle N., Cui, Hongchang (Professor of Biological Science), McGinnis, Karen M., Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Tissue integrity and homeostasis are accomplished through strict spatial and temporal regulation of cell growth and proliferation during development. A derailment of such balance may lead to tumorigenesis. Various signaling pathways have emerged as major growth regulators across metazoans; yet, how differential growth within a tissue is spatiotemporally coordinated remains largely unclear. In the first half of my dissertation, I answered a fundamental question: How cell growth and...
Show moreTissue integrity and homeostasis are accomplished through strict spatial and temporal regulation of cell growth and proliferation during development. A derailment of such balance may lead to tumorigenesis. Various signaling pathways have emerged as major growth regulators across metazoans; yet, how differential growth within a tissue is spatiotemporally coordinated remains largely unclear. In the first half of my dissertation, I answered a fundamental question: How cell growth and proliferation are differentially regulated in the wing imaginal disc. I report a role of a growth modulator Yorkie (Yki), the Drosophila homolog of Yes-associated protein (YAP), differentially regulates its targets in Drosophila wing imaginal discs, whereby Yki interacts with its transcriptional partner, Scalloped (Sd), the homolog of the TEAD/TEF family transcription factor in mammals, to control an essential cell-cycle regulator Cyclin E (CycE). Interestingly, when Yki was coexpressed with Fizzy-related (Fzr), a Drosophila endocycle inducer and homolog of Cdh1 in mammals, surrounding hinge cells displayed larger nuclear size than distal pouch cells. The observed size difference is attributable to differential regulation of CycE, a target of Yki and Sd, the latter of which can directly bind to CycE regulatory sequences, and is expressed only in the pouch region of the wing disc starting from the late second-instar larval stage. During earlier stages of larval development, when Sd expression was not detected in the wing disc, coexpression of Fzr and Yki did not cause size differences between cells along the proximal-distal axis of the disc. I show that ectopic CycE promoted cell proliferation and apoptosis, and inhibited transcriptional activity of Yki targets. These findings suggest that spatiotemporal expression of transcription factor Sd induces differential growth regulation by Yki during wing disc development, highlighting coordination between Yki and CycE to control growth and maintain homeostasis. In the second half of my dissertation, I studied the roles of an important class of noncoding RNAs miRNAs in cancers. Despite their emergence as an important class of noncoding RNAs involved in cancer cell transformation, invasion, and migration, the precise role of microRNAs (miRNAs) in tumorigenesis remains elusive. To gain insight into how miRNAs contribute to primary tumor formation, I conducted an RNA sequencing (RNA-Seq) analysis of Drosophila wing disc epithelial tumors induced by knockdown of a neoplastic tumor-suppressor gene (nTSG) lethal giant larvae (lgl), combined with overexpression of an active form of oncogene Ras (RasV12), and identified 51 mature miRNAs that changed significantly in tumorous discs. Followed by an in vivo tumor enhancer and suppressor screen in sensitized genetic background, I identified 10 tumor-enhancing miRNAs and 11 tumor-suppressing miRNAs that contributed to the nTSG defect-induced tumorigenesis. Among these, four tumor-enhancing and four tumor-suppressing miRNAs have human homologs, which have been reported to actively contribute to various human cancers. From this study, I also identified 29 miRNAs that individually had no obvious role in enhancing (18) or alleviating (12) tumorigenesis despite their changed expression levels in nTSG-tumors. Further studies showed that several signaling pathways, including EGFR, JNK, JAK/STAT, and Hippo signaling, may collaborate with the nTSG defect to induce tumors. This systematic analysis, which include both RNA-seq and in vivo functional studies, helps to categorize miRNAs into different groups based on their expression profile and function relevance in epithelial tumorigenesis, whereas the evolutionarily conserved tumor-enhancing and -suppressing miRNAs could provide potential therapeutic targets for epithelial tumor treatment.
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Date Issued: 2017
Identifier: FSU_SUMMER2017_Shu_fsu_0071E_13966
Format: Thesis

Title: Altered Nucleosome Positions at Transcription Start Sites in Maize Haplotypes and Mutants of Putative Chromatin Remodelers.
Creator: Stroud, Linda Kozma, McGinnis, Karen M., Hurt, Myra M., Bass, Hank W., Chadwick, Brian P., Dennis, Jonathan Hancock, Florida State University, College of Arts and Sciences,...
Show moreStroud, Linda Kozma, McGinnis, Karen M., Hurt, Myra M., Bass, Hank W., Chadwick, Brian P., Dennis, Jonathan Hancock, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Chromatin remodelers alter DNA-histone interactions in eukaryotic organisms, and have been well characterized in yeast and Arabidopsis. While there are maize proteins with similar domains as known remodelers, the ability of the maize proteins to alter nucleosome position has not been reported. Mutant alleles of genes encoding several maize proteins (RMR1, CHR101, CHR106, CHR127, CHR156, CHB102, and CHR120) with similar functional domains to known chromatin remodelers were identified. Altered...
Show moreChromatin remodelers alter DNA-histone interactions in eukaryotic organisms, and have been well characterized in yeast and Arabidopsis. While there are maize proteins with similar domains as known remodelers, the ability of the maize proteins to alter nucleosome position has not been reported. Mutant alleles of genes encoding several maize proteins (RMR1, CHR101, CHR106, CHR127, CHR156, CHB102, and CHR120) with similar functional domains to known chromatin remodelers were identified. Altered expression of Chr101, Chr106, Chr127, Chr156, Chb102, and Chr120 was demonstrated in plants homozygous for the mutant alleles. These mutant genotypes were subjected to nucleosome position analysis to determine if misregulation of putative maize chromatin proteins would lead to altered DNA-histone interactions. Nucleosome position changes were observed in plants homozygous for chr101, chr106, chr127, chr156, chb102, and chr120 mutant alleles, suggesting that CHR101, CHR106, CHR127, CHR156, CHB102, and CHR120 may affect chromatin structure. The role of RNA polymerases in altering DNA-histone interactions was also tested. Changes in nucleosome position were demonstrated in homozygous mop2-1 individuals. These changes were demonstrated at the b1 tandem repeats and at newly identified loci. While the α-amanitin-inhibited RNA polymerase II demonstrated reduced expression of an RNA polymerase II transcribed gene, no changes in nucleosome position were detected in the α-amanitin-treated plants. Additionally, differential DNA-histone interactions and altered expression of putative chromatin remodelers in different maize haplotypes suggest a role for differentially expressed chromatin proteins in haplotype-specific variation.
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Date Issued: 2017
Identifier: FSU_SUMMER2017_Stroud_fsu_0071E_13987
Format: Thesis

Title: Replication Timing Regulation and Chromatin Structure Dynamics during the Cell Cycle and Development.
Creator: Dileep, Vishnu, Gilbert, David M., Lemmon, Alan R., Bass, Hank W., Chadwick, Brian P., Dennis, Jonathan Hancock, Florida State University, College of Arts and Sciences,...
Show moreDileep, Vishnu, Gilbert, David M., Lemmon, Alan R., Bass, Hank W., Chadwick, Brian P., Dennis, Jonathan Hancock, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Eukaryotic genomes replicate via the synchronous firing of clusters of origins that together produce multi-replicon domains, each of which replicates at a defined time during S-phase. This temporal program is termed the DNA replication-timing program. Replication Timing (RT) is a stable epigenetic property that is cell type specific and is extensively regulated during differentiation in units that range from 400-800kb called replication domains. DNA that replicates at distinct times during S...
Show moreEukaryotic genomes replicate via the synchronous firing of clusters of origins that together produce multi-replicon domains, each of which replicates at a defined time during S-phase. This temporal program is termed the DNA replication-timing program. Replication Timing (RT) is a stable epigenetic property that is cell type specific and is extensively regulated during differentiation in units that range from 400-800kb called replication domains. DNA that replicates at distinct times during S-phase is also spatially separated in the nucleus. Consistent with this, the binary nuclear compartments defined by chromatin spatial proximity maps, align precisely with the replication-timing program. But the dynamics of this relationship during differentiation and cell cycle have been poorly understood. To this end, we first showed that there is a coordinated switch in nuclear compartment along with a switch in replication timing during differentiation. It was also observed that regions of the genome that switch replication timing and nuclear compartment continue to maintain their structural boundaries. Genome-wide analysis of replication domains revealed that they are indeed stable structural units corresponding to Topologically-Associating Domains (TADs) defined by Hi-C. Next we showed that the interphase chromatin structure consisting of TADs and their long-range contacts are established during early G1 coincident with the establishment of the replication-timing program. We also show that developmentally regulated regions of the genome have fundamentally different higher order structure. In G2 phase, the replication timing-program is lost while inter-phase chromatin structure acquired in early G1 was retained. This shows that interphase chromatin structure is not sufficient to dictate RT and lead us to hypothesize that the chromatin structure set-up during early G1 may act as a scaffold to seed the assembly of some factor capable of setting replication initiation thresholds. The de-coupling of chromatin structure and RT could then be due to the removal of this factor during S-phase. Consistent with this hypothesis, we discovered a protein Rif1 that enters the nucleus right after mitosis and its knockout has a profound disruptive effect on RT in both mouse and human cells. Lastly, we explored the conservation of replication timing at single cell level that revealed a highly conserved yet stochastic regulation of replication timing. Surprisingly, the intrinsic (within cell) stochasticity and the extrinsic (cell-to-cell) stochasticity were similar. This is consistent with a model of replication timing regulation where the timing is the outcome of stochastic origin firing and is not affected by the precise environment within a cell. In summary, the work descried in this thesis uncovers a model where replication-timing is regulated at the unit of chromatin structure called TADs, which are generally stable across cell-types, but the compartment that they reside in corresponds to the time of their replication. Interphase chromatin structure is established along with the establishment of RT and may act as scaffold for replication regulation factors like Rif1. Finally, replication timing and its association with chromatin structure are highly conserved and are observed even at the single chromosome level.
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Date Issued: 2017
Identifier: FSU_SUMMER2017_Dileep_fsu_0071E_14038
Format: Thesis

Title: Neuromodulation of Mitral Cells by Serotonin and GLP-1 Neurons in the Olfactory Bulb and the Consequences of Gene Deletion of Kv1.3.
Creator: Huang, Zhenbo, Fadool, Debra Ann, Logan, Timothy M., Gilbert, David M., Lyons, Lisa C., Wang, Zhi, Wang, Zuoxin, Florida State University, College of Arts and Sciences,...
Show moreHuang, Zhenbo, Fadool, Debra Ann, Logan, Timothy M., Gilbert, David M., Lyons, Lisa C., Wang, Zhi, Wang, Zuoxin, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Neuromodulation plays important roles in adjusting our nervous system to produce behaviors. The same neuromodulator could have different effects on different targets, or the same target could be modulated by multiple neuromodulators. In the first project of my dissertation I investigated differential modulation of mitral cells (MCs) contained in the main (MOB) and accessory (AOB) olfactory bulb by serotonin (5-HT) using an in vitro, brain slice approach in postnatal (P15-30) day mice. In the...
Show moreNeuromodulation plays important roles in adjusting our nervous system to produce behaviors. The same neuromodulator could have different effects on different targets, or the same target could be modulated by multiple neuromodulators. In the first project of my dissertation I investigated differential modulation of mitral cells (MCs) contained in the main (MOB) and accessory (AOB) olfactory bulb by serotonin (5-HT) using an in vitro, brain slice approach in postnatal (P15-30) day mice. In the MOB, 5-HT elicited three types of responses in 94% of 158 cells tested. Cells were either directly excited (73%, n = 115), inhibited (9%, n = 15), or showed a mixed response −first inhibition followed by excitation (12%, n = 19). In the AOB, 83% of 115 cells were inhibited with 17% of cells showing no response. Albeit located in parallel partitions of the olfactory system, 5-HT largely elicited excitation of MOB MCs while it evoked two different kinetic rates of inhibition in MCs of the AOB. Using a combination of pharmacological agents, I found that the excitatory responses in MOB MCs were mediated by 5-HT2A receptors through a direct activation. In comparison, 5-HT-evoked inhibitory responses in the AOB arose due to a polysynaptic, slow-onset inhibition attributed to 5-HT2 receptor activation exciting GABAergic interneurons. The second type of inhibition had a rapid onset as a result of direct inhibition mediated by the 5-HT1 class of receptors. The distinct serotonergic modulation of MCs between the MOB and AOB could provide a molecular basis for differential chemosensory behaviors driven by the brainstem raphe nuclei into these parallel systems. In the second project of my dissertation, I explored the modulation of glucagon-like peptide-1 (GLP-1) neurons in the olfactory bulb (OB). A population of GLP-1 neurons was recently discovered in the OB. The functions of these neurons remain incompletely understood. Herein, I used an in vitro, brain slice approach to investigate the modulations of GLP-1 neurons. Juvenile mice (P20 to P45) of both sexes were used to examine the involvement of centrifugal projections from higher brain areas including serotonergic, cholinergic, and noradrenergic afferents. Bath application of serotonin (40 µM, n = 4) and norepinephrine (100 µM, n = 4) had no effect on the evoked firing frequency. Acetylcholine (ACh; 100 µM), however, led to either inhibition or excitation of GLP-1 neurons. For inhibition, ACh induced a small outward current (5.1 ± 1.8 pA, n = 9) recorded by voltage-clamp when neurons were held at −70 mV. When recorded in current-clamp mode, ACh delayed the latency to first spike (control: 253 ± 30 ms, ACh: 396 ± 4 ms; n = 2). For excitation, bath application of ACh resulted in 1.9 ± 0.6-fold increase in firing frequency (n = 21). Previous evidence showed that GLP-1 neurons in the brainstem could be modulated by metabolic-related hormones such as leptin and cholecystokinin (CCK). I found that GLP-1 neurons could be modulated by CCK, but not by leptin. Bath application of CCK (0.8 µM) led to either cessation of firing (n = 10) or an increase in firing of 1.7 ± 0.4-fold (n = 11). Lastly, mice were injected intraperitoneally with the GLP-1 analogue Exendin-4 (0.4 µM /kg) or control saline and tested 30 minutes post injection in a habituation-dishabituation odor test. Mice receiving Exendin-4 failed to show significant dishabituation, demonstrating impaired ability to discriminate a novel odor from a familiar odor. One primary target of neuromodulation is ion channels. Depending on which group of neurons and in which brain region it is expressed, the same type of ion channel can contribute to multiple functions. In the third project of my dissertation I examined the consequences of loss of function of voltage-gated potassium channel Kv1.3. It has long been recognized that olfaction and emotion are linked. My study aimed to investigate the roles of olfaction in modulating anxiety. Kv1.3 knockout mice (Kv1.3-/-), which have heightened olfaction, and wild-type (WT) mice were examined for anxiety-like behaviors. Because Kv1.3-/- mice have also been observed to show increased locomotor activity, which is one behavior reported in animal models of attention-deficit/hyperactivity disorder (ADHD), inattentive behavior was quantified for both genotypes. Kv1.3-/- mice showed increased anxiety levels compared to their WT counterparts and administration of methylphenidate (MPH) via oral gavage alleviated their increased anxiety. Object-based attention testing indicated Kv1.3-/- mice had attention deficits and treatment with MPH also ameliorated this condition. My data suggest that heightened olfaction does not necessarily lead to decreased anxiety levels, and that Kv1.3-/- mice may be used as a behavioral model of the inattentive subtype of ADHD.
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Date Issued: 2017
Identifier: FSU_FALL2017_Huang_fsu_0071E_14226
Format: Thesis

Title: Functional Circuitry of the Medial Amygdala and Main Intercalated Nucleus in the Golden Hamster.
Creator: Biggs, Lindsey Marie, Meredith, Michael, Kelley, Colleen M., DuVal, Emily H., Hull, Elaine M., Trombley, Paul Q., Florida State University, College of Arts and Sciences,...
Show moreBiggs, Lindsey Marie, Meredith, Michael, Kelley, Colleen M., DuVal, Emily H., Hull, Elaine M., Trombley, Paul Q., Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Chemical signals are important for social communication in many rodent species. Detection and processing of these chemosignals is necessary for the production of reproductive and defensive behaviors that are important for species survival. The medial amygdala is the first site of convergence for input regarding these chemosignals, thus it is vital to investigate its role in chemosignal processing. Previous studies using immediate early gene (IEG) expression indicative of neuronal activation...
Show moreChemical signals are important for social communication in many rodent species. Detection and processing of these chemosignals is necessary for the production of reproductive and defensive behaviors that are important for species survival. The medial amygdala is the first site of convergence for input regarding these chemosignals, thus it is vital to investigate its role in chemosignal processing. Previous studies using immediate early gene (IEG) expression indicative of neuronal activation have demonstrated a categorical response in the medial amygdala subareas to different types of chemosignals. Both the anterior and posterior medial amygdala (MeA and MeP, respectively) show higher activation after exposure to conspecific odors, while only MeA has a higher response to heterospecific odors. These experiments also suggest that the primarily GABAergic, main intercalated nucleus (m-ICNc) may be involved in modulating the MeP response since there is a negative correlation in the IEG responses between these two areas after exposure to heterospecific odors. These data suggest that the medial amygdala and possibly the m-ICNc are involved in the processing of chemosignals, however little is known about the functional circuitry underlying chemosignal processing within and between these two areas. Using whole-cell patch clamp electrophysiology and immunohistochemical staining, the experiments included in this dissertation investigated the functional circuitry between the medial amygdala areas; the connections between the medial amygdala and m-ICNc; how this circuit may be modulated by input from other brain areas; and the potential involvement of phenotypically distinct subpopulations during chemosignal processing. Consistent with previous tract-tracing studies, I demonstrated functional excitatory and inhibitory connections between the anterior-dorsal (MeAd) and posterior dorsal (MePd) regions of the medial amygdala in electrophysiology experiments. These diverse connections may provide a means by which MeAd can directly affect MePd activity during chemosignal processing consistent with previously published IEG responses. Further electrophysiology experiments provide evidence for an indirect pathway allowing for even further modulation of MePd by MeAd. In these experiments, I found excitatory projections from MeAd to m-ICNc that were strong enough to drive action potential firing in my thin tissue slices. Projections from m-ICNc to MePd were also documented and stimulation of m-ICNc often resulted in hyperpolarization of MePd neurons. The m-ICNc-evoked hyperpolarization of MePd persisted during glutamate receptor blockade suggesting that there are direct inhibitory connections from m-ICNc to MePd. These data suggest that MeAd may also modulate MePd indirectly providing an even greater diversity of medial amygdala output in order to produce appropriate behavioral responses to various chemosignals. The neurotransmitter dopamine may also be involved in chemosignal processing. Dopamine decreased the excitability of m-ICNc neurons and decreased the m-ICNc-evoked hyperpolarization of MePd neurons, suggesting that this indirect pathway may also be modulated by other brain areas. I also found evidence of projections from infralimbic cortex and the localization of mu-opioid receptors to the m-ICNc. These two inputs may provide even further modulation of the circuitry and greater diversity in medial amygdala output depending on the brain state of the animal during chemosignal processing. Lastly, I investigated the potential role of phenotypically distinct subpopulations of presumably GABAergic medial amygdala neurons characterized by expression of calcium binding proteins in chemosignal processing. I found differential expression patterns of parvalbumin, calbindin and calretinin neurons throughout the medial amygdala areas. Parvalbumin was not expressed in the medial amygdala but was found in other amygdalar areas surrounding the medial amygala. Calbindin and calretinin neurons were found throughout the medial amygdala with different densities across subdivisions. Overall, very few calretinin or calbindin neurons were activated (as indicated by IEG expression) after exposure to the female conspecific odors. However, the overall pattern of activation of calbindin and calretinin-immunoreactive neurons was similar to the overall IEG expression, suggesting that these neuronal subpopulations may be involved in the inhibitory feedback networks within the medial amygdala.
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Date Issued: 2017
Identifier: FSU_2017SP_Biggs_fsu_0071E_13704
Format: Thesis

Title: Structural and Functional Characterization of Escherichia Coli Assimilatory Sulfite Reductase.
Creator: Del Carmen, Isabel Askenasy Flores, Stroupe, M. Elizabeth (Margaret Elizabeth), Stagg, Scott, Dennis, Jonathan Hancock, Jones, Kathryn M., Yu, Hong-Guo, Florida State University...
Show moreDel Carmen, Isabel Askenasy Flores, Stroupe, M. Elizabeth (Margaret Elizabeth), Stagg, Scott, Dennis, Jonathan Hancock, Jones, Kathryn M., Yu, Hong-Guo, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Assimilatory NADPH-dependent sulfite reductase (SiR) is the enzyme responsible for the six-electron reduction of sulfite to sulfide. In Enterobacteria, SiR is a dodecameric complex with two subunits: an octameric flavin-containing SiRFP, and four copies of a monomeric iron-containing subunit (SiRHP). SiRFP is a homologous to cytochrome P-450 reductase (CYP), each of which has three main domains: NADPH/FAD-binding domain, FMN-binding domain and a connecting domain that is responsible of the...
Show moreAssimilatory NADPH-dependent sulfite reductase (SiR) is the enzyme responsible for the six-electron reduction of sulfite to sulfide. In Enterobacteria, SiR is a dodecameric complex with two subunits: an octameric flavin-containing SiRFP, and four copies of a monomeric iron-containing subunit (SiRHP). SiRFP is a homologous to cytochrome P-450 reductase (CYP), each of which has three main domains: NADPH/FAD-binding domain, FMN-binding domain and a connecting domain that is responsible of the relative orientation of the other two domains. However, SiRFP differs from CYP because SiRFP is a soluble protein that forms an octamer through its first 51 residues and is a stable complex with the oxidase subunit. SiRHP has a Fe4S4 cluster and a siroheme cofactor in its active site. In the SiR holoenzyme electrons flow from NADPH to FAD to FMN in SiRFP and from the FMN cofactor to SiRHP. SiRFP, SiRHP, and PAPS sulfotransferase, another enzyme required for sulfate assimilation, are encoded by three genes in the cysJIH operon, therefore, they can be independently expressed and purified. In this way, each subunit has been characterized structural and functionally, however the structure of the SiR holoenzyme remains ill defined. Therefore, the main aim of this dissertation is to understand SiR assembly and the mechanism of electron transfer between subunits. I made important advances in understanding SiR’s subunit interactions by the use of a variety of biochemical and structural techniques. First, we confirmed the stoichiometry of the holoenzyme as an α8β4 complex. I further discovered that in this complex, SiRFP has two interfaces with SiRHP. The Region 1, is in SiRFP’s C-terminus and it is responsible for forming the stable interaction between each SiR subunits. Region 2 is in SiRFP’s N-terminus and we hypothesize that it is involved in a transient interaction with SiRHP and, such as, is the one required for electron transfer between subunits. My more detailed characterization shows that complex formation depends on hydrophobic interactions between the subunits. In further characterization this interaction, I identified three regions of predicted intrinsically disorder/disorder-based binding sites in each subunit that are required for SiR assemble and function. First, the N-terminus of SiRHP has characteristics of a disorder-base binding region, and it is required for holoenzyme complex formation. Second, the N-terminus of SiRFP is required for octamerization of this subunit. Third, the linker between the FMN-binding domain and FAD/NADPH binding domain is also an intrinsically disorder region that orients the FMN-domain relative to rest of the complex. As result of my experiments, I propose a model for electron transfer in which all SiRFP subunits participate in electron transfer to SiRHP. This model explains the high efficiency of the complex that is observed by the absence of partially reduced sulfur-oxygen intermediates. Additionally, advances were made to prepare cryo-electron microscopy samples of SiR based in the new insights into SiR’s structural characteristics. Additionally, a new reductionist approach using x-ray crystallography is proposed to better study SiR subunits interactions.
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Date Issued: 2017
Identifier: FSU_FALL2017_AskenasyFlores_fsu_0071E_14225
Format: Thesis

Title: The Genetics of Adaptation of ssRNA Viruses.
Creator: Sackman, Andrew Michael, Rokyta, Darin R., Beerli, Peter, Houle, David C., Hughes, Kimberley A., Inouye, Brian D., Florida State University, College of Arts and Sciences,...
Show moreSackman, Andrew Michael, Rokyta, Darin R., Beerli, Peter, Houle, David C., Hughes, Kimberley A., Inouye, Brian D., Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: The process of adaptive evolution is complex, determined in part by the set of beneficial mutations available to adapting populations, the distribution of their effects, and the extents to which pleiotropy and epistasis impact the relationships between genotype, phenotype, and fitness. Assumptions about epistasis, pleiotropy, and the availability of beneficial mutations are made by theories and models of adaptation and determine their predictions. Through the experimental evolution of...
Show moreThe process of adaptive evolution is complex, determined in part by the set of beneficial mutations available to adapting populations, the distribution of their effects, and the extents to which pleiotropy and epistasis impact the relationships between genotype, phenotype, and fitness. Assumptions about epistasis, pleiotropy, and the availability of beneficial mutations are made by theories and models of adaptation and determine their predictions. Through the experimental evolution of bacteriophages, we tested model assumptions and predictions about epistasis, hybrid viability, the causes of parallel evolution, the distribution of beneficial fitness effects, the genotype-fitness landscape, antagonistic pleiotropy, and the cost of complexity. We did this in an effort to answer standing questions in the study of evolution regarding the process of adaptation and the forces that govern it. To characterize the effects of epistasis on determining the viability and persistence of novel hybrids, we generated experimental hybrids of highly divergent and highly related pairs of phage strains. We found that incompatibilities resulting from disruption of favorable epistatic interactions within the genome manifested as fitness costs, even in very closely related genotypes, but that ancestral fitness levels could be recovered through only a handful of compensatory mutations. Remarkably, we found that by permitting movement over wide expanses of sequence space in a single step—coupled with subsequent adaptation—hybridization allowed one phage genotype to explore a substantially higher and previously inaccessible peak in its fitness landscape. Thus, if conditions permitted hybrid genotypes to persist long enough to allow for compensatory evolution, even very low fitness hybrids could be viable in populations with their ancestral genotypes, potentially facilitating the establishment of stable hybrid zones and the creation of new species. Competing selectionist and mutationist viewpoints argue that parallel evolution is the result of either selection repeatedly fixing the same, optimal adaptive solution to a recurring evolutionary problem or a product of mutational constraint presenting selection with a biased subset of potentially suboptimal adaptive strategies. To characterize the rate of parallel evolution and the underlying forces driving parallelism, we performed 20 replicate first steps of adaptation for four phage genotypes. We found parallel and convergent adaptation to be common at every level of biological organization both within and among genotypes, from the level of mutational effects to the levels of protein, gene, amino acid, and nucleotide substitution. We also found that contrary to expectations, the mutation of greatest benefit in each genotype was never the most frequently fixed, and only fixed once in each of two genotypes. Patterns of parallel evolution are often offered as strong evidence of adaptation. However, we found that parallelism was driven at least as much by mutational biases and constraints as by selection, indicating that frequent observations of par- allel adaptive evolution in natural and experimental populations may not be driven primarily by selection, but rather by selection acting on a biased subset of all available mutations, possibly, as in the case with our results, yielding suboptimal adaptive outcomes. We also fit a distribution to the set of fitness effects for each set of 20 replicates. The form of the distribution of beneficial fitness effects shapes the predictions of models of adaptation and affects the properties of adaptive walks, particularly the average size of mutational steps and the likelihood of parallel evolution. We found that all four genotypes were best described by a distribution of beneficial fitness effects with a finite upper bound, conflicting with the assumption of some models that this distribution is exponential. In another experiment, we constructed double-mutant genotypes containing all possible pairings of three sets of five beneficial mutations generated under three different selective regimes: selection on growth rate only, selection on growth rate and thermal stability, and selection on growth rate and pH stability. The purpose of this experiment was to characterize patterns of pairwise epistasis for beneficial mutations, and in particular to measure interactions for each of the phenotypes underlying fitness. We found that mutations interacted antagonistically with regard to growth rate and fitness, in line with other recent work which has generally found a trend of antagonistic interactions between beneficial mutations. However, we found that mutations behaved on average more additively with regard to capsid stability. We concluded that mutations interact additively with regard to phenotype when considered at a basic, biophysical level, and that epistasis for fitness emerges from an intermediate phenotypic optimum and pleiotropy between its underlying phenotypes, manifested in adapting populations as a pattern of diminishing returns and antagonism between beneficial mutations. Finally, we performed adaptive walks for two pairs of wild-type and growth-optimized phage genotypes under selection on growth rate and capsid stability to test the hypotheses that adaptation of these two traits is constrained by antagonistic pleiotropy and that organisms evolving under complex environmental or selective pressures incur a cost of complexity manifested as a lower rate of adaptation. Despite expectations that antagonism would prevent optimization of growth rate and stability, both traits were significantly improved over the course of replicate adaptive walks. Additionally, we found no evidence for a cost of complexity, as the rate of adaptation under complex selection was actually higher than under simple selective conditions and did not require any additional mutational steps relative to one-trait selection. Our results indicated that increased organismal complexity, or an increase in the number of traits under selection, may not decrease an organism's rate of adaptation, even when mutations affect all of the traits under selection simultaneously, contrary to model predictions.
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Date Issued: 2017
Identifier: FSU_2017SP_Sackman_fsu_0071E_13708
Format: Thesis

Title: Modulation of Kinase Signaling by ORF45 during the Lytic Cycle of Kaposi's Sarcoma-Associated Herpesvirus.
Creator: Avey, Denis R., Zhu, Fanxiu, Sang, Qing-Xiang Amy, Tang, Hengli, Gilbert, David M., Dennis, Jonathan Hancock, Florida State University, College of Arts and Sciences, Department...
Show moreAvey, Denis R., Zhu, Fanxiu, Sang, Qing-Xiang Amy, Tang, Hengli, Gilbert, David M., Dennis, Jonathan Hancock, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Kaposi's Sarcoma-Associated Herpesvirus (KSHV) is an oncogenic virus that has adapted unique mechanisms to modulate the cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling pathways, including the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. We have previously shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK,...
Show moreKaposi's Sarcoma-Associated Herpesvirus (KSHV) is an oncogenic virus that has adapted unique mechanisms to modulate the cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling pathways, including the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. We have previously shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK, a major functional mediator of ERK/MAPK signaling) during KSHV lytic replication. ORF45-activated RSK is required for optimal KSHV lytic gene expression and progeny virion production, though the underlying mechanisms downstream of this activation are still unclear. We hypothesized that the activation of RSK by ORF45 causes differential phosphorylation of cellular and viral substrates, affecting biological processes essential for efficient KSHV lytic replication. Accordingly, we observed widespread and significant differences in protein phosphorylation upon induction of lytic replication. Mass-spectrometry-based phosphoproteomic screening identified putative substrates of ORF45-activated RSK in KSHV-infected cells. Bioinformatic analyses revealed that nuclear proteins, including several transcriptional regulators, were overrepresented among these candidates. We validated the ORF45/RSK-dependent phosphorylation of several putative substrates by employing KSHV BAC mutagenesis, kinase inhibitor treatments, and/or CRISPR-mediated knockout of RSK in KSHV-infected cells. Furthermore, we assessed the consequences of knocking out these substrates on KSHV progeny virion production. Importantly, we investigated the regulation of gene expression by ORF45-actvated RSK by performing RNA-seq of KSHV-infected cells. We show data to support that ORF45 regulates the translational efficiency of a subset of viral/cellular genes with complex secondary structure in their 5' UTR. One of the few viral substrates of ORF45-activated identified by our mass spectrometry analysis was ORF36. KSHV ORF36 encodes a serine/threonine viral protein kinase, which is conserved throughout all herpesviruses. Although several studies have identified the viral and cellular substrates of conserved herpesvirus protein kinases (CHPKs), the precise functions of KSHV ORF36 during lytic replication remain elusive. We report that ORF36 interacts with another lytic protein, ORF45, in a manner dependent on ORF36 kinase activity. We mapped the regions of ORF36 and ORF45 involved in their binding. Their association appears to be mediated by electrostatic interactions, since deletion of either the highly basic N-terminus of ORF36 or an acidic patch of ORF45 abolished the binding. Additionally, dephosphorylation of ORF45 protein dramatically reduced its association with ORF36. Importantly, ORF45 enhances both the stability and kinase activity of ORF36. Consistent with previous studies of CHPK homologs, we detected ORF36 protein in extracellular virions. To investigate the roles of ORF36 in the context of KSHV lytic replication, we employed BAC mutagenesis to engineer both ORF36-null and kinase-dead (KD) mutants. We found that ORF36-null/mutant virions are moderately defective in viral particle production and are further deficient in primary infection. In summary, our results uncover a functionally important interaction between ORF36 and ORF45, and indicate a significant role of ORF36 in the production of infectious progeny virions. Altogether, these data shed light on the mechanisms by which KSHV ORF45 manipulates viral and cellular kinase signaling to optimize lytic replication. The findings reported here have important implications for the pathobiology of KSHV and other diseases in which RSK activity is missregulated.
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Date Issued: 2016
Identifier: FSU_2016SP_AVEY_fsu_0071E_13145
Format: Thesis

Title: A View of Rhynchosporeae (Cyperaceae) Diversification before and after the Application of Anchored Phylogenomics Across the Angiosperms.
Creator: Buddenhagen, Christopher E. (Christopher Evan), Mast, Austin R., Parker, William C., Winn, Alice A., Steppan, Scott J., Inouye, Brian D., Florida State University, College of...
Show moreBuddenhagen, Christopher E. (Christopher Evan), Mast, Austin R., Parker, William C., Winn, Alice A., Steppan, Scott J., Inouye, Brian D., Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: This study examines the evolutionary history of the cosmopolitan beaksedge tribe Rhynchosporeae (ca. 386 spp.; Cyperaceae) using phylogenetics. Taxon sampling covers 25 of the 28 taxonomic sections proposed for the tribe. I compare a history inferred for Rhynchosporeae using a single plastid gene (Chapter 2) with one inferred using hundreds of loci (Chapter 4). The latter involves a sequencing methodology I develop with collaborators that can be applied across angiosperms (Chapter 3). Chapter...
Show moreThis study examines the evolutionary history of the cosmopolitan beaksedge tribe Rhynchosporeae (ca. 386 spp.; Cyperaceae) using phylogenetics. Taxon sampling covers 25 of the 28 taxonomic sections proposed for the tribe. I compare a history inferred for Rhynchosporeae using a single plastid gene (Chapter 2) with one inferred using hundreds of loci (Chapter 4). The latter involves a sequencing methodology I develop with collaborators that can be applied across angiosperms (Chapter 3). Chapter 2 recognizes that Rhynchosporeae has high levels of endemicity (≥ 44%) in tropical and subtropical American savannas and can provide insights into the diversification of their biotas. Wind pollination, occupation of a savanna habitat, and a C3 photosynthetic pathway are common in the tribe, but showy (presumably insect-pollinated) inflorescences, occupation of forest habitat, and a C4 pathway also occur. I reconstructed a dated phylogenetic hypothesis for 79 taxa, using the trnL/F plastid region, inferring a mean crown-group age of 56 million years. Fitch parsimony infers the most recent common ancestor (MRCA) to have occupied a savanna habitat with eight or more shifts to forest. Features associated with insect pollination—white bracts and spikelets—were shown to evolve six or more times but were not correlated with the shifts to forest habitat where wind pollination is likely to be less effective. I found evolutionary correlations in the pairwise comparisons of bract color versus spikelet color and bract positioning versus bract color. Members with anatomies associated with C4, though anatomically variable, form a clade with a crown age of 19 million years. In Chapter 3, with collaborators I develop a robust probe design process to identify 499 low-copy nuclear regions and 18 high-copy functional genes for hybrid enrichment. We obtained >90% enrichment success for target regions. Between 159 and 488 orthologs were retained in alignments used for phylogenetic inference at deep and shallow levels across the angiosperms. A sampling strategy focusing on incremental removal of incongruent loci in combination with removal of sites with high rates of change produced 196 alignments for phylogenetic inference. The phylogenetic hypotheses at each sample level represent outcomes under different regions of parameter space. These outcomes were presented using heatmaps that depict bootstrap support at all nodes for those 196 levels of parameter space. This provided a new approach for sensitivity analyses and for testing the robustness of any hypotheses. A randomization methodology for hypotheses testing at specific nodes takes advantage of the heatmap approach. Focusing on the difficult-to-resolve eudicot, monocot, Magnoliid nodes the analysis revealed that the supermatrix approach produced a spuriously confident yet conflicting result in some regions of parameter space. With >97% of the data, supermatrix analyses supported eudicot and Magnoliids as sister. Support switched to strongly support the eudicot and monocot sister relationship at higher levels of data removal. In contrast the coalescent model consistently supported the latter relationship across most of the parameter space. Overall the eudicot and monocot sister relationship is robustly favored. In Chapter 4, I reexamine beaksedge (tribe Rhynchosporeae ) diversification but employ the anchored hybrid enrichment protocols developed in Chapter 3. A dated phylogenetic hypothesis for 115 taxa in the tribe and 11 outgroup taxa inferred a mean crown-group age for the tribe of 43.2 million years. Ancestral state reconstruction using stochastic mapping infers an open (savanna) habitat for the MRCA. This was the common state along 77% of the total branch lengths. However, there was an average of 22 independent shifts from open habitats into forest understory or edges in its descendants. The common state was the typical seasonally wet savanna soils. The state associated with their occurrence in dry, well drained soils was reconstructed for 4% of the total branch lengths, but there was an average of 11.2 transitions to that state. There were 3.7 transitions to the state where plants typically occur in standing or flowing water. An average of 5.9 transitions from nondescript brown or green inflorescences associated with wind pollination to those associated with insect pollination (white spikelets and/or bracts) were inferred but these were not correlated with the shifts to forest habitat. Members with C4 anatomy formed a clade that diverged from a sister clade containing taxa with C3 photosynthetic anatomies 26 MYA; this is earlier than previously thought. Most of the taxonomic sections described by Shirley Gale and Georg Kükenthal for Rhynchospora and Pleurostachys were not monophyletic. I also briefly discuss the possible significance of detecting a recently described repetitive satellite DNA element known to be associated with the centromeric protein CENH3.
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Date Issued: 2016
Identifier: FSU_FA2016_Buddenhagen_fsu_0071E_13553
Format: Thesis

Title: The Dynamics of Replication Timing, Chromatin Compartments, and Gene Expression Changes during Lineage Specification of Stem Cells.
Creator: Wilson, Korey, Gilbert, David M., Megraw, Timothy, Bass, Hank W., Chadwick, Brian P., Dennis, Jonathan Hancock, Florida State University, College of Arts and Sciences,...
Show moreWilson, Korey, Gilbert, David M., Megraw, Timothy, Bass, Hank W., Chadwick, Brian P., Dennis, Jonathan Hancock, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: The temporal order in which segments of the genome are duplicated is referred to as the replication timing (RT) program. RT is established in each cell cycle coincident with the repositioning and anchorage of chromosomes in early G1. In general, segments that replicate in early S are organized into transcriptionally permissive chromatin, and segments that replicate in late S are assembled into repressive chromatin. During human embryonic stem cell (hESC) differentiation, segments of the...
Show moreThe temporal order in which segments of the genome are duplicated is referred to as the replication timing (RT) program. RT is established in each cell cycle coincident with the repositioning and anchorage of chromosomes in early G1. In general, segments that replicate in early S are organized into transcriptionally permissive chromatin, and segments that replicate in late S are assembled into repressive chromatin. During human embryonic stem cell (hESC) differentiation, segments of the genome undergo changes in RT, which are accompanied by changes in chromatin compartments, and transcriptional activity. Determining the order these changes occur during hESC differentiation required defining cell cycle parameters for hESCs. First, we demonstrate that the fluorescence ubiquitination cell cycle indicator (Fucci) system is incapable of demarcating G1/S cell cycle transitions. Instead, we employed a combination of fluorescent PCNA to monitor S phase progression, cytokinesis to demarcate mitosis, and fluorescent nucleotides to label early and late replicating DNA and track 3D organization. We find that re-localization and anchorage of chromosomes were completed prior to the onset of S phase, even in the context of an abbreviated G1 phase. Furthermore, we find that single hESCs preferentially differentiate from G1. We show changes in RT are remarkably coincident with transcription; although, neither is sufficient for the other to occur. We also show changes in RT accompany cell commitment during the first cell cycle and precede changes in chromatin compartments. Finally, we find that in hESCs, domains that switch from early to late replication interact more frequently with late replicating chromatin, suggesting hESCs may be poised to quickly repress early to late switching domains upon stimulation.
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Date Issued: 2016
Identifier: FSU_FA2016_Wilson_fsu_0071E_13444
Format: Thesis

Title: The Genetics of Adaptation of Island Rattlesnakes.
Creator: Margres, Mark J., Rokyta, Darin R, Beerli, Peter, Erickson, Gregory, Travis, Joseph, Winn, Alice A., Florida State University, College of Arts and Sciences, Department of...
Show moreMargres, Mark J., Rokyta, Darin R, Beerli, Peter, Erickson, Gregory, Travis, Joseph, Winn, Alice A., Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: The study of adaptive molecular evolution in natural populations has been severely limited by the difficulty of linking genetic variation to phenotypic variation to fitness effects. Most studies connecting genotype, phenotype, and fitness have used reverse genetic approaches to measure the functional effects of specific mutations in the laboratory because this relationship is difficult to measure in natural populations, particularly for complex traits because of the "many-to-one" mapping of...
Show moreThe study of adaptive molecular evolution in natural populations has been severely limited by the difficulty of linking genetic variation to phenotypic variation to fitness effects. Most studies connecting genotype, phenotype, and fitness have used reverse genetic approaches to measure the functional effects of specific mutations in the laboratory because this relationship is difficult to measure in natural populations, particularly for complex traits because of the "many-to-one" mapping of genotype to phenotype. Many of the fundamental features of evolving systems, such as evolvability, epistasis, and pleiotropy, however, may be stronger determinants of evolutionary outcomes in natural populations than in the laboratory because artificial selection and breeding schemes are generally more simplistic relative to selection and demographic effects in natural settings. Snake venoms have emerged as a system for the study of the genetics of adaptation in complex, polygenic traits because of their genetic tractability and role in feeding, digestion, and defense, all of which are directly relevant to fitness. Because venom gene expression is tissue-specific (i.e., no pleiotropic constraints) and toxin protein abundance directly influences venom efficacy, venoms are not inherently biased toward a particular mutational pathway, enabling a systematic comparison of the molecular mechanisms underlying adaptive evolution. Venom phenotypes are manifest only upon injection into another animal, and venom functions are directly measurable through various assays, allowing direct tests of adaptive hypotheses in natural prey populations. In this work, we sought to create a genotype-phenotype-fitness map for the venom system of the eastern diamondback rattlesnake (Crotalus adamanteus) and, for the first time, identify the genetic basis of adaptation for a complex, polygenic trait in natural populations. Crotalus adamanteus is the largest species of rattlesnake and exclusively consumes endotherms. Crotalus adamanteus is historically native to seven states in the southeastern United States but has recently been extirpated from Louisiana, is endangered in North Carolina, and is currently under consideration for listing as threatened under the Endangered Species Act. In Chapter 1, we sequenced the venom-gland transcriptome and integrated mass spectrometry data to construct a transcriptome-proteome map for the venom system. We then used this map to identify significant toxin-gene expression differentiation across the range of C. adamanteus, providing candidate-genes for which to test the functional and evolutionary significance of the identified variation. In Chapter 2, we used a similar approach and identified significant ontogenetic differentiation in toxin gene expression; further analyses determined that ontogenetic effects explained more variation in toxin expression than geographic effects, although both juvenile and adult expression patterns varied geographically, and time-series experiments in lab-raised individuals demonstrated that geographic and ontogenetic expression differentiation were not environmentally induced but rather under genetic control. In Chapter 3, we used in vitro functional assays to verify that the expression differences found in the previous two chapters corresponded to differences in venom function. We found that, overall, the statistical differences in toxin expression outlined in the first two chapters equated to functional differences in toxic activities in a predictable, tractable manner, suggesting that the differences identified in the first two chapters were, in fact, biologically relevant. In Chapter 4, we used a target-enrichment approach to sequence the exons of all identified toxins in the venom-gland transcriptome as well as several thousand neutral loci to ascertain the relative roles of expression versus coding-sequence changes in a trait not inherently biased towards either mutational pathway. We found evidence for adaptive changes at both the expression and sequence levels across the entire range, although expression differentiation did appear to be the more frequent molecular mechanism. But, without functional characterizations of the identified sequence and expression evolution, it was difficult to characterize the relative roles demography, selection, and drift played in generating the identified sequence and expression divergence. Although Chapter 3 did link expression variation to functional variation, these assays were not conducted in the actual target of venoms, natural prey. To address these issues, we examined toxin sequence and expression evolution and estimated venom toxicity (i.e., fitness) in sympatric and allopatric natural prey across an island-mainland population pair in Chapter 5 to, for the first time, construct a genotype-phenotype-fitness map for a complex trait in natural populations. We found that expression differentiation was predominantly, or exclusively, the genetic basis of polygenic adaptation, suggesting that over ecological timescales complex traits may preferentially evolve through mutations affecting expression because more molecular mechanisms exist for altering the amount of protein produced than for altering their functions through their primary sequences. In Chapter 1, we found significant expression differentiation in both high- and low-abundance proteins across the range and over 1 million years of divergence, and in Chapter 4, we found both sequence and expression differentiation across the same temporal and spatial scales. In Chapter 5, however, we only identified expression differentiation, and found that this expression differentiation was restricted to low-expression proteins because of physiological and selective constraints on high-expression proteins. These differences in the molecular mechanism underlying adaptive evolution were most likely the result of temporal constraints on generating beneficial variation; because more molecular mechanisms exist for altering protein amounts than protein function, the probability of generating a beneficial expression variant is greater than the probability of generating a beneficial point mutation in the coding-region of a specific protein, and these differences in probability would be most pronounced over extremely short timescales. Given enough time, however, both mutational pathways and proteins expressed at all levels can generate beneficial variation, and these results provide qualitative predictions regarding the process of adaptation for a complex trait.
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Date Issued: 2016
Identifier: FSU_FA2016_Margres_fsu_0071E_13496
Format: Thesis

Title: Implications of Population Growth Rate Projections and Pollen Limitation for the Conservation of a Threatened Dioecious Plant.
Creator: Ramirez-Bullon, Natali Rubi, Winn, Alice A., Travis, Joseph, Underwood, Nora, Florida State University, College of Arts and Sciences, Department of Biological Science
Abstract/Description: The effective conservation of threatened and endangered plants requires an understanding of population dynamics and the evaluation of factors that could reduce population growth. I constructed and analyzed a stage structured demographic model for Euphorbia telephioides, a threatened dioecious perennial herb, to determine the current status of three populations, compare projections of population growth using different methods, and determine the effects of pollen limitation in the population...
Show moreThe effective conservation of threatened and endangered plants requires an understanding of population dynamics and the evaluation of factors that could reduce population growth. I constructed and analyzed a stage structured demographic model for Euphorbia telephioides, a threatened dioecious perennial herb, to determine the current status of three populations, compare projections of population growth using different methods, and determine the effects of pollen limitation in the population dynamics of this species. Dioecious plants are prone to pollen limitation due to their inability to self-pollinate. Studies indicate that pollen limitation reduces seed set in plants due to insufficient quantity or quality of pollen, which can reduce population growth rate due to the decrease in fecundity. I combined experimental tests for pollen limitation with construction and analysis of structured demographic models, to examine how increased levels of pollen limitation would affect population growth rates. Determining the current status of populations, and simulating the consequences of possible threats, such as pollen limitation, provides a quantitative basis for conservation actions. I compared deterministic and stochastic projections of a stage structured demographic model to examine how environmental variation affects population growth rates, and I examined the effects of parameterizing the model excluding demographic measures of randomly marked individuals in the population growth rates (Lambda). The majority of estimated lambdas and their 95% confidence intervals indicate that these three populations are projected to decline. Lambda estimated excluding randomly marked individuals overestimated population growth because adult plants had 100% survival. I did not find evidence of significant pollen limitation of fruit or seed production, and simulations of increased levels of pollen limitation reduce Lambda at a modest rate between 0.17% to 1.91%. The main advantage of constructing a structured demographic model is that these models allow us to integrate data on different stages of a complex life cycle. In the case of E. telephioides elasticity analysis indicates that increasing stasis of non-flowering plants could lead to increasing population growth rates.
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Date Issued: 2016
Identifier: FSU_FA2016_RamirezBullon_fsu_0071N_13551
Format: Thesis

Title: The Cryo-EM 3D Reconstruction of Isolated Lethocerus Z-Discs.
Creator: Summerill, Corinne Alethea Oriana, Taylor, Kenneth A., Stroupe, Margaret Elizabeth, Chase, P. Bryant, Deng, Wu-Min, Florida State University, College of Arts and Sciences,...
Show moreSummerill, Corinne Alethea Oriana, Taylor, Kenneth A., Stroupe, Margaret Elizabeth, Chase, P. Bryant, Deng, Wu-Min, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Electron microscopy is an important technique for observing macromolecular structures, such as DNA and viruses, which would be too small to see under light microscopy. This type of microscopy utilizes electrons as its illumination source, produced by an electron gun, to generate an image of the specimen which is captured by either a charged coupled device (CCD) or direct electron detector (DED) camera. Specimens in electron microscopy can either be stained with heavy atom, embedded in plastic...
Show moreElectron microscopy is an important technique for observing macromolecular structures, such as DNA and viruses, which would be too small to see under light microscopy. This type of microscopy utilizes electrons as its illumination source, produced by an electron gun, to generate an image of the specimen which is captured by either a charged coupled device (CCD) or direct electron detector (DED) camera. Specimens in electron microscopy can either be stained with heavy atom, embedded in plastic, or embedded in vitrified ice. Cryo-electron microscopy (cryo-EM) embeds specimens in a vitreous ice layer that resembles the specimen’s natural environment and increases the overall resolution of the specimen. In conjunction with cryo-electron tomography (cryo-ET), cryo-EM specimens can be tilted on a specimen holder to collect multiple 2D views in order to generate a 3D reconstruction through a weighted back-projection algorithm. The projections are first corrected to counter the effects of contrast transfer function (CTF), which can filter out high resolution information. The resulting tomogram undergoes cycles of image processing steps such as multivariate data analysis, classification, and subvolume averaging to bring out the features of the specimen. At the borders of the striated muscle sarcomere, there exists an electron dense structure called the Z-Disc. The arrangement of thin filaments in the Z-Disc differs between vertebrates and invertebrates. Z-Discs of vertebrates have a tetragonal lattice that contrasts with the hexagonal lattice seen in the A-Band, which might be caused by arrangement of an elastic protein named Titin from the A-Band to the Z-Disc. The tetragonal lattice in the vertebrate Z-Disc has two structural states, small-square and basket-weave, depending on the contraction state of the muscle. Invertebrate Z-Discs have a hexagonal lattice that contains five connecting densities that form large and small solvent channels. Z-Discs contain many proteins that are important for structural stability and signaling functions. Three Z-Disc proteins that are structurally important in invertebrate Z-Discs are α-actinin, an actin crosslinker, Kettin and Projectin, the latter being components of the elastic connecting filament. Alternative Z-Disc isolation methods were explored using Wild-type (WT) Drosophila and Sls-RNAi knockdown Drosophila for the possibility of using the specimens for cryo-EM. The insect flight muscle (IFM) was dissected from the thorax of WT Drosophila and the individual myofibrils were obtained through a homogenization and cleaning process. The Z-Discs were isolated from the myofibrils by exposing them to high salt buffers, to remove thick filaments, and gelsolin, to remove thin filaments. The isolated Z-Discs were negatively stained and observed under an electron microscope. The lattice arrangement of the thin filaments could not be seen due to the stain, but this method produced many Z-Discs on the EM grids. Cryo-EM samples of the isolated WT Drosophila Z-Discs could not be obtained due to problems pertaining to the plunge freezing method. Sls-RNAi Drosophila was obtained using the GAL4/UAS method to generate smaller Z-Discs and decrease the width of the myofibrils due to a decrease in the presence of Kettin. The IFM was extracted from the thorax, but the myofibrils were not exposed to high salt buffers and gelsolin. Under negative stain, the myofibrils observed produced Z-Discs about 500 nm in width, which is ideal for cryo-ET conditions. However, a width of 200-300 nm would produce higher resolution images for a 3D reconstruction. A cryo-EM 3D reconstruction was generated from isolated Lethocerus Z-Discs to confirm the structural features seen in plastic-embedded sections of Apis. Projections for cryo-ET were collected using a DED camera and underwent CTF correction. The tilt series images were coarse-aligned and went through cycles of refinement using an Appion-based database with Protomo. A 3D reconstruction was generated using a weighted back-projection algorithm, filtered to bring out structural features (subvolumes), and then the subvolumes were averaged through single- and multi-reference alignment. The results were visualized in CHIMERA which confirmed the hexagonal lattice arrangement of thin filaments as reported previously in Apis Z-Discs. The location of connecting densities, C1 and C2, were confirmed as forming apices and bases of the large solvent channel, while C3 and C5 were confirmed to be connecting thin filaments of opposite orientation at the tapered end of the small solvent channel. C4 connecting density/three-wheel spoke was seen linking the ends of three thin filaments in the same orientation that form the small solvent channel. C1 and C2 were proposed to contain α-actinin, especially in C2 where an atomic model of F-actin with CH1 domains closely interacted with an atomic model of α-actinin in the C2 density map. The results of this experiment confirmed what was currently known about the invertebrate Z-Disc structure, but the locations of Z-Disc proteins, Kettin and Projectin, are yet to be determined.
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Date Issued: 2016
Identifier: FSU_FA2016_Summerill_fsu_0071N_13636
Format: Thesis

Title: The Role of Biocrusts in Coastal Dune Plant Communities.
Creator: Lauck, Marina, Miller, Thomas E., Winn, Alice A., Inouye, Brian D., Florida State University, College of Arts and Sciences, Department of Biological Science
Abstract/Description: Biocrusts have been shown to affect the ecological conditions in a variety of habitats, involving abiotic mechanisms such as soil moisture retention, nitrogen-rich biomass additions, changing soil physical properties, as well as direct biotic mechanisms such as impeding seedling growth. In coastal dunes, low-lying interdune habitats experience occasional flooding, which promotes the formation of microbial biocrusts. While storm patterns are known to be effective drivers of coastal dune plant...
Show moreBiocrusts have been shown to affect the ecological conditions in a variety of habitats, involving abiotic mechanisms such as soil moisture retention, nitrogen-rich biomass additions, changing soil physical properties, as well as direct biotic mechanisms such as impeding seedling growth. In coastal dunes, low-lying interdune habitats experience occasional flooding, which promotes the formation of microbial biocrusts. While storm patterns are known to be effective drivers of coastal dune plant community composition, the interaction between microbial communities and vegetation in the context of storm patterns is not well understood. I investigated the role of biocrusts on native coastal dune community, how environmental conditions, particularly storm patterns, affect interactions between crusts and vegetation, as well as how the presence of crusts affects plant-plant interactions. To explore correlative patterns between biocrusts, plants and abiotic factors in the field, I analyzed a long term dataset of a coastal dune plant community on St George Island, FL. The presence of several plant species was correlated with crust, and included both negative and positive associations. Additionally, I found correlative relationships with crusts and total plant cover, as well as soil moisture; the latter supports previous research in other systems on the effects of crusts on soil characteristics. To test the effect of crusts on plant species, I conducted a greenhouse experiment that simulated various environmental conditions, including several water and salt treatments, with and without biocrust in a factorial design. Crusts did have an effect on the growth and survival of some of these species, and this effect varied from negative to positive depending on the species. Additionally, I found trends in the data which suggest that crusts may interact with rain and salt, and this interaction may change the effect of crust on plant species in various environmental conditions. Lastly, I performed a target-neighbor competition experiment using four dominant coastal dune plant species in a full factorial design to test the effect of crusts on plant-plant interactions. I found that the selected species often strongly compete, and these competition interactions vary among species. I also found a significant effect of crust on one species, Schizachyrium maritimum.
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Date Issued: 2016
Identifier: FSU_2016SU_Lauck_fsu_0071N_13466
Format: Thesis

Title: Ecology, Evolution, and Conservation of the Pine Barrens Treefrog (Hyla andersonii).
Creator: Warwick, Alexa, Lemmon, Emily C., Travis, Joseph, Beerli, Peter, Steppan, Scott J., Means, D. Bruce, Florida State University, College of Arts and Sciences, Department of...
Show moreWarwick, Alexa, Lemmon, Emily C., Travis, Joseph, Beerli, Peter, Steppan, Scott J., Means, D. Bruce, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: The Pine Barrens treefrog (Hyla andersonii) is restricted to three isolated (disjunct) regions in the eastern United States: New Jersey, North and South Carolina, and the Florida panhandle and southern Alabama. It is a seepage bog specialist and a species of conservation concern in each state in which it occurs. Previous work showed some differentiation among the three regions; however, these studies had small sample sizes, measured few variables, and/or did not include all three regions....
Show moreThe Pine Barrens treefrog (Hyla andersonii) is restricted to three isolated (disjunct) regions in the eastern United States: New Jersey, North and South Carolina, and the Florida panhandle and southern Alabama. It is a seepage bog specialist and a species of conservation concern in each state in which it occurs. Previous work showed some differentiation among the three regions; however, these studies had small sample sizes, measured few variables, and/or did not include all three regions. Moreover, the only genetic study of H. andersonii, using allozyme data, did not resolve clear relationships among the three regions. Conservation management of this species requires a clearer understanding of how populations in these three regions may differ from one another and how populations within regions are structured. To extend previous work on this species, I measured differentiation among regions using morphometric, acoustic signal, ecological, and genetic data. I developed 15 microsatellite markers and used targeted sequence capture to collect large-scale nuclear and mitochondrial genomic data to test models of its evolutionary history. It is hypothesized that the species shifted southward during the last glacial maximum (LGM) into one or more refugia, then expanded northward as the glaciers receded. Overall I find a strikingly concordant pattern in which the first axis of variation for each of the data types distinguishes populations along a latitudinal and longitudinal gradient and the second axis distinguishes the set of populations occurring in the Carolinas (CL) from those occurring in the New Jersey (NJ) and Alabama/Florida (AF) regions. I know of no comparable data set that displays such concordance among different types of data across so large a geographic range. The overlap in trait values (i.e. exchangeability) between neighboring regions, however, is substantial in all types of data, except genetic, which supports continued consideration of this taxon as a single species. Using a phylogenetic framework with large-scale genomic data, I found that AF forms a single clade in both the nuclear and mitochondrial trees and that AF is sister to the rest of the Atlantic clade (CL, NJ). Climate models suggest that the distribution of the species has been repeatedly disjunct since at least the last interglacial, but probably even earlier given the genetic divergence time estimates. All three regions also showed little overlap in broad-scale (climate) environmental data, although niche modeling using climate data alone closely matched the known distribution. Fine-scale environmental data (abiotic and biotic) show greater overlap between CL and AF, with some divergence from NJ. I found some association of genetic and morphometric measures with ecological values, mostly for the broad-scale data. Projections for 2050 suggest habitat suitability will be greatly reduced in CL and AF, with only a small area of NJ available. Finally, I found very different patterns of population genetic structure within each region. Little evidence for isolation by distance was found for all regions, suggesting isolation via environment or other factors may be important for connectivity between populations. The results of my research can be applied to the conservation and management of H. andersonii because it is the most comprehensive study of the species using both an ecological and evolutionary perspective across different temporal and spatial scales.
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Date Issued: 2016
Identifier: FSU_2016SU_Warwick_fsu_0071E_13389
Format: Thesis

Title: Social Nesting Behvior of the Bahama Parrot on Abaco Island and Its Conservation Implications.
Creator: Walker, Caroline S. (Caroline Stahala), DuVal, Emily H., Beerli, Peter, Hughes, Kimberly A., Inouye, Brian D., James, Frances C., Miller, Thomas E., Florida State University,...
Show moreWalker, Caroline S. (Caroline Stahala), DuVal, Emily H., Beerli, Peter, Hughes, Kimberly A., Inouye, Brian D., James, Frances C., Miller, Thomas E., Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Nesting distributions of animals vary from isolated individuals to large colonies of breeding individuals and they can be influenced by numerous factors, including environmental conditions, relatedness and social interactions. Parrots are one of the most threatened orders of birds. Factors that influence their nesting success in natural habitat have been the focus of previous research. However, these past studies operated in systems limited in nest sites, which may also have influenced...
Show moreNesting distributions of animals vary from isolated individuals to large colonies of breeding individuals and they can be influenced by numerous factors, including environmental conditions, relatedness and social interactions. Parrots are one of the most threatened orders of birds. Factors that influence their nesting success in natural habitat have been the focus of previous research. However, these past studies operated in systems limited in nest sites, which may also have influenced population dynamics. In contrast, the Bahama Parrot nests underground in abundant limestone solution cavities in the karst pine forest of Abaco Island, The Bahamas. This situation allows exploration of environmental and social factors that may influence nest success and breeding productivity without the limitation of the availability of nesting site resources. The objective of this study is to investigate the causes and consequences of semi-colonial nesting aggregations in the Bahama Parrot, addressing the influence of local habitat, relatedness of nesting neighbors, social behavior, and success of neighboring nests. The results of this information can be used in the management of habitat and parrot populations on Abaco Island, The Bahamas. The research was carried out on Great Abaco Island during the parrot breeding season (April - September) in 2010 – 2013. Nests were found by traversing logging roads. A number of ecological features were measured around nesting cavities and unused limestone cavities with the same dimensions as parrot cavities to identify favored characteristics. Nearest neighbors were identified for a subset of nests. Behavioral observations included time budgets of vocalization and movement to and from nests at focal and comparison nests (nearest neighbors and more distant nests) to determine if parrots were synchronizing behavior with close-nesting individuals. Predator simulations were conducted to elicit defense responses from focal and nearest neighbor nesting pairs. Using DNA collected from adults and chicks, I assessed whether spatially aggregated nests reflected kin clusters. Bahama Parrots on Abaco were distributed in aggregated spatial patterns in the nesting area. They nested in cavities found in more open areas. One contributing factor for the nest distribution was the uneven distribution of limestone cavities. Greater numbers of nests were found in areas with higher cavity concentrations. However, areas with high cavity concentrations but no parrot nests indicated that additional factors also were involved in concentrating nests in an area. Relatedness did not influence the aggregated nesting pattern. Close nesting neighbors were not more closely related than nesting individuals at other nest sites. No evidence of extra pair paternity was found within the small sample of nests that had full families sampled. However, genotypes of chicks raised in the same cavity in different years did provide support for the general belief that monogamous parrot pairs often reused the same nesting cavities over multiple years. Distances between neighbors had no effect on vocal synchrony. When a predator was introduced to a nest, the vocal response by the nesting pair and the nearest neighbor nesting pair increased, however no other behaviors provided nest defense. An effective defense against feral cats as predators was the predator control program carried out by the Bahamas National Trust. Nest success was higher in years with the predator control program underway. I did detect a relationships between nest success at a focal nest and its neighbor. Finally, I reviewed the taxonomic status of the Cuban parrot (Amazona leucocephala) complex. My review suggested that the two Bahama populations of the Cuban parrot (Amazona leucocephala bahamensis) should be classified as separate species (Amazona abaconensis and Amazona inaguanensis) and that the two Cuban populations should be a single subspecies (Amazona leucocephala leucocephala). These parrots use limestone cavities in the ground in open pine woodland as nest sites, and areas with higher densities of these cavities are used to a greater extent. As populations are expected to increase due to the success of a predator control program, sites with high cavity density are expected to be prime habitat for new nesting individuals. I therefore recommend the continuation of the prescribed burning to maintain the open understory that these parrots select. Furthermore, given the effectiveness of a current feral cat removal program in increasing nesting success, I recommend that predator control continue.
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Date Issued: 2016
Identifier: FSU_2016SU_Walker_fsu_0071E_13477
Format: Thesis

Title: Role of Indirect and Direct Genetic Effects in Modification of Behavior and Maintenance of Color Polymorphism in Male Gambusia Holbrooki.
Creator: Kraft, Brittany Harrison, Hughes, Kimberly A., Kabbaj, Mohamed, Travis, Joseph, DuVal, Emily H., Levitan, Don R., Florida State University, College of Arts and Sciences,...
Show moreKraft, Brittany Harrison, Hughes, Kimberly A., Kabbaj, Mohamed, Travis, Joseph, DuVal, Emily H., Levitan, Don R., Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Indirect genetic effects (IGEs) on traits are present if variation in the social environment provided by others is at least partially due to genetic variation among those individuals. IGEs can result from a variety of interactions between conspecifics and may contribute to the high phenotypic variation in and rapid evolution of behavior. "GxG" epistasis", also known as direct-by-indirect genetic interaction, is a potentially widespread type of IGE that occurs when the expression of behavior...
Show moreIndirect genetic effects (IGEs) on traits are present if variation in the social environment provided by others is at least partially due to genetic variation among those individuals. IGEs can result from a variety of interactions between conspecifics and may contribute to the high phenotypic variation in and rapid evolution of behavior. "GxG" epistasis", also known as direct-by-indirect genetic interaction, is a potentially widespread type of IGE that occurs when the expression of behavior in a social context depends on the genotypic combination of the individuals(s) creating the social contexts for a behavior and the individual expressing that behavior. Despite the potential widespread importance of IGEs, few studies have evaluated IGEs on natural genetic variation in behavior occurring in its natural context. The Eastern mosquitofish, Gambusia holbrooki, is a tractable system in which to study IGEs on behavior. Male Eastern mosquitofish exhibit a discrete natural color polymorphism: a majority of males are silver (S), while a minority is mottled black-and-white (M). This color polymorphism is associated with variation in behavior, responses from conspecifics, survival rate, and selective advantages in the presence of predators. These findings suggest that variation in the social environment provided by others may affect the behavioral differences between M and S male morphs. To determine whether variation in the social environment (IGEs) is a critical influence on differences in male morph behavior, we conducted three studies. In the first study, we compared association with social partners of M and S males in both laboratory and artificially constructed social groups. We found that M and S males associated nonrandomly with different partners, suggesting that these male morphs experience different immediate social environments that could result in IGEs on behavior. We evaluated the role of IGEs on differences in M and S behavior in the second study, in which we compared differences in behavior of male morphs placed in social contexts that varied in the genotypic composition of social partners. We found that social context affected some behavioral differences between M and S males, but that the genotype of the focal individual also influenced focal male behavior. In the third study, we evaluated how M and S juvenile behavior changed during ontogeny in response to different social environments, and whether IGEs seen in adult mosquitofish have their developmental origin in social experiences during development. Results showed that IGEs strongly affected juvenile behavior and maturation: juveniles were more interactive with social partners, fed more, and reached maturity faster when reared with S male adults compared to those reared with M males. There was additional evidence that M and S juveniles behaved differently depended on their rearing context. In a fourth study, we also evaluated M and S male responses to predator and conspecific odor cues; male morphs may respond to odor cues in a way that promotes differences in their relative survival and informs association with social partners. While we found some evidence that males respond differently to odor cues, this evidence was limited to one replicate of the study. In two studies, we also compared association and interactions with social partners in both freely interacting wild social groups and constructed laboratory environments to evaluate whether IGEs influenced differences in male morph behavior similarly in natural and artificial conditions. As described above, we examined patterns of association with social partners between M and S male morphs; we found similar nonrandom association trends between both laboratory and wild social groups, though these trends were nonsignificant for field data. We also compared the behavior of freely interacting individuals in two wild populations using underwater video to behavior of male morphs in a gradient of laboratory social environments. Results showed that both the genotype of the focal individual, and the genotype of their social partner, affected behavior in both wild and laboratory settings. These results implicate that both IGEs and GxG epistasis are important influences on behavior across both settings. In conclusion, this work is some of the first to describe the role of IGEs on differences in behavior associated with a natural polymorphism in its natural context. We establish that males differing in a genetically determined color pattern associate nonrandomly with social partners, and that the behavior of these males depends on both their own genotype and their genotypes of their social partners. We draw comparisons between behavior in laboratory and natural social environments, suggesting that the Eastern mosquitofish is a tractable system in which to continue to explore the role of the social environment on behavior.
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Date Issued: 2016
Identifier: FSU_2016SP_Kraft_fsu_0071E_13035
Format: Thesis

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