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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: 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: The Impact of Estradiol on Food Intake, Cell Signaling, and Diet-Induced Inflammation.
Creator: Butler, Michael James, Eckel, Lisa A., Overton, J. Michael (James Michael), Hyson, Richard Lee, Levenson, Cathy W., Maner, Jon K., Florida State University, College of Arts and...
Show moreButler, Michael James, Eckel, Lisa A., Overton, J. Michael (James Michael), Hyson, Richard Lee, Levenson, Cathy W., Maner, Jon K., Florida State University, College of Arts and Sciences, Department of Psychology
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Abstract/Description: There is a well-documented sexual dimorphism in the neural control of food intake and body weight regulation. Estradiol is a leading candidate as a potential biological factor contributing to these sex differences due to its robust anorexigenic effect. However, the cellular and molecular mechanisms driving estradiol's anorexigenic effect are poorly understood. The goal of this dissertation was to investigate several potential mechanisms underlying estradiol's effect on energy homeostasis in...
Show moreThere is a well-documented sexual dimorphism in the neural control of food intake and body weight regulation. Estradiol is a leading candidate as a potential biological factor contributing to these sex differences due to its robust anorexigenic effect. However, the cellular and molecular mechanisms driving estradiol's anorexigenic effect are poorly understood. The goal of this dissertation was to investigate several potential mechanisms underlying estradiol's effect on energy homeostasis in female rats. Many of estradiol's behavioral effects are mediated, at least partially, via extra-nuclear estradiol signaling. Here, we investigated whether two estrogen receptor (ER) agonists, targeting ERα and G protein-coupled ER-1 (GPER-1), could promote rapid anorexigenic effects in ovariectomized (OVX) rats. Our findings demonstrated that selective activation of ERα produces a rapid (within 1 h) decrease in food intake that is best explained by a non-genomic signaling pathway and thus implicates the involvement of extra-nuclear ERα. We also found that activation of GPER-1 is both sufficient to suppress feeding and necessary for ERa agonist, PPT's, rapid anorexigenic effect. Next, we investigated estradiol's impact on the Janus kinase – signal transducer and activator of transcription (JAK-STAT) pathway in the hypothalamus of OVX rats and in cultured proopiomelanocortin (POMC) neurons. The JAK-STAT pathway mediates leptin's anorexigenic effect and previous work has shown that estradiol also activates this pathway in male mice. However, the specific estrogen receptor subtype and neuronal phenotype has not been investigated in the rat. Here, we show that activation of ERa in OVX rats increases the expression of phosphorylated STAT3 in the hypothalamic arcuate nucleus (ARC) and activation of both ERa and GPER-1 increases the nuclear translocation of phosphorylated STAT3 in cultured POMC neurons. In addition to investigating the effects of estradiol under normal, chow-fed, conditions, we wanted to investigate the effects of estradiol in animals consuming a palatable high fat diet (HFD). Previous work has shown consumption of a HFD increases inflammation in the hypothalamus in male mice and rats, but little to no work has been conducted in females. Here, we showed for the first time that acute HFD exposure increases microgliosis, as measured by an increase in the number of cells expressing the microglia-specific protein Iba1, and decreased microglial branching and complexity in the hypothalamus and nucleus of the solitary tract (NTS) of OVX rats. These data suggest that HFD increased microglial accumulation and activation in the hypothalamus and hindbrain. Estradiol replacement blocked the HFD-induced increase in microglia in the hypothalamus and hindbrain and reduced microglia activation in the hypothalamus. These data provide the first in vivo evidence that estradiol may play a protective role in diet-induced inflammation in female rats. In addition to increasing microglial activity, consumption of a HFD has been shown to negatively impact neuronal health in the hypothalamus. In vitro studies have shown that treatment of hypothalamic neurons with palmitate, a common dietary saturated fat, increases markers of both inflammation and endoplasmic reticulum stress. In our study, we showed that treating cultured POMC neurons with palmitate increased mRNA expression of interleukin-6 (IL-6), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B), and CCAAT-enhancer-binding protein homologous protein (CHOP). Pre-treatment with estradiol attenuated the palmitate-induced increase in IL-6 and treatment with the selective GPER-1 agonist, G-1, attenuated increases in IL-6 and NF-kB mRNA caused by palmitate. These data suggest that estradiol attenuates markers of inflammation caused by saturated fat, but has no effect on a marker of endoplasmic reticulum stress. Taken together, these studies demonstrate that estradiol affects energy homeostasis via activation of extra-nuclear receptors, JAK-STAT activity, and decreasing the neuroimmune response to diet.
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Date Issued: 2019
Identifier: 2019_Summer_Butler_fsu_0071E_15351
Format: Thesis

Title: Insights into Proteasome Quality Control: A Mechanistic Analysis of Proteasome Biogenesis and Clearance.
Creator: Howell, Lauren Anne, Zhu, Fanxiu, Wang, Yanchang, Megraw, Timothy L., Florida State University, College of Medicine, Department of Biomedical Sciences
Abstract/Description: Maintaining the balance of protein synthesis and degradation is essential for efficient proteostasis and organismal health. The 26S proteasome is a large, multisubunit proteolytic complex that functions as the primary mechanism for regulated protein degradation in eukaryotes. Nearly every biological pathway depends on proteasome-mediated catabolism to perform cellular functions or ensure the integrity of its components, making the maintenance of proteasome quality critical. The elaborate...
Show moreMaintaining the balance of protein synthesis and degradation is essential for efficient proteostasis and organismal health. The 26S proteasome is a large, multisubunit proteolytic complex that functions as the primary mechanism for regulated protein degradation in eukaryotes. Nearly every biological pathway depends on proteasome-mediated catabolism to perform cellular functions or ensure the integrity of its components, making the maintenance of proteasome quality critical. The elaborate architecture and large size of the proteasome necessitates dedicated quality control mechanisms to ensure the integrity, abundance, and functionality of the complex. The mechanisms governing proteasome quality control have been implicated in numerous disease pathologies, emphasizing the importance of gaining insight into these cellular processes, which remain poorly understood. Toward this goal, the work set forth in this dissertation aims to elucidate the mechanistic underpinnings of proteasome quality control, focusing on the regulation of proteasome assembly and destruction. A cooperative network of regulatory mechanisms function to rapidly and faithfully assemble dozens of canonical subunits into a highly-organized, functional proteasome. Several alternative proteasome species with non-canonical subunit arrangements have been identified and are linked to diverse physiological and pathophysiological processes. The mechanisms that control canonical versus non-canonical proteasome biogenesis remain largely unknown. The first chapter of this dissertation aims to clarify the quality control mechanisms governing subunit incorporation during proteasome biogenesis. Failure of proteasome quality control can compromise the integrity of the complex and result in the accumulation of aberrant species that can disrupt the proteostatic balance, necessitating the critical requirement for a clearance pathway that has only recently been identified and remains poorly understood. The second chapter of this dissertation aims to study the spatial control of the proteasome during autophagic clearance and identify new factors mediating proteasome destruction. Together, the work presented in this dissertation provides new insight into the mechanisms of proteasome quality control and furthers our understanding into how these processes may be compromised to contribute to disease pathology.
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Date Issued: 2019
Identifier: 2019_Summer_Howell_fsu_0071E_15411
Format: Thesis

Title: Mechanisms of Oxytocin Regulation of Sensory Processing and Sociality in Mice and Humans.
Creator: Tabbaa, Manal, Hammock, Elizabeth Anne Dunn, Gunjan, Akash, Patrick, Christopher J., Wang, Zuoxin, Stanwood, Gregg, Florida State University, College of Arts and Sciences,...
Show moreTabbaa, Manal, Hammock, Elizabeth Anne Dunn, Gunjan, Akash, Patrick, Christopher J., Wang, Zuoxin, Stanwood, Gregg, Florida State University, College of Arts and Sciences, Department of Psychology
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Abstract/Description: Social behaviors are foundational to our society and quality of life while social behavioral deficits are core symptoms in a variety of psychopathologies. Yet, despite the significance for human health, the neural mechanisms that regulate social behavior are incompletely characterized. Oxytocin (OXT) is a neuro-active hormone that has been well-studied over the last several decades and shown to regulate social behaviors. The OXT system interacts with early life environment and an individual's...
Show moreSocial behaviors are foundational to our society and quality of life while social behavioral deficits are core symptoms in a variety of psychopathologies. Yet, despite the significance for human health, the neural mechanisms that regulate social behavior are incompletely characterized. Oxytocin (OXT) is a neuro-active hormone that has been well-studied over the last several decades and shown to regulate social behaviors. The OXT system interacts with early life environment and an individual's genotype to regulate social behaviors in a sexually dimorphic manner. Chapter 1 describes background information on the role of OXT in social behaviors. Thereafter, research on the developmental role of OXT is discussed. Chapter 1 ends with the proposal of a novel mechanism by which socially acquired OXT may regulate social behaviors. In chapters 2-5, experiments are described which aim to empirically test this central hypothesis. In chapter 2, a functional role for oxytocin receptors (OXTR) in the oronasal cavity were investigated in developing mice as a potential mechanism by which OXT can interact with early life sensory experience to regulate brain activity and behavior. OXTRs have recently been characterized in the periphery of developing mice that appear to be located on sensory apparatuses. We tested the effects of orally applied OXT, compared to saline, on a marker of neural activation, c-Fos, in sensory processing brain regions and the paraventricular nucleus of the hypothalamus (PVN). To test the effects of orally applied OXT on sensory-dependent brain activity, we also brushed the whiskers of mice after OXT or saline treatment in a separate experiment. Orally administered OXT, compared to saline, increased c-Fos in the PVN, and decreased c-Fos activity in sensory processing brain regions after whisker brushing. Additionally, orally applied OXT, compared to saline, with whisker brushing decreased Fos in the trigeminal motor nucleus as well as oral-motor behavior of females. Compared to saline, oral OXT with whisker brushing also increased males long distance locomotor behavior. These data support the possibility of a functional role for OXT acting on oronasal OXTR to modulate the brain and behavior in developing mice. In chapter 3, the idea of OXT exchange between conspecifics as a potential driver of social behaviors was tested by examining the preference of male and female mice towards OXT containing social stimuli. Male and female mice showed a preference to investigate same-sex mice that contained OXT versus OXT knock-out (KO) mice. Next, we tested if mice prefer OXT KO bedding containing OXT versus saline and found that males prefer female OXT KO bedding with OXT compared to saline. Finally, we tested the role of OXTR in sensory ganglia in the social preference of live mice by using transgenic mice breeding strategies to selectively knock out OXTR in sensory ganglia. We found that female mice lacking sensory OXTR (OXTRsensory KO) had reduced social investigation levels compared to their wild-type (OXTRsensory WT) litter mates. The role of OXTR in sensory ganglia in social behaviors was further tested in chapter 4 by examining sociability, preference for social novelty, and aggression in OXTRsensory WT and KO mice. OXTRsensory KO females had reduced approach behaviors during the sociability test, compared to OXTRsensory WT females. Altogether, data from chapters 2-4 indicate that environmentally acquired OXT can regulate brain activity and influence behaviors. In addition, mice preferred other mice that contain OXT over OXT KO mice, but not same-sex OXT KO bedding with OXT over saline. These data suggest that preference for an OXT containing live same-sex conspecific may be due to an interaction between OXT and social sensory signals rather than OXT alone. Lastly, these data indicate that OXTRs in sensory ganglia are an important regulator of social behaviors, particularly for females. Finally, in chapter 5, we aimed to explore the translational significance of these findings by examining if variation in the OXTR gene is associated with variation in social sensory processing in humans, with significance for trait meanness. We genotyped the common OXTR single nucleotide polymorphisms (SNPs) rs1042778 and rs53576 in male and female participants that had brain event related potentials (ERP) recorded in response to viewing affective faces. We found that females homozygous for the OXTR SNP rs1042778 G allele had enhanced fear-specific brain responses compared to T allele carriers. Additionally, the rs53576 A allele was associated with higher ERP amplitudes to face stimuli, but not scrambled faces, compared to GG allele carriers. Further, OXTR SNPs rs1042778 and rs53576 interacted to predict trait meanness in females. These data implicate OXTR gene variability in the etiology of sensory processing variation in the brain as well as trait meanness. A summary of results as well as implications and suggestions for future research are discussed in chapter 6.
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Date Issued: 2019
Identifier: 2019_Summer_Tabbaa_fsu_0071E_15376
Format: Thesis

Title: Human Pluripotent Stem Cells on Cellular Behaviors of Isogenic Cortical Spheroids.
Creator: Marzano, Mark Cole, Li, Yan, Ma, Teng, Guan, Jingjiao, Florida State University, FAMU-FSU College of Engineering (Tallahassee, Fla.), Department of Chemical and Biomedical...
Show moreMarzano, Mark Cole, Li, Yan, Ma, Teng, Guan, Jingjiao, Florida State University, FAMU-FSU College of Engineering (Tallahassee, Fla.), Department of Chemical and Biomedical Engineering
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Abstract/Description: Extracellular vesicles (EVs) or exosomes are responsible for a variety of signaling processes and overall physiological and pathological states of stem cells and tissues. Human induced pluripotent stem cells (hiPSCs) have unique characteristics that can mimic embryonic tissue development. EVs derived from hiPSCs can be used as therapeutics, biomarkers, and drug delivery vehicles. One issue is that little is known about the characteristics of secreted EVs/exosomes by hiPSCs during tissue...
Show moreExtracellular vesicles (EVs) or exosomes are responsible for a variety of signaling processes and overall physiological and pathological states of stem cells and tissues. Human induced pluripotent stem cells (hiPSCs) have unique characteristics that can mimic embryonic tissue development. EVs derived from hiPSCs can be used as therapeutics, biomarkers, and drug delivery vehicles. One issue is that little is known about the characteristics of secreted EVs/exosomes by hiPSCs during tissue morphogenesis due to paracrine signaling. In this study, EVs derived from hiPSC-derived neural progenitors (ectoderm), hiPSC-derived cardiac cells (mesoderm), and the undifferentiated hiPSCs (healthy iPSK3 and Alzheimer's associated SY-UBH lines) were analyzed. Nanoparticle tracking analysis and electron microscopy results showed that the derived EVs had the average size of 100-250 nm. Western blot revealed that exosomal markers ALIX, CD63, and TSG 101 were expressed in the derived EVs. miRNAs including miR-133 and miR-155 were differently expressed in different EV groups. Treating the cortical spheroids with different EVs in vitro showed the differential abilities of increasing cell proliferation (indicated by BrdU assay) and axonal growth (indicated by β-tubulin III staining). For the Aβ42 oligomer treated cultures, the derived EVs increased cell viability and reduced oxidative stress differentially, showing neural protective ability. This study should advance our understanding of cell-cell communications in stem cell microenvironment and provide possible therapeutic options for treating neural degeneration.
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Date Issued: 2019
Identifier: 2019_Summer_Marzano_fsu_0071N_15211
Format: Thesis

Title: On the Use of Conformal Mappings, Invariants and Warpings in Investigations of the Cortical Surface.
Creator: Eady, Carolyn M. (Carolyn Marie), Cogan, Nicholas G., Stroupe, M. Elizabeth (Margaret Elizabeth), Bowers, Philip L., Mio, Washington, Florida State University, College of Arts...
Show moreEady, Carolyn M. (Carolyn Marie), Cogan, Nicholas G., Stroupe, M. Elizabeth (Margaret Elizabeth), Bowers, Philip L., Mio, Washington, Florida State University, College of Arts and Sciences, Department of Mathematics
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Abstract/Description: Brain mapping studies the structure and function of the brain through imaging techniques in an effort to relate function to specific anatomical regions. The highly folded nature of the human brain makes visualization difficult; thus neuroscientists desire quantitative methods to assist with characterization of anatomical differences between subjects. As cortical folding patterns are unique to each person, methods for quantifying differences across subjects are lacking. A first step in...
Show moreBrain mapping studies the structure and function of the brain through imaging techniques in an effort to relate function to specific anatomical regions. The highly folded nature of the human brain makes visualization difficult; thus neuroscientists desire quantitative methods to assist with characterization of anatomical differences between subjects. As cortical folding patterns are unique to each person, methods for quantifying differences across subjects are lacking. A first step in quantifying data is to define a normal, healthy brain in terms of both shape and function. Establishing spatial characteristics of a healthy brain, or an aggregate of healthy brains, will allow for better diagnosis and treatment of brain injuries and diseases. The research discussed in this thesis uses a two-dimensional, or ``flat", map of the cortical surface based on a three-dimensional reconstruction from medical images acquired using magnetic resonance imaging. Such mappings can be used to measure cross-subject differences, as well as the development of abnormalities in longitudinal studies of diseased patient brains. The ``flat" mappings we discuss are constructed using a mathematical approach known as circle packing. Our research makes use of conformal mappings and the properties preserved under them, defining ways to apply them to brain data. These conformally invariant properties take into account aspects of the three-dimensional shape of the surface which are not easily visualized, and allow us to quantify them in a two-dimensional result. In particular, we develop methods for calculation of extremal length and harmonic measure. We also give various forms of visualization of each invariant, and in some cases visualization of biological data in conjunction with our calculations. After calculating methods of comparison to be used indirectly, we offer two adaptations to an existing program which allow for the direct comparison of cortical surfaces. The data we use does not fit specific criteria of the program, so these adaptations are necessary to generalize the usage of the existing program. We again use the concept of circle packing for ``flat" mappings; however, the ingenuity of our adaptations lies in the connecting between these ``flat" maps. We follow a procedure for transforming the coordinates of a source surface into those of a destination surface, through a system of weighting vertices by barycentric or gyrobarycentric coordinates. Though our research focuses on a small region of the human brain, we proceed with the expectation that all methods can be generalized to other regions of the brain. As many psychological disorders have structural manifestations, the research can be used to investigate anomalies in brain data for such illnesses. Additionally, the methods we provide can be further generalized to surfaces other than the brain, laying groundwork for use in general investigations of two and three-dimensional surfaces.
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Date Issued: 2019
Identifier: 2019_Summer_Eady_fsu_0071E_15275
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: 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: Assembly Mechanisms and Functions of Centrosomal and Non-Centrosomal Microtubule-Organizing Centers.
Creator: Zheng, Yiming, Megraw, Timothy L., Yu, Hong-Guo, Wang, Yanchang, Tomko, Robert J., Florida State University, College of Medicine, Department of Biomedical Sciences
Abstract/Description: This dissertation focuses on microtubule assembly mechanisms and functions in dividing and non-dividing cells. In dividing cells, centrosome is the major microtubule-organizing center (MTOC). Ninein (Nin) is a centrosomal protein whose gene is mutated in Seckel syndrome, an inherited recessive disease that results in primordial dwarfism, cognitive deficiencies, and increased sensitivity to genotoxic stress. Nin is evolutionarily conserved, yet its role in cell division and development has not...
Show moreThis dissertation focuses on microtubule assembly mechanisms and functions in dividing and non-dividing cells. In dividing cells, centrosome is the major microtubule-organizing center (MTOC). Ninein (Nin) is a centrosomal protein whose gene is mutated in Seckel syndrome, an inherited recessive disease that results in primordial dwarfism, cognitive deficiencies, and increased sensitivity to genotoxic stress. Nin is evolutionarily conserved, yet its role in cell division and development has not been investigated in a model organism. Here, we characterize the single Nin ortholog in Drosophila. Drosophila Nin localizes to the periphery of the centrosome, but not at centriolar structures as in mammals. However, Nin shares the property of its mammalian ortholog of promoting microtubule assembly. In neural and germline stem cells, Nin localizes asymmetrically to the younger (daughter) centrosome, yet it is not required for the asymmetric division of dividing stem cells. Surprisingly, loss of nin expression from a nin mutant does not significantly impact embryonic and brain development, fertility, or locomotor performance of mutant flies, nor their survival upon exposure to DNA damaging agents. While not essential, Nin localizes to non-centrosomal MTOCs (ncMTOCs) in wing epithelia and muscle, two types of specialized and differentiated cell types, suggesting that Nin plays a supportive role in non-centrosomal microtubule organization. ncMTOCs have a variety of roles presumed to serve the diverse functions of the range of non-dividing cell types in which they are found. ncMTOCs are diverse in their composition, subcellular localization, and function. Here we report a novel perinuclear MTOC in another differentiated Drosophila cell type, fat body cells. This perinuclear ncMTOC in fat body cells is anchored by Msp300/Nesprin at the cytoplasmic surface of the nucleus. Msp300 recruits the MT minus-end protein Patronin/CAMSAP, which functions redundantly with Nin to assemble non-centrosomal MTs and does so independently of the widespread MT nucleation factor -tubulin. Patronin does not antagonize with known MT depolymerases in fat body ncMTOC, but acts cooperatively with Ninein to assemble circumferential MTs, and also recruit MT polymerase Msps to promote elongation of radial MTs. Functionally, the fat body ncMTOC is essential for retrograde dynein-dependent endosomal trafficking to restrict plasma membrane growth and for the secretion of basement membrane proteins. Together, we identify an ncMTOC with novel architecture and MT regulation properties that serves a vital secretory function.
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Date Issued: 2019
Identifier: 2019_Fall_Zheng_fsu_0071E_15516
Format: Thesis

Title: The Molecular-Genetic Basis of Sex-Specific Behaviors.
Creator: Newell, Nicole R., Arbeitman, Michelle N. (Michelle Nina), Hughes, Kimberly A., Megraw, Timothy L., Horabin, Jamila I., Florida State University, College of Medicine, Department...
Show moreNewell, Nicole R., Arbeitman, Michelle N. (Michelle Nina), Hughes, Kimberly A., Megraw, Timothy L., Horabin, Jamila I., Florida State University, College of Medicine, Department of Biomedical Sciences
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Abstract/Description: Understanding the neural and genetic basis of complex behaviors is a major outstanding question in neurobiology. The fruit fly, Drosophila melanogaster, displays innate, complex reproductive behaviors. These behaviors are regulated by transcription factors encoded by fruitless (fru) and doublesex (dsx). Both fru and dsx are sex-specifically spliced downstream of the sex determination hierarchy, resulting in production of male-specific isoforms of Fru and Dsx and a female-specific isoform of...
Show moreUnderstanding the neural and genetic basis of complex behaviors is a major outstanding question in neurobiology. The fruit fly, Drosophila melanogaster, displays innate, complex reproductive behaviors. These behaviors are regulated by transcription factors encoded by fruitless (fru) and doublesex (dsx). Both fru and dsx are sex-specifically spliced downstream of the sex determination hierarchy, resulting in production of male-specific isoforms of Fru and Dsx and a female-specific isoform of Dsx. These transcription factors direct sex differences in behavior by regulating expression of downstream genes. fru is a complex locus and only transcripts from the P1 promoter (fru P1) are sex-specifically spliced to generate male-specific isoforms (FruM). To gain insight into gene expression differences in fru P1-expressing neurons between males and females, we used Translating Ribosome Affinity Purification (TRAP). The results of this study and other studies led us to examine a family of proteins with known functions in synaptic connectivity encoded by defective proboscis extension response (dpr) and Dpr Interacting Protein (DIP) genes. We examined the co-expression of dprs/DIPs with fru P1 neurons. We found the majority of dprs/DIPs are co-expressed with fru P1-expressing neurons in both males and females, but with distinct patterns. When we activate and silence subsets of fru P1 neurons that overlap with dpr/DIP- expressing neurons, particular courtship phenotypes are observed, allowing us to assign functions to subpopulations of neurons. Overall, this work contributes to our understanding of how FruM is specifying and maintaining the neural circuitry for male courtship behavior, with a focus on the regulation and function of the dprs and DIPs. We also focus on the regulation of female reproductive behaviors, by examining interactions between the germline and female head tissues. We examined gene expression changes in head tissues of females with and without a germline, as virgins and at one- and three- day(s) post-mating, as well as in females that had been mated to males lacking a germline. A set of female post-mating behaviors was also characterized. Additionally, we performed a genome-wide association study which led to the identification of polymorphisms that contribute to natural variation of female re-mating behavior. Together, these studies provide insight into the molecular-genetic control of female reproduction.
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Date Issued: 2019
Identifier: 2019_Fall_Newell_fsu_0071E_15487
Format: Thesis

Title: Effects of Developmental Nicotine Exposure on the Brain and Behavior.
Creator: Martin, Melissa M., Bhide, Pradeep, Dennis, Jonathan Hancock, Nowakowski, Richard S., Stanwood, Gregg, Metin, Christine, Florida State University, College of Medicine,...
Show moreMartin, Melissa M., Bhide, Pradeep, Dennis, Jonathan Hancock, Nowakowski, Richard S., Stanwood, Gregg, Metin, Christine, Florida State University, College of Medicine, Department of Biomedical Sciences
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Abstract/Description: Cigarette smoking during pregnancy is a major public health concern, resulting in detrimental health effects in the mother and her offspring. The adverse behavioral consequences for children with developmental nicotine exposure include increased risk for attention deficit hyperactivity disorder, working memory deficits, epilepsy, novelty-seeking, and risk-taking behaviors. Interestingly, these behavioral conditions are consistent with altered inhibitory (GABA) neurotransmitter signaling....
Show moreCigarette smoking during pregnancy is a major public health concern, resulting in detrimental health effects in the mother and her offspring. The adverse behavioral consequences for children with developmental nicotine exposure include increased risk for attention deficit hyperactivity disorder, working memory deficits, epilepsy, novelty-seeking, and risk-taking behaviors. Interestingly, these behavioral conditions are consistent with altered inhibitory (GABA) neurotransmitter signaling. Therefore, the goal of my dissertation research was to test the hypothesis that early exposure to nicotine alters the development of the GABA system, which can produce functional changes in the brain. In order to test the hypothesis that developmental nicotine exposure produces long-lasting effects in the adult mouse brain, specifically the GABA system, I used a GAD67-GFP knock-in mouse in which GFP is intrinsically expressed under the GAD67 promoter. I first examined GABA neuron numerical densities, non-GABA neuron numerical densities, as well as the GABA-to-non-GABA neuron ratio for male and female offspring that received plain drinking water (WATER) or drinking water containing either 100 µg/ml (Nic100) or 200 µg/ml (Nic200) nicotine during development. I performed these analyses in the prefrontal and medial prefrontal cortices, two brain regions which are important for executive function and regulate behaviors known to be altered in developmental disorders such as attention deficit/hyperactivity disorder. In addition, these frontal cortical brain regions were subdivided into cortical layers (II-III, V, and VI) since each cortical layer is defined based on their cortical connections and hence have different functions. Overall, I found that developmental nicotine exposure does not alter the numerical density of GABA or non-GABA cells in the frontal cortex, however, I saw a significant reduction in the GABA-to-non-GABA ratio in the Nic200 treatment group compared to the WATER and Nic100 groups across both brain regions (prefrontal and medial prefrontal cortices) and across all cortical layers. Next, in order to test the hypothesis that developmental nicotine exposure produces long-lasting effects on behavioral phenotypes, I performed a battery of tests that examined locomotor activity, approach-avoidance behavior, exploratory behavior, spatial working memory, and object-based attention beginning at 90 days of age. Since I found a significant reduction in the GABA-to-non-GABA ratio for the Nic200 treatment group and not for the Nic100 group, I performed behavioral analyses in the WATER and Nic200 treatment groups. For these analyses, I used both male and female offspring as well as GAD67-GFP and wild-type offspring. I found that developmental nicotine exposure increases novelty-induced locomotor activity, increases exploratory behavior, and alters approach-avoidance behavior in favor of the approach behavior. During embryonic development, the majority of the GABA neurons originate in the medial ganglionic eminence of the basal forebrain and migrate tangentially to regions of the dorsal forebrain. At the same time, neurogenesis in the ventricular and subventricular zones is occurring in the dorsal forebrain. Therefore, I wanted to examine the immediate effects of developmental nicotine exposure on GABA neuron migration and neurogenesis in the embryonic brain. To do this, I collected the brains from embryonic day 13 and 15 embryos; two time points during embryonic development in which GABA neurons are migrating through regions of the dorsal forebrain and neurogenesis is occurring in the dorsal forebrain. Using GAD67-GFP embryos, I found that nicotine exposure significantly increases the number of GABA neurons in the prefrontal and medial prefrontal cortices. Similarly, using an in-vitro culture assay, nicotine exposure increased the number of GABA neurons migrating out from a medial ganglionic eminence explant. Taken together these results suggest that nicotine exposure during embryonic development alters GABA neuron migration. In addition, using wild-type embryos, I found that nicotine exposure significantly reduces the cell output in the future prefrontal and medial prefrontal cortices. Overall, developmental nicotine exposure produced dose-dependent decreases in GABA-to-non-GABA neuron ratios in the prefrontal and medial prefrontal cortices without perturbing the intrinsic differences in cortical thickness and laminar distribution of GABA or non-GABA neurons between these regions. A significant increase in exploratory behavior and a shift toward “approach” in the approach-avoidance paradigm were also observed. Thus, developmental nicotine exposure shifts the cortical excitation-inhibition balance toward excitation and produces behavioral changes consistent with novelty-seeking behavior. Lastly, I found that nicotine exposure during embryonic development produces a significant increase in GABA neuron migration and reduces the cell output in the dorsal forebrain. Collectively, these results suggest that developmental nicotine exposure alters embryonic brain development and that continued exposure during the embryonic and early postnatal period produces long-lasting structural and functional changes in the brain.
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Date Issued: 2019
Identifier: 2019_Fall_Martin_fsu_0071E_15481
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: Foraging Ecology and Diet Selection of Juvenile Green Turtles (Chelonia mydas) in the Western Bahamas: Insights from Stable Isotope Analysis and Prey Mapping.
Creator: Gillis, Anthony John, Fuentes, Mariana, Chanton, Jeffrey P., Spencer, Robert G. M., Seminoff, Jeffrey A., Florida State University, College of Arts and Sciences, Department of...
Show moreGillis, Anthony John, Fuentes, Mariana, Chanton, Jeffrey P., Spencer, Robert G. M., Seminoff, Jeffrey A., Florida State University, College of Arts and Sciences, Department of Earth, Ocean and Atmospheric Science
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Abstract/Description: Species’ foraging choices influences their somatic growth rates, age at maturity, and time spent in vulnerable early life stages. Thus, differences in population demographics are often attributed to variability either in diet type, quality or quantity ingested. Knowledge of species diet selection, though currently limited, particularly in marine environments, can enhance our understanding of the roles of species in marine ecosystem and, at a finer scale, elucidate how nutrition and diet...
Show moreSpecies’ foraging choices influences their somatic growth rates, age at maturity, and time spent in vulnerable early life stages. Thus, differences in population demographics are often attributed to variability either in diet type, quality or quantity ingested. Knowledge of species diet selection, though currently limited, particularly in marine environments, can enhance our understanding of the roles of species in marine ecosystem and, at a finer scale, elucidate how nutrition and diet influences their growth and productivity. Marine green turtles (Chelonia mydas) are considered to be herbivores, predominantly consuming seagrass and algae. However, recent studies have suggested that they may exhibit omnivory in certain forage areas. Using juvenile green turtles as a case study, I coupled stable isotope analysis with a diet preference index to provide insights into the selection and plasticity of their diet. The study was conducted within two sites (Bonefish Hole and South Bimini) in Bimini, Bahamas in 2016. Habitat surveys were conducted to gather habitat data and determine resource availability. A dichotomy in diet was found between the sites: at Bonefish Hole, turtles exhibited a more generalist omnivorous diet, selecting for sessile filters feeders and green algae, whereas turtles in South Bimini had a more specialist herbivorous diet, primarily consuming seagrasses and selecting for red algae, when available. The foraging dichotomy found in this study by green turtles expands our understanding of the spatial differences in their biology in the Bahamas and provides novel information for turtle foraging in Bimini. Knowledge about differences in intra-specific diet, with a focus on diet selection and potential drivers, can elucidate the factors that influence critical life history traits and ultimately inform species management.
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Date Issued: 2018
Identifier: 2018_Sp_Gillis_fsu_0071N_14539
Format: Thesis

Title: Traits, Species, and Communities: Integrative Bayesian Approaches to Ecological Biogeography across Geographic, Environmental, Phylogenetic, and Morphological Space.
Creator: Humphreys, John M., Elsner, James B., Steppan, Scott J., Mesev, Victor, Pau, Stephanie, Florida State University, College of Social Sciences and Public Policy, Department of...
Show moreHumphreys, John M., Elsner, James B., Steppan, Scott J., Mesev, Victor, Pau, Stephanie, Florida State University, College of Social Sciences and Public Policy, Department of Geography
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Abstract/Description: Assuming a methodological perspective, this dissertation proceeds through a series of studies that cover levels of biological organization ranging from the morphological traits of individual specimens to community assemblages. The presented research explores geographic extents ranging from local to global scales, examines both plants and animals, and explores relationships among species with common ancestry. The research appraises and then proposes solutions to a variety of yet unresolved...
Show moreAssuming a methodological perspective, this dissertation proceeds through a series of studies that cover levels of biological organization ranging from the morphological traits of individual specimens to community assemblages. The presented research explores geographic extents ranging from local to global scales, examines both plants and animals, and explores relationships among species with common ancestry. The research appraises and then proposes solutions to a variety of yet unresolved issues in species distribution modeling; including, preferential sampling, spatial dependency, multi-scaled spatial processes, niche equilibrium assumptions, data structure arising from shared evolutionary history, and correlations between predictor variables. Approaching the geographic distribution of wetlands as an applied concern, the study presented in Chapter 2 emphasizes that the identication and inventory of wetlands are essential components of water resource management. To be eective in these endeavors, it is critical that the process used to detect and document wetlands be time ecient, accurate, and repeatable as new environmental information becomes available. Approaches dependent on aerial photographic interpretation of land cover by individual human analysts necessitate hours of assessment, introduce human error, and fail to include the best available soils and hydrologic data. The goal of Chapter 2 is to apply hierarchical modeling and Bayesian inference to predict the probability of wetland presence as a continuous gradient with the explicit consideration of spatial structure. The presented spatial statistical model can evaluate 100 km2 at a 50 x 50 meter resolution in approximately 50 minutes while simultaneously incorporating ancillary data and accounting for latent spatial processes. Model results demonstrate an ability to consistently capture wetlands identied through aerial interpretation with greater than 90% accuracy (scaled Brier Score) and to identify wetland extents, ecotones, and hydrologic connections not identied through use of other modeling and mapping techniques. The provided model is reasonably robust to changes in resolution, areal extents between 100 km2 and 300 km2, and region-specic physical conditions. As with modeling wetland occurrence, species distribution modeling aimed at forecasting the spread of invasive species under projected global warming also oers land managers an important tool for assessing future ecological risk and for prioritizing management actions. Chapter 3 applies Bayesian inference and newly available geostatistical tools to forecast global range expansion for the ecosystem altering invasive climbing fern Lygodium microphyllum. The presented modeling framework emphasizes the need to account for spatial processes at both the individual and aggregate levels, the necessity of modeling non-linear responses to environmental gradients, and the explanatory power of biotic covariates. Results indicate that Lygodium microphyllum will undergo global range expansion in concert with anthropogenic global warming and that the species is likely temperature and dispersal limited. Predictions are presented for current and future climate conditions assuming both limited and unlimited dispersal scenarios. Finally, Chapter 4 provides a novel framework to combine multi-species joint modeling techniques with spatially explicit phylogenetic regression to simultaneously predict the probability of species occurrence and the geographic distribution of interspecic continuous morphological traits. Choosing the South American leaf-eared mice (genus: Phyllotis) as an empirical example, a threetiered phylogenetic coregionalization trait biogeography model (PhyCoRTBio) is constructed. The conditionally dependent structure of the PhyCoRTBio model enables information from multiple species and from multiple specimen-specic trait metrics to be leveraged towards estimation of a focal species distribution. I hypothesize that, relative to other commonly used species distribution modeling methods, the PhyCoRTBio approach will exhibit improved performance in predicting occurrence for species within the genus Phyllotis. After describing its statistical implementation, this hypothesis is assessed by constructing PhyCoRTBio models for six dierent Phyllotis species and then comparing results to those derived using maximum entropy methods, random forest clustering, Gaussian random eld species distribution models, and Hierarchical Bayesian species distribution models. To judge the relative performance of each modeling approach, model sensitivity (proportion of correctly predicted presences), specicity (proportion of correctly predicted absences), the area under the receiver operating characteristic curve (AUC), and the True Skill Statistic (TSS) are calculated. Findings indicate that trait-based covariates improve model performance and highlight the need to consider spatial processes and phylogenetic information during multi-species distribution modeling.
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Date Issued: 2018
Identifier: 2018_Sp_Humphreys_fsu_0071E_14298
Format: Thesis

Title: Using Mathematical Tools to Investigate the Autoimmune Hair Loss Disease Alopecia Areata.
Creator: Dobreva, Atanaska, Cogan, Nicholas G., Stroupe, M. Elizabeth, Bertram, R., Hurdal, Monica K., Florida State University, College of Arts and Sciences, Department of Mathematics
Abstract/Description: Alopecia areata is an autoimmune condition where the immune system attacks hair follicles and disrupts their natural cycle through phases of growth, regression, and rest. The disease manifests with distinct hair loss patterns, and what causes it and how to treat it are open questions. We first construct an ODE model for alopecia areata in follicles which are in stage of growth. The dynamical system describes the behavior of immune cells and signals highlighted by experimental studies as...
Show moreAlopecia areata is an autoimmune condition where the immune system attacks hair follicles and disrupts their natural cycle through phases of growth, regression, and rest. The disease manifests with distinct hair loss patterns, and what causes it and how to treat it are open questions. We first construct an ODE model for alopecia areata in follicles which are in stage of growth. The dynamical system describes the behavior of immune cells and signals highlighted by experimental studies as primarily involved in the disease development. We perform sensitivity analysis and linear stability and bifurcation analysis to investigate the importance of processes in relation to the levels of immune cells. Our findings indicate that the pro-inflammatory pathway via the messenger protein interferon-gamma and the immunosuppressive pathway via hair follicle immune privilege agents are crucial. Next, we incorporate follicle cycling into the model and explore what processes have the greatest impact on the duration of hair growth in healthy versus diseased follicles. The results suggest that some processes matter in both cases, but there are differences, as well. Finally, the study presents and analyzes a PDE model which captures patterns characteristic of hair loss in alopecia areata.
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Date Issued: 2018
Identifier: 2018_Sp_Dobreva_fsu_0071E_14479
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: Exploration of the Role of Disinfection Timing, Duration, and Other Control Parameters on Bacterial Populations Using a Mathematical Model.
Creator: Acar, Nihan, Cogan, Nicholas G., Keller, Thomas C. S., Bertram, R., Mio, Washington, Florida State University, College of Arts and Sciences, Department of Mathematics
Abstract/Description: Tolerant bacteria enmeshed in a biofilm causes several difficult to treat illnesses like tuberculosis, chronic pneumonia, and chronic inner ear infections. These diseases typically respond poorly to antibiotics due to high tolerance. Bacterial tolerance can be genotypic (resistance-e.g. MRSA), phenotypic (non-heritable) or environmental (e.g. nutrient gradients). Persister formation is phenotypic tolerance that is highly tolerant to disinfection. Constant dosing is typically ineffective in...
Show moreTolerant bacteria enmeshed in a biofilm causes several difficult to treat illnesses like tuberculosis, chronic pneumonia, and chronic inner ear infections. These diseases typically respond poorly to antibiotics due to high tolerance. Bacterial tolerance can be genotypic (resistance-e.g. MRSA), phenotypic (non-heritable) or environmental (e.g. nutrient gradients). Persister formation is phenotypic tolerance that is highly tolerant to disinfection. Constant dosing is typically ineffective in eliminating persister cells. To generate an effective treatment protocol, more research must examine the dynamics of persister cells. This study investigates how manipulating the application of antibiotics and the addition of nutrient may enhance the disinfection of a bacterial population in batch culture. Previous studies focused on the antimicrobial agent as a control variable to eliminate the bacterial population. In addition to antibiotic treatments, we consider the significance of the nutrient in eradicating the susceptible and persister cells since the disinfection of the susceptible population is dependent on nutrient intake. We present a mathematical model that captures the dynamics between susceptible and persister bacteria with antibiotic and nutrient as control variables. We investigate the optimal dose-withdrawal timing of antibiotic in several cases including: constant nutrient in time, dynamic nutrient in time, and piecewise constant nutrient in time. Also a global sensitivity analysis method, Partial Rank Correlation Coefficient (PRCC), is applied to determine the significance of model parameters for a quantity of interest. The highlights of this study are; 1.) Constant dosing is not an effective disinfection protocol. 2.) Nutrient plays a significant role such that in the presence of nutrient, bacterial population is eliminated much faster. 3.) Checking the eigenvalues of the established Poincaré map gives us information on how to choose withdraw-dose timing for the nonlinear system. 4.) Periodic dose-withdraw offers a more efficient disinfection provided dose time is longer than withdrawal of antibiotic. 5.) As duration of dose decreases, the elimination of bacteria decreases and the death rate becomes insignificant.
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Date Issued: 2018
Identifier: 2018_Su_Acar_fsu_0071E_14749
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: 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: Philosophical, Historical, and Empirical Investigations into the Concept of Biological Fitness.
Creator: Takacs, Peter, Ruse, Michael, Travis, Joseph, Bishop, Michael A., Justus, James, Florida State University, College of Arts and Sciences, Department of Philosophy
Abstract/Description: While undeniably one of the central explanatory concepts in biology, fitness is deployed in an ambiguous or even inconsistent manner by evolutionary biologists as well as philosophers. This sort of foundational confusion is a plea for conceptual clarity and has, thereby, presented a wonderful opportunity for philosophers of science to ply their trade. After engaging with the topic, however, several influential philosophers of science (e.g., Mohan Matthen, Dennis Walsh, and Andre Ariew) and...
Show moreWhile undeniably one of the central explanatory concepts in biology, fitness is deployed in an ambiguous or even inconsistent manner by evolutionary biologists as well as philosophers. This sort of foundational confusion is a plea for conceptual clarity and has, thereby, presented a wonderful opportunity for philosophers of science to ply their trade. After engaging with the topic, however, several influential philosophers of science (e.g., Mohan Matthen, Dennis Walsh, and Andre Ariew) and biologists (Richard Lewontin and Massimo Pigliucci) have reached the conclusion that biological fitness is not in fact the cause of natural selection but instead a mere statistical artifact or redescription of systematic transgenerational change. It is, as they see matters, a label best reserved for abstract trait types rather than the organisms that bear such traits. This poses a serious challenge to the working intuitions of most biologists and many philosophers of biology. Moreover, it is but one of many challenges to the explanatory and ontological primacy of natural selection in recent memory. For at least three decades, some practitioners in the burgeoning subdiscipline of evolutionary developmental biology have been outspoken in insisting that the tools of population biology are insufficient for describing or explaining observations of adaptive evolutionary change both past and present. In this dissertation, I examine these recent challenges to orthodox conceptions of fitness and natural selection, as well as the rejoinders given in defense. Ultimately, I defend a conception of fitness as a probabilistic dispositional property (i.e., a propensity) of token organisms that causes natural selection.
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Date Issued: 2017
Identifier: FSU_FALL2017_Takacs_fsu_0071E_14240
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: 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: Sex Differences in the Addiction-like Properties of Ketamine.
Creator: Wright, Katherine Nicole, Kabbaj, Mohamed, Keller, Laura R., Bhide, Pradeep, Horabin, Jamila I., Feng, Jian, Florida State University, College of Medicine, Department of...
Show moreWright, Katherine Nicole, Kabbaj, Mohamed, Keller, Laura R., Bhide, Pradeep, Horabin, Jamila I., Feng, Jian, Florida State University, College of Medicine, Department of Biomedical Sciences
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Abstract/Description: Depression is a devastating disease that is the leading cause of disability worldwide. One reason for its massive disease burden is that classical antidepressants require several weeks of administration to take effect, and they are only effective in roughly half of patients. Ketamine, previously known as primarily a veterinary anesthetic, rapidly alleviates treatment-resistant depressive symptoms at sub-anesthetic doses. Indeed, a single intravenous (i.v.) infusion of ketamine elicits an...
Show moreDepression is a devastating disease that is the leading cause of disability worldwide. One reason for its massive disease burden is that classical antidepressants require several weeks of administration to take effect, and they are only effective in roughly half of patients. Ketamine, previously known as primarily a veterinary anesthetic, rapidly alleviates treatment-resistant depressive symptoms at sub-anesthetic doses. Indeed, a single intravenous (i.v.) infusion of ketamine elicits an antidepressant effect within 2 hours and can last up to 2 weeks. Furthermore, this therapeutic effect of ketamine can be prolonged with repeated intermittent treatments. However, there are still many unanswered questions regarding ketamine’s safety, especially with respects to the long-term effects of prolonged, repeated exposure. Since ketamine is also a popular club drug with addictive properties, it is critical to characterize the safety and abuse liability of this drug. Additionally, women have twice the prevalence rates of depression as compared to men, and they progress more rapidly through the phases of drug addiction than men. Despite these known sex differences, females have been heretofore underrepresented in clinical and pre-clinical research. We sought to determine if intermittent self-administration of ketamine in rats can trigger drug-seeking behavior, at a dose that is close to the therapeutic human dose, and if the stage of the four-day estrous cycle can influence this behavior, as antidepressant-like effects of ketamine in females depends on estrous cycle stage (Dossat 2016). To that end, rats were trained to self-administer ketamine (0.1 mg/kg/infusion) in an operant chamber once every fourth day for males, while females’ self-administration sessions coincided with either diestrus 1 (when estradiol and progesterone are low), or proestrus (when these hormones are high). Ketamine intake in diestrus-trained females rapidly declined, while proestrus-trained females and males were stable across the acquisition phase of the experiment. After extinction training, proestrus-trained females and males (but not diestrus-trained females) displayed reinstated ketamine-seeking behavior when re-exposed to discrete cues that were previously paired to ketamine availability. Interestingly, a ketamine priming injection in the absence of cues did not reinstate ketamine-seeking behavior as is consistently seen with a priming injection of cocaine, a drug with very high abuse potential. Together, this indicates not only that ketamine-paired cues are more salient precipitators of relapse than the pharmacological effects of ketamine on its own, but also that the stage of estrous cycle associated with high levels of gonadal hormones supports female’s ketamine intake and subsequent ketamine relapse. To address comorbid depression and ketamine addiction, we assessed ketamine addictive-like behaviors in males and females previously exposed to unpredictable chronic mild stress (CMS), a procedure shown to induce a depressive-like phenotype in rodents. They were treated with four intermittent therapeutic i.v. ketamine infusions, designed to mimic infusion protocols used in clinics. They were then given access to self-administer 0.5 mg/kg/infusion ketamine and tested for their motivation to obtain ketamine using progressive ratio schedule (PR) and persistence of incubated ketamine craving after a period of forced abstinence, which is a major factor in the precipitation of relapse. CMS decreased sucrose intake in both sexes, but ketamine had no effect on anhedonia like-behaviors. Ketamine treatment reduced anxiety-induced neophagia, measured by the novelty-suppressed feeding test, suggesting that i.v. ketamine’s antidepressant-like effects may be symptom- and species-specific. While prior ketamine exposure and CMS had no effect on subsequent ketamine addiction-like behavior in males, females that underwent CMS displayed more addiction-like behavior than non-stressed females, suggesting that chronic stress can increase the risk for ketamine abuse in females. Additionally, ketamine pre-exposed females displayed lower ketamine intake, with no alterations in motivated responding or craving. Finally, dendritic spine density and morphology was assessed in the nucleus accumbens (NAc), the central processor for rewarding stimuli that is affected by both depression and addiction. Females with prior ketamine exposure regardless of stress condition had an increase in spine density that was primarily driven by the formation of immature thin spines; there were no changes in males. Together this suggests that although ketamine pre-treatment may alter NAc plasticity in a sex-dependent manner, it does not potentiate ketamine addiction-like behavior. Taken together, this work demonstrates that although ketamine and drug-paired cues have strong reinforcing effects, prior exposure to therapeutic ketamine infusions do not increase the risk of abuse. Estrous cycle stage and prior exposure to chronic stress influences the reinforcing properties of ketamine in females, suggesting an influence of ovarian hormones. The fact that ketamine-seeking behavior was dependent on the drug-paired cues rather than the acute pharmacological effects of ketamine, and that prior therapeutic ketamine did not increase the subsequent development of addiction-like behavior, suggests that there may be little overlap in ketamine’s antidepressant effects and addictive effects. It is possible that ketamine, if administered under the appropriate conditions, may have limited abuse potential, but research is necessary to determine if these sex-specific effects in rats translate to humans.
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Date Issued: 2017
Identifier: FSU_FALL2017_Wright_fsu_0071E_14202
Format: Thesis

Title: Insulin Secretion Rhythms: Calcium Regulation of Beta-Cell Metabolism and Rescue of Islet Oscillations.
Creator: McKenna, Joseph P., Bertram, R. (Richard), Roper, Michael Gabriel, Muslimani, Ziad H., Moore, M. Nicholas J. (Matthew Nicholas J.), Miller, Brian G., Florida State University,...
Show moreMcKenna, Joseph P., Bertram, R. (Richard), Roper, Michael Gabriel, Muslimani, Ziad H., Moore, M. Nicholas J. (Matthew Nicholas J.), Miller, Brian G., Florida State University, College of Arts and Sciences, Department of Mathematics
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Abstract/Description: Pancreatic islet beta-cells play a vital role in regulating blood glucose levels by releasing insulin into the bloodstream. Insulin is released in pulses that parallel interacting beta-cell rhythms, including oscillatory glucose metabolism and periodic calcium influx. We present concurrent time series records of metabolic variables and intracellular calcium levels in glucose-stimulated beta-cells that support regulation of mitochondrial dehydrogenases is the dominant calcium feedback effect...
Show morePancreatic islet beta-cells play a vital role in regulating blood glucose levels by releasing insulin into the bloodstream. Insulin is released in pulses that parallel interacting beta-cell rhythms, including oscillatory glucose metabolism and periodic calcium influx. We present concurrent time series records of metabolic variables and intracellular calcium levels in glucose-stimulated beta-cells that support regulation of mitochondrial dehydrogenases is the dominant calcium feedback effect onto metabolism in the insulin secretory pathway. We include this effect into the beta-cell Dual Oscillator Model to reconcile model simulations with experimental data, then we determine the oscillation mechanism in the modified model. Islets lose the rhythms that govern insulin pulses when glucose is elevated to hyperglycemic levels. We demonstrate with modeling and experiments that oscillations lost to elevated glucose can be recovered by converting the elevated glucose stimulus to a sinusoidal wave. We predict with modeling which periodic glucose stimuli can recover islet oscillations.
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Date Issued: 2017
Identifier: FSU_2017SP_McKenna_fsu_0071E_13864
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: Establishment of Novel Techniques for the Analysis of the Yin Yang 1 Interactome and the Role of Serine 247 Phosphorylation.
Creator: Conlon, Meghan, Hurt, Myra M, Lee, Choogon, Olcese, James, Tang, Hengli, Florida State University, College of Medicine, Department of Biomedical Sciences
Abstract/Description: Yin Yang 1 (YY1) is a highly conserved, multifunctional zinc finger transcription factor. It has been shown to regulate genes involved in cell cycle regulation, apoptosis, cell growth and proliferation, differentiation, and development. YY1 is ubiquitously expressed, leading to the hypothesis that post-translational modifications, specifically phosphorylation, and protein-protein interactions play a crucial role in regulating its various functions. It was therefore important to develop new...
Show moreYin Yang 1 (YY1) is a highly conserved, multifunctional zinc finger transcription factor. It has been shown to regulate genes involved in cell cycle regulation, apoptosis, cell growth and proliferation, differentiation, and development. YY1 is ubiquitously expressed, leading to the hypothesis that post-translational modifications, specifically phosphorylation, and protein-protein interactions play a crucial role in regulating its various functions. It was therefore important to develop new tools in order to better understand these two regulatory mechanisms. A new system using the BioID approach was established to further understand the interactions of YY1 and identify interactions brought about by phosphorylation of serine 247. This phosphorylation is inhibited by a cyclin-dependent kinase inhibitor, flavopiridol. After further analysis, the phosphorylation of YY1 at serine 247 increased in response to DNA damage induced by etoposide, however, this increase was not inhibited by flavopiridol. This leads to the possibility of two separate pathways responsible for phosphorylating serine 247. A lentiviral expression system was developed in order to further explore the functional role of serine 247. This system is extremely versatile and has the ability to rescue YY1 expression after siRNA knockdown. As a whole, the tools developed here will allow for an in depth understanding of serine 247 phosphorylation and the kinases responsible for this modification. Furthermore, the BioID and the lentiviral expression systems aid in uncovering novel YY1 interactions in normal cellular conditions as well as under DNA damage at both the transcriptional and translational level. This research has the possibility of improving our understanding of chemotherapeutic drugs as well as providing novel drug targets for disease treatment.
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Date Issued: 2016
Identifier: FSU_FA2016_Conlon_fsu_0071N_13627
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: Sex Differences in Molecular Pathology in the 5XFAD Mouse Model of Alzheimer’s Disease.
Creator: Bundy, Joseph L. (Joseph Ligon), Nowakowski, Richard S., Houpt, Thomas, Arbeitman, Michelle N., Inouye, Brian D., Olcese, James, Florida State University, College of Medicine,...
Show moreBundy, Joseph L. (Joseph Ligon), Nowakowski, Richard S., Houpt, Thomas, Arbeitman, Michelle N., Inouye, Brian D., Olcese, James, Florida State University, College of Medicine, Department of Biomedical Sciences
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Abstract/Description: Alzheimer’s disease is a progressive neurodegenerative disorder and the most common form of dementia. Like many neurological disorders, Alzheimer’s disease has a sex-biased epidemiological profile, affecting approximately twice as many women as men. The cause of this sex difference has yet to be elucidated. To identify molecular correlates of this sex bias, we investigated molecular sex differences in the hippocampus of healthy female and male mice at 1, 2, and 4 months of age. This analysis...
Show moreAlzheimer’s disease is a progressive neurodegenerative disorder and the most common form of dementia. Like many neurological disorders, Alzheimer’s disease has a sex-biased epidemiological profile, affecting approximately twice as many women as men. The cause of this sex difference has yet to be elucidated. To identify molecular correlates of this sex bias, we investigated molecular sex differences in the hippocampus of healthy female and male mice at 1, 2, and 4 months of age. This analysis identifies a host of genes that display sex-biased expression in the developing mouse hippocampus, many of which are heat shock proteins. Using this dataset as a baseline, we investigated molecular pathology in both sexes using the 5XFAD transgenic mouse model of Alzheimer’s disease. We profiled the transcriptome of the mouse hippocampus during early stages of disease development with RNA-sequencing. To supplement our transcriptomic analysis, we performed a series of liquid-chromatography mass spectrometry analyses of protein abundance. This proteomic investigation was refined by an extensive methods-oriented analysis of sample fractionation. The analysis of 5XFAD transgenic mice reveals pathological differences in transcript abundance as early as 2 months of age, prior to observable plaque deposition. At 4 months of age, we detect wide-spread up regulation of transcripts associated with immune function in diseased animals. Interestingly, our data indicate that female transgenic mice show a stronger disease phenotype than their male counterparts as measured by number of differentially expressed genes. We also find elevated expression of the 5XFAD transgenes in females relative to males, which likely accounts for a portion of sex-biased molecular pathology observed in this dataset. Taken together, our analyses identify both innate molecular sex differences in the rodent brain, as well as molecular correlates of sex-biased disease features. The findings enhance our understanding of neural sex-differences, and present potential candidate biomarkers for pharmacological intervention for Alzheimer’s disease.
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Date Issued: 2016
Identifier: FSU_FA2016_Bundy_fsu_0071E_13588
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: Investigating Persistent Infections Using Mathematical Modeling and Analyses.
Creator: Jarrett, Angela Michelle, Cogan, Nicholas G., Hussaini, M. Yousuff, Bass, Hank W., Bertram, R. (Richard), Case, Bettye Anne, Hurdal, Monica K., Florida State University, College...
Show moreJarrett, Angela Michelle, Cogan, Nicholas G., Hussaini, M. Yousuff, Bass, Hank W., Bertram, R. (Richard), Case, Bettye Anne, Hurdal, Monica K., Florida State University, College of Arts and Sciences, Department of Mathematics
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Abstract/Description: While the immune system is extraordinarily complex and powerful, and medical advancements are more spectacular than ever, in recent history we have seen the unfortunate failure of both processes (immune system and drugs) in the increasing levels of persistent infections. This work is an example of a collaborative effort to study multiple forms of persistent infections using mathematical tools and analyses. We will discuss the biological backgrounds for the immune system's components and...
Show moreWhile the immune system is extraordinarily complex and powerful, and medical advancements are more spectacular than ever, in recent history we have seen the unfortunate failure of both processes (immune system and drugs) in the increasing levels of persistent infections. This work is an example of a collaborative effort to study multiple forms of persistent infections using mathematical tools and analyses. We will discuss the biological backgrounds for the immune system's components and functions, the bacterial and viral resistance mechanisms for methicillin-resistant Staphylococcus aureus and the human immunodeficiency virus, respectively, and some of the current methods for treating these diseases. Then, using ordinary and partial differential equations we present the results of models that were created to study specific infections—namely, methicillin-resistant Staphylococcus aureus osteomyelitis, Staphylococcus aureus nasal carriage, and human immunodeficiency virus prophylactic gel. These models are shown to be in good agreement with the biology by looking at, when possible, their analytical solutions and numerical results when compared to experimental evidence. We further explore these models using several different computational analyses that can be classified as at least one of the following methods: uncertainty quantification, sensitivity analysis, and data assimilation. We give an overview of each of these topics and delve into the technicalities and caveats of each of the analyses we apply. We show that all of the methods presented, individually and in concert, are valuable tools for not only revealing details about the model structure and verifying model robustness, but they can also bring to light elements of the biological phenomena that the model represents. While considering all these details, throughout the manuscript we consider the philosophical perspective of biological modeling and modeling in general.
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Date Issued: 2016
Identifier: FSU_2016SP_Jarrett_fsu_0071E_13046
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

Title: The Sine Oculis Homologue Six7 Maintains Photoreceptor Diversity and Patterning in the Diurnal Zebrafish Retina.
Creator: Sotolongo-Lopez, Mailin, Fadool, James Michael, Horabin, Jamila I., Chadwick, Brian P., Bass, Hank W., Deng, Wu-Min, Florida State University, College of Arts and Sciences,...
Show moreSotolongo-Lopez, Mailin, Fadool, James Michael, Horabin, Jamila I., Chadwick, Brian P., Bass, Hank W., Deng, Wu-Min, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: During the age of non-avian dinosaurs, ancestors of present-day mammals were likely small insectivores, relegated to nocturnal and subterranean niches. This nocturnal "bottle-neck" is postulated as a driving force of numerous physiological and sensory adaptations including those of the visual system. The consequences of structural mutations of opsins upon spectral sensitivity and environmental adaptation have been studied in great detail, but lacking is knowledge of the potential influence of...
Show moreDuring the age of non-avian dinosaurs, ancestors of present-day mammals were likely small insectivores, relegated to nocturnal and subterranean niches. This nocturnal "bottle-neck" is postulated as a driving force of numerous physiological and sensory adaptations including those of the visual system. The consequences of structural mutations of opsins upon spectral sensitivity and environmental adaptation have been studied in great detail, but lacking is knowledge of the potential influence of alterations in gene regulatory networks upon the diversity of cone subtypes and the variation in the ratio of rods and cones observed in diurnal and nocturnal species. Exploiting photoreceptor patterning in cone-dominated zebrafish, we uncovered two independent mechanisms by which the sine oculis homeobox homolog 7 (six7) regulates photoreceptor development. In a genetic screen, we isolated the lots-of-rods-junior (ljrp23ahub) mutation that resulted in an increased number and uniform distribution of rods in otherwise normal appearing larvae. Sequence analysis, genome editing using TALENs and knockdown strategies confirm ljrp23ahub as a hypomorphic allele of six7, a teleost orthologue of six3, with known roles in forebrain patterning and expression of opsins. Based on the lack of protein-coding changes and an identified deletion of a conserved sequence about 40 kb upstream of six7 loci, a cis-regulatory mutation is proposed as the basis of the reduced expression in ljrp23ahub. Comparison of the hypomorphic and knock-out alleles provides evidence of two independent roles in photoreceptor development. EdU and PH3 labeling show that the increase in rod number and uniform distribution is associated with extended mitosis of photoreceptor progenitors, and TUNEL suggest that the lack of green cones is the result of cell death of the cone precursor. These data add six7 to the small but growing list of genes essential for specification and patterning of photoreceptors in non-mammalian vertebrates, and form the basis of a model that underscores the potential of alterations in transcriptional regulation as a mechanism underpinning photoreceptor variation across species.
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Date Issued: 2016
Identifier: FSU_2016SP_SotolongoLopez_fsu_0071E_13092
Format: Thesis

Title: Post-Release Mortality of Deep Sea Bycatch Species.
Creator: Talwar, Brendan Suneel, Grubbs, R. Dean (Ralph Dean), Brooks, Edward J., Levitan, Don R., Travis, Joseph, Florida State University, College of Arts and Sciences, Department of...
Show moreTalwar, Brendan Suneel, Grubbs, R. Dean (Ralph Dean), Brooks, Edward J., Levitan, Don R., Travis, Joseph, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Deep-sea organisms are increasingly subject to bycatch interactions worldwide. Recent studies have shown that discard mortality can lead to significant declines in deep sea fish stocks, and highlight the inherent vulnerability of deep sea organisms to overexploitation due to their shared suite of conservative life history characteristics. Estimating the post-release mortality (PRM) rates of these deep-sea organisms is a necessary step towards responsible fisheries management, particularly as...
Show moreDeep-sea organisms are increasingly subject to bycatch interactions worldwide. Recent studies have shown that discard mortality can lead to significant declines in deep sea fish stocks, and highlight the inherent vulnerability of deep sea organisms to overexploitation due to their shared suite of conservative life history characteristics. Estimating the post-release mortality (PRM) rates of these deep-sea organisms is a necessary step towards responsible fisheries management, particularly as PRM represents a substantial source of uncertainty when estimating total fishery mortality. The deep-sea giant isopod Bathynomus giganteus and its relatives are captured as bycatch in numerous fisheries, although knowledge is limited regarding their population trends or response to capture and release. In order to assess and predict PRM in B. giganteus, we used reflex action mortality predictors (RAMP) whereby the presence or absence of target reflexes was used to create a delayed mortality model, and considered factors affecting mortality. Mortality rates five days post-capture ranged from 50-100% and both RAMP scores and time at the surface were significant predictors of mortality, although our conclusions regarding the effect of surface time are limited. In-cage video documented little movement within the 24 h monitoring period following cage deployment, and it appeared that surviving individuals often fed within the holding period after cage deployment. Our results suggest that PRM in B. giganteus is common and that this unaccounted source of mortality should be quantified and investigated for other deep-sea crustaceans as well. Similarly, bycatch interactions with deep-sea elasmobranchs can lead to dramatic declines in abundance over short time scales. Sharks hooked in the deep sea could face a higher likelihood of severe physiological disturbance, at-vessel mortality, and PRM than their shallower counterparts. Unfortunately, robust PRM rates have not yet been estimated for deep-sea elasmobranchs and as such are not currently incorporated into total fishery mortality estimates or bycatch assessments, limiting the effectiveness of conservation or management initiatives. We empirically estimated PRM for two focal species of deep-sea shark, the Cuban dogfish Squalus cubensis and the gulper shark Centrophorus sp. using post-release cages deployed at-depth. We calculated 24 h PRM rates of 49.7% (± 8.5 SE) for S. cubensis and 83% (± 16 SE) for Centrophorus sp. and identified shark size (total length), blood lactate, blood pH, and vitality scores as predictors of PRM in Squalus cubensis. We also observed all PRM within 11 h post-capture and demonstrated the effects of capture and recovery depth on stress and behavior. Our results suggest that PRM rates of deep-sea sharks are higher than previously assumed, and highlight the need for filling in this gap in fishery mortality estimates for other common deep-sea discards in the future.
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Date Issued: 2016
Identifier: FSU_2016SP_Talwar_fsu_0071N_13088
Format: Thesis

Title: Reproductive Interference Explains the Competitive Disparity Between Congeneric Beanweevils Callosobruchus Maculatus and Callosobruchus Chinensis.
Creator: Takacs, Peter, Inouye, Brian D., Underwood, Nora, Travis, Joseph, Florida State University, College of Arts and Sciences, Department of Biological Science
Abstract/Description: The system including the congeners C. maculatus and C. chinensis has long been a model for competition studies. Members of these two pest species often cohabit facilities designed for the storage of dried legumes, a crucial nutritional resource for the larvae of both species. In both natural and laboratory settings it has been observed that C. chinensis routinely drives C. maculatus to extinction. These observations convinced many ecologists that the trophic relationship between the two...
Show moreThe system including the congeners C. maculatus and C. chinensis has long been a model for competition studies. Members of these two pest species often cohabit facilities designed for the storage of dried legumes, a crucial nutritional resource for the larvae of both species. In both natural and laboratory settings it has been observed that C. chinensis routinely drives C. maculatus to extinction. These observations convinced many ecologists that the trophic relationship between the two species presents an unequivocal case of competitive exclusion. Several predictively successful mathematical models based on resource competition alone accordingly estimated the competitive disparity between these two bean beetle species, suggesting that individual C. chinensis are approximately four times better competitors for a shared resource than their average C. maculatus counterparts. Recent studies (Kishi et al. 2009; Kyogoku and Nishida 2012, 2013; Kishi and Nakazawa 2013; Kishi and Tsubaki 2014) have called into question this apparently straightforward conclusion since there are few if any characteristic life history traits or behavioral mechanisms associated with exploitative or interference competition that would explain the competitive disparity. The authors of these studies contend that reproductive interference—a negative interspecific sexual interaction that reduces the fitness of at least one of the species involved—is a largely ignored but equally consequential factor when it comes to explaining interspecific population dynamics. This study tests for the presence and explanatory importance of reproductive interference on per capita female fitness by simultaneously manipulating conspecific density and operational sex ratio in the presence (and absence) of heterospecific males. The experimental design also reveals the operative mechanism(s) behind reproductive interference. Results of this study clearly support outstanding claims for the causal significance of reproductive interference in this system.
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Date Issued: 2016
Identifier: FSU_2016SP_Takacs_fsu_0071N_13173
Format: Thesis

Title: Towards the Development of Lipid Multilayer Microarrays for Dose Dependent in Vitro Delivery and Screening.
Creator: Kusi-Appiah, Aubrey Emmanuel, Lenhert, Steven, Guan, Jingjiao, Keller, Thomas C. S., Chase, P. Bryant, Ma, Teng, Florida State University, College of Arts and Sciences,...
Show moreKusi-Appiah, Aubrey Emmanuel, Lenhert, Steven, Guan, Jingjiao, Keller, Thomas C. S., Chase, P. Bryant, Ma, Teng, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Screening for effects of small molecules on cells grown in culture is a well-established method for drug discovery and testing, and faster throughput at lower cost is needed especially for lipophilic materials. Small-molecule arrays present a promising approach. However, it has been a challenge to use them to obtain quantitative surface based dose-response curves in vitro, especially for lipophilic compounds. This thesis first introduces a simple novel method of surface-mediated delivery of...
Show moreScreening for effects of small molecules on cells grown in culture is a well-established method for drug discovery and testing, and faster throughput at lower cost is needed especially for lipophilic materials. Small-molecule arrays present a promising approach. However, it has been a challenge to use them to obtain quantitative surface based dose-response curves in vitro, especially for lipophilic compounds. This thesis first introduces a simple novel method of surface-mediated delivery of drugs to cells from a microarray of phospholipid multilayers (layers thicker than a bilayer) encapsulating small molecules. The capability of controlling the dosage of the lipophilic molecules delivered to cells using the lipid multilayer microarray assay is further demonstrated using the nanointaglio printing method. This control enabled the variation of the volumes of surface supported lipid micro- and nanostructure arrays fabricated with nanointaglio. The volumes of the lipophilic drug-containing nanostructures were determined using a fluorescence microscope calibrated by atomic-force microscopy. The surface supported lipid volume information was used to obtain EC-50 values for the response of HeLa cells to treatment with three FDA-approved lipophilic anticancer drugs, docetaxel, imiquimod and triethylenemelamine, which were found to be significantly different from neat lipid controls. Features with sub-cellular lateral dimensions were found to be necessary to obtain normal cell adhesion with HeLa cells. Comparison of the microarray data to dose-response curves for the same drugs delivered liposomally from solution revealed quantitative differences in the efficacy values, which may be explained in terms of cell-adhesion playing a more important role in the surface-based assay. Finally, solution encapsulation was done for a library of hydrophilic silicon nanocrystals in order to set a solution standard for comparison with future surface supported delivery of the library. The work presented here opens the door for the possible benchtop high throughput and high content screening of hydrophobic materials that were previously difficult or impossible to readily screen.
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Date Issued: 2016
Identifier: FSU_2016SP_KusiAppiah_fsu_0071E_13194
Format: Thesis

Title: Characterization of Cardiac Troponin C FRET Constructs to Analyze Structural Changes Induced by Divalent Cation Binding.
Creator: Lentsch, Cassidy, Chase, P. Bryant, Pinto, Jose R. (Jose Renato), Bates, George W. (George Wesley), Meredith, Michael, Florida State University, College of Arts and Sciences,...
Show moreLentsch, Cassidy, Chase, P. Bryant, Pinto, Jose R. (Jose Renato), Bates, George W. (George Wesley), Meredith, Michael, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: This research focuses on the characterization of three isoforms of the human cardiac Troponin C protein previously designed and created by Myriam Badr. These isoforms differ both in the fluorophores bound to the N and C termini and the size of the linker sequences between the fluorophores and TnC protein. Troponin C is notable for its function in Ca2+ binding, which, in functioning muscle, induces a conformational change. This reveals the hidden myosin head binding site and allows contraction...
Show moreThis research focuses on the characterization of three isoforms of the human cardiac Troponin C protein previously designed and created by Myriam Badr. These isoforms differ both in the fluorophores bound to the N and C termini and the size of the linker sequences between the fluorophores and TnC protein. Troponin C is notable for its function in Ca2+ binding, which, in functioning muscle, induces a conformational change. This reveals the hidden myosin head binding site and allows contraction to proceed. Using fluorometer analysis to measure changes in FRET, the structural changes each undergoes in the presence of calcium, magnesium, and calcium in the presence of magnesium were analyzed to better understand the functioning of each of these proteins under physiological conditions. Since deficits in cardiac troponin C function have been implicated in some cases of familial hypertrophic cardiomyopathy, it is important to better understand how function changes with the size and shape of the cardiac troponin C protein. With this in mind, we hope to gain a more complete understanding of human cardiac troponin C function.
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Date Issued: 2016
Identifier: FSU_2016SP_Lentsch_fsu_0071N_13055
Format: Thesis

Title: Symmetry Solutions of the Multiphase Model with Biological Applications.
Creator: Ekrut, David, Cogan, Nicholas G., Keller, Thomas C. S., Quine, J. R. (John R.), Hurdal, Monica K., Jain, Harsh Vardhan, Florida State University, College of Arts and Sciences,...
Show moreEkrut, David, Cogan, Nicholas G., Keller, Thomas C. S., Quine, J. R. (John R.), Hurdal, Monica K., Jain, Harsh Vardhan, Florida State University, College of Arts and Sciences, Department of Mathematics
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Abstract/Description: The multiphase model has given keen insights into many aspects of biology, from crawling cells to bio-gel morphology and tumor angiogenesis [1, 2, 3, 4, 5, 6, 7, 8, 9, 10]. Phases are averaged fractions of a control volume, treated as viscoelastic fluid-like or visco-elastic solids. Derived by conservation laws, the governing equations of the multiphase model are nonlinear partial dierential equations (PDEs). Nonlinear PDEs are often difficult to solve. For this reason, asymptotic and...
Show moreThe multiphase model has given keen insights into many aspects of biology, from crawling cells to bio-gel morphology and tumor angiogenesis [1, 2, 3, 4, 5, 6, 7, 8, 9, 10]. Phases are averaged fractions of a control volume, treated as viscoelastic fluid-like or visco-elastic solids. Derived by conservation laws, the governing equations of the multiphase model are nonlinear partial dierential equations (PDEs). Nonlinear PDEs are often difficult to solve. For this reason, asymptotic and numerical methods have been the predominant tool to analyze these problems, but this is not the only approach to yield results. Reduction transformations can take a system of nonlinear PDEs and reduce it to a system of ordinary differential equations (ODEs), which are typically less difficult to solve. Finding and using such reductions was the focus of this thesis. In this work, we provide a framework for producing analytic solutions to the multiphase model by way of transformations. We begin by deriving exact solutions to a free boundary problem with a sharp interface, a sharp interface being the discontinuity occurring in gel dynamics where a boundary layer separates a mixture of phases from a region of pure solvent. The main focus of study is to track the dynamic interface between the gel and the pure solvent as the gels swell and deswell. These questions arise in designing drug delivery methods, for example [11]. In the event of no mass production, we are able to use this analytic solution to replicate the numerical results provided by others with a closed form solution. Further, this solution yields additional information not recovered by other methods. In addition to being able to track the front velocity, we recover the time dependency lost by asymptotic methods. Next, we explore the multiphase system with sources and sinks. Once more, the reduction transformation we develop provides closed form solutions for the multiphase problem. Assessing the characteristic curves for a smooth boundary, we find shocks and rarefactions arise with logistic growth. Shocks occur when multi-valued solutions are present for certain boundary conditions, and rarefactions occur when solutions vanish on the boundary. In gel dynamics, this "disturbance'' occurs when there is a disruption from phases are attempting to occupy the same space. Finally, we develop a general reduction transformation for an arbitrary number of phases and dimensions. We show the effectiveness of this transformation by reducing two known biological examples. The first model has spatially one dimension with three phases and describes tumor encapsulation and transcapular spread. The second has two spatial dimensions with four phases and describes vascular tumor growth. In addition to the novel contribution of mapping the multiphase system in j spatial dimensions of n phases, there are many directions for this research. We have made progress in the formal analysis of these complicated, nonlinear PDEs. More interestingly, we have opened several directions that appear to be fruitful avenues for future study. During the analysis of the two-phase model, we discovered many exact solutions without clear significance. We could explore the nature of these solutions and attempt to uncover biological relevance. When we added the growth function, we discovered shock and rarefaction waves for logistic growth. We could pursue alternative forms for growth. After designing a general reduction transformation, we show the reduction of two multiphase systems. We could derive solutions from either of the above examples by solving the reduced ODE systems. Also, now that we have a better understanding of the analytical solution structure of the multiphase system, it is possible to seek compatible forms for mass redistribution and pressure terms in the equations. Essentially, we can seek to derive mathematical forms for hydrostatic pressure, a phenomenon which cannot be captured empirically, by imposing specific source/sinks functions seen in numerical models.
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Date Issued: 2016
Identifier: FSU_2016SP_Ekrut_fsu_0071E_13019
Format: Thesis

Title: Characterization of Role of LARP6 Phosphorylation in Regulating Type I Collagen Biosynthesis in Liver Fibrosis.
Creator: Zhang, Yujie, Stefanovic, Branko, Tang, Hengli, Gunjan, Akash, Hurt, Myra M., Wang, Yanchang, Florida State University, College of Medicine, Department of Biomedical Sciences
Abstract/Description: Liver fibrosis is the common end stage of all chronic liver diseases, such as chronic viral hepatitis, alcoholism, nonalcoholic fatty liver disease, autoimmune hepatitis, alpha 1 anti-trypsin deficiency and some rare metabolic diseases. Fibrosis it is a major cause of morbidity and mortality worldwide. However, the specific and efficient anti-fibrotic therapy is still lacking. Thus, better understanding the underlying mechanism of liver fibrosis is critical in order to find a cure. Liver...
Show moreLiver fibrosis is the common end stage of all chronic liver diseases, such as chronic viral hepatitis, alcoholism, nonalcoholic fatty liver disease, autoimmune hepatitis, alpha 1 anti-trypsin deficiency and some rare metabolic diseases. Fibrosis it is a major cause of morbidity and mortality worldwide. However, the specific and efficient anti-fibrotic therapy is still lacking. Thus, better understanding the underlying mechanism of liver fibrosis is critical in order to find a cure. Liver fibrosis is histologically characterized by excessive deposition of extracellular matrix composed primarily of type I collagen. Type I collagen is a complex protein composed by folding by two α1(I) and one α2(I) polypeptides into triple helix. The production of collagen polypeptides is regulated by the cis-acting sequence of their respective mRNAs, the 5' stem loop (5'SL). In the 5' untranslated region of collagen α1(I) and α2(I) mRNAs, there is a secondary structure forming a stem loop (5'SL). This cis-acting element regulates type I collagen expression in fibrosis by binding an RNA binding protein, La ribonucleoprotein domain family, member 6 (LARP6). LARP6 specifically binds to 5'SL of collagen mRNAs with high affinity and sequence specificity. The binding recruits several effector proteins to stimulate type I collagen production in fibrosis. LARP6 is a phosphor-protein, however, how the phosphorylation of LARP6 is involved in the process of collagen biosynthesis has not been studied before. My dissertation focuses on the role of LARP6 phosphorylation in biosynthesis of type I collagen in fibrosis. I have identified eight serines of LARP6 that undergo phosphorylation and six of these serines have never been reported to be phosphorylated before. I have characterized the functional consequence of phosphorylation of these serines, identified the responsible kinases, and analyzed the role in collagen biosynthesis. These studies are presented in the dissertation as three logically connected chapters. In the chapter two provide evidence that phosphorylation of LARP6 follows a hierarchical order; namely, that phosphorylation of S451 is the initial event, which is required for phosphorylations of other serines. Phosphorylation of S451depends on the activity of PI3K/Akt signaling pathway. Akt inhibitor, GSK-2141795, which is in clinical trials for treatment of solid tumors, reduced collagen production with EC50 of 150 nM. This effect is explained by inhibition of LARP6 phosphorylation and suggests that Akt inhibitors may be effective in treatment of xi various forms of fibrosis. The S451A mutant of LARP6 lacks phosphorylation, not only at 451 position, but also at several other serines. Its overexpression has a dominant negative effect on collagen biosynthesis; the S451A mutant drastically reduces secretion of type I collagen and induces synthesis of aberrant and over-modified collagen polypeptides. This indicates that LARP6 phosphorylation at S451 is critical for activation of the protein in translation and folding of collagen polypeptides. In the chapter three I have characterized two other phosphorylations of LARP6, the phosphorylation of S348 and S409. These sites are phosphorylated by mTORC1 and are redundant. Mutation of both of these serines is required to inactivate LARP6. The double mutant, S348A/S409A, acts as a dominant negative protein in collagen biosynthesis, which retards secretion of type I collagen and causes excessive posttranslational modifications. Similar effects are seen using mTORC1 inhibitor rapamycin or by knocking down mTORC1 function by siRNA. The phosphorylation of S348A or S409A is needed for two processes: 1. To recruit an accessory protein STRAP to collagen mRNAs and 2. To enable normal subcellular trafficking of LARP6. STRAP is needed to coordinate translation of collagen α1(I) and α2(I) mRNAs, what becomes critical in fibrosis. In the absence of S348/S409 phosphorylation LARP6 is also sequestered in increasing amounts at the ER membrane. The mechanistic details and significance of the S348/S409 phosphorylation are described in chapter 2. The role of TGF-β1 in LARP6 phosphorylation is described in the fourth chapter. TGF-β is the most potent profibrotic cytokine and this discovery provides the link between the TGF-β activity and the LARP6 dependent mechanism of collagen synthesis. The phosphorylation of LARP6 at S396 is stimulated by TGF-β and it promotes the distribution of LARP6 into the nucleus. This is necessary for binding of the newly transcribed collagen mRNAs and their inclusion in the LARP6 dependent metabolic pathway, resulting in more efficient type I collagen expression. In conclusion, my dissertation work has characterized different phosphorylation events of LARP6 and how they are involved in regulating the function of LARP6 in type I collagen biosynthesis. These findings will contribute to better understanding of the underlying mechanism of overproduction of type I collagen in fibrosis, and provide the rationale of using kinase inhibitors for treating fibrotic disorders.
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Date Issued: 2016
Identifier: FSU_2016SP_Zhang_fsu_0071E_13114
Format: Thesis

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