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Title: A 9.4 Ghz EPR Investigation of the Non-Heme Iron Active Site of Single Soybean Lipoxygenase-1 Crystals.
Creator: Ottenberg, Gregory Kenneth, Gaffney, Betty Jean, Blaber, Michael, Keller, Thomas, Department of Biological Science, Florida State University
Abstract/Description: Lipid oxidation pathways and nitric oxide signaling pathways are interrelating pathways with significance both separately and in conjunction (references 1-2). Lipid oxygenating pathways involve the formation of hydroperoxides, alkanes, alkenes, aldehydes (notably nonenal), epoxides, alcohols and other species from polyunsaturated fatty acids that are readily oxidized. The nitric oxide signaling pathway has been of great interest in the last several years and involves the formation of nitric...
Show moreLipid oxidation pathways and nitric oxide signaling pathways are interrelating pathways with significance both separately and in conjunction (references 1-2). Lipid oxygenating pathways involve the formation of hydroperoxides, alkanes, alkenes, aldehydes (notably nonenal), epoxides, alcohols and other species from polyunsaturated fatty acids that are readily oxidized. The nitric oxide signaling pathway has been of great interest in the last several years and involves the formation of nitric oxide near the site of inflammation and its transport to other tissues to function as a messenger. Oxidized lipid signaling pathways are an active area of research, and many interactions with the nitric oxide pathway are left open to discussion. The interactions of nitric oxide and lipid oxidizing enzymes have been demonstrated, and these interactions are of particular significance in the regulation of vascular homeostasis. The studies presented here investigate the specific interactions of soybean lipoxygenase-1, a lipid-oxygenating enzyme, and nitric oxide by electron paramagnetic resonance (EPR) analyses of single lipoxygenase crystals complexed with nitric oxide. Nitric oxide is known to bind with high affinity to the lipoxygenase active site iron. Though nitric oxide is often used as a dioxygen analog, molecular oxygen binds to a fatty acid radical and not directly to the active site iron in the lipoxygenase mechanism, so nitric oxide was not used as a dioxygen analog in our studies. Rather, we expected the electron configuration and any differences in coordination to be a reasonable model of a transition state that mimics a peroxyl radical formed during catalysis. The aim of this project was to determine the local structure of the active site of a transition state analog with mechanistic significance. To complete this line of experimentation, I obtained a number of lipoxygenase crystals similar to those used for x-ray analysis. The crystals were then complexed with nitric oxide using protocols that are similar to those used in previous studies of lipoxygenase and other iron proteins. X-band (9.26 GHz) EPR experiments were performed and analyzed to determine the suitability of the experimental methodology presented here for observation of changes in the electronic orbital structure of the active site. Differences between the structure of the resting enzyme and the structure of the NO–LOX complex that may be observed with EPR include both the positions of the electron orbitals and the spatial orientation of the nitric oxide-iron bond, from which one may be able to infer the positions and coordination of the other iron ligands. While no conclusions about the electronic structure of the lipoxygenase iron-nitric oxide bond could be drawn from these experiments, the suitability of the experimental conditions for further studies was proven. This project also represents an advancement in the area of EPR studies of small protein crystals, similar in size to those used in x-ray diffraction experiments. Further studies of the complex that were not included in the masters project may include W-band (92.4 GHz) EPR studies and X-ray crystallography of crystals of the nitric oxide-lipoxygenase complex.
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Date Issued: 2004
Identifier: FSU_migr_etd-2421
Format: Thesis

Title: ABA Contents in the Guard-Cell Symplast and Guard-Cell Apoplast Are Not Correlated with Stomatal Aperture Size under Three Conditions of Water Sufficiency.
Creator: Meng, Fanxia, Outlaw, William H., Logan, Timothy M., Bass, Hank W., Bates, George W., Epstein, Lloyd M., Keller, Laura R., Department of Biological Science, Florida State...
Show moreMeng, Fanxia, Outlaw, William H., Logan, Timothy M., Bass, Hank W., Bates, George W., Epstein, Lloyd M., Keller, Laura R., Department of Biological Science, Florida State University
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Abstract/Description: Guard cells respond to environmental stimuli by opening and closing stomata, which balance CO2 uptake and water conservation. Stomatal closure under water deficiency and the involvement of abscisic acid (ABA) in this response are well-known. However, whether ABA plays a role in stomatal regulation under some water-sufficient conditions, such as diurnal changes, humidity shift (i.e. transpiration-rate change) and brief flooding, is not clear. Using an enzyme-linked immunosorbent assay (ELISA)...
Show moreGuard cells respond to environmental stimuli by opening and closing stomata, which balance CO2 uptake and water conservation. Stomatal closure under water deficiency and the involvement of abscisic acid (ABA) in this response are well-known. However, whether ABA plays a role in stomatal regulation under some water-sufficient conditions, such as diurnal changes, humidity shift (i.e. transpiration-rate change) and brief flooding, is not clear. Using an enzyme-linked immunosorbent assay (ELISA) with sub-femtomole sensitivity for ABA assays, we studied the relationships of stomatal aperture size with the ABA contents in the symplast and apoplast of guard cells, as well as those in the leaf and the leaf apoplast in Vicia faba under the following three conditions. (1) Diurnal changes. Stomata opened in the morning, reached a maximum opening at 1400 h, and closed at 1800 h. Neither the leaf nor the leaf apoplastic ABA content strongly correlated with stomatal aperture sizes. The ABA contents of the guard-cell compartments did not change over the course of the day, providing evidence that ABA is not involved in diurnal stomatal regulation. (2) Humidity-induced transpiration-rate changes. The transpiration rate of intact plants and that of detached leaves infused with 1µM ABA was decreased by shifting RH from 60% to 90%. The ABA contents of the four compartments were not changed by this humidity shift, in spite of an increase of 2–3 µm in stomatal aperture sizes. Thus, the guard-cell-apoplastic ABA content is not affected by transpiration rate, and ABA may not participate in the stomatal response to transpiration rate. (3) Brief flooding. Stomata closed after brief (4-h) flooding, when the leaf and the leaf apoplastic ABA increased 2–3 fold and the xylem pH increased 0.2 pH units. The leaf ABA increase did not correlate strongly with stomatal aperture size and the xylem ABA delivery rate remained unchanged. The ABA contents in the guard-cell compartments of flooded plants were not different from those of non-flooded plants. Therefore, ABA may not be an initiator of stomatal closure under brief flooding, and xylem alkalinization probably does not induce leaf ABA redistribution to guard cells.
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Date Issued: 2007
Identifier: FSU_migr_etd-2485
Format: Thesis

Title: Acclimation of Red Tide Dinoflagellate Karenia Brevis to Higher Temperatures Results in Abnormal Morphology and Changes in Growth Rates.
Creator: Owen, Daniel P., Department of Biological Science
Abstract/Description: This paper addresses the effects of increased temperature on the Red Tide dinoflagellate, Karenia brevis. A clonal strain of Karenia brevis was acclimated to the currently estimated increase in Gulf temperatures over a period of time long enough to ensure proper acclimation of the experimental cultures. A long acclimation time was used to avoid temperature shock conditions for the culture and to more closely mimic natural temperature increases, such as those seen during seasonal transitions....
Show moreThis paper addresses the effects of increased temperature on the Red Tide dinoflagellate, Karenia brevis. A clonal strain of Karenia brevis was acclimated to the currently estimated increase in Gulf temperatures over a period of time long enough to ensure proper acclimation of the experimental cultures. A long acclimation time was used to avoid temperature shock conditions for the culture and to more closely mimic natural temperature increases, such as those seen during seasonal transitions. Over the course of the experiment, K. brevis cultures were acclimated from 25° C to 31° C. An abnormal, rounded, cell morphology was produced in K. brevis cultures acclimated to 28° C and persisted in cultures acclimated through 28° C to 31° C. As well, specific growth rates of cultures growing at 25° C and acclimated to 30° C differed depending on whether the average growth rates were derived from culture cell density or RFU measurements. K. brevis cultures grown in GP/2 media had significantly higher average growth rates based on RFU measurements than cultures growing in L1-Si media. Cultures growing at 25° C and 30° C did not have significantly different chlorophyll a content per cell. In conjunction with the rounded cell morphology, the reported higher maximum temperature range, and future physiological observations, the result of this experiment aim to help researchers understand what may be happening to populations of K. brevis throughout seasonal temperature variations.
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Date Issued: 2015
Identifier: FSU_migr_uhm-0515
Format: Set of related objects

Title: Adaptation, Diversification, and Desert Ecology of the Most Diverse Order of Mammals (Mammalia, Rodentia).
Creator: Alhajeri, Bader H., Steppan, Scott J., Parker, William C., Erickson, Gregory M., Travis, Joseph, Miller, Thomas E., Department of Biological Science, Florida State University
Abstract/Description: Globally, species diversity is regulated by speciation and extinction, and regionally it is regulated by competition, niche, colonization, emigration, and extinction, and more locally, by environmental tolerance and species interactions which filter out non-adapted species based on intrinsic characteristics, or their Hutchinsonian niche. In this dissertation, I examined some of the mechanisms that govern biodiversity patterns in order to determine the main causes of uneven diversity in muroid...
Show moreGlobally, species diversity is regulated by speciation and extinction, and regionally it is regulated by competition, niche, colonization, emigration, and extinction, and more locally, by environmental tolerance and species interactions which filter out non-adapted species based on intrinsic characteristics, or their Hutchinsonian niche. In this dissertation, I examined some of the mechanisms that govern biodiversity patterns in order to determine the main causes of uneven diversity in muroid rodent clades, the most diverse superfamily of mammals, comprising 28% of all mammal species. This extensive diversity, in addition to the remarkable eco-morphological adaptability which facilitated their colonization of all terrestrial biomes make muroids an ideal system to study this fundamental question in evolutionary ecology. In addition, the use of robust phylogenies that have recently been developed in muroids and non-muroid rodents makes the order an especially attractive model system to understand the process of mammalian adaptation to arid environments and the ecological interactions that shaped patterns of coexistence within desert communities, the second main goal of the dissertation. The use of a combination of molecular phylogenetics and geometric morphometrics allows for a robust investigation of general patterns that shape the ecological evolution of this group within and without desert habitats, warranting a reinterpretation of classical studies in evolutionary biology, desert ecology, and the traditional systematics of desert rodent clades.
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Date Issued: 2014
Identifier: FSU_migr_etd-8930
Format: Thesis

Title: Allergenic Cross-Reactivity Between Cashew and Pistachio Nuts.
Creator: Tawde, Pallavi D., Roux, Kenneth, Sathe, Shridhar, Keller, Tom, Department of Biological Science, Florida State University
Abstract/Description: Title: Allergenic cross-reactivity between cashew and pistachio nuts Rationale: Cashew and pistachio belong to Anacardiaceae family and strong allergenic cross-reactivity between nuts of these two species has been reported. The aim of our study was to identify the cross-reactive allergenic proteins from cashew and pistachio nuts. Methods: Extracted cashew and pistachio nut proteins were separated by means of 1- and 2-dimensional PAGE. Pooled human sera from cashew-allergic patients was tested...
Show moreTitle: Allergenic cross-reactivity between cashew and pistachio nuts Rationale: Cashew and pistachio belong to Anacardiaceae family and strong allergenic cross-reactivity between nuts of these two species has been reported. The aim of our study was to identify the cross-reactive allergenic proteins from cashew and pistachio nuts. Methods: Extracted cashew and pistachio nut proteins were separated by means of 1- and 2-dimensional PAGE. Pooled human sera from cashew-allergic patients was tested for reactivity to soluble cashew and pistachio proteins by IgE immunoblotting after one-dimensional (1-D) and 2-D electrophoresis. The identities of the IgE-reactive bands from the pistachio immunoblot were further analyzed by means of N-terminal amino acid sequencing and comparison to previously published data from the cashew. ELISAs were performed using individual sera from subjects with cashew and tree nut allergy to assess the degree of IgE reactivity to cashew and pistachio nut extracts. Inhibition ELISA studies were conducted to assess the degree of allergenic cross-reactivity between cashew and pistachio nuts. Results: IgE immunoblots of cashew and pistachio extract probed with cashew-allergic sera identified proteins of 35kDa, 22kDa, and 7-9kDa. N-terminal amino acid sequencing of the IgE-reactive spots from pistachio immunoblot identified them as the acidic subunit, basic subunit of 11S globulin and 2S albumin seed storage proteins respectively. Seed storage proteins are known food allergens in cashew and have been designated as Ana o 1 (7S globulin), Ana o 2 (11S globulin) and Ana o 3 (2S albumin). ELISA results with ten individual cashew-allergic sera (two out of the ten patients have pistachio allergy, and the remaining eight patients had never eaten pistachio) showed IgE reactivity to both cashew and pistachio nut. Inhibition ELISA demonstrated that pre-incubation of sera with pistachio extract resulted in a marked decrease in IgE binding to cashew extract, and vice versa indicating allergenic cross-reactivity. Conclusion: The results demonstrate the presence of cross-reactive B cell epitopes on cashew and pistachio nut allergens. The plant taxonomic classification of cashew and pistachio nuts does appear to predict allergenic cross-reactivity.
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Date Issued: 2004
Identifier: FSU_migr_etd-0358
Format: Thesis

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

Title: Amygdala Response to Artificial Olfactory and Chemosensory Input: Modulation by Neurohormones.
Creator: Blake, Camille Bond, Meredith, Michael, Maner, Jon, Chase, P. Bryant, Hyson, Richard, Wang, Zuoxin, Department of Biological Science, Florida State University
Abstract/Description: In male hamsters mating behavior is dependent on sufficient androgens and chemosensory input from the main olfactory and vomeronasal systems, whose central pathways contain cell bodies and fibers of gonadotropin-releasing hormone (GnRH) neurons. Regions of the medial amygdala (vomeronasal amygdala) contain androgen receptors and differentially process chemosignals with different social implications. According to published reports of "categorical" patterns of response, conspecific chemosensory...
Show moreIn male hamsters mating behavior is dependent on sufficient androgens and chemosensory input from the main olfactory and vomeronasal systems, whose central pathways contain cell bodies and fibers of gonadotropin-releasing hormone (GnRH) neurons. Regions of the medial amygdala (vomeronasal amygdala) contain androgen receptors and differentially process chemosignals with different social implications. According to published reports of "categorical" patterns of response, conspecific chemosensory stimuli activate the anterior (MeA) and posterior (MeP) medial amygdala, while heterospecific stimuli only activate MeA, in male hamsters (and male mice). Furthermore, chemosignals with distinct social implications differentially activate the dorsal and ventral subregions of MeA and MeP (MeAd/v, MePd/v). In sexually-naïve male hamsters, lesions of the vomeronasal organ (VNX), but not the main olfactory bulb, impair mating behavior. Intracerebroventricular (icv)-GnRH restores mating in sexually-naïve VNX males and enhances medial amygdala (Me) activation by chemosensory stimulation. In sexually-experienced males, VNX does not impair mating and icv-GnRH suppresses Me activation. Thus, main olfactory input is sufficient for mating in experienced- but not naïve-VNX males. I tested whether GnRH enhances access of main olfactory input to the amygdala using icv-GnRH and either electrical or pharmacological stimulation of the main olfactory bulb (MOB), and then examined immediate early gene (IEG) expression there. Electrical stimulation of the MOB did not significantly activate the ipsilateral main olfactory cortex or amygdala in intact or VNX animals. When the IEG counts from both sides of the brain were averaged together, GnRH appeared to enhance activation in the medial amygdala in naïve-intact males, but appeared to decrease activation in naïve-VNX males. I concluded that electrical stimulation was not a sufficient means of driving main olfactory input to downstream brain regions, possibly due to activation of intra-bulbar inhibitory circuits. To alleviate this possible confound, I pharmacologically stimulated the MOB with a mixture of bicuculline methiodide and d,l Homocysteic acid. In sexually-naïve intact-males, MOB stimulation produced significant activation in MeAv and MePv. MePv activation is also characteristic of chemosensory stimuli from potential competitors and predators. In sexually-naïve VNX-males, in which GnRH facilitates mating, GnRH enhanced activation by MOB stimulation in posterodorsal medial amygdala (MePd), a region known to be rich in androgen resceptors and activated by conspecific reproductive chemosignals. Conversely, in sexually-experienced VNX-males, animals that do not require exogenous GnRH to mate normally after VNX, there is a depression in activation in MePd due to GnRH and stimulation in MePd, similar to its response to natural chemosensory stimulation. There also appeared to be a possible effect of VNX due to the difference in selective activation of GnRH in naïve-intact vs. naïve-VNX animals. MeP is also rich in steroid receptors and many chemosensory behaviors are steroid dependent. Therefore, I also tested the activation of androgen receptor (AR)-containing cells in Me after conspecific or heterospecific chemosensory stimulation. Conspecific and heterospecific chemosensory stimuli significantly activated AR-containing cells in Me and significantly increased the number of AR-positive cells in Me above control. The increase in the number of AR-ir cells produced by conspecific stimuli was also significantly above the numbers of AR-ir cells produced by the heterospecific stimulus. These effects may be due to increases of testosterone in response to chemosignals or circuit activity dependent on steroid levels. Future studies on castrated testosterone-replaced males will test these possibilities. The studies of this dissertation provide important information about the neurohormonal regulation of chemosensory and olfactory input to the medial amygdala. The integration of hormonal and chemosensory factors is vital to mating and other social behaviors, and thus species survival. The amygdala is crucial to this process in many vertebrate species, including the hamsters, which use chemicals to communicate with one another. This dissertation suggests, and provides some evidence for a part of the mechanism by which the amygdala accomplishes this integration.
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Date Issued: 2009
Identifier: FSU_migr_etd-3669
Format: Thesis

Title: Analysis of Gene Expression in the Amygdala during Conditioned Taste Aversion Learning.
Creator: Kwon, Bumsup, Houpt, Thomas A., Grant, Samuel C., Ouimet, Charles C., Johnson, Frank, Bass, Hank W., Department of Biological Science, Florida State University
Abstract/Description: Conditioned taste aversion (CTA) learning occurs after the pairing of a novel taste with a toxin (e.g. sucrose taste with LiCl toxin). The immediate early gene c-Fos is necessary for CTA learning, but c-Fos alone cannot be sufficient for CTA consolidation. The expression of other activator protein 1 (AP-1) proteins from the Fos (c-Fos, FosB, Fra-1 and Fra-2)- and Jun (c-Jun, JunB and JunD)-families may also be required shortly after conditioning for CTA consolidation. To screen for the...
Show moreConditioned taste aversion (CTA) learning occurs after the pairing of a novel taste with a toxin (e.g. sucrose taste with LiCl toxin). The immediate early gene c-Fos is necessary for CTA learning, but c-Fos alone cannot be sufficient for CTA consolidation. The expression of other activator protein 1 (AP-1) proteins from the Fos (c-Fos, FosB, Fra-1 and Fra-2)- and Jun (c-Jun, JunB and JunD)-families may also be required shortly after conditioning for CTA consolidation. To screen for the expression of AP-1 transcription factors within small subregions, RT-PCR analysis was used after laser capture microdissection (LCM) of the amygdala. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization and immunohistochemistry showed that changes in Fra-2 and c-Fos expression in the BLA and CeA at the time of conditioning, together with constitutive expression of c-Jun and JunD, may contribute to CTA learning. Using double immunolabeling, I confirmed that c-Fos co-localized with Fra-2 in a majority of LiCl-induced c-Fos-positive cells in the CeA. This co-localization of c-Fos and Fra-2 following LiCl suggests that the transcriptional regulation of AP-1 dimeric complexes of c-Fos and Fra-2 may occur in a subset of cells in the CeA. AP-1 family members bind each other to make homo- or hetero-dimers that regulate expression of target genes. For example, c-Fos needs to dimerize with a complementary member of other AP-1 proteins, specially Jun members. Therefore, it can be postulated that there may be transcriptional modulation of a set of AP-1 genes to form a functional AP-1 complex in a neuronal unit that is expressing c-Fos during CTA learning. I examined changes in mRNA expression of immediate early genes including AP-1 family members in c-Fos-specific neurons of the amygdala during CTA learning. Using X-Gal staining, single-cell LCM and RT-PCR, I detected mRNA expression of c-fos and β-actin genes in LiCl-induced lacZ-positive cells in the CeA, cortex and hippocampus of c-fos-lacZ transgenic mice. This result confirmed that endogenous c-fos gene and the c-fos-lacZ transgene were expressed in same cells of the brain. LiCl administration increases c-Fos expression in some brain regions including the CeA. Recent studies show that c-Fos expression in the brain after certain stimuli may be affected by histone modification such as acetylation and phosphorylation. Continuing our studies on gene expression in the amygdala in CTA learning, I investigated if LiCl-induced c-Fos expression in the amygdala is correlated with histone acetylation and phospho-acetylation. LiCl significantly increased the level of acetylated histone H3 (AcH3) in the CeA at 0.5 h and the number of phospho-acetyl-histone H3 (pAcH3)-positive cells in the CeA at 0.5 and 1 h, and the timecourse of LiCl-induced AcH3 and pAcH3 corresponded to LiCl-induced c-Fos timecourse in the CeA. Double immunolabeling results showed that c-Fos co-localized with pAcH3 in a majority of LiCl-induced c-Fos-positive cells in the CeA. These results suggest a possible correlation between LiCl-induced c-Fos expression and modifications of histone H3 in the CeA.
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Date Issued: 2008
Identifier: FSU_migr_etd-3034
Format: Thesis

Title: Analysis of IgE Reactivity to Pru Du 6, an 11S Globulin from Almond Nut, and Identification of Both Sequential and Conformational Epitopes.
Creator: Willison, Leanna N., Roux, Kenneth H., Sathe, Shridhar K., Keller, Thomas C. S., Keller, Laura R., Hsieh, Yun-Hwa Peggy, Department of Biological Science, Florida State University
Abstract/Description: Tree nuts are a widely consumed food and include walnut, cashew, almond, hazelnut, pistachio, pecan, chestnut, macadamia, and Brazil nut. Although enjoyed safely by most individuals, allergic reactions are common and ~0.6% of the US population is allergic to one or more tree nuts. An important IgE-reactive protein in almonds is prunin (Pru du 6), a legumin, which represents a major component of the seed and has been identified as an important allergen. Two prunin isoforms have been identified...
Show moreTree nuts are a widely consumed food and include walnut, cashew, almond, hazelnut, pistachio, pecan, chestnut, macadamia, and Brazil nut. Although enjoyed safely by most individuals, allergic reactions are common and ~0.6% of the US population is allergic to one or more tree nuts. An important IgE-reactive protein in almonds is prunin (Pru du 6), a legumin, which represents a major component of the seed and has been identified as an important allergen. Two prunin isoforms have been identified: prunin 1 (Pru du 6.01) and prunin 2 (Pru du 6.02). IgE reactivity to recombinant Pru du 6.01 and Pru du 6.02 was found in the sera of 9 of 18 (50%) and 5 of 18 (28%) patients, respectively. To test for epitope stability, the recombinant proteins (rPru du 6) were treated with various denaturants. Immunodot blotting assays revealed the presence of both stable (sequential) and unstable (conformational) rPru du 6 epitopes. The sequential IgE binding epitopes on Pru du 6 were identified by solid-phase overlapping peptide analysis (SPOTs assay). Six IgE-binding sequential epitope-bearing peptides were found on Pru du 6.01 and eight on Pru du 6.02 using almond-allergic sera. Murine anti-almond IgG mAbs also showed greater reactivity to rPru du 6.01 than to rPru du 6.02. The sequential epitopes targeted for several mAbs (4G2, aa 353LQQERQQ359 ; 2B4 and 5D1, aa 120QQGRQQ125) were identified by SPOTs assay. The 5D1 and 2B4 epitopes were found to directly overlap with an IgE binding epitope on Pru du 6.01. Immunoblots, immunodot blots, and inhibition immunoblots indicate that Pru du 6.01 is the predominate isoform in the nut. To investigate conformational epitopes on prunin (Pru du 6), phage display technology was used to identify IgE-binding epitopes. Total IgE was purified from an almond-allergic patient by affinity chromatography and used to capture phage displaying 12 aa-long peptides that mimic prunin epitopes. The phage sequences were analyzed using the Pepitope program and three conformational epitopes were identified (epitope 1, E49,N78, L80, L82, P83, L244, A245, N290, R312, L314, G316, N322, I325, Q326; epitope 2, V104, F105, H190, Q191, T193, P205, A206, G207, V208, V327, R328, G329, N330, L331, D332, F333; epitope 3, H227,S229, S230, D231, H232, F411, W431, V433, N434, H436, V451, Q473, N474, H475, G476, T493, N496, A497, F498, L502). One of these epitopes partially overlaps a sequential epitope previously identified by the SPOTs assay. Conformational epitope mapping studies were also performed using a murine monoclonal antibody (mAb) 4C10. This mAb reacts exclusively with non-reduced native prunin in immunoblotting assays, indicating that 4C10 binds to a conformational epitope expressed on prunin that is dependent on the large and small subunit association. Inhibition ELISA assays found that human IgE and IgG binding epitopes (sequential and/or conformational) overlap or sterically hinder 4C10 binding to prunin. To identify the epitope, hydrogen/deuterium exchange (HDX) monitored by 14.5 T Fourier transform ion cyclotron resonance mass spectrometry was performed on prunin and the 4C10-prunin complex. Comparison of deuterium uptake between the free vs. mAb-bound prunin identified several epitope candidate peptides that differ in deuterium uptake, suggesting that these peptides are part of the 4C10 epitope. Analyses of chimeric molecules using the homologous soybean allergen, Gly m 6, and alanine mutants further localized the epitope to three discontinuous strands (aa 21-45, 320-328, and 460-465). These data demonstrates that HDX-MS is a useful technique to aid in the identification of unknown conformational epitopes on native tree nut allergens.
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Date Issued: 2013
Identifier: FSU_migr_etd-7663
Format: Thesis

Title: Ant (Formica Pallidefulva) Nest Architecture: Structure and Rules of Excavation.
Creator: Mikheyev, Alexander S., Tschinkel, Walter R., Houle, David, Levitan, Don R., Department of Biological Science, Florida State University
Abstract/Description: The nest architecture of underground ant nests was studied in Formica pallidefulva. These ants build shallow (30-45 cm deep) nests, which consisted of more or less vertical shafts that bear chambers. Shafts appeared to be modular units of nest growth; nests were enlarged by adding more shafts or extending previously existing ones. The nests were top-heavy, their volume declining exponentially with depth. The total volume of the nest was strongly correlated with the number of worker occupying...
Show moreThe nest architecture of underground ant nests was studied in Formica pallidefulva. These ants build shallow (30-45 cm deep) nests, which consisted of more or less vertical shafts that bear chambers. Shafts appeared to be modular units of nest growth; nests were enlarged by adding more shafts or extending previously existing ones. The nests were top-heavy, their volume declining exponentially with depth. The total volume of the nest was strongly correlated with the number of worker occupying the nest. Several rules and templates that may be used by workers for nest construction were determined: (a) chambers are formed in the direction of the tunnels leading up to them, (b) the amount of soil excavated per unit time was related to the soil temperature and the moisture content of soil. The amount of time and energy required to construct a typical nest were estimated using digging ability parameters estimated in the lab. It was found that if a colony was to move twice a year, it would expend 21% of its energy intake and 6% of its worker time on nest excavation.
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Date Issued: 2002
Identifier: FSU_migr_etd-2444
Format: Thesis

Title: Aspects of Antipredation in Panulirus Argus and Panulirus Guttatus: Behavior, Morphology, and Ontogeny.
Creator: Bouwma, Peter Edward, Herrnkind, William F., Thistle, David E., Travis, Joseph, Tschinkel, Walter R., Houpt, Thomas A., Department of Biological Science, Florida State University
Abstract/Description: Spiny lobsters (Family Palinuridae) are large, diverse, and abundant marine crustaceans, which have conquered tropical, subtropical, and temperate coastal waters around the globe despite strong predation pressure. The mechanisms and function of antipredation strategies for most species in this highly successful taxon, encompassing behavior, morphology, and life-history characteristics, are poorly understood, particularly against natural predators. I investigate mechanisms of antipredation in...
Show moreSpiny lobsters (Family Palinuridae) are large, diverse, and abundant marine crustaceans, which have conquered tropical, subtropical, and temperate coastal waters around the globe despite strong predation pressure. The mechanisms and function of antipredation strategies for most species in this highly successful taxon, encompassing behavior, morphology, and life-history characteristics, are poorly understood, particularly against natural predators. I investigate mechanisms of antipredation in spiny lobster Panulirus argus in the open during the day, at night, and while sheltering diurnally in natural dens. I also examine the function of putatively defensive acoustic signals produced by P. argus during diurnal attacks by piscine predators and while escaping octopuses at night. I also compare and contrast the mechanism and survival value of antipredator behavior and morphology between sympatric Panulirus argus and P. guttatus. Finally, I investigate ontogenetic changes in defensive behavior by diurnally sheltered P. argus to chemically-mediated predator cues. Nearly 40 species of spiny lobsters produce a characteristic sound (termed stridulation), speculated to deter predation. The occurrence and efficacy of stridulation has not been documented quantitatively during encounters with natural predators. I examined sound production in the sympatric spiny lobsters Panulirus argus and P. guttatus during attacks by their common predator, gray triggerfish Balistes capriscus, to determine if lobsters produce sound during defense, how stridulation integrates with behavioral and morphological defenses, and how interspecific differences in sound production relate to efficacy in repelling predators. Both lobster species stridulated coincident with specific defensive actions during triggerfish attack. In P. argus, stridulation occurred both during antennal lunging and during escape attempts (rapid retreat by tailflips). Panulirus guttatus stridulated only coincident with tailflips and did not lunge. Same-sized individuals of P. guttatus were subdued ~3 times more quickly on average than P. argus. The two species differed also in the relative size of the primary defensive weapons, the spinose 2nd antennae, which were far more robust in P. argus, particularly at larger body sizes. These results suggest that stridulation is an integral component of aggressive defense and escape behavior in spiny lobsters. The timing of sound production during aggressive, retaliatory defensive behavior (lunging) by P. argus suggests an aposematic role for stridulation against triggerfish. Using staged encounters of P. argus with B. capriscus, I examined whether stridulation, coincident with thrusting spines during aggressive defense, functions aposematically or simply renders a defending lobster more difficult to subdue without playing an aposematic role. I demonstrate, by disabling the stridulating organ in some lobsters (muting), that sound plays a vital role in defense against inexperienced (naïve) triggerfish, resulting in fewer successful attacks in subsequent encounters. Choice experiments with triggerfish that previously bypassed defenses and consumed lobsters show that experienced attackers do not choose muted lobsters over stridulating individuals. I propose that stridulation by P. argus against triggerfish is aposematic, as part of a multi-modal display, advertising the lobster's spiny defenses to predators. It is widely documented that sound production in P. argus and other spiny lobsters accompanies grasping of the carapace or other disturbance by human captors. Additionally, stridulation accompanies tailflip escape attempts during attacks by triggerfish. Although sound production during daytime attacks does not appear to increase survival against triggerfish, stridulating during escape may be more effective against grasping predators like octopus. Here, I investigate P. argus defensive behavior during nighttime encounters with Caribbean reef octopus Octopus briareus to determine whether P. argus stridulate during octopus attacks, how stridulation is used along with other defensive behavior (e.g. tailflips), and whether sound production improves survival in stridulating individuals. Lobsters stridulate both during grasping attacks by octopus and during escape attempts after being captured. Stridulating lobsters are also more likely to escape from attacking octopuses and remain uncaptured longer during encounters. I suggest that improving the efficacy of tailflip escapes against octopus may have been the function for which the stridulating organ initially evolved in the Stridentes clade of the Palinuridae. Benthic stages of P. argus reside in shelters during the day as a primary means of antipredation. However, when an active predator approaches and/or successfully attacks a nearby conspecific, these individuals must decide whether to emigrate quickly from the area or remain in shelter (dens or macroalgae) and rely on crypticity, defensive behavior, or the presence conspecifics to avoid attack, injury, or death. In this study, I examine how the three benthic juvenile phases of Caribbean spiny lobster Panulirus argus respond to exposure to fresh conspecific body fluid and how antipredator behavior, particularly the decision to stay or leave the area, changes during ontogeny. Additionally, I examined how the presence of conspecifics affects the decision to stay or leave in gregarious juvenile stages of P. argus. Although all size classes of P. argus respond to alarm odor, the decision to stay or leave dens changes unexpectedly with increasing body size and in the presence of conspecifics. Once shelters were abandoned, body size was a strong indicator of distance traveled in response to alarm odor. This indicates that Panulirus argus undergo an ontogenetic shift in defensive behavior, more frequently leaving dens in response to alarm odor and traveling across open substrate during the day, but only after reaching a body size at which they can effectively defend against predators.
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Date Issued: 2006
Identifier: FSU_migr_etd-3466
Format: Thesis

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

Title: The Association of Pectobacterium, a Plant Pathogen, with the Carapace of the Caribbean Spiny Lobster, Panulirus Argus.
Creator: Lowenberg, Megan M., Reeves, Robert H., Bass, Hank W., Herrnkind, William F., Department of Biological Science, Florida State University
Abstract/Description: The Caribbean spiny lobster, Panulirus argus, is found throughout the Florida Keys and Dry Tortugas. In 1998, lobsters were discovered with small black, necrotic lesions on the carapace some associated with trauma. Since then the number of occurrences of shell disease has increased, a potential problem for the lobster fishery in the southeast. In an effort to determine the etiology of shell disease, bacterial samples were obtained from healthy and diseased lobsters from 28 different locations...
Show moreThe Caribbean spiny lobster, Panulirus argus, is found throughout the Florida Keys and Dry Tortugas. In 1998, lobsters were discovered with small black, necrotic lesions on the carapace some associated with trauma. Since then the number of occurrences of shell disease has increased, a potential problem for the lobster fishery in the southeast. In an effort to determine the etiology of shell disease, bacterial samples were obtained from healthy and diseased lobsters from 28 different locations in the Florida Keys and Dry Tortugas. DNA fingerprinting of the 16s-23s rRNA intergene region (IGR) placed 487 isolates into five major groups of gamma-Proteobacteria, one group of Gram-positive organisms, and individuals of low occurrence were placed in an "Other" group. Sequencing of the 16s rRNA gene and GenBank BLAST analysis identified the genus Erwinia was the second largest group that consisted of 89 isolates. Out of the 89 isolates, 88 were identified as Erwinia cypripedii. The E. cypripedii sequences were aligned with sequences from known strains of marine bacteria; members of the Erwinia, Brenneria, Pectobacterium, Pantoea genera; and other members from the gamma-Proteobacteria group. After performing phylogenetic analyses using the program PAUP*, the lobster isolates were placed into a monophyletic group distinct from, but closely related to, E. cypripedii. Erwinia spp. are normally associated with plants and rotting vegetation, and therefore, not seen in a marine environment especially on the carapace of an invertebrate. Since the isolates showed little diversity by 16S rRNA analysis, two protein coding genes, recombinase A (recA) and recombination-dependent growth C (rdgC), were picked to further analyze the Erwinia isolates. Once again the recA and rdgC gene sequences showed very little diversity among the isolates. Each isolate was also screened for extracellular enzyme production to see if they could break down proteins, lipids, and/or chitin, to establish a possible pathogenic role.
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Date Issued: 2009
Identifier: FSU_migr_etd-1028
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: Assortative Mating in the Tropical Sea Urchin Lytechinus Variegatus.
Creator: Moscoso, Jose A., Department of Biological Science
Abstract/Description: Lytechinus variegatus, a widely known species of sea urchins, exist in different color morphs. Previous studies have shown that gamete recognition proteins are related to color expression in Echinoderms. In this study, crosses within and between color morphs of L. variegatus were done to assess assortative fertilization, and field measurements of aggregation and color frequency to determine behavioral and genetic components of assortative mating. Differences in fertilization success were...
Show moreLytechinus variegatus, a widely known species of sea urchins, exist in different color morphs. Previous studies have shown that gamete recognition proteins are related to color expression in Echinoderms. In this study, crosses within and between color morphs of L. variegatus were done to assess assortative fertilization, and field measurements of aggregation and color frequency to determine behavioral and genetic components of assortative mating. Differences in fertilization success were discovered between color morphs and evidence was found for color aggregation as well as phenotypic effects in the distribution of colors in a population of L. variegatus.
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Date Issued: 2015
Identifier: FSU_migr_uhm-0490
Format: Thesis

Title: Attention Deficit/Hyperactivity Disorder (ADHD) and Kv1.3: Evaluating a Potential Disease Model.
Creator: Hoffman, Carlie, Department of Biological Science
Abstract/Description: Attention deficit/hyperactivity disorder (ADHD) is a condition characterized by excessive levels of impulsivity, inattention, and hyperactivity. Mice subjected to deletion of the Kv1.3 potassium ion channel (knockout, KO) were observed to display behavioral symptoms of ADHD, indicating they may model this disorder. Due to the comorbidity between ADHD and anxiety disorders, the anxiety levels of KO and wildtype (WT) mice were determined as a precursor for the presence of additional ADHD-type...
Show moreAttention deficit/hyperactivity disorder (ADHD) is a condition characterized by excessive levels of impulsivity, inattention, and hyperactivity. Mice subjected to deletion of the Kv1.3 potassium ion channel (knockout, KO) were observed to display behavioral symptoms of ADHD, indicating they may model this disorder. Due to the comorbidity between ADHD and anxiety disorders, the anxiety levels of KO and wildtype (WT) mice were determined as a precursor for the presence of additional ADHD-type behaviors using marble burying, elevated plus maze, and light-dark box testing. Because ADHD affects children and adults alike, inattentive and hyperactive behaviors were quantified for young and aged mice of both genotypes. Methylphenidate (MPH) or saline were also administered to the mice via oral gavage to determine the influence of MPH on behavior. Anxiety testing indicated that KO mice trended towards having decreased anxiety levels compared to their WT counterparts. Object-based attention testing indicated young and aged KO mice had attention deficits and treatment with MPH ameliorated this symptom. However, metabolic chamber testing revealed that WT and KO mice had equivalent activity levels and MPH treatment had no influence on locomotor activity. Based on these findings, there is a link between enhanced olfactory ability and decreased anxiety, and KO mice may be used as behavioral models of the inattentive subtype of ADHD.
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Date Issued: 2014
Identifier: FSU_migr_uhm-0316
Format: Thesis

Title: The Batoid Tree of Life: Recovering the Patterns and Timing of the Evolution of Skates, Rays and Allies (Chondrichthyes: Batoidea).
Creator: Aschliman, Neil C., Naylor, Gavin J.P., Steppan, Scott J., Parker, William C., Mast, Austin R., Department of Biological Science, Florida State University
Abstract/Description: Batoid fishes (skates, stingrays and allies) comprise the majority of species diversity and morphological disparity among chondrichthyans, one of the two primary divisions of extant jawed vertebrates. The largely recent and growing interest in batoid evolution has emphasized the need for a well-supported phylogeny against which evolutionary changes in traits can be interpreted. While batoids are morphologically well characterized and have an excellent fossil record, there is currently no...
Show moreBatoid fishes (skates, stingrays and allies) comprise the majority of species diversity and morphological disparity among chondrichthyans, one of the two primary divisions of extant jawed vertebrates. The largely recent and growing interest in batoid evolution has emphasized the need for a well-supported phylogeny against which evolutionary changes in traits can be interpreted. While batoids are morphologically well characterized and have an excellent fossil record, there is currently no consensus on the interrelationships of family-level taxa. Patterns of evolution within the two largest groups of batoids, skates and stingrays, also remain obscure. This dissertation presents novel frameworks for interpreting the patterns and timing of batoid evolution based on molecular data, morphology, and fossils. I recovered a resolved and time-calibrated phylogeny of the major extant groups of batoids using mitochondrial genomes, two independent nuclear markers, and fossil ages. Taxon sampling included 37 ingroup species from 22 of 23 families. Data partitioning schemes, potential biases in the sequence data, the relative informativeness of each fossil, and ancestral state reconstructions were explored. The molecular data set was then expanded with additional species in order to address questions at a finer taxonomic scale, in particular among skates and stingrays. Two nuclear and two mitochondrial markers were sequenced for 87 batoid species across 52 of 81 genera. A similar analytical approach was applied to this larger data set. I also performed a morphology-based phylogenetic analysis in order to interpret accurately coded morphological characters against the molecular frameworks. I updated the data set of McEachran and Aschliman (2004) with a number of corrections and modifications, and added new characters from the synarcual and other chondroskeletal structures. The molecular phylogenies indicate that the major lineages of batoids originated in relatively rapid sequence, followed by long periods of independent evolution. These trees corroborate morphology-based hypotheses in many respects, but have strongly divergent implications in others. Lineages inferred to be distantly related, such as skates and stingrays, or sawfishes and sawsharks, are indicated to have achieved very similar, specialized body plans through convergence. Skates and stingrays are unique among batoids in exhibiting a highly depressed disc supported to the apex by fin rays, and swim by passing waves along the lateral margin of the pectoral fin without additional propulsion by lateral motion of the tail and caudal fin. The plesiomorphic mode of locomotion and body plan for most batoid groups was probably not shark-like, as in sawfishes. Instead, it is inferred to be a combination of pectoral fin undulation and shark-like axial locomotion, which is correlated with a broader pectoral disc and reduced tail compared to the sawfish body plan. The higher-resolution molecular phylogeny is in many cases congruent with previous morphological studies or biogeography. Exceptions typically suggested a potential weakness in the molecular data, suggested that a morphology-based classification scheme is likely based on convergent characters or ignores a highly divergent morphotype nested within otherwise similar taxa, or helped resolve taxonomic confusion based on informal re-assignment by authority. Potentially non-monophyletic families and genera were identified for future study with expanded taxon sampling, additional sequence data and morphological re-evaluation. The origin of Batoidea is estimated to have occurred in the Late Triassic, with the major groups diverging throughout the Jurassic and possibly into the Cretaceous. Radiations of each major crown group are indicated to have occurred from the Late Cretaceous to the Cenozoic. The tree shape recovered for all major batoid groups, with long internal branches subtending subsequent radiations around the Cretaceous/Tertiary boundary, suggests that batoid standing diversity may be due in large part to lineage pruning and/or rapid radiation into vacated niche space. This is consistent with the fossil record, which suggests that batoids were more severely affected by the end-Cretaceous extinction event than were the other neoselachians. The updated morphological phylogeny included several key changes from earlier hypotheses and more closely approximates the molecular frameworks. There remain conflicts between morphological and molecular trees, such as the phylogenetic placements of skates and thornbacks, which currently appear to be difficult to reconcile. Future attempts to reconcile molecules and morphology will require expanded data sets and investigation of potential sources of error in each.
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Date Issued: 2011
Identifier: FSU_migr_etd-0242
Format: Thesis

Title: Behavioral and Neural Characterization of Conditioned Flavor-Taste Preferences.
Creator: Golden, Glen J. (Glen Jesse), Houpt, Thomas A., Licht, Barbara G., Meredith, Michael, Wang, Zuoxin, Fadool, James M., Steppan, Scott J., Department of Biological Science,...
Show moreGolden, Glen J. (Glen Jesse), Houpt, Thomas A., Licht, Barbara G., Meredith, Michael, Wang, Zuoxin, Fadool, James M., Steppan, Scott J., Department of Biological Science, Florida State University
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Abstract/Description: Many animals, including humans, choose their source of nutrition based on the nutritive value and the flavor (i.e, odor, taste and texture) of available foods. Sweet taste is one of the more potent orosensory stimuli that contributes to food choice. Laboratory animals develop preferences for neutral or aversive tastes and flavors by associating them with the taste of sugars and non-caloric sweeteners. Learning how these preferences develop would aid in understanding how and why specific foods...
Show moreMany animals, including humans, choose their source of nutrition based on the nutritive value and the flavor (i.e, odor, taste and texture) of available foods. Sweet taste is one of the more potent orosensory stimuli that contributes to food choice. Laboratory animals develop preferences for neutral or aversive tastes and flavors by associating them with the taste of sugars and non-caloric sweeteners. Learning how these preferences develop would aid in understanding how and why specific foods are selected over others. Given the wide availability and variety of sweetened foods and beverages in modern society, the formation and persistence of learned food preferences by consumers may contribute to health issues such as diabetes and obesity. Conditioned flavor-taste preference (CFTP) learning is a form of associative learning in which a rat comes to prefer a neutral flavor paired with a preferred taste. Experimentally, one flavor (the conditioned stimulus or CS+; e.g., cherry or grape Kool-Aid) is paired with the sweet and highly preferred taste of fructose (F; the unconditioned stimulus or US) while a second flavor (the CS-) is paired with the less preferred taste of saccharin (S) on 1-bottle conditioning days (CS+/F or CS-/S). The acquisition of the learned preference is then assessed with a 2-bottle preference test in which both flavors mixed with saccharin (CS+/S and CS-/S) are presented simultaneously. While CFTP learning is well known, it has not been well characterized. The olfactory and gustatory associative brain regions necessary for CFTP learning are unknown. Dopamine receptors have been implicated, but otherwise it is not known which neurotransmitters or receptors mediate CFTP. In order to identify the associative neural substrates that are involved in CFTP learning, three approaches were taken; behavioral, pharmacological and molecular assays. To precisely characterize the behavioral acquisition and expression of a CFTP, lickometers were used to determine the pattern of drinking in rats. During 1 or 2-bottle preference tests, total intake, bout size, bout number, lick rate and first minute licks were analyzed. The pattern of drinking was examined under 3 conditions: 1. During expression of unconditioned preferences for 8% fructose over 0.2% saccharin. The unconditioned preference for fructose over saccharin was slow to develop, but was seen in significantly greater total intake, bout size, and first minute licks for fructose by the fourth preference test. 2. During the pairing of Kool-Aid flavors with either 8% fructose or 0.2% saccharin. CS-/S total intake and bout size was significantly greater than CS+/F during conditioning, but a preference for the CS+ flavor was seen in the third and fourth 2-bottle preference test days with significantly greater total intake and bout size of CS+/S vs. CS-/S. 3. During long-term presentations of Kool-Aid mixed with two different concentrations of saccharin (0.2% vs 0.05%) as the unconditioned stimuli. Total intake, bout size and bout number were significantly greater for the flavor mixed with the high concentration of saccharin over the low concentration of saccharin. during conditioning. During 2-bottle preference tests when both flavors were mixed with the low concentration of saccharin, total intake and bout size were significantly greater for the CS+. The increases in lick rate and bout size observed in all 3 experiments suggest that fructose is more palatable than saccharin, and a high concentration of saccharin is more palatable than a low concentration. A change in relative palatability of the Kool-Aid flavors is conditioned by association with the high palatability tastes; greater intake of the conditioned flavor is mediated by increased bout size. These results suggest that flavor preference learning interacts with both orosensory processes and satiety processes (i.e. prolonged bout size) to elevate intake of the preferred flavor. The N-methyl-D-aspartate (NMDA) glutamate receptor (NR) is a candidate mediator in olfactory and taste learning (Barkai and Saar, 2001; Jimenez and Tapia, 2004). To determine if NR is involved in CFTP, systemic MK-801, a non-competitive NR antagonist, was administered prior to conditioning and prior to expression. To determine the glycinergic contribution to NR activation in CFTP, systemic D-cycloserine, an agonist at the NR glycine-binding site, was administered prior to conditioning and reversal learning. While vehicle-treated rats acquired a preference for CS+/S over CS-/S, CFTP learning was completely blocked in MK-801-treated rats. The effect of MK-801 was specific to CFTP acquisition, because follow-up experiments demonstrated that MK-801 did not induce a conditioned taste aversion, cause state-dependent learning, or affect CFTP expression. In a second approach, rats were injected with DCS (15 mg/kg) 60 min prior to daily conditioning. In contrast to MK-801, administration of DCS prior to conditioning enhanced CFTP learning (but not reversal conditioning). These results demonstrate that NR neurotransmission is critical for CFTP learning. Furthermore, enhancement of CFTP learning by DCS suggests that endogenous levels of glycine or D-serine may be a limiting factor in CFTP learning. To determine the activation of neural populations during associative CFTP learning, c-Fos immunohistochemistry was used to illuminate the differential patterns of cellular activation. To eliminate the potential confounds of food restriction, restricted drinking sessions and potential postingestive effects that may effect c-Fos activation, rats were conditioned using a highly preferred concentration (0.2% saccharin) and a lesser preferred concentration of saccharin (0.05% saccharin) as the unconditioned stimuli. In order to standardize exposures, stimuli were applied by intraoral infusion. C-Fos immunolabeling was visualized within the brain after an intraoral infusion of either CS+ or CS- flavors (e.g. grape and cherry Kool-Aid) in combination with greater or lesser preferred taste US (e.g. 0.2% or 0.05% saccharin). Neuronal activation was assessed in forebrain sites (e.g. gustatory cortex, amygdala, and lateral hypothalamus). There was no difference in intraoral intake between all experimental groups in both conditioned and unconditioned rats, and extensive c-Fos activation was evoked in the olfactory, gustatory and learning relays of all experimental groups. Analysis of the differential patterns of c-Fos immunolabeling among unconditioned rats revealed a significant increase in c-Fos immunolabeling in the basolateral nuclei of the amygdala after intraoral infusions of dH2O compared to unconditioned rats after intraoral infusions of CS+/0.2% saccharin or CS-/ 0.055 saccharin. Therefore, the basolateral amygdala may be involved in the unconditioned response to sweetened flavors, or in the association of flavor with sweet tastes. Among conditioned rats, there was a trend towards greater in c-Fos immunolabeling in the lateral habenula of rats after intraoral infusions of CS+ vs the CS- or saccharin alone. Therefore, the lateral habenula, which is part of the accumbens-ventral tegmental area reward pathway, may be involved in the discrimination of learned preferences.
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Date Issued: 2007
Identifier: FSU_migr_etd-4199
Format: Thesis

Title: Behavioral Types and Sexual Selection in the Bluefin Killifish.
Creator: McGhee, Katie E., Travis, Joseph, Johnson, Frank, Fadool, James, Hansen, Thomas, Houle, David, Department of Biological Science, Florida State University
Abstract/Description: In many animal species there is pronounced and repeatable variation among individuals in their expression of suites of behaviors, resulting in consistent behavioral types. These behavioral types are characterized by measures like the degree of boldness, aggression, activity, and exploratory behavior. While the prevalence of behavioral types and their importance in terms of natural selection have become apparent in recent years, their importance in determining the outcome of sexual selection...
Show moreIn many animal species there is pronounced and repeatable variation among individuals in their expression of suites of behaviors, resulting in consistent behavioral types. These behavioral types are characterized by measures like the degree of boldness, aggression, activity, and exploratory behavior. While the prevalence of behavioral types and their importance in terms of natural selection have become apparent in recent years, their importance in determining the outcome of sexual selection remains imperfectly understood. In this dissertation I examined the role of behavioral types in sexual selection in the bluefin killifish, Lucania goodei, as well as the influence of environmental and genetic factors on the expression of behavioral types. In chapter two I investigated how female choice and male competition interact in the bluefin killifish in a 3-staged experiment where (1) females could choose between two males, (2) those males could interact in the presence of that female, and (3) females and males could freely interact and spawn. In the pairwise stages (1 and 2), females displayed pronounced preferences between males and male competition produced a distinctly dominant individual. None of the morphological traits, including color, measured in males were associated with either female preference or male dominance. When all three fish interacted (stage 3), male mating activity level (behavioral type) was the sole predictor of spawning success. Males with elevated activity levels were more aggressive toward males and females, exhibited intensified courtship, and obtained more spawns. Female preference did not predict the number of spawns with a male but it did predict her latency to spawn; females spawned more quickly with preferred males. Thus, male competition and female choice interact to determine reproductive success but there is evidence for conflict and a cost to females of associating with dominant males. Reproductive success in this species is not easily predicted from simple measures of morphology or female preference, and is influenced by complex social interactions, both between males, and between males and females. The outcomes of these social interactions are in turn influenced by the behavioral types of the participants. In chapter three I examined whether repeatable personality differences among males are associated with repeatable outcomes of male-male interactions within the mating context. Specifically I examined the repeatability of individual differences in mating behaviors and the stability of dominance status, which partially determines mating success in the bluefin killifish. The expression of male behaviors in competition between males and female courtship was significantly repeatable over a five-week time period; the number of aggressive behaviors to males, to females, and the number of courtship bouts had significant repeatabilities of 0.71, 0.72, and 0.65 respectively. A male's behavioral type within the mating context, as measured by a composite measure of the overall level of mating behavior activity, was significantly repeatable at 0.75. Males showed repeatable, linear dominance hierarchies and a male's rank in the hierarchy was highly correlated with his behavioral type. Neither behavioral type nor dominance status was associated with body size or body condition. The repeatability of behavioral types and stability in the outcomes of aggressive interactions, suggest that these behavioral types are inherent characteristics of individuals rather than short-term responses to recent social experience or daily levels of food or stress. In chapter four, I examined how differences between individuals in behavioral type arise. Specifically I examined whether nutritional and social conditions experienced by individuals early in life affect the development of behavioral type and behavioral expression later in life in the bluefin killifish. As fry, individuals experienced either high food or low food levels and within each food treatment, individuals experienced one of three social environments: they were reared with one adult male, one adult female, or no adult, producing six treatment combinations. Social treatments were removed at 5 months and food manipulations were ceased at 6 months, after which individuals were fed ad libitum until behavioral testing at 10 months. Males reared on high food and low food were paired with one another within a social treatment and tested in a female choice trial and male dominance trial. Food and social treatments produced strong, synergistic effects on growth; individuals raised at high food were larger than those raised at low food and high food individuals reared with an adult female grew faster than those in all other treatments. There was no evidence for compensatory growth after release from treatments imparting slow growth. Despite the effects of early experience on growth and body size, I was unable to detect any comparable effect on behavioral variation. Male behavioral type and behavioral expression were not significantly affected by food level and this did not vary across social treatments. High food and low food males did not differ significantly in their likelihood of becoming dominant as adults or of being preferred by females. These results suggest that an individual's early rearing environment, at least the particular aspects manipulated here, have little influence on the development of behavioral type in this species. Given that personalities can affect fitness, understanding the environmental and genetic factors that influence personalities, as well as the associations among behaviors across different contexts are crucial to understanding the evolutionary response of personality to direct and indirect selection. In chapter five, I performed a series of paternal half-sib crosses to estimate the heritability of a number of distinct behaviors within and across three different contexts (mating, response to a predator, recovery from stress) as well as whether there are phenotypic and genetic correlations among behaviors exhibited within and across contexts. Despite the presence of strong phenotypic correlations among the behaviors exhibited in the mating context, my results indicate that only courtship behaviors appear to be significantly heritable and there is little evidence for significant additive genetic variance in aggressive behaviors. In addition, I found no evidence for phenotypic correlations among traits expressed outside the mating context, nor evidence for additive genetic variation for those behavioral traits. The results of my dissertation point to more complex sources of individual variation in personality and, concomitantly, more complex patterns of selection. I found that while variation in three behaviors formed an integrated personality, in only one behavior was that variation based on substantial heritable genetic variation. What emerges as individual personality could be the result of individual variation in a single behavior and the network of provocation and response that is built by social interactions. This complexity opens new avenues for future empirical work and new possibilities for understanding the subtle interplay of genetic and environmental foundations for the behaviors seen in complex social systems.
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Date Issued: 2009
Identifier: FSU_migr_etd-2560
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: Biomechanics and the Ontogeny of Feeding in the American Alligator (Alligator Mississippiensis): Reconciling Factors Contributing to Intraspecific Niche Differentiation in a Large Bodied Vertebrate.
Creator: Gignac, Paul Michael, Erickson, Gregory, Parker, William, Chase, P. Bryant, Inouye, Brian, DuVal, Emily, Hollis, Patrick, Department of Biological Science, Florida State University
Abstract/Description: As vertebrates attain a larger body size, the relative growth of various body parts results in differential rates of growth across the organism and may result in substantial impacts on mechanical performance. To deal with this problem, shape changes often accompany somatic growth, and the nature of these changes is thought to reflect a tight relationship between the morphological form of an organism's anatomy and its function within a given environment. In this context, the feeding anatomy of...
Show moreAs vertebrates attain a larger body size, the relative growth of various body parts results in differential rates of growth across the organism and may result in substantial impacts on mechanical performance. To deal with this problem, shape changes often accompany somatic growth, and the nature of these changes is thought to reflect a tight relationship between the morphological form of an organism's anatomy and its function within a given environment. In this context, the feeding anatomy of vertebrates is a consummate form-function relationship. An extreme example of a taxon that undergoes ontogenetic dietary shifts is the American alligator, Alligator mississippiensis, which traverses a 5000-fold increase in mass during its lifetime. As a consequence, this species must cope with changes in its feeding functional morphology (i.e., size and shape of dental and musculoskeletal attributes) while exploiting varying ecological feeding niches. Hatchlings start out as insectivorous neonates but abandon that feeding niche as larger body size allows them access to a wider range of prey items, first frogs and fish and other small, compliant prey before finally reaching the adult ecomorphology where they access more robust quarry. Absolute body size and bite-force positive allometry play an important role in these transitions. Therefore, to understand the nature of the anatomical changes that underpin these factors and thus facilitate dietary niche transitions, I dissected a growth series of wild A. mississippiensis. I standardized the topology, attachment points, and naming scheme for the jaw adductor musculature and quantified the growth of the cranial skeleton, jaw adductor muscle system, and dental form throughout ontogeny. I derived mathematical models of bite-force and hold-force generation based on these data, and tested them against additional developmental series of known bite-force A. mississippiensis. I developed and implemented a novel technique to identify the aerobic capacity of the jaw adductor muscles, called Muscle Oxidative Inference Analysis (MOIA). Finally, dental pressures were quantified using bite forces and dental morphology. These data demonstrate that after hatching, larger body size (25 cm snout-vent length, SVL) initially allows A. mississippiensis access to increasingly larger, compliant prey. At 45 cm SVL positive allometry in the post-orbital growth of the skull and M. Pterygoideus ventralis muscles in addition to substantial changes in the aerobic capacity of some jaw adductor muscles facilitates access elusive terrestrial prey such as birds and small mammals. By 75 cm SVL, subtle changes in dental form along with bite-force positive allometry make it possible for this taxon to generate tooth pressures that can fracture and mechanically fail the bony carapaces of turtles. Finally, large adult body size (150+ cm SVL) and continued positive allometry in the musculoskeletal attributes of its jaw adductor system and further augmented oxidative capacity of its adductor muscles allow A. mississippiensis to secure large game mammals such as deer, boar, and cattle.
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Date Issued: 2010
Identifier: FSU_migr_etd-7143
Format: Thesis

Title: Bite-Force Generation and Feeding Biomechanics in the Loggerhead Musk Turtle, Sternotherus Minor: Implications for the Ontogeny of Performance.
Creator: Pfaller, Joseph Bryce, Erickson, Gregory M., Inouye, Brian D., Oates, William S., Department of Biological Science, Florida State University
Abstract/Description: Ontogenetic growth can profoundly affect the ability of organisms to perform ecologically-relevant feeding tasks that ultimately impact survival. In particular, bite-force generation is exceedingly important for vertebrates that process and consume robust prey (i.e. durophagy). Consequently, bite-force generation used in durophagy is a suitable parameter to investigate the functional relationships between musculoskeletal biomechanics, feeding performance, and ecology. I studied the ontogeny...
Show moreOntogenetic growth can profoundly affect the ability of organisms to perform ecologically-relevant feeding tasks that ultimately impact survival. In particular, bite-force generation is exceedingly important for vertebrates that process and consume robust prey (i.e. durophagy). Consequently, bite-force generation used in durophagy is a suitable parameter to investigate the functional relationships between musculoskeletal biomechanics, feeding performance, and ecology. I studied the ontogeny of bite-force generation and feeding biomechanics in the durophagous turtle, Sternotherus minor. Across an ontogenetic series of 75 S. minor, craniofacial growth was characteristized by allometric increases (i.e. positive allometry) in the dimensions of the head and beak. Moreover, bite-force generation increased with positive allometry relative to body and head dimensions. These results indicate that ontogenetic modifications to the lever mechanics of the jaw system, and/or changes in the size (i.e. mass) and/or physiology (e.g., fiber lengths, degree of pennation) of the jaw adductor musculature have more explanatory power for bite-force generation than external measures. A detailed, quantitative examination of the musculoskeletal biomechanics was performed to elucidate how these animals are capable of generating disproportionately high bite forces throughout ontogeny. Mechanical levers, muscle masses, and muscle architecture (fiber lengths and pennation angles) were measured from an ontogenetic series of 30 S. minor. With these data, a biomechanical model of the feeding apparatus was developed that accurately predicts individual and ontogenetic scaling of bite forces. Increasing muscle masses and changing the muscle architecture resulted in an increase in total physiological cross-sectional area (PCSA) of the jaw adductor muscles that was proportional to changes in bite-force generation. These results indicate that the disproportionate increase in bite-force generation relative to skull length found in S. minor is explained by allometric changes in muscle size and architecture that collectively act to allometrically elevate the PCSA and muscle force. The alternative explanation of improving the mechanical leverage was not supported. Dietary data indicated that the positive allometry in bite-force generation observed in S. minor is tightly linked to the incorporation of exponentially larger snails into the diet and positive allometry of the forces required to fracture the largest dietary items. These forces were found to be greater than the observed and theoretical bite forces, which suggested that fatigue failure resulting from multiple bite-force loadings may allow S. minor to fracture snails at lower compressive forces and access large snails that are apparently outside the range of their bite-force capacity. Moreover, age-based growth patterns for bite-force generation fit a logistic growth curve and reveal a close relationship with the forces required to fracture snails found in the diet. The results of this study provide empirical evidence that ontogenetic changes to musculoskeletal morphology and ecology are inextricably linked by the performance of the feeding apparatus in S. minor. Such results are exceedingly important for establishing a baseline from which to explore the mechanistic and functional issues that underlie the evolution of phenotypic traits among vertebrates.
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Date Issued: 2009
Identifier: FSU_migr_etd-1973
Format: Thesis

Title: Brain-Derived Neurotrophic Factor (BDNF) Modulation of Kv1.3 in the Olfactory Bulb.
Creator: Colley, Beverly Shelley, Fadool, Debra Ann, Logan, Timothy M., Freeman, Marc E., Levenson, Cathy W., Kabbaj, Mohamed, Hyson, Richard L., Department of Biological Science,...
Show moreColley, Beverly Shelley, Fadool, Debra Ann, Logan, Timothy M., Freeman, Marc E., Levenson, Cathy W., Kabbaj, Mohamed, Hyson, Richard L., Department of Biological Science, Florida State University
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Abstract/Description: Voltage-gated potassium ion channels such as Kv1.3 have a role in altering excitability of neurons. The neuron has to have a sophisticated mechanism to regulate the modulation, expression, turnover and distribution of ion channels. Ion channels, like Kv1.3, become crucial in affording the neuron one of a symphony of players that can strategically play their part in transmitting electrical and chemical signals into meaning. This dissertation uses electrophysiology and biochemistry to...
Show moreVoltage-gated potassium ion channels such as Kv1.3 have a role in altering excitability of neurons. The neuron has to have a sophisticated mechanism to regulate the modulation, expression, turnover and distribution of ion channels. Ion channels, like Kv1.3, become crucial in affording the neuron one of a symphony of players that can strategically play their part in transmitting electrical and chemical signals into meaning. This dissertation uses electrophysiology and biochemistry to investigate how brain-derived neurotrophic factor (BDNF) and TrkB utilize a very simple aspect of the biochemistry of Kv1.3 to specifically modulate the biophysical properties, expression and turnover of Kv1.3. Acute BDNF application suppresses Kv1.3 current and results in phosphorylation of tyrosine residues 111-113, 137 and 449. There is a delicate balance of other downstream cellular components; N-Shc, Grb10 and PSD95 that disrupt this BDNF induced current suppression. N-Shc disrupts a post-phosphorylation event that usually leads to BDNF-evoked Kv1.3 current suppression, and N-Shc causes phosphorylated Kv1.3 to be retained in the membrane. Grb10 and PSD95 left-shift the voltage at half-activation of Kv1.3 and this effect may not be phosphorylation-dependent. Grb10 also reduces Kv1.3 expression and causes redistribution of membrane inserted Kv1.3. The presence of BDNF activated tropomyosin-related kinase B (TrkB) increases the phosphorylation of Kv1.3 tyrosine residues and increases Kv1.3 expression by two fold. TrkB also increases the half-life of Kv1.3 and this can account for the increase in protein expression. Kv1.5 is another member of the Shaker family but TrkB decreases the expression of Kv1.5. The insulin receptor (IR) is also a tyrosine kinase like TrkB however, IR decreases Kv1.3 expression and has no effect on Kv1.5 expression. These effects demonstrate that the TrkB and IR mediated regulation of Kv1.3 expression is not a promiscuous interaction of Shaker channels with receptor tyrosine kinases. Given the prominence of Kv1.3 in the olfactory bulb, one can hypothesize that the above interactions can play a part in modulating the function of Kv1.3 during development, learning and injury in the olfactory bulb. The neuron can utilize these interactions to regulate Kv1.3 and change how Kv1.3 contributes to shaping the neuron's response.
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Date Issued: 2006
Identifier: FSU_migr_etd-3501
Format: Thesis

Title: A Brief Natural History on the Ant Odontomachus Brunneus (Patton).
Creator: Hart, Lauren M., Tschinkel, Walter R., Stallins, Tony, Inouye, Brian, Department of Biological Science, Florida State University
Abstract/Description: A population of Odontomachus brunneus, a primitive species of Ponerine ants, located in north Florida was studied for a one-year period. Through nest excavation and colony census, the annual cycle of reproduction and colony growth was determined with nests exiting a period of winter inactivity in late April and beginning brood production. Early brood is a combination of both sexuals and workers, with sexuals present in nests during June and July. After production of sexuals in May and June,...
Show moreA population of Odontomachus brunneus, a primitive species of Ponerine ants, located in north Florida was studied for a one-year period. Through nest excavation and colony census, the annual cycle of reproduction and colony growth was determined with nests exiting a period of winter inactivity in late April and beginning brood production. Early brood is a combination of both sexuals and workers, with sexuals present in nests during June and July. After production of sexuals in May and June, all subsequent brood produced were found to be workers through dissection of pupal cocoons. Brood production ceased in October, with the final pupae eclosing in November at which time the colonies began a four month period of relative inactivity. Within-nest seasonal energy allocation was determined by fat extraction. Seasonal energy stores coincided with the annual cycle of reproduction, with workers declining in energy stores (fat) during initial brood production and regaining these stores after production of brood in preparation for the winter season during which colonies are primarily inactive. Comparison of body fat provided the relative ages of worker ants, which suggested that O. brunneus nests display internal age stratification throughout the majority of the year with older, leaner workers being found in the upper chambers of most nests and younger, fatter workers in the lower chambers. Using a combination of mark-recapture of foragers and nest excavation, the proportion of foragers per colony was shown to include a mean of 77% (S.D. 22) of the workforce. This proportion was not related to colony size. Female alates were also found to be a part of the foraging population of colonies.
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Date Issued: 2009
Identifier: FSU_migr_etd-4225
Format: Thesis

Title: Broad, an Active Participant in Drosophila Oogenesis with Broad Functions.
Creator: Jia, Dongyu, Deng, Wu-Min, Horabin, Jamila I., Dennis, Jonathan Hancock, Megraw, Timothy L., Lenhert, Steven, Florida State University, College of Arts and Sciences, Department...
Show moreJia, Dongyu, Deng, Wu-Min, Horabin, Jamila I., Dennis, Jonathan Hancock, Megraw, Timothy L., Lenhert, Steven, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: The follicular epithelium (FE) of the Drosophila egg chamber is an excellent model system to study cell-cycle regulation, cell differentiation and cell migration in development. During oogenesis, follicle cells sequentially undergo three distinct cell-cycle programs: the mitotic cycle (stage 1-6), endocycle (stage 7-10a), and gene amplification (stage 10b-13). Notch signaling plays a central role in regulating follicle-cell differentiation and cell-cycle switches; its activation and...
Show moreThe follicular epithelium (FE) of the Drosophila egg chamber is an excellent model system to study cell-cycle regulation, cell differentiation and cell migration in development. During oogenesis, follicle cells sequentially undergo three distinct cell-cycle programs: the mitotic cycle (stage 1-6), endocycle (stage 7-10a), and gene amplification (stage 10b-13). Notch signaling plays a central role in regulating follicle-cell differentiation and cell-cycle switches; its activation and inactivation in follicle cells are essential for the mitotic cycle/endocycle (M/E) and the endocycle/gene amplification (E/A) switches, respectively. In my dissertation, I mainly focus on Notch signaling and its downstream target broad (br). In the first part of the dissertation, I introduce the background information of the egg chamber system, Notch signaling and other associated factors. In the second part, I describe a screen strategy to identify novel genes involved in Notch-mediated follicle cell differentiation and cell cycle switches. In the third part, I select a Notch target gene br from the above-mentioned screen and study its regulation and functions. I will show br, encoding a small group of zinc-finger transcription factors resulting from alternative splicing, is a transcriptional target of Notch nuclear effector Suppressor of Hairless (Su(H)). The early pattern of Br in the FE, uniformly expressed except in the polar cells, is established by Notch signaling around stage 6, through the binding of Su(H) to the br early enhancer (brE) region. My findings also suggest an important role of br in the timing of follicle cell development during the M/E switch. In the fourth part, I report the uniform pattern of Br in the follicular epithelium is gradually lost in the anterior follicle cells (stretched cells and border cells) from stage 9 to 10a during oogenesis. This downregulation of Br is functionally significant for proper stretched-cell stretching. I also find ecdysone and JAK/STAT signaling mediate the downregulation of Notch-maintained Br. Together, My research investigates the complex Notch signaling network, and reveal that Notch-directly-regulated Br interacts with the ecdysone and JAK/STAT pathways, serving as an important spatiotemporal cue for proper cell differentiation and morphogenetic movement during Drosophila oogenesis.
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Date Issued: 2015
Identifier: FSU_migr_etd-9361
Format: Thesis

Title: Cardiac Thin Filament Regulatory Proteins Familial Hypertrophic Cardiomyopathy Mutations and Post Translational Modifications.
Creator: Compton, Lisa A., Chase, P. Bryant, Overton, J. Michael, Fajer, Peter G., Keller, Thomas C. S., Moerland, Timothy S., Department of Biological Science, Florida State University
Abstract/Description: The cardiac thin filament is a highly ordered regulatory system for cardiac contraction. Regulatory proteins, a-tropomyosin (a-Tm) and troponin (cTn, composed of cTnT, cTnI, and cTnC), provide a Ca2+-dependent mechanism for controlling actin-myosin interactions that underlie cardiac muscle contraction and relaxation. These regulatory proteins may be altered or modified in a variety of ways in vivo, e.g. by mutation or post-translational modifications, which can alter the function and possibly...
Show moreThe cardiac thin filament is a highly ordered regulatory system for cardiac contraction. Regulatory proteins, a-tropomyosin (a-Tm) and troponin (cTn, composed of cTnT, cTnI, and cTnC), provide a Ca2+-dependent mechanism for controlling actin-myosin interactions that underlie cardiac muscle contraction and relaxation. These regulatory proteins may be altered or modified in a variety of ways in vivo, e.g. by mutation or post-translational modifications, which can alter the function and possibly the structure of the whole heart over time scales both short (beat-to-beat) and long (years). Familial Hypertrophic Cardiomyopathy (FHC) is a disease caused by genetic mutations in sarcomeric genes, notably cTnI and a-Tm, that result in left and/or right ventricular hypertrophy. Three cTnI FHC mutations (P82S, D196N, and S199N) are characterized in Chapter 1 in relation to the modulation of their respective Ca2+-sensitivities of individual actin filaments in the in vitro motility assay. One a-Tm FHC mutant (E180G) that causes an increase in Ca2+-sensitivity in the in vitro motility assay is further studied in Chapter 2. Chapter 3 characterizes a commercially available cTn sample from Research Diagnostics for post-translational modifications.
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Date Issued: 2006
Identifier: FSU_migr_etd-3490
Format: Thesis

Title: Categorization of Pheromonal Chemosignals by Medial Amygdala.
Creator: Westberry, Jenne M., Meredith, Michael, Wang, Zuoxin, Freeman, Marc, Houpt, Thomas, Keller, Laura, Department of Biological Science, Florida State University
Abstract/Description: Individual members of many species use chemical signals detected by the vomeronasal system to communicate with other members of the same species. Over the past two decades, research in the chemical senses field has focused on peripheral detection and processing of chemosensory signals by the vomeronasal organ (VNO). Not as much focus has been placed on central processing of these signals once they are detected and communicated to the accessory olfactory bulb (AOB). The studies included in...
Show moreIndividual members of many species use chemical signals detected by the vomeronasal system to communicate with other members of the same species. Over the past two decades, research in the chemical senses field has focused on peripheral detection and processing of chemosensory signals by the vomeronasal organ (VNO). Not as much focus has been placed on central processing of these signals once they are detected and communicated to the accessory olfactory bulb (AOB). The studies included in this dissertation were designed to investigate neuronal activation in the medial amygdala, an area of the brain that gets direct input from the accessory olfactory bulb (AOB). Based on immediate early gene (IEG) expression in activated neurons, I found that the medial amygdala responded differently to pheromonal chemosignals from animals that were conspecific (same species) versus heterospecific (different species). The anterior medial amygdala (MeA) responded to both, but posterior medial amygdala (MeP) responded only to conspecific stimuli and was suppressed during responses to heterospecific stimuli or artificial (non-biological) stimuli. These data provide the first report of categorical discrimination of chemosensory signals in the medial amygdala. This categorization was not apparent in the AOB so it appears to reflect a second level of sensory analysis. Additional studies indicated a reciprocal relationship between activation in MeP and in adjacent inhibitory intercalated nucleus (ICN) cells. MeP is the brain area that only responded to conspecific stimuli. Its lack of activation in MeP with heterospecific stimuli is accompanied by selective suppression in MeP neurons expressing GABA-a Receptor and occurs concurrently with significant activation in GABA immunoreactive cells of the adjacent ICN. The ability of neurons in medial amygdala to show a discrimination between conspecific and heterospecific stimuli was not dependent on main olfactory input and MeA is the first place in the vomeronasal pathway where all the information from rostral and caudal accessory olfactory bulb comes together. Immediate early gene expression does not reveal all the neural activity of the brain, but within the limits of this method all indications are that discrimination occurs in the anterior medial amygdala.
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Date Issued: 2003
Identifier: FSU_migr_etd-1145
Format: Thesis

Title: Cell Adhesion and Motility on Biocompatible Polyelectrolyte Multilayers.
Creator: Martinez, Jessica Susanne, Keller, Thomas C. S., Schlenoff, Joseph B., Keller, Laura R., Ma, Teng, Lenhert, Steven, Florida State University, College of Arts and Sciences,...
Show moreMartinez, Jessica Susanne, Keller, Thomas C. S., Schlenoff, Joseph B., Keller, Laura R., Ma, Teng, Lenhert, Steven, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: To improve the design of prostheses surfaces, our research group investigates how biocompatible polyelectrolyte multilayers (PEMUs) can be constructed to serve as coatings for biomedical implants, providing a versatile, inexpensive, and potentially efficient solution to create anti-bacterial, anti-inflammatory, and biologically selective surfaces. More specifically, this dissertation research investigates how individual cells and cell sheets adhere and migrate on PEMUs constructed to have...
Show moreTo improve the design of prostheses surfaces, our research group investigates how biocompatible polyelectrolyte multilayers (PEMUs) can be constructed to serve as coatings for biomedical implants, providing a versatile, inexpensive, and potentially efficient solution to create anti-bacterial, anti-inflammatory, and biologically selective surfaces. More specifically, this dissertation research investigates how individual cells and cell sheets adhere and migrate on PEMUs constructed to have uniform and gradients of modulus and how individual cells and gram negative bacteria, Escherichia coli, adhere to PEMUs constructed to have an anti-adhesive surface chemistry. In this investigation, PAH/PAA PEMUs are shown to be biocompatible compared to the soluble polycation PAH at concentrations above 0.1mM. Soluble PAH concentrations at 1 and 10mM cause irreversible damage to the plasma membrane of smooth muscle, A7r5, and bone, U2OS, cells. Additionally, adhesive and motile responses of cells are dependent on PEMU surface chemistry. Cells on PEMUs terminated with the polycation PAH relocalize their focal adhesions to their cell periphery and are highly motile compared to cells cultured on PAA terminated PEMUs and uncoated glass coverslips. To investigate effects of PEMU modulus on cell adhesion and motility, PEMUs were made with the polyanion PAA (poly(acrylic acid)) modified with a photosensitive 4-(2-Hydroxyethoxy) benzophenone (PAABp) and the polycation PAH (poly(allylamine hydrochloride)). UV irradiating PAH/ PAABp PEMUs forms covalent bonds between PE layers and consequently increases its Young's Elastic Modulus, while retaining innate surface chemistry. Individual cells and cell sheets detect differences in PEMU modulus and respond by varying morphology and behavior. These PAH/PAABp PEMUs modulate the adhesion, spreading, and migration of individual cells, specifically smooth muscle, bone, and fibroblast cells. PAABp containing PEMUs were constructed to have either a shallow (~5MPa mm-1) or a steep (~50MPa mm-1) modulus gradient. Only smooth muscle cells durotax along steep modulus gradients toward increasing modulus and orient toward increasing modulus on shallow modulus gradients. In contrast, bone cells discriminately adhere to the stiffest region of both steep and shallow modulus gradients and fibroblasts show no difference in behavior along any region of the gradients. Epithelial sheets, isolated as primary explants of fish epithelial tissue from the scales of fish Poecilia sphenops (Black Molly) and Carassius auratus (Comet Goldfish), orient toward increasing modulus on steep modulus gradient. Cell sheets collectively durotax near the ~90MPa region of the gradient toward increasing modulus. Surfaces with substantial zwitterionic functionality (possessing a net neutral surface charge due to equal contribution of both positive and negative charges in polymer side groups) have been shown to effectively prevent cell and protein attachment. PEMUs built with PAH (poly(allylamine hydrochloride)) and PAA (poly(acrylic acid)) containing the AEDAPS zwitterionic group 3-(2-(acrylamido)-ethyldimethyl ammonio) propane sulfonate (PAH/PAA-co-AEDAPS PEMUs) and a new benzophenone crosslinker to stiffen the thin film were shown to prevent rat aortic smooth muscle (A7r5) and mouse fibroblast (3T3) cells attachment, but failed to prevent irreversible attachment of biofilm-forming gram-negative bacteria Escherichia coli, strain ATCC-8739. AEDAPS containing PEMUs are hydrophilic and have increased nanoroughness of ~10nm. 'Super soaking' AEDAPS PEMUs incorporates more zwitterions into the PEMU and significantly maximizes the surface presentation of PAA-co-AEDAPS, which promotes early attachment of bacteria, but eventually, causes a gradual decrease in bacteria attachment with increasing incubation time. This investigation provides further insight into the possible application of PEMUs as bioselective thin film coatings, which may have potential for use in biomedical applications.
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Date Issued: 2015
Identifier: FSU_2015fall_Martinez_fsu_0071E_12894
Format: Thesis

Title: Cell and Disease Type Specific Fingerprints in the Mammalian Replication Program.
Creator: Ryba, Tyrone, Gilbert, David M., Bertram, Richard, Bass, Hank, Zhang, Jinfeng, Dennis, Jonathan, Department of Biological Science, Florida State University
Abstract/Description: The time at which DNA replicates during S-phase (replication timing; RT) is a precisely orchestrated, yet large-scale epigenetic property that offers an unparalleled window into the structure and regulation of the genome. While the timing program has been studied in many contexts and its significance is now well-established, the mechanisms that establish it have proven elusive, and recent studies have shed light on temporal and spatial aspects to replication control that were previously...
Show moreThe time at which DNA replicates during S-phase (replication timing; RT) is a precisely orchestrated, yet large-scale epigenetic property that offers an unparalleled window into the structure and regulation of the genome. While the timing program has been studied in many contexts and its significance is now well-established, the mechanisms that establish it have proven elusive, and recent studies have shed light on temporal and spatial aspects to replication control that were previously unanticipated. In this work, I and other members of the Gilbert laboratory worked to characterize the replication program in mammalian development, and described its structure, developmental regulation, and potential applications to medicine. In genome-wide studies of replication timing in mice, we found that replication timing profiles are both remarkably stable and cell type-specific, and are composed of coordinately regulated units (replication domains) that span one to several megabases. Changes in replication time typically occurred in 400-800kb units, and encompassed roughly 20% of the genome upon differentiation of embryonic stem cells (ESCs) to neural precursor cells (NPCs). These changes remarkably aligned domain timing values to genomic GC content and LINE-1 retrotransposon density. Consistent with previous results at individual loci, early replication was significantly (but not perfectly) associated with active transcription and active histone marks, and switches to later and earlier replication were accompanied by chromatin movement toward and away from the nuclear periphery respectively. Since H3K9 dimethylation was the only repressive histone mark with a moderate relationship to late replication, we next studied the regulation of the replication program with a cell line harboring an inducible conditional knockout of histone methyltransferase G9a. However, by comparison to the typical amount of timing differences between replicates (roughly 2-4% of the genome), we found no regions exhibiting unusually large timing changes upon G9a knockout in ESCs, or after differentiation of G9aCKO cells into NPCs. Nevertheless, many late-replicating H3K9me2-marked genes were transcriptionally upregulated, providing evidence that partially uncouples expression and histone mark changes from replication timing and nuclear location. To determine the extent of conservation between mouse and human replication program, we profiled several human ESC lines, differentiated NPCs, and lymphoblasts. Nearly all of the major properties from mouse were consistent in human cells, including domains sizes, timing changes in 400-800kb units, and relationships to activating histone marks and transcription. We also demonstrated that the replication program is well-conserved in regions syntenic between human and mouse. Importantly, hESCs aligned most closely not to mESCs, but to mouse EpiSCs, a more advanced population of cells derived from the epiblast and with comparatively limited plasticity. In studying the relation to histone marks we observed a peak of active marks 100kb within the border of most early replicating domains, but most remarkably (and unexpectedly), we found a correlation between replication timing profiles and Hi-C chromatin interactions stronger than any other genomic property, despite the Hi-C data deriving from an abstract computational model. As the robust cell type specificity of replication profiles suggested their potential for use in cell typing and studies of disease, I created (in collaboration with Jinfeng Zhang) a computational method to define "replication fingerprints"--collections of genomic regions with unique patterns of replication in defined collections of samples. Using these regions, 67/67 (34 mouse and 31 human) datasets could be correctly classified among 11 mouse and 9 human tissue types using a simple nearest-neighbor approach, and these results were confirmed through cross-validation and independent PCR assays. As a biological application, we created a fingerprint to isolate regions with common timing changes between pluripotent and committed cells, which revealed a conserved switch to later replication in the major histone H1 cluster that may help to explain the chromatin compaction previously observed during differentiation. To apply what we have learned about the replication program to the study of human disease, we collaborated with Drs. Bill Chang and Brian Druker to profile cell lines and pediatric patients with acute lymphoblastic leukemia (ALL). In contrast with normal B and T lymphocytes, leukemic samples displayed a high level of heterogeneity in replication profiles that offered intriguing potential for epigenetic fingerprints. Therefore, we applied the fingerprinting method to define regions with unique replication timing in high-risk patients and various genetic subtypes. To confirm the identity of leukemic samples and ability to detect known genetic lesions, we identified translocations and copy number variants in cell lines and patient samples known from CGH or karyotype information. These studies have opened paths to study less well-characterized subtypes of leukemia such as AML, which we plan to explore in future work.
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Date Issued: 2012
Identifier: FSU_migr_etd-5153
Format: Thesis

Title: Cell Competition in Metabolic Pathway Mosaics.
Creator: Acuna, Juan, Department of Biological Science
Abstract/Description: In Drosophila melanogaster, cells that are normally viable in homogeneous tissue can be eliminated when surrounded by cells with a different level of expression of a certain gene, such a phenomenon has been termed cell competition. A handful of genes have been shown to create cell competition, and based on the ability for these genes to affect the level of total protein production, it is now believed that cell competition is produced by a difference in protein production between cells. Here...
Show moreIn Drosophila melanogaster, cells that are normally viable in homogeneous tissue can be eliminated when surrounded by cells with a different level of expression of a certain gene, such a phenomenon has been termed cell competition. A handful of genes have been shown to create cell competition, and based on the ability for these genes to affect the level of total protein production, it is now believed that cell competition is produced by a difference in protein production between cells. Here we demonstrate that the knockdown of certain genes involved in glycolysis and oxidative phosphorylation can create a phenotype similar to cell competition, adding further support for the hypothesis that it is lower protein production that creates cell competition.
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Date Issued: 2014
Identifier: FSU_migr_uhm-0369
Format: Thesis

Title: Cell Signaling and the Regulation of Axis Formation, Cell Proliferation, and Differentiation in Drosophila Melanogaster.
Creator: Poulton, John, Deng, Wu-Min, Horabin, Jamila I., Epstein, Lloyd, III, Thomas Keller, Tang, Hengli, Department of Biological Science, Florida State University
Abstract/Description: The development of multicellular animals involves a diverse array of cellular processes, including cell differentiation, proliferation, and polarization. The control of these processes is largely governed by communication between different cells. This intercellular communication, known as cell signaling, is therefore a fundamental aspect of developmental and cellular biology. Despite a wealth of knowledge regarding the canonical cell signaling pathways, many questions remain regarding the...
Show moreThe development of multicellular animals involves a diverse array of cellular processes, including cell differentiation, proliferation, and polarization. The control of these processes is largely governed by communication between different cells. This intercellular communication, known as cell signaling, is therefore a fundamental aspect of developmental and cellular biology. Despite a wealth of knowledge regarding the canonical cell signaling pathways, many questions remain regarding the mechanistic nature of the communication taking place during specific developmental events, as well as questions regarding the control of activation of cell signaling. In this dissertation I will use the egg chamber of Drosophila melanogaster as a model system to investigate the genetics and cellular biology surrounding two important developmental events involving cell signaling. In the first part I describe a role for an adhesion molecule, Dystroglycan (DG), in the communication between two important cell types present in the egg chamber (the follicle cells and the oocyte). This communication is of great developmental significance because it creates the foundation for the polarization of the oocyte. The finding that DG is involved in this process suggests that changes in cell adhesion are important in the communication that establishes oocyte polarity. In the second part of the dissertation I identify a novel role for the gene, Belle (Bel), in controlling the activation of a key cell signaling pathway known as Notch. Notch activation in the follicle cells is essential for many aspects of egg chamber development. I also demonstrate that the regulation of Notch by Bel occurs through Bel's role in the microRNA pathway, possibly through regulation of levels of another protein, Delta. Together my research sheds new light on two key facets of egg chamber development that will potentially elucidate similar mechanisms present in other aspects of development in Drosophila, as well as other organisms.
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Date Issued: 2009
Identifier: FSU_migr_etd-0459
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: Characterization of Dopamine and Kainate Receptors in Olfactory Bulb Neurons and Their Efffects on Glutamatergic Transmission.
Creator: Davila, Gabriel Nestor, Trombley, Paul, Ouimet, Charles, Freeman, Marc, Gaffney, Betty, Meredith, Michael, Department of Biological Science, Florida State University
Abstract/Description: The main olfactory bulb (OB) receives odorant information from the nasal epithelium, interprets much of that information, and transmits the results to higher cortical regions. The predominant excitatory neurotransmitter in the OB and throughout the brain is glutamate. Modulators of glutamatergic activity influence synaptic transmission of intrabulbar circuits profoundly; therefore, the effects of neuromodulators must be thoroughly characterized in order to understand fully how OB circuits...
Show moreThe main olfactory bulb (OB) receives odorant information from the nasal epithelium, interprets much of that information, and transmits the results to higher cortical regions. The predominant excitatory neurotransmitter in the OB and throughout the brain is glutamate. Modulators of glutamatergic activity influence synaptic transmission of intrabulbar circuits profoundly; therefore, the effects of neuromodulators must be thoroughly characterized in order to understand fully how OB circuits function. Investigations performed here address the capacity of dopamine receptor (DAR) and kainate receptor (KAR) activation to modulate glutamate transmission from principal cells to interneurons in OB primary cultures. Initially, I obtained immunocytochemical evidence for DARs expressed in principal cells. Subsequent electrophysiological analyses revealed that the D2-like receptor subtype (D2Rs) attenuated both spontaneous and evoked glutamatergic transmission. Information gleaned from studies of input resistances and calcium currents allowed me to determine that the site of modulation is located on the presynaptic cell. My research into KARs demonstrated the existence of functional KARs in OB neurons and began to elucidate their physiological roles in OB neurotransmission. First, I gathered immunocytochemical evidence to visualize KARs expressed both at and near synapses. In situ hybridization (ISH) was employed to map which OB neurons express mRNA for each KAR subunit. Expression levels for each subunit were quantified in parallel studies using the reverse transcriptase-polymerase chain reaction (RT-PCR). Electrophysiological approaches were used to determine whether or not KARs participate in synaptic transmission between OB neurons in primary cultures. I provide evidence for KAR-mediated modulation of both spontaneous and evoked glutamatergic transmission between OB neurons. Taken together, this work supports the notion that synaptic transmission of OB neurons can be modulated by either metabotropic or ionotropic ligand-gated ion channels. In addition, this is the first thorough characterization of KAR expression and physiology in OB neurons.
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Date Issued: 2003
Identifier: FSU_migr_etd-0817
Format: Thesis

Title: Characterization of Germ-Line Endopolyploid Chromatin Dispersal in Drosophila Oogenesis.
Creator: Klusza, Stephen, Deng, Wu-Min, Sang, Qiang-Xing (Amy), Bass, Hank W., Keller, Laura R., Horabin, Jamila, Department of Biological Science, Florida State University
Abstract/Description: Drosophila melanogasterfemale fruit flies possess a pair of ovaries in which many egg chambers are produced, each containing an oocyte that houses the haploid female gamete. In the process of oogenesis, the multi-cellular egg chamber is composed of somatic follicle cells which encapsulate the germ-cells (1 oocyte and 15 nurse cells); the germ-line nurse cells produce an enormous amount of RNAs and proteins that are needed for growth and morphological changes of the egg chamber to aid in the...
Show moreDrosophila melanogasterfemale fruit flies possess a pair of ovaries in which many egg chambers are produced, each containing an oocyte that houses the haploid female gamete. In the process of oogenesis, the multi-cellular egg chamber is composed of somatic follicle cells which encapsulate the germ-cells (1 oocyte and 15 nurse cells); the germ-line nurse cells produce an enormous amount of RNAs and proteins that are needed for growth and morphological changes of the egg chamber to aid in the development of the oocyte in preparation for fertilization and embryogenesis. Intriguingly, nurse-cell (NC) nuclei undergo the endocycle in which DNA is re-replicated in the absence of mitosis, creating visible chromatin structures. During stages 4-6 of oogenesis, the NC nuclei undergo a dramatic change in morphology from a visible polytenic state to a diffuse state via a transient condensation phase. Mutations in many genes involved in various transcriptional, splicing, and translational processes routinely retain nurse-cell chromatin dispersal (NCCD) failure phenotypes, in which NC chromatin never disperse in the later stages of oogenesis. However, the significance of NCCD remains elusive in terms of its effect on essential processes like ribosomal synthesis of proteins or oocyte polarization. In order to investigate in greater detail the conditions required for NCCD, I first identify the novel genepolythrough a traditional FLP-FRT mosaic clone screen and characterize its requirement in the germ-line for NCCD and oocyte polarization. Later on, I also demonstrate that that NCCD itself does not affect oocyte polarity, through studies of anovarian tumormutation haploinsufficient for NCCD. However, performing a genetic modifier screen with theovarian tumormutation yields many loci that affect the morphology of NC chromatin, suggesting that NCCD is sensitive to genetic background. In the third part, I characterize mutations inpeanuts, a spliceosomal protein isolated from the screen, and its role in mediating spliceosomal function and NCCD, thus validating the screen as a tool for further identification of genes that affect chromatin dynamics.
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Date Issued: 2011
Identifier: FSU_migr_etd-5746
Format: Thesis

Title: Characterization of HDAC4's Role in Brain.
Creator: Darcy, Michael, Ouimet, Charles C., Kelley, Colleen, Kabbaj, Mohamed, Bolaños, Carlos, Keller, Laura, Department of Biological Science, Florida State University
Abstract/Description: Epigenetic regulation of gene expression involves a steady-state balance of acetylation carried about by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HATs act as transcriptional co-activators and HDACs interact with large multi-protein complexes to promote transcriptional repression. HDACs have only recently been characterized in mammalian cells, and most work has focused on the function of HDACs in vitro using biochemical analysis, inhibitors, and cultured cell types....
Show moreEpigenetic regulation of gene expression involves a steady-state balance of acetylation carried about by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HATs act as transcriptional co-activators and HDACs interact with large multi-protein complexes to promote transcriptional repression. HDACs have only recently been characterized in mammalian cells, and most work has focused on the function of HDACs in vitro using biochemical analysis, inhibitors, and cultured cell types. HDAC4, a class II HDAC, displays the ability to shuttle between the cytoplasm and the nucleus where it can regulate transcriptional programs. HDAC4 plays a key role in calcium-dependent transcriptional regulation of many non-neuronal cell processes including cardiac hypertrophy and bone formation. HDAC4 mRNA is also highly expressed in brain; however protein expression and its underlying biological role in brain is still unclear. HDAC4 localization in cultured neurons is dependent on neural activity and calcium-dependent signaling pathways. Mechanisms governing long-term changes in synaptic plasticity and learning and memory take place on dendritic spines, a site affected by many cognitive disorders. Dendritic spines act to compartmentalize calcium signaling and second messenger cascades leading to activation of enzymes and proteins associated with transcriptional regulation. Inhibition of HDACs has become a prevalent tool in exploring the role of HDACs in brain and has proven useful in many models of psychiatric and neurodegenerative disorders with more recent implication in the recovery or enhancement of synaptic plasticity and learning and memory. HDAC inhibition, however, is non-specific, and the localization of specific HDACs in brain and their role in these neuronal functions needs to be addressed. The similarity between HDAC4 regulation in non-neuronal cells and the processes initiated within a dendritic spine led to the hypothesis that HDAC4 may be present at the dendritic spine, where it can relay alterations of synaptic activity to the nucleus in order to regulate transcriptional programs affecting synaptic plasticity or other cell function. For this dissertation, I report findings which establish the regional and novel subcellular localization pattern of HDAC4 expression in brain, identify a mechanism specific to synaptic activity at the dendritic spine which results in HDAC4 trafficking, and attempt to establish a direct interaction of HDAC4 to a key member of the scaffolding network within a dendritic spine. In additional studies, I report the effects of HDAC inhibition on learning and memory and lesion size using a model of traumatic brain injury (TBI) as well as the effects of amyloid plaque level on the localization pattern of HDAC4 in the hippocampus. These studies failed to illicit a significant change in the conditions tested and are not discussed in the main text, however, useful information regarding the role of HDAC inhibition and HDAC4 was obtained. In brief, I report the localization of HDAC4 across brain regions germane to many pathological conditions such as Huntington's, Parkinson's, and Alzheimer's disease. HDAC4 was found to be present in dendritic spines, enriched at the level of the post-synaptic density (PSD), and partially colocalized with post-synaptic density protein 95 (PSD-95), a key scaffolding protein for the formation and maintenance of dendritic spines. Furthermore, using hippocampal slice cultures to more closely represent in vivo synaptic connections, exogenous overexpression of HDAC4 localized to the cytoplasm and in dendritic spines. Dendritic spines, synaptic activity, and the ability to form memories are tightly regulated through the activation of N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Blockade of both NMDA and AMPA receptors together was necessary to induce the nuclear localization of HDAC4 in these cultures, a shift that was reversed upon removal of the antagonists or reduced by HDAC inhibition. Finally, HDAC4 was expressed along with PSD-95 in vitro as well as extracted from hippocampal tissue to explore whether HDAC4 was a direct member of the PSD-95 scaffolding network in vivo. HDAC4 failed to show a complex with PSD-95, however, indirect interactions may still exist which anchor HDAC4 to the PSD. Together, these results suggest HDAC4 can act as a synaptic monitor, translocating to the nucleus during synaptic blockade where it can alter transcriptional programs and gene expression. Isolating the biological role for individual HDAC isoforms remains a critical step in understanding the mechanisms behind therapeutic candidates such as HDAC inhibitors, which have been used clinically in non-neuronal disruption of cancerous cells, and show much promise in the alleviation of many symptoms resulting from various psychiatric and neurodegenerative disorders.
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Date Issued: 2010
Identifier: FSU_migr_etd-0848
Format: Thesis

Title: Characterization of Hepatitis C Virus Subgenomic Replicon Resistance to Cyclosporine in Vitro.
Creator: Robida, John, Tang, Hengli, Keller, Thomas C. S., Zhu, Fanxiu, Department of Biological Science, Florida State University
Abstract/Description: The current treatment for hepatitis C virus (HCV) consists of a combination therapy of alpha interferon (IFN-alpha) and ribivirin (RBV). Due to IFN resistance and side effects, new classes of drugs are needed to combat HCV infection. Cyclosporine A (CsA), an immunosuppressive and anti-inflammatory drug, has been shown to suppress HCV via a mechanism independent of the IFN pathway. In order to study the mechanism of CsA action on HCV, CsA resistant strains of HCV subgenomic replicon were...
Show moreThe current treatment for hepatitis C virus (HCV) consists of a combination therapy of alpha interferon (IFN-alpha) and ribivirin (RBV). Due to IFN resistance and side effects, new classes of drugs are needed to combat HCV infection. Cyclosporine A (CsA), an immunosuppressive and anti-inflammatory drug, has been shown to suppress HCV via a mechanism independent of the IFN pathway. In order to study the mechanism of CsA action on HCV, CsA resistant strains of HCV subgenomic replicon were selected and characterized. Here we report that different levels of resistance can be seen in different replicons and that different sets of mutations are associated with the different levels of resistance. Several different single cell clones with varying levels of CsA resistance contained mutations in the nonstructural protein 5B (NS5B), the HCV-encoded polymerase. When engineered into wildtype replicon these mutations were sufficient to confer a certain degree of resistance, but not to the original levels of selected replicons. Furthermore, these mutations, both individually and in groups, were able to rescue the lethal phenotype of a point mutation in NS5B (P540A) that has been previously implicated in the blockade of cyclophilins binding. These results demonstrate that CsA exerts selective pressure on the HCV genome despite being known to act on a cellular protein and identify a major target of CsA-mediated inhibition of HCV replication.
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Date Issued: 2009
Identifier: FSU_migr_etd-1814
Format: Thesis

Title: Characterization of Human MacroH2A Through Gene Targeting.
Creator: Moseley, Shawn C., Chadwick, Brian, Dennis, Jonathan, McGinnis, Karen, Department of Biological Science, Florida State University
Abstract/Description: Macro histone H2A (macroH2A) is a variant of core histone H2A that differs primarily by an extensive carboxy-terminal tail of unknown function that makes up two-thirds of the protein's mass. The histone variant is distributed throughout the nucleus, but in female mammalian cells, it has been found to be associated with the inactive X chromosome (Xi) in a local accumulation referred to as a macrochromatin body. The association of macroH2A with facultative heterochromatin of the Xi is...
Show moreMacro histone H2A (macroH2A) is a variant of core histone H2A that differs primarily by an extensive carboxy-terminal tail of unknown function that makes up two-thirds of the protein's mass. The histone variant is distributed throughout the nucleus, but in female mammalian cells, it has been found to be associated with the inactive X chromosome (Xi) in a local accumulation referred to as a macrochromatin body. The association of macroH2A with facultative heterochromatin of the Xi is suggestive of a role for the variant in gene silencing. MacroH2A1 was the first form of macroH2A discovered and is encoded by the H2AFY gene. Two splice isoforms exist due to two alternate versions of exon-6, giving rise to macroH2A1.1 and macroH2A1.2. A second form of macroH2A encoded by H2AFY2 gene, known as macroH2A2, shares 80% amino acid identity with macroH2A1 and also accumulates at Xi, suggesting the possibility of functional redundancy between the two proteins. In order to further investigate macroH2A, we have generated knockouts of macroH2A1 and targeted a single allele of macroH2A2 in a human female telomerase immortalized retinal pigment epithelial cell line (RPE1). Targeted clones were generated by exchanging exon-2 of one or both alleles with a promoter-trap construct containing a promoterless neomycin selection cassette flanked by arms of homology designed to the intronic sequences immediately adjacent to exon-2. The selection cassette contains a splice-acceptor, internal ribosome entry site and polyadenylation signal, which when exchanged with exon-2 results in the inclusion of the neomycin coding sequence in the resulting truncated messenger RNA and its subsequent translation, allowing for correct targeting to be selected for through neomycin resistance. To enhance targeting, Zinc Finger Nucleases (ZFNs) were engineered to create a double strand break at or close to exon-2. In order to target the genes, cells were co-nucleofected with the targeting construct and ZFNs before seeding cells in media containing neomycin. Single cell clones were screened for correct targeting and loss of macroH2A assessed by Western blotting. In order to explore the impact of macroH2A1 loss, RNA was extracted from a macroH2A1 knockout as well as parental RPE1 and changes to the transcriptome assessed by massively paralleled sequencing of complementary DNA (RNAseq). Genes that showed a significant change in expression between the wildtype and knockout cells were selected for further study. Quantitative Chromatin Immunoprecipitation (qChIP) was performed on an affected gene to evaluate any local chromatin changes due to macroH2A1 loss. Additionally, to examine the possibility that macroH2A1 splice isoforms fulfill different roles, full-length MYC-tagged expression constructs for macroH2A1.1 and macroH2A1.2 were reintroduced into cells and rescue of wild-type expression assessed for genes that displayed altered expression in response to macroH2A1 loss.
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Date Issued: 2014
Identifier: FSU_migr_etd-8853
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: Characterization of the Cytosolic Proteins Involved in the Amoeboid Motility of Ascaris Sperm.
Creator: Buttery, Shawnna Marie, Roberts, Thomas M., Cross, Timothy A., Keller, Thomas C. S., Hurt, Myra M., Moerland, Timothy S., Department of Biological Science, Florida State University
Abstract/Description: The amoeboid sperm of Ascaris crawl through a cycle of protrusion, adhesion, and retraction, similar to that seen in conventional actin-based cells. However, instead of actin, these cells power their movement through modulation of the major sperm protein (MSP) cytoskeleton. MSP forms dense filament meshworks that pack the sperm lamellipod. Protrusion is associated with the assembly of MSP filaments at the leading edge of the lamellipod, and retraction is connected with the disassembly of the...
Show moreThe amoeboid sperm of Ascaris crawl through a cycle of protrusion, adhesion, and retraction, similar to that seen in conventional actin-based cells. However, instead of actin, these cells power their movement through modulation of the major sperm protein (MSP) cytoskeleton. MSP forms dense filament meshworks that pack the sperm lamellipod. Protrusion is associated with the assembly of MSP filaments at the leading edge of the lamellipod, and retraction is connected with the disassembly of the MSP network at the base of the lamellipod. The motility of Ascaris sperm can be reconstituted in cell-free extracts. In vitro, plasma membrane vesicles are pushed forward by the elongation of fibers constructed from a columnar meshwork of MSP filaments. This in vitro motility requires components from both the cytosol and the vesicle. LeClaire et al. (2003) recently identified the 48 kDa membrane protein required to orchestrate MSP cytoskeletal assembly at the leading edge of the lamellipod. In this study, I describe the first cytosolic proteins that are components of the MSP locomotory machinery. I fractionated cytosol with a range of biochemical techniques and reconstituted fiber assembly with a limited subset of cytosolic components. Thus, this fraction contains all the cytosolic accessory proteins required to build fibers. Several of the components in this active fraction were used to generate antibodies, which labeled the cytoskeleton in Ascaris sperm and in fibers grown in vitro and thus, identified six proteins, p43, p42, p40, p38, p34, and p16, as part of the MSP cytoskeleton. Sequence analysis showed that each protein is novel to nematode sperm and has a homolog of unknown function in C. elegans that exhibits sperm-enriched expression (Reinke et al., 2000; Hill et al., 2001). The predicted protein sequence of p43 is based on a tandem array of repeats with serine phosphorylation sites. P38, p40, and p42 appear to be a family of closely related polypeptides. The sequences of p38 and p40 contain a domain duplication as well as proline-rich repeats. P34 shows homology to serine/threonine kinases. The p16 triplet is a family of closely related polypeptides. Two of the proteins had reciprocal effects on fiber growth: p38 increased fiber growth rate and p16 decreased fiber growth rate. The effects of both p38 and p16 were concentration-dependent and antagonistic. Since the rate-enhancement by p38 was not potentiated by MSP, another cytosolic protein is involved in up-regulation of the rate of MSP elongation. Additionally, p16 altered the number of filaments polymerized at the vesicle surface and thus may regulate MSP nucleation. Immunoprecipitations and affinity chromatography established that these two proteins bind to MSP under conditions that promote in vitro assembly. Based on these data, I present a revised model for the mechanism of membrane-associated MSP polymerization, which proposes that p38 influences the rate of MSP elongation by recruiting MSP to the vesicle surface and that p16 incorporates into MSP filaments and blocks further assembly by acting as a capping protein. My results imply that although the specific components differ, actin-based and MSP-based systems both rely on the interplay of positive and negative regulators of cytoskeletal assembly to maintain the pace of locomotion.
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Date Issued: 2003
Identifier: FSU_migr_etd-2403
Format: Thesis

Title: Characterization of the Interaction Between Titn Kinase Domain and Enigma/Pdlim7.
Creator: Fazel, Arif, Keller, Thomas C. S., Roberts, Thomas M., Deng, Wu Min, Department of Biological Science, Florida State University
Abstract/Description: Titin is a very large protein that contributes to sarcomere structure and mechanosensing in striated muscle. Our lab discovered isoforms of titin in nonmuscle cells (Eilersten and Keller, 1992). Nonmuscle cell titin (c-titin) contains an alpha-actinin binding Z-repeat region, a distinctly PEVK region, a myosin filament-binding region, and the kinase domain also present in striated muscle titin isoforms. In striated muscle, the titin kinase domain (TKD) functions as a mechanosensor that...
Show moreTitin is a very large protein that contributes to sarcomere structure and mechanosensing in striated muscle. Our lab discovered isoforms of titin in nonmuscle cells (Eilersten and Keller, 1992). Nonmuscle cell titin (c-titin) contains an alpha-actinin binding Z-repeat region, a distinctly PEVK region, a myosin filament-binding region, and the kinase domain also present in striated muscle titin isoforms. In striated muscle, the titin kinase domain (TKD) functions as a mechanosensor that signals changes in gene expression through interaction with nbr1 and p62. A previous yeast two hybrid (Y2H) screen to identify proteins that interact with the TKD in nonmuscle cells revealed an interaction with the ubiquitously expressed scaffold protein Enigma/PDLIM7. Enigma/PDLIM7 consists of an N-terminal PDZ domain that binds to β-tropomyosin on actin filaments, a Mid piece, and C-terminal region containing three LIM domains. The work described here further characterizes the interaction between the TKD and Enigma/PDLIM7. Y2H analysis with cloned TKD and Enigma/PDLIM7 fragments demonstrated that a region of the Enigma/PDLIM7 Mid piece and LIM1 and LIM3 domains interact with TKD. In vitro pull-down assays with bacterially expressed protein confirmed the interaction between TKD and Enigma LIM3. Immunolocalization of the TKD and Enigma/PDLIM7 in cultured human mesenchymal stem cells containing robust stress fibers revealed that both TKD and Enigma localized along stress fibers where they could interact, but there was little direct overlap in the cells under the standard culture conditions tested. These results support the possibility that Enigma/PDLIM7 functions as a scaffold to localize the TKD near actin filaments in the cytoskeleton of nonmuscle cells.
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Date Issued: 2011
Identifier: FSU_migr_etd-4487
Format: Thesis

Title: Characterization of the Lung Seven Transmembrane (LUSTR) Protein Family.
Creator: Middlebrooks, Jennifer Ivey, Keller, Laura R., Levenson, Cathy, Keller, Tom, McGinnis, Karen, Deng, Wumin, Department of Biological Science, Florida State University
Abstract/Description: Cilia are recognized as an important sensory structure for the cell. However, much about the signaling involved in ciliary outgrowth remains a mystery. Chlamydomonas, a green algae, is a model for ciliary and flagellar studies owing to its pair of anterior flagella and haploid genome. In a previous study of gene expression during flagellar outgrowth in Chlamydomonas after environmental shock, the gene encoding a predicted seven transmembrane protein (Cr7TM) was differentially expressed....
Show moreCilia are recognized as an important sensory structure for the cell. However, much about the signaling involved in ciliary outgrowth remains a mystery. Chlamydomonas, a green algae, is a model for ciliary and flagellar studies owing to its pair of anterior flagella and haploid genome. In a previous study of gene expression during flagellar outgrowth in Chlamydomonas after environmental shock, the gene encoding a predicted seven transmembrane protein (Cr7TM) was differentially expressed. Subsequent experiments described here have attempted to elucidate the function of Cr7TM in the cell and specifically in the flagella. Bioinformatic search techniques and predictions of the evolutionary history of Cr7TM and its homologues imply that this LUSTR protein family is unique in both its primary structure and mechanisms for protein interaction. It is also highly conserved and likely involved in a critical cellular function. LUSTR proteins are ubiquitously expressed across metazoan organisms and tissue types. Also, Cr7TM is differentially expressed during outgrowth of cilia/flagella but not during oxidative stress, indicating Cr7TM's involvement in flagellar outgrowth rather than the stress response. Fluorescence microscopy indicates that Cr7TM co-localizes with acetylated á-tubulin in the flagella. Creation of an inducible knockdown mutant line of Cr7TM allowed for comparison of the phenotypes between induced and uninduced RNAi knockdown mutant lines. The Cr7TM knockdown line demonstrates a diminished cell size along with a rapid increase in the density of cell cultures as compared to the control, indicating a possible cellular role for Cr7TM in cell cycling or mitotic regulation. Additionally, imaging of human embryonic kidney cells shows colocalization of LUSTR proteins near the highly conserved centriole and basal body organelles. When taken together the high degree of sequence conservation, knockdown phenotype and cellular localization of LUSTR proteins provide evidence for LUSTR involvement in the regulation of cell cycle.
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Date Issued: 2012
Identifier: FSU_migr_etd-5800
Format: Thesis

Title: Characterization of the Role of Eco1 in Chromosome Segregation During Meiosis in Budding Yeast.
Creator: Reynolds, Torrie, Department of Biological Science
Abstract/Description: To ensure accurate chromosome segregation, sister chromatid cohesion must be properly established at S-phase when DNA is replicated. The protein Eco1/Ctf7 is responsible for establishing this cohesion, and is the focus of this Thesis work. The conserved protein complex cohesin acts as the "molecular glue" that mediates this sister chromatid cohesion. Cohesin is loaded onto the chromosomes before DNA replication, and when the acetyltransferase Eco1 acetylates cohesin at S-phase, the sister...
Show moreTo ensure accurate chromosome segregation, sister chromatid cohesion must be properly established at S-phase when DNA is replicated. The protein Eco1/Ctf7 is responsible for establishing this cohesion, and is the focus of this Thesis work. The conserved protein complex cohesin acts as the "molecular glue" that mediates this sister chromatid cohesion. Cohesin is loaded onto the chromosomes before DNA replication, and when the acetyltransferase Eco1 acetylates cohesin at S-phase, the sister chromatids become entrapped in the cohesin ring. This Thesis aimed to elucidate the roles of Eco1 in cohesin-mediated sister chromatid cohesion, specifically during meiosis. A novel eco1 meiosis-specific mutant was constructed in the budding yeast Saccharomyces cerevisiae. Fluorescence microscopy techniques were used to assay sister chromatid cohesion, nuclear division, and chromosome structure in cells depleted of Eco1 in meiosis. This work shows that meiotic Eco1 and its establishment of sister chromatid cohesion regulates recombination and chromosome segregation during meiosis.
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Date Issued: 2012
Identifier: FSU_migr_uhm-0137
Format: Thesis

Title: The Characterization of the Roles of Ipl1 and Cdc5 in Spindle Pole Body Duplication in Yeast Meiosis.
Creator: Shirk, Katelan, Yu, Hong-Guo, Wang, Yanchang, Chase, P. Bryant, Megraw, Timothy, Department of Biological Science, Florida State University
Abstract/Description: The centrosome is the microtubule organizing center (MTOC) in higher eukaryotes. During meiosis, proper duplication and separation of the centrosomes are necessary for accurate chromosome segregation and leads to the production of gametes containing half of the parental genome. During meiotic interphase I, centrosomes are duplicated when chromosomes replicate. A pair of centrosomes establish a bipolar microtubule spindle that facilitates the segregation of homologs during meiosis I....
Show moreThe centrosome is the microtubule organizing center (MTOC) in higher eukaryotes. During meiosis, proper duplication and separation of the centrosomes are necessary for accurate chromosome segregation and leads to the production of gametes containing half of the parental genome. During meiotic interphase I, centrosomes are duplicated when chromosomes replicate. A pair of centrosomes establish a bipolar microtubule spindle that facilitates the segregation of homologs during meiosis I. Centrosomes duplicate once more at interphase II, when DNA duplication is absent, and form two independent spindles for sister chromatid separation during meiosis II. The centrosome in yeast is called the spindle pole body (SPB). Here we show that the Aurora Kinase Ipl1, which protects sister chromatid cohesion, is also required for the maintenance of a tight association between duplicated sister SPBs, referred to here as SPB cohesion. Premature loss of cohesion leads to SPB over-duplication and the formation of multipolar spindles during meiosis II. The Polo-like kinase Cdc5 is a licensing factor for SPB duplication at interphase II and promotes SPB separation during meiosis II. Our data suggests Ipl1 and Cdc5 interact antagonistically at the SPB to maintain proper duplication and separation of SPBs during meiosis.
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Date Issued: 2011
Identifier: FSU_migr_etd-1746
Format: Thesis

Title: Characterizing Gene Networks and RNA-Mediated Gene Regulation in Maize.
Creator: Huang, Ji, McGinnis, Karen M., Lemmon, Alan R, Jones, Kathryn M., Chadwick, Brian P., Dennis, Jonathan Hancock, Florida State University, College of Arts and Sciences,...
Show moreHuang, Ji, McGinnis, Karen M., Lemmon, Alan R, Jones, Kathryn M., Chadwick, Brian P., Dennis, Jonathan Hancock, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Controlling spatial-temporal gene expression patterns is a fundamental task for maize growth and development. With the emergence of massively parallel sequencing, genome-wide expression data production has reached an unprecedented level. This abundance of data has greatly facilitated maize research, but may not be amenable to traditional analysis techniques that were optimized for other data types. In one project, using publicly available data, a Gene Co-expression Network (GCN) was...
Show moreControlling spatial-temporal gene expression patterns is a fundamental task for maize growth and development. With the emergence of massively parallel sequencing, genome-wide expression data production has reached an unprecedented level. This abundance of data has greatly facilitated maize research, but may not be amenable to traditional analysis techniques that were optimized for other data types. In one project, using publicly available data, a Gene Co-expression Network (GCN) was constructed and used for gene function prediction, candidate gene selection and improving understanding of regulatory pathways. To build an optimal GCN from plant materials RNA-Seq data, parameters for expression data normalization and network inference were evaluated. A comprehensive evaluation of these two parameters and ranked aggregation strategy on network performance using libraries from 1266 maize samples was conducted. Three normalization methods (VST, CPM, RPKM) and ten inference methods, including six correlation and four mutual information (MI) methods, were tested. The three normalization methods had very similar performance. For network inference, correlation methods performed better than MI methods at some genes. Increasing sample size also had a positive effect on GCN. Aggregating single networks together resulted in improved performance compared to single networks. In another project, a maize mutant, transgene reactivated 9-1 (tgr9-1) in the transcriptional gene silencing (TGS) pathway, was cloned. The B-A translocation lines were used to map tgr9-1 on chromosome 3 and this result was consistent with molecular markers. To further locate tgr9-1, next-generation sequencing (NGS) combined with bulk segregant analysis was applied to the tgr9-1 mapping population. Using coexpression analysis, our result indicates a maize dicer-like3a (Zmdcl3a) gene is a high-confidence candidate gene for tgr9. Zmdcl3a is involved in the RNA-directed DNA methylation (RdDM) pathway. This pathway is driven by two plant-specific DNA-dependent RNA polymerases, Polymerase IV (Pol IV) and Polymerase V (Pol V). Several kinds of non-coding RNAs are involved, including long single-stranded RNAs, double-stranded RNAs, and small interfering RNAs. The identification of tgr9-1 uncovered the role of non-coding RNAs in TGS and revealed the diversity of TGS pathways in maize. One primary focus of gene regulation study is by studying transcription factors (TFs). Transcription factors (TFs) are proteins that can bind to DNA sequences and regulate gene expression. Many TFs are master regulators in cells that contribute to tissue-specific and cell-type-specific gene expression patterns in eukaryotes. Little is known about tissue-specific gene regulation through TFs in maize. In this project, a network approach was applied to elucidate gene regulatory networks (GRNs) in four tissues (leaf, root, shoot apical meristem and seed) in maize. We used GENIE3 machine-learning algorithm combined with the large quantity of RNA-Seq expression data to construct four tissue-specific GRNs. Although many TFs were expressed across multiple tissues, a multi-tiered analysis predicted tissue-specific regulatory functions for many transcription factors. Some well-studied TFs emerged within the four tissue-specific GRNs, and the GRN predictions matched expectations based upon published results for many of these examples. The GRNs were also validated by ChIP-Seq datasets (KN1, FEA4, and O2). Key TFs were identified for each tissue and matched expectations for key regulators in each tissue, including GO enrichment and identity with known regulatory factors for that tissue.
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Date Issued: 2018
Identifier: 2018_Sp_Huang_fsu_0071E_14421_comp
Format: Set of related objects

Title: Characterizing the Epigenetic Regulation of ABA-Induced Transcriptional Responses in Zea Mays.
Creator: Vendramin Alegre, Stefania, McGinnis, Karen M., Li, Hong, Bass, Hank W., Chadwick, Brian P., Dennis, Jonathan Hancock, Florida State University, College of Arts and Sciences,...
Show moreVendramin Alegre, Stefania, McGinnis, Karen M., Li, Hong, Bass, Hank W., Chadwick, Brian P., Dennis, Jonathan Hancock, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Plants are often subjected to extreme environmental conditions and must adapt rapidly. The phytohormone abscisic acid (ABA) accumulates under abiotic stress conditions, signaling transcriptional changes that trigger physiological responses. Epigenetic modifications are also required to facilitate transcription, particularly at genes exhibiting temporal, tissue-specific and environmentally induced expression. In maize (Zea mays), MEDIATOR OF PARAMUTATION 1 (MOP1) is required for progression of...
Show morePlants are often subjected to extreme environmental conditions and must adapt rapidly. The phytohormone abscisic acid (ABA) accumulates under abiotic stress conditions, signaling transcriptional changes that trigger physiological responses. Epigenetic modifications are also required to facilitate transcription, particularly at genes exhibiting temporal, tissue-specific and environmentally induced expression. In maize (Zea mays), MEDIATOR OF PARAMUTATION 1 (MOP1) is required for progression of an RNA-dependent epigenetic pathway that regulates transcriptional silencing of loci across the genome. As critical regulators of gene expression, MOP1 and ABA pathways predictably regulate specific genes in a coordinated manner. In one project, the amino acid sequence of DNG103 and the gene promoter region were analyzed for conserved domains and cis-responsive elements, respectively. DNG103 is similar to the Arabidopsis ROS1, contains the conserved domains of a DNA glycosylase with DNA demethylase activity, and contains ABA-responsive elements (ABREs) in its promoter region. Transcript levels of Dng103, and the ABA-responsive gene Viviparous 1 (Vp1), were monitored in maturing embryos from two genotypes placed in culture under different conditions. Expression of both genes decreased after culture in hormone-free medium and was induced by ABA in Mop1 wildtype. Dng103 and Mop1 showed decreased expression in mop1-1 and dng103 mutants, respectively. Dng103 is not responsive to ABA in the mop1-1 mutant and Vp1 has reduced sensitivity. Therefore, DNG103, MOP1 and ABA might have common regulatory targets. Protoplast isolation and transfection protocols were standardized alongside the production of reporter constructs containing the Dng103 and Vp1 promoters. This technology will allow for the promoter characterization of genes of interest through quantifiable luciferase expression, using different conditions and genotypes. To identify genome-wide ABA-induced, MOP1-dependent and independent transcriptional responses, mop1-1 and Mop1 homozygous seedlings were subjected to exogenous ABA and RNA-sequencing. A total of 3,242 differentially expressed genes (DEGs) were identified in four pairwise comparisons. Overall, the loss of MOP1 exaggerated some ABA-induced changes in gene expression. The highest number of DEGs were identified in ABA-induced mop1-1 mutants, including many transcription factors. A gene regulatory network was used to predict relationships between DEGs; suggesting multifaceted regulatory scenarios including direct and indirect transcriptional responses to genetic disruption (mop1-1) and/or stimulus-induction of a hierarchical, cascading network of responsive genes. Additionally, a modest increase in CHH methylation at putative MOP1-RdDM loci in response to ABA was observed in some genotypes, suggesting that MOP1 might be necessary to achieve environmentally induced transcriptional responses in maize. To understand the multistep ABA response of identified ABA-induced genes, an ABA-time course was carried out in another project using four different time points. The previously predicted ABA-responsive transcription factors Hb41 and Bzip4 showed an early induction to ABA and the late embryogenesis abundant Rab17 gene was induced during the transition from early to late response. Together, these results indicate that MOP1 and ABA act at multiple levels within complex, connected transcriptional networks to mediate tissue-specific growth and responses to some abiotic stresses. LIST OF SUPPLEMENTARY FILES 1. Significant differentially expressed genes in analysis groups I-VIII. 2. GO term enrichment in groups I, II, V and VI. 3. Arabidopsis and maize homologous transcription factors and target genes in ABA transcription factor hierarchical network and their corresponding gene expression. 4. Transcription factors and target genes in ABA transcription factor hierarchical network separated by transcriptional levels. 5. Group model parameters per gene. 6. Genome-wide siRNA changes in mop1-1 mutant. 7. TGS2 target genes. 8. Sequence Capture (SeqCap) DNA methylation ratios in all sequence contexts. 9. Promoter DNA methylation for Mop1 wildtype ABA-responsive DEGs. 10. MOP1-ABA targets with a loss of siRNA and DNA methylation at ABRE sites.
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Date Issued: 2019
Identifier: 2019_Fall_VendraminAlegre_fsu_0071E_15503_P
Format: Set of related objects

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

Title: Characterizing the Relationship Between Williams Syndrome Transcription Factor and Heterochromatin Maintenance Through the Targeted Disruption of the BAZ1B Gene.
Creator: Culver-Cochran, Ashley E., Chadwick, Brian P., Hurt, Myra M., Dennis, Jonathan H., McGinnis, Karen M., Tang, Hengli, Department of Biological Science, Florida State University
Abstract/Description: Maintaining genome integrity and epigenetic programming is essential to avoid disease and retain the identity and proper function of the multitude of specialized differentiated cell types in the body. Much progress has been made in the past 10 years toward identifying and understanding the range of proteins and complexes involved in these processes. Many of these are chromatin-remodeling complexes, including those that contain the Williams syndrome transcription factor (WSTF). This protein is...
Show moreMaintaining genome integrity and epigenetic programming is essential to avoid disease and retain the identity and proper function of the multitude of specialized differentiated cell types in the body. Much progress has been made in the past 10 years toward identifying and understanding the range of proteins and complexes involved in these processes. Many of these are chromatin-remodeling complexes, including those that contain the Williams syndrome transcription factor (WSTF). This protein is particularly interesting for two reasons. First, it functions in many central nuclear processes, such as DNA replication, transcription, and DNA repair. Second, WSTF is haploinsufficient in Williams-Beuren syndrome (WBS) patients. This is because the WSTF gene, BAZ1B, is deleted, along with approximately 27 other genes, from one copy of chromosome 7 in affected individuals. Prior research has implicated WSTF in contributing to several of the phenotypes exhibited in WBS, yet many aspects concerning the function of WSTF remain unclear. A more detailed understanding of WSTF function is necessary to appreciate how this versatile protein contributes to general nuclear processes, and how perturbation of these roles contribute to the symptoms displayed in WBS patients. This study examines the function of WSTF in several ways. First, this research identifies and characterizes the relationship between WSTF and heterochromatin, with a particular focus on facultative heterochromatin of the human inactive X chromosome (Xi). Next, it describes the generation of human cell lines that either lack or are happloinsufficient for WSTF through the generation of heterozygous and homozygous mutant BAZ1B alleles. Using these invaluable model cell lines, this research explores the impact of WSTF reduction or loss on several processes, including heterochromatin maintenance, the DNA damage response, and vitamin D induced gene expression. This work reveals that WSTF is necessary to maintain appropriate expression of a substantial number of genes, and describes a novel nuclear phenotype in BAZ1B knockout cells, characterized by the spontaneous formation and subsequent resolution of extensive regions of heterochromatin throughout the nucleus. This research contributes to and extends current understanding of WSTF function, and provides BAZ1B knockout cells to further investigate WSTF mechanism, as well as providing BAZ1B heterozygous knockouts that will serve as a model to examine how WSTF haploinsufficiency contribute to WBS in the absence of the effects of the other 27 genes that are typically deleted in the disorder. Analyses found in this dissertation link WSTF function to maintenance of chromatin and transcriptional states. Through the examination of BAZ1B knockout cells, this work also underscores that the current understanding of WSTF is not as clear as anticipated, given that processes expected to be disrupted in the absence of WSTF were unaffected. This dissertation concludes with a discussion of these findings as well as future implications.
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Date Issued: 2013
Identifier: FSU_migr_etd-7752
Format: Thesis

Title: Chemosensory Processing in the Amygdala.
Creator: Samuelsen, Chad L., Meredith, Michael, Kelley, Colleen, Hull, Elaine, Keller, Laura, Wang, Zuoxin, Department of Biological Science, Florida State University
Abstract/Description: Rodents depend on the main olfactory and accessory olfactory systems to detect and process chemical-communication signals important for complex reproductive and social behaviors. As the medial amygdala is the first site of convergence of these two pathways in the brain, examining its role in chemosensory processing is of primary importance. Previous experiments have shown that the hamster medial amygdala exhibits a categorical response to different types of chemical signals. The use of...
Show moreRodents depend on the main olfactory and accessory olfactory systems to detect and process chemical-communication signals important for complex reproductive and social behaviors. As the medial amygdala is the first site of convergence of these two pathways in the brain, examining its role in chemosensory processing is of primary importance. Previous experiments have shown that the hamster medial amygdala exhibits a categorical response to different types of chemical signals. The use of immediate early gene (IEG) expression to assess neural activity in different brain regions showed that chemical signals from the same species (conspecific) activated anterior and posterior medial amygdala, while chemical signals from other species (heterospecific) increased activity in only the anterior medial amygdala. The experiments discussed below aim to provide information about how the mouse medial amygdala responds to categories of chemical signals, how important the main and accessory olfactory systems are to the medial amygdala response and whether oxytocin (which has been shown to be important in a variety of social behaviors) is necessary for the medial amygdala categorization of chemical signals. Upon exposure of male mice to chemical-communication signals, I found that conspecific chemosignals (male, female mouse urine) increased immediate early gene-protein (IEG) expression in both anterior and posterior medial amygdala of male mice, whereas most heterospecific chemosignals (e.g.: hamster vaginal fluid, steer urine) increased expression only in anterior medial amygdala. This categorization of responses in medial amygdala conforms to the previously reported findings in male hamsters. The same characteristic pattern of IEG expression appeared in the medial amygdala of each species in response to conspecific stimuli for that species. These results suggested that the amygdala categorizes stimuli according to the biological relevance for the tested species. Thus, a heterospecific predator (cat collar) stimulus, which elicited behavioral avoidance in mice, increased IEG expression in mouse posterior medial amygdala (like conspecific stimuli). Further analysis suggests reproduction related and potentially threatening stimuli produce increased IEG expression in different sub-regions of posterior medial amygdala (dorsal and ventral, respectively). These patterns of IEG expression in medial amygdala may provide glimpses of a higher-level processing of chemosensory signals beyond the primary-level selectivity of chemosensory neurons, the secondary sorting in main and accessory olfactory bulbs and the tertiary sorting by the medial amygdala into "biologically relevant and non-relevant" categories. Both non-volatile and volatile chemical-communication signals may be detected by the vomeronasal organ, which sends projections to the accessory olfactory bulb and on to the medial amygdala. Results of the first experiment (above) argue that the mouse medial amygdala sorts complex chemosensory information categorically, according to its biological relevance (salience). In order to determine the role of AOS in categorization, male mice underwent vomeronasal removal surgery (VNX) or a sham-operation (SHAM) and then were exposed to conspecific (male and female mouse urine) or heterospecific (hamster vaginal fluid and worn cat collar) chemical stimuli. As with the mice in the above experiment, SHAM mice exhibited different IEG expression patterns in the medial amygdala dependent upon the biological relevance of the chemical stimuli. However, regardless of biological relevance, vomeronasal organ removal eliminated the different IEG response patterns in the medial amygdala to any of the chemical stimuli. Interestingly, VNX also disrupted the avoidance of (an unfamiliar) predator odor, worn cat collar. These experiments show that the medial amygdala response to these tested chemical signals is dependent upon an intact vomeronasal organ. Normal function of the neuropeptide oxytocin (OT) in the medial amygdala is necessary for social recognition. In the final set of experiments, male mice having undergone intracerebroventricular cannulation (i.c.v.) were injected with either PBS (control) or oxytocin antagonist (OTA) and exposed to conspecific (female mouse urine) and heterospecific (steer urine and worn cat collar) chemical stimuli. As in the above experiments, PBS-injected mice exhibited different IEG expression patterns in the medial amygdala dependent upon the biological relevance of the chemical stimuli. However, OTA injection eliminated the increase in IEG expression in the medial amygdala to all of the tested conspecific or heterospecific stimuli. Importantly, OTA injection disrupted defensive and non-defensive behaviors after exposure to the unfamiliar predator odor, worn cat collar. The disruption of the social/individual recognition behavior in male mice deficient in OT may be due to the inability of the medial amygdala to process all biologically relevant chemical-communication signals.
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Date Issued: 2009
Identifier: FSU_migr_etd-2098
Format: Thesis

Title: Chromatin Structural Changes in the Innate Immune Response.
Creator: Welch, Alexandra, Department of Biological Science
Abstract/Description: Eukaryotic nucleosomes have critical regulatory roles in mediating access to DNA. Differential regulation of nucleosome distribution is necessary for generating a rapid response to pathogens in the innate immune response and other stimuli. However, this genomic reorganization event is largely uncharacterized. Our lab has proposed that early, widespread, and transient reorganization of the genomic structure potentiates the appropriate responses of the cell to insult. We have identified...
Show moreEukaryotic nucleosomes have critical regulatory roles in mediating access to DNA. Differential regulation of nucleosome distribution is necessary for generating a rapid response to pathogens in the innate immune response and other stimuli. However, this genomic reorganization event is largely uncharacterized. Our lab has proposed that early, widespread, and transient reorganization of the genomic structure potentiates the appropriate responses of the cell to insult. We have identified chromatin structural changes in THP-1 human macrophage-like cell line induced by S. aureus stimulation. We measured nucleosome distribution high temporal resolution through the course of infection at more than 800 inflammation and immunity related genes. Herein we show that S. aureus indeed does induce rapid widespread changes in nucleosome distribution. This work supports a new model in which widespread changes potentiate a robust immune response.
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Date Issued: 2014
Identifier: FSU_migr_uhm-0416
Format: Thesis

Title: Chromatin-Based Regulation and Maintenance of the Human Genome.
Creator: Sexton, Brittany Sloane, Dennis, Jonathan Hancock, Roper, Michael Gabriel, Bass, Hank W., Chase, P. Bryant, Fadool, Debra Ann, Florida State University, College of Arts and...
Show moreSexton, Brittany Sloane, Dennis, Jonathan Hancock, Roper, Michael Gabriel, Bass, Hank W., Chase, P. Bryant, Fadool, Debra Ann, Florida State University, College of Arts and Sciences, Department of Biological Science
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Abstract/Description: Nucleosome distributions are critically important in regulating access to the eukaryotic genome. Cells with different physiologies have strikingly similar nucleosome distributions. Few studies in human cells have measured genome-wide nucleosome distributions at high temporal resolution during a response to a common stimulus. Factors regulating the maintenance of the basal state as well as changes in nucleosome distribution following a response must be investigated. In our first set of...
Show moreNucleosome distributions are critically important in regulating access to the eukaryotic genome. Cells with different physiologies have strikingly similar nucleosome distributions. Few studies in human cells have measured genome-wide nucleosome distributions at high temporal resolution during a response to a common stimulus. Factors regulating the maintenance of the basal state as well as changes in nucleosome distribution following a response must be investigated. In our first set of experiments we used the reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) as a model system for stimulus-induced nucleosome distribution changes. We measured nucleosome distribution at high temporal resolution in human cells at the 2 kb flanking the transcription start sites (TSSs) of hundreds immunity-related loci, using microarray technology, during the reactivation of KSHV. We show that nucleosome redistribution peaks at 24 hours post KSHV reactivation and that the nucleosomal redistributions are widespread and transient. To clarify the role of DNA sequence in these nucleosomal redistributions, we compared the genes with altered nucleosome distribution to a sequence-based computer model and in vitro assembled nucleosomes. We demonstrate that both the predicted model and the assembled nucleosome distributions are concordant with the majority of nucleosome redistributions at 24 hours post KSHV reactivation. We suggest a model in which loci are held in an unfavorable chromatin architecture and "spring" to a transient intermediate state directed by DNA sequence information. We propose that DNA sequence plays a more considerable role in the regulation of nucleosome positions than was previously appreciated. The surprising findings that nucleosome redistributions are widespread, transient, and DNA-directed shift the current perspective regarding regulation of nucleosome distribution in humans. We next wanted to affirm and extend our previous observations regarding the widespread and transient nature of nucleosome redistributions during viral reactivation. We tested if this widespread nucleosome remodeling was a genome wide event or limited solely to the hundreds of immunity-related loci measured by microarray. We measured nucleosome distributions at high temporal resolution following KSHV reactivation using our newly developed mTSS-seq technology, which maps nucleosome distribution at the TSS of all human genes. Nucleosomes underwent widespread changes in organization 24 hours after KSHV reactivation and returned to their basal nucleosomal architecture 48 hours after KSHV reactivation. 72% of the loci with translationally remodeled nucleosomes have nucleosomes that moved to positions encoded by the sophisticated underlying DNA sequence. We demonstrated that these widespread alterations in nucleosomal architecture potentiated regulatory factor binding. These descriptions of nucleosomal architecture changes have allowed us to propose a new hierarchical model for chromatin-based regulation of genome response. Given that we discovered that nucleosome distributions are widespread and transient, it was important for us to understand the forces maintaining the basal state. We It would be interesting to understand the forces and factors that maintain nucleosome architecture in a basal state and regenerate it following a response, such as KSHV reactivation. An appealing group of candidates that might maintain and regenerate the nucleosome architecture in its basal state is the transcriptional machinery. We identified RNA polymerase II (RNA Pol II) as a likely candidate as it is found throughout the genome and not always associated with transcription. We next were interested in the role RNA Pol II plays in the maintenance of chromatin structure. We measured nucleosome distributions in response to RNA Pol II inhibition by δ-amanitin treatment. Nucleosome distribution changes, following RNA Pol II inhibition, were widespread and the TSSs with nucleosome distribution changes were enriched for RNA Pol II independent of it's role it plays in active transcription. This work gives new insight into understanding the role of chromatin structure regulates and maintains the human genome.
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Date Issued: 2015
Identifier: FSU_migr_etd-9449
Format: Thesis

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