Search results

Title: Protecting Against Rod Degeneration In A Model Of Retinitis Pigmentosa.
Creator: Lebowitz, Joseph, Department of Biological Science
Abstract/Description: Retinitis Pigmentosa (RP) is a heritable disease that affects the retina, the photosensitive neural tissue lining the back of the eye. RP results in the gradual degeneration of rod photoreceptor cells, but secondarily cone cells degenerate and all visual input is lost. Mutations in nearly 100 genes have been associated with this disease and related disorders, however little is known about the series of cellular events that precede photoreceptor death. Taking advantage of a transgenic...
Show moreRetinitis Pigmentosa (RP) is a heritable disease that affects the retina, the photosensitive neural tissue lining the back of the eye. RP results in the gradual degeneration of rod photoreceptor cells, but secondarily cone cells degenerate and all visual input is lost. Mutations in nearly 100 genes have been associated with this disease and related disorders, however little is known about the series of cellular events that precede photoreceptor death. Taking advantage of a transgenic zebrafish model of rod degeneration, a small molecule screen and morpholino injections were used to identify modifiers of the cell death typically observed in RP. Rod photoreceptors in the developing zebrafish larvae express a fluorescent reporter gene. This leads to rod degeneration similar to that seen in RP, but cell death can easily be observed under a microscope. Live larvae were observed under the fluorescence microscope to determine if a compound has slowed or altered the degeneration of the rod cells at 4 days post fertilization. A gene-dosage dependent rate of degeneration was uncovered when comparing larvae that were hetero- and homozygous for the transgene. Modulation of the retinoic acid pathway modestly altered cell death. Knocking down Nr2e3 and p53 expression also showed protective effects that were stronger than those following chemical treatment. p53 codes for a key regulator of apoptosis, and Nr2e3 codes for a transcription factor of the rhodopsin gene. These results suggest further study of genetic and chemical manipulation of photoreceptor degeneration, and genotype-phenotype correlations in such diseases.
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Date Issued: 2013
Identifier: FSU_migr_uhm-0269
Format: Thesis

Title: Phylogeographic Patterns in the Eastern Diamondback Rattlesnake (Crotalus adamanteus).
Creator: Aronow, Karalyn, Biological Science
Abstract/Description: The aim of this project was to analyze relationships among populations of Crotalus adamanteus (Eastern Diamondback Rattlesnake) and to compare phylogeographic patterns across different classes of genes. The objective was to determine the effect of selective pressures on the detection of barriers to gene flow. For example, venom genes are under strong positive selection and allow an analysis of the rate of allele exchange in a gene that directly affects the fitness of an individual. This...
Show moreThe aim of this project was to analyze relationships among populations of Crotalus adamanteus (Eastern Diamondback Rattlesnake) and to compare phylogeographic patterns across different classes of genes. The objective was to determine the effect of selective pressures on the detection of barriers to gene flow. For example, venom genes are under strong positive selection and allow an analysis of the rate of allele exchange in a gene that directly affects the fitness of an individual. This selection might drive alleles through phylogeographic barriers that limit the spread of neutral alleles. The relationship between nontoxin genes and fitness is not as clear and can be difficult to establish. However, most variation in these genes is neutral and determined by stochastic events. The type of selection on a gene will affect the phylogeographic pattern displayed. Prior phylogeographic studies have used either neutral markers (e.g., microsatellites) or genes not directly involved in specific adaptations (e.g., mitochondrial genes). One venom gene, two mitochondrial genes, one nuclear gene, and one pseudogene were compared for patterns of adaptation and the presence of geographic barriers to gene flow. This novel approach to studying gene flow could have an impact on defining taxonomic groups and conservation efforts, as well as serve as a guide for choosing appropriate genetic markers for other species. In the venom gene that was analyzed, it appeared the high level of selection led to alleles being driven across the barriers to gene flow of alleles under neutral selection.
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Date Issued: 2011
Identifier: FSU_migr_uhm-0002
Format: Thesis

Title: Retinal gene profile.
Creator: Thornton, Kimberley
Date Issued: 2005-08-03
Identifier: 160469, FSDT160469, fsu:18925
Format: Document (PDF)

Title: Histone Deacetylase Inhibitors Prevent Nucleosome Redistribution at Transcription Start Sites.
Creator: Pickeral, Crystal, Biological Science
Date Issued: 2011
Identifier: FSU_migr_uhm-0034
Format: Thesis

Title: Analysis of the length control mechanism in Chlamydomonas reinhardtii.
Creator: Smith, Felicia
Date Issued: 2005-08-03
Identifier: 160529, FSDT160529, fsu:18953
Format: Document (PDF)

Title: Patterns of genetic variation and covariation in Viola septemloba.
Creator: Ransom, David Glenn
Date Issued: 2007-07-13
Identifier: 160757, FSDT160757, fsu:19061
Format: Document (PDF)

Title: The linking of coral disturbance, specifically the formation of "chimneys" on Acropora palmata, to the threespot damselfish Stegastes planifrons.
Creator: Clapp, Francis
Date Issued: 2005-12-09
Identifier: 160587, FSDT160587, fsu:18983
Format: Document (PDF)

Title: Amygdala Mechanisms Involved in Chemosensory Communication.
Creator: Stroe, Ioana, Biological Science
Abstract/Description: The amygdala is an almond-shaped region of the brain present in many vertebrates such as human, hamsters and mice. The amygdala is composed of several parts, of which the following are most relevant to my research: the basolateral amygdala (BLA) and the posterior medial amygdala (MeP). The BLA functions in learning and shows increased activation after the mouse has learned a behavior. The lateral paracapsular nuclei of the intercalated nucleus (ICNlpcn) project inhibitory neurons into the BLA...
Show moreThe amygdala is an almond-shaped region of the brain present in many vertebrates such as human, hamsters and mice. The amygdala is composed of several parts, of which the following are most relevant to my research: the basolateral amygdala (BLA) and the posterior medial amygdala (MeP). The BLA functions in learning and shows increased activation after the mouse has learned a behavior. The lateral paracapsular nuclei of the intercalated nucleus (ICNlpcn) project inhibitory neurons into the BLA and mediate its activity. Dopamine has been shown to inhibit the lpcn group and therefore relieve inhibition of the BLA (disinhibition). Dopamine also acts directly in the BLA to increase activation. Therefore, if a mouse is conditioned to prefer a particular odor (in this case steer urine), dopamine injection will cause an increase in BLA activation after learning due to both disinhibition and direct excitatory response. The medial posterior amygdala in the mouse mostly responds to olfactory stimuli from the same species (conspecific stimuli). The MeP is also thought to be under control of another ICN group, the caudal ICN (ICNc). If the same inhibitory relationship is seen between ICNc and MeP, the dopamine injection may cause disinhibition in the MeP and therefore the conditioned steer urine stimulus may show increased activation in the MeP despite being a stimulus from another species (heterospecific stimulus). My experiment tests whether the ICNc has inhibitory control over the MeP, and whether dopamine will cause inhibition in the ICNc and therefore cause disinhibition in the MeP.
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Date Issued: 2011
Identifier: FSU_migr_uhm-0040
Format: Thesis

Title: The Cost of Safety: Negotiating the Trade-Off between Safety and Growth.
Creator: Matechik, Christopher, Biological Science
Abstract/Description: To avoid the threat of predation, prey can alter their behavior such as moving from habitats where predation risk is high to those where it is low. However, such behaviors may involve a tradeoff if the low risk habitats are suboptimal feeding locations for the prey compared to the high risk ones. In seagrass, pinfish (Lagodon rhomboides) often move from low- to high-blade density patches in the presence of the predatory Gulf flounder (Paralichthys albigutta). However, restricted movements in...
Show moreTo avoid the threat of predation, prey can alter their behavior such as moving from habitats where predation risk is high to those where it is low. However, such behaviors may involve a tradeoff if the low risk habitats are suboptimal feeding locations for the prey compared to the high risk ones. In seagrass, pinfish (Lagodon rhomboides) often move from low- to high-blade density patches in the presence of the predatory Gulf flounder (Paralichthys albigutta). However, restricted movements in high-blade habitats may lead to reduced foraging efficiency and growth rates. I used a field experiment that ran for 28 days to examine this potential tradeoff between survival and growth. Neither predator presence nor seagrass blade density affected survival or growth in pinfish. However, after accounting for the loss of experimental replicates at lower grass-blade densities, the change in pinfish biomass was greater in the predator-present treatment compared to the controls, suggesting higher mortality, increased vigilance or both when flounder were present. Unfortunately, loss of experimental replicates and high variance in the measured responses hindered my ability to address the potential tradeoff, but suggestive results support further examination.
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Date Issued: 2011
Identifier: FSU_migr_uhm-0025
Format: Thesis

Title: Characterization of Maize Transgene-Reactivated Mutants.
Creator: Mills, Erica, Biological Science
Abstract/Description: Epigenetic gene regulation results from heritable changes in chromatin structure, DNA methylation, and/or histone modifications associated with different expression levels for endogenous genes and transgenes. The silent b1 transgene (Btg-s) can be used to study epigenetic gene regulation in maize. A forward genetics screen based on the reactivation of Btg-s was conducted using EMS mutagenized maize. Through this screen, several mutants reactivating the transgene, designated transgene...
Show moreEpigenetic gene regulation results from heritable changes in chromatin structure, DNA methylation, and/or histone modifications associated with different expression levels for endogenous genes and transgenes. The silent b1 transgene (Btg-s) can be used to study epigenetic gene regulation in maize. A forward genetics screen based on the reactivation of Btg-s was conducted using EMS mutagenized maize. Through this screen, several mutants reactivating the transgene, designated transgene reactivated (tgr), were identified. Published work demonstrates that mutations affecting proteins involved in the RNA-dependent DNA methylation pathway cause transgene reactivation. Such mutations result in characteristic phenotypes, such as a loss of DNA methylation, loss of siRNA accumulation, and loss of transcriptional silencing. To investigate Btg promoter methylation in the tgr mutants, bisulfite sequencing was performed on plants with silenced and reactivated transgenes from four lines of tgr mutants (tgr1-3,11). Results show that in three mutant lines, the promoter region had higher methylation levels in silenced plants compared to reactivated plants, while another mutant had similar levels of methylation in both phenotypes. For tgr2, siRNA levels were assayed and transcript abundance was evaluated for genes encoding proteins possibly involved in the methylation pathway. It was found that tgr2 showed no loss of siRNA production and no difference in transcript levels were observed between silenced and reactivated individuals for the genes evaluated. This work aided in characterizing the tgr mutants which are potentially strong tools for understanding epigenetic regulation of transgenes.
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Date Issued: 2011
Identifier: FSU_migr_uhm-0028
Format: Thesis

Title: Chromatin Structural Changes Linking Retinoic Acid Function to Genomic Targets.
Creator: Hood, Preston
Abstract/Description: Eukaryotic DNA is organized into chromatin, which serves as the ultimate substrate for all nuclear events including transcription, replication, recombination and repair. Alterations to chromatin and its modes of remodeling serve as the basis for understanding gene regulation. Chromatin structure can be analyzed to identify the mechanics and regulation associated to genome response. All-trans retinoic acid (atRA), a metabolite of Vitamin A, uses a direct route to control gene expression within...
Show moreEukaryotic DNA is organized into chromatin, which serves as the ultimate substrate for all nuclear events including transcription, replication, recombination and repair. Alterations to chromatin and its modes of remodeling serve as the basis for understanding gene regulation. Chromatin structure can be analyzed to identify the mechanics and regulation associated to genome response. All-trans retinoic acid (atRA), a metabolite of Vitamin A, uses a direct route to control gene expression within the nucleus. Retinoic acid (RA) binds DNA via coactivating RA receptors that function through a type 2 nuclear receptor pathway. This binding is believed to stimulate a shift in the structure of DNA that could possibly result from alterations to proteins associated with specific locations (Wan et al., 2012). The GM12878 cell line was treated with atRA for different lengths of time and the resulting changes in chromatin structure were mapped. We sought to understand if this chromatin structure mechanism is more broadly used to facilitate RA-induced regulation in the human genome. Here we show that changes can be mapped using the R console computer program created by the Dennis Lab. Genes were analyzed and can be categorized into three different categories of change: consistent changes through all three time points in the same location; changes through all three time points in different locations; and transient stage changes that do not appear in the early or late treatment times.
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Date Issued: 2012
Identifier: FSU_migr_uhm-0082
Format: Thesis

Title: Rescue of a Chlamdydomonas Reinhardtii Mutant.
Creator: Crumbley, Christine, Biological Science
Abstract/Description: This research focuses on the signal transduction pathway involved in acid-induced deflagellation in the green algae Chlamydomonas reinhardtii. A wild type copy of the 2L15 gene will be amplified and used to create a plasmid. This plasmid will contain a wild type copy of the nitrate reductase gene of Chlamydomonas reinhardtii, which will serve as a selectable marker, as well as the 2L15 gene. The plasmid will be used to transform Chlamydomonas reinhardtii mutants with the hope of rescuing the...
Show moreThis research focuses on the signal transduction pathway involved in acid-induced deflagellation in the green algae Chlamydomonas reinhardtii. A wild type copy of the 2L15 gene will be amplified and used to create a plasmid. This plasmid will contain a wild type copy of the nitrate reductase gene of Chlamydomonas reinhardtii, which will serve as a selectable marker, as well as the 2L15 gene. The plasmid will be used to transform Chlamydomonas reinhardtii mutants with the hope of rescuing the mutant phenotype.
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Date Issued: 2007
Identifier: FSU_migr_uhm-0044
Format: Thesis

Title: Restoration and Control of Circadian Rhythmicity in a Novel Transgenic Mouse.
Creator: Abich, Estela, Department of Biological Science
Abstract/Description: Previous biochemical findings from our lab revealed that PERIOD (PER) proteins globally regulate the molecular oscillator through both transcriptional and posttranslational mechanisms. Furthermore, our lab demonstrated that PER rhythms define a critical rhythmic nodal point in the generation of circadian rhythms. Deciphering the phase shifting mechanism is important for developing effective pharmacological or environmental (e.g., phototherapy) interventions to treat many circadian diseases,...
Show morePrevious biochemical findings from our lab revealed that PERIOD (PER) proteins globally regulate the molecular oscillator through both transcriptional and posttranslational mechanisms. Furthermore, our lab demonstrated that PER rhythms define a critical rhythmic nodal point in the generation of circadian rhythms. Deciphering the phase shifting mechanism is important for developing effective pharmacological or environmental (e.g., phototherapy) interventions to treat many circadian diseases, including sleep disorders in shift workers. Our novel inducible Per2 transgenic mouse in Per1/2-null background provides a unique opportunity to address how perturbations in Per expression can cause phase shifts and the opportunity to delve into whether the clock can be reset to a desirable phase by manipulating PER rhythms in a calculated manner. Although photic stimuli acutely induces both Per1 and Per2, manipulation of Per2 alone should be enough to mimic light-induced phase shifts, because Per-deficient mice are similar to wild type mice in their phase delays and advances in response to light pulses. This experiment aimed to demonstrate how we could use this novel genetic construct to gain an in-depth understanding of how we can artificially regulate rhythms in humans.
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Date Issued: 2014
Identifier: FSU_migr_uhm-0288
Format: Thesis

Title: Quantifying locomotor activity by computer video analysis.
Creator: Liu, Yang
Date Issued: 2005-04-18
Identifier: 160778, FSDT160778, fsu:19070
Format: Document (PDF)

Title: The varnished conk and the forked fungus beetle: predation vs. mutualism in the relationship between Ganoderma lucidum and Bolitotherus cornutus.
Creator: Seitzman, Brian H.
Date Issued: 2007-07-13
Identifier: 160747, FSDT160747, fsu:19057
Format: Document (PDF)

Title: Crosstalk between Chromatin Structure and DNA Methylation and the Regulation of DNA Templated Processes.
Creator: Noori, Tarreq, Biological Science
Abstract/Description: Methylation of DNA is one of the earliest described epigenetic modifications. Hypermethylation is associated with gene silencing, while the inhibition of methylation is generally associated with reactivating silenced genes. The packaging of DNA in the nucleus into chromatin also plays a role in regulating gene expression. We sought to understand the crosstalk between changes in methylation status of the genome and changes in chromatin structure. 5-azacytidine (5-azaC), a potent DNA...
Show moreMethylation of DNA is one of the earliest described epigenetic modifications. Hypermethylation is associated with gene silencing, while the inhibition of methylation is generally associated with reactivating silenced genes. The packaging of DNA in the nucleus into chromatin also plays a role in regulating gene expression. We sought to understand the crosstalk between changes in methylation status of the genome and changes in chromatin structure. 5-azacytidine (5-azaC), a potent DNA methytransferase inhibitor, has recently generated interest as a potential anti-cancer drug, possibly functioning by reactivating silenced tumor suppressor genes. We treated the hematologic cancer cell lines U-937 and T-HP1 with 5-aza for varying lengths of time. We then harvested DNA for methylation studies, RNA for gene expression studies and chromatin for nuclease accessibility studies. The chromatin accessibility was further measured at two different levels of resolution; the domain level (10s of kb) and nucleosome position (10s of bp). This was achieved using an innovative DNA microarray assay. Results were analyzed to correlate changes in chromatin structure with changes in methylation and gene expression. We have identified that the class II, major histocompatibility complex, transactivator (CIITA) shows chromatin structural changes. The results provide a model for understanding the regulatory chromatin structure involved in the immune response.
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Date Issued: 2011
Identifier: FSU_migr_uhm-0032
Format: Thesis

Title: Identification of residues in budding yeast RAD53 kinase that are crucial for regulating histone protein level S.
Creator: Adair, Abigail
Date Issued: 2007-02-01
Identifier: 160306, FSDT160306, fsu:18849
Format: Document (PDF)

Title: Binding interactions between intron RNA and the spliceosomal proteins SF3b155 and p14.
Creator: Cherkis, Karen A.
Date Issued: 2006-05-18
Identifier: 160576, FSDT160576, fsu:18977
Format: Document (PDF)

Title: Exploring the Function of 14-3-3 Proteins in Neurodegenerative Disease.
Creator: Vogt, Peggy, Biological Science
Abstract/Description: The two pathological hallmarks of Parkinson's are neurodegeneration in the substantia nigra parc compacta and the presence of Lewy bodies in the surviving neurons. Recent evidence indicates that Lewy bodies share biochemical and morphological similarities with aggresomes, a large juxtanuclear aggregate. In previous studies, we observed that 14-3-3 proteins promote the aggresome formation of α-Synuclein, the main component of Lewy bodies. In this study, we determined 14-3-3 to have a...
Show moreThe two pathological hallmarks of Parkinson's are neurodegeneration in the substantia nigra parc compacta and the presence of Lewy bodies in the surviving neurons. Recent evidence indicates that Lewy bodies share biochemical and morphological similarities with aggresomes, a large juxtanuclear aggregate. In previous studies, we observed that 14-3-3 proteins promote the aggresome formation of α-Synuclein, the main component of Lewy bodies. In this study, we determined 14-3-3 to have a generalized aggresome promoting function by examining two known aggregation-prone proteins, including a mutant form of CFTR and the GFP-250 protein. To assess the isoform specificity of 14-3-3 proteins in this process, we compared the extent of increase in aggresome formation promoted by overexpression of two different 14-3-3 isoforms. Furthermore, we utilized two different mutants to demonstrate that dimerization of 14-3-3 may not be required for its function in the aggresome formation cascade. Taken together, our data indicate that 14-3-3 proteins may play essential critical role in the aggresome formation process. The discovery of the mechanism by which 14-3-3 participates in the aggresome formation cascade may help us to understand pathogenesis of neurodegenerative diseases, such as Parkinson's disease.
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Date Issued: 2011
Identifier: FSU_migr_uhm-0042
Format: Thesis

Title: Indirect Genetic Effects and the Growth of Juvenile Eastern Mosquitofish (Gambusia holbrooki).
Creator: Buncy, Adam Timothy, Department of Biological Science
Abstract/Description: The purpose of this study was to discover if indirect genetic effects (IGEs) affect male mosquitofish juvenile growth. IGEs can be defined as the environmental effect on an individual's phenotype due to the variation in the genotypes of its social partners (Wolf et al 1998). Indirect genetic effects can impact a variety of phenotypic traits. Specifically, I asked: what are the effects of IGEs on the growth (length and weight) of male juvenile siblings reared in different social environments?...
Show moreThe purpose of this study was to discover if indirect genetic effects (IGEs) affect male mosquitofish juvenile growth. IGEs can be defined as the environmental effect on an individual's phenotype due to the variation in the genotypes of its social partners (Wolf et al 1998). Indirect genetic effects can impact a variety of phenotypic traits. Specifically, I asked: what are the effects of IGEs on the growth (length and weight) of male juvenile siblings reared in different social environments? I tested the hypothesis that indirect genetic effects act during development to affect the growth of juvenile male mosquitofish. The weight and standard length of pairs of sibling males reared in the same physical environment, but different social environments, were measured once per month to assess physical condition. If physical condition differs in response to social environment, this is evidence that indirect genetic effects do play a role. Specifically, mottled and silver juvenile males were reared with two adult females and two adult males, both of which were either silver or mottled. I measured the length and weight of these pairs of sibling males monthly. I found that focal males in silver social environments grew the longest, while the focal males in mottled social environments were shorter. Juvenile male weight was only significantly affected by age. However, trends for weight mirrored those for length. For instance, in the length experiment, mottled focal males in a silver context grew the longest. In the weight experiment, we also saw that mottled focal males in a silver context grew the biggest. Based on these results, I concluded that the growth of juvenile mosquitofish depends upon the genetic composition of their social environment, independent of the genotype of the focal male. This suggests that scientists should take IGEs into consideration when measuring the effects of genotype and environment on traits such as growth, and that IGEs may be an important factor contributing to the rapid evolution of growth in some species.
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Date Issued: 2015
Identifier: FSU_migr_uhm-0529
Format: Thesis

Title: Ecology of an Invasive Lupine.
Creator: Luscuskie, Lauren P., Department of Biological Science
Abstract/Description: Since humans began settling in Iceland, the native vegetation has been slowly degrading. Today, Iceland is one of the most heavily environmentally damaged countries due to human impacts. When Iceland was settled, approximately 60% of the land area was vegetated (Arnalds, 2001). Even as recently as two centuries ago, the plant cover of Iceland was far more extensive than it is at the present. Today, vegetation cover is estimated at 25% of the land area, with trees occupying only 1% (Arnalds,...
Show moreSince humans began settling in Iceland, the native vegetation has been slowly degrading. Today, Iceland is one of the most heavily environmentally damaged countries due to human impacts. When Iceland was settled, approximately 60% of the land area was vegetated (Arnalds, 2001). Even as recently as two centuries ago, the plant cover of Iceland was far more extensive than it is at the present. Today, vegetation cover is estimated at 25% of the land area, with trees occupying only 1% (Arnalds, Aradóttir and Thorsteinsson, 1987; Arnalds, 2004). There are many factors that combine to affect the degradation of Iceland’s vegetated areas. The soils are derived from volcanic activity, are mostly classified as andisols, and are very inclined to erosion (Arnalds, 2004; Ólafsdóttir, Schlyter and Haraldsson, 2001). The cold, windy conditions accelerate soil erosion when plant cover is initially removed by human activity, decreasing the likelihood that vegetative cover can be re-established. The cold, wet soils experience regular frosts that further inhibit the ability of plants to colonize and re-establish themselves. Lupinus nootkatensis, the Nootka Lupine, is native to Western North America. This species of lupine has been shown to ameliorate soil characteristics in other locations due to its ability to fix nitrogen and grow in dense monocultures, subsequently influencing successional dynamics. A legume, the Nootka Lupine fixes nitrogen by a symbiosis with a rhizobium bacteria. This facilitates lupine growth in nitrogen-deficient and highly disturbed soils, such as those of volcanic origin present in Iceland (Riege and Sigurgeirsson, 2009; Myrold and Huss-Danell, 2003). In fixing nitrogen, lupine also supplements the surrounding soil with excess nitrogen. Dense monocultures of lupine, as tall as 120cm, increase litter production and decomposition as well as the trapping of detritus, increasing soil organic matter content (Myrold and Huss-Danell, 2003). Both of these methods allow lupine to improve soil characteristics in unvegetated areas, over time increasing nitrogen and organic matter content in the soils to allow for growth of native successional plant species.--Introduction.
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Date Issued: 2015
Identifier: FSU_migr_uhm-0481
Format: Thesis

Title: The Dynamics of the Distribution of Predeators in an Enclosed Reserve in South Africa.
Creator: Engbretsen, Kirstin, Department of Biological Sciences
Abstract/Description: Siyafunda Conservation Initiative conducts wildlife research on the Greater Makalali Private Game Reserve, a 61,000 acre fenced reserve in South Africa. Their objective is to reintroduce native species to the reserve and reestablish the natural ecosystem that was once present across much of South Africa. The purpose of my research trip was to examine the activities of mammalian predators and create GIS-based maps that show predator distribution in the reserve. The predators I focused on were...
Show moreSiyafunda Conservation Initiative conducts wildlife research on the Greater Makalali Private Game Reserve, a 61,000 acre fenced reserve in South Africa. Their objective is to reintroduce native species to the reserve and reestablish the natural ecosystem that was once present across much of South Africa. The purpose of my research trip was to examine the activities of mammalian predators and create GIS-based maps that show predator distribution in the reserve. The predators I focused on were lion, cheetah, leopard and hyena. My goals were to receive training and field experience in conservation research and to provide Siyafunda with information that could help with decisions for future management. During my six weeks living in the reserve, we took daily monitoring drives to collect information, such as demographic statistics, prey selection, reproductive behavior, and habitat usage for multiple species. Using the predator information I collected in six weeks, as well as eight years of Siyafunda's previous data, I created maps to visualize predatory home ranges in the reserve. I also helped with updating the identification system for individual animals and creating family trees that help identify the bloodline of individual predators. I studied the past data to help analyze predator movement and population dynamics throughout the years. I also investigated the ways that adding or removing animals from the system affects already established populations. I am working with the director of Siyafunda, Michael Job, to develop a GIS-based distribution model that can be continuously updated. My research should assist the managers of the GMPGR as they continue to assure the health of the ecosystem, especially regarding the challenges within a closed system of overpopulation or species decline from competition.
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Date Issued: 2013
Identifier: FSU_migr_undergradresearch-0011
Format: Citation

Title: Protecting Against Photoreceptor Degeneration in a Model of Retinitis Pigmentosa.
Creator: Lebowitz, Joseph, Department of Biological Sciences
Date Issued: 2013
Identifier: FSU_migr_undergradresearch-0023
Format: Citation

Title: Zymosan Fungal Infection Induces Necleosome Distributions During the Innate Immune Response on a Time Dependent Manner.
Creator: Gruder, Olivia, Department of Biological Sciences
Abstract/Description: Chromatin structure plays a critical role in the regulation of the human genome. An understanding of the role of chromatin structure and its relationship to gene regulation is critical to developing new strategies to prevent and treat diseases. We chose to investigate the anti-inflammatory response of human macrophage-like cell line (THP1) to Zymosan, in order to elucidate the regulation of chromatin. Zymosan is a component of the fungal cell wall that induces an innate immune response. After...
Show moreChromatin structure plays a critical role in the regulation of the human genome. An understanding of the role of chromatin structure and its relationship to gene regulation is critical to developing new strategies to prevent and treat diseases. We chose to investigate the anti-inflammatory response of human macrophage-like cell line (THP1) to Zymosan, in order to elucidate the regulation of chromatin. Zymosan is a component of the fungal cell wall that induces an innate immune response. After THP1 were treated with Zymosan, we hypothesized that the fungal infection would initiate an inflammatory response by altering nucleosome redistribution and/or altering chromatin structure in a time dependent manner. Based on previous results that showed rapid, widespread, transient changes in nucleosome distribution in the innate immune response, we chose to look at multiple time points at high temporal resolution: 0 (control), 20', 40', 60', 80', 100', 2h, 3h, 4h and 12h. We measured nucleosome distribution at each of these time points at hundreds of genes transcription start sites involved in the immune response. We saw the greatest changes in nucleosome positioning from 20 to 60 minutes, and it appeared that these changes were transient since they reverted back to their original after the 60-minute time point. These results support our prediction that all cells have the same nucleosome distributions during their resting states, but can be altered with the addition of an insult. In response to a stimulus, a biochemical "yawn" occurs to provide accessibility to genes needed to provide a response. The data indicates that widespread but transient changes occur to the entire genome upon response to an environmental stimulus.
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Date Issued: 2013
Identifier: FSU_migr_undergradresearch-0006
Format: Citation

Title: Determining by use of Nested Deletions what Maintains Proper Replication Timing.
Creator: Davis, Caroline, Department of Biological Sciences
Date Issued: 2013
Identifier: FSU_migr_undergradresearch-0020
Format: Citation

Title: Analysis of Chromatin Structure Indicates a Role for Fluoxetine in Altering Nucleosome Distribution.
Creator: Gracia, Ely
Abstract/Description: An interesting stimulus for chromatin structural changes is the generic and popular anti-depressant drug Fluoxetine, commonly known as Prozac. Generally accepted as a Selective Serotonin Reuptake Inhibitors (SSRI's), recent work has emerged suggesting that this antidepressant also functions as a Histone Deaceylase Inhibitors (HDIs). Studies have also come out indicating that Fluoxetine acts as an immunosuppressant drug. Treatment with Fluoxetine is believed to reduce the over-activation of...
Show moreAn interesting stimulus for chromatin structural changes is the generic and popular anti-depressant drug Fluoxetine, commonly known as Prozac. Generally accepted as a Selective Serotonin Reuptake Inhibitors (SSRI's), recent work has emerged suggesting that this antidepressant also functions as a Histone Deaceylase Inhibitors (HDIs). Studies have also come out indicating that Fluoxetine acts as an immunosuppressant drug. Treatment with Fluoxetine is believed to reduce the over-activation of the immune system associated with depression. We have used an innovative microarray technology to measure changes in nucleosomal positioning that stem from Fluoxetine treatment. With the use of the microarray, we were able to show that Fluoxetine regulated chromatin structure, that Fluoxetine induced nucleosomal changes show time-dependent kinetics, and targeted genes responsible for the regulation of immune system processes. These results give new and important insights into non-SSRI roles of this highly prescribed class of drugs.
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Date Issued: 2012
Identifier: FSU_migr_uhm-0087
Format: Thesis

Title: The Genetic Studies of Cell Cycle Transition in Budding Yeast.
Creator: Liu, Hong, Wang, Yanchang, Bass, Hank W., Sang, Qing-Xiang Amy, Bates, George W., Hurt, Myra M., Deng, Wu-Min, Department of Biological Science, Florida State University
Abstract/Description: Budding yeast, Saccharomyces cerevisiae, has a unique cell cycle. During its S-phase, DNA replication occurs, but spindle formation and chromosome-microtubule attachment also take place. Thus, how to couple spindle formation and chromosome attachment with DNA replication during S-phase is a challenge to yeast cells. In this dissertation, we investigated the differential regulation of cyclin-dependent kinase (Cdk) activity in response to DNA replication stress. In addition, we addressed how...
Show moreBudding yeast, Saccharomyces cerevisiae, has a unique cell cycle. During its S-phase, DNA replication occurs, but spindle formation and chromosome-microtubule attachment also take place. Thus, how to couple spindle formation and chromosome attachment with DNA replication during S-phase is a challenge to yeast cells. In this dissertation, we investigated the differential regulation of cyclin-dependent kinase (Cdk) activity in response to DNA replication stress. In addition, we addressed how the shorter spindle structure contributes to kinetochore-microtubule interaction. Finally, the function of a kinesin complex in tension generation across sister kinetochores and kinetochore-microtubule (KT-MT) interaction has also been investigated. Periodically regulated Cdk is required for DNA synthesis and mitosis. Hydroxyurea (HU) inhibits DNA synthesis by depleting dNTPs, the basic unit for DNA synthesis. HU treatment triggers the S-phase checkpoint, which arrests cells at S-phase, inhibits late origin firing and stabilizes replication forks. Using budding yeast as a model system, we found that Swe1, a negative regulator of Cdk, is stabilized in HU-treated cells. Interestingly, this accumulation is not dependent on S-phase checkpoint. hsl1Δ, hsl7Δ, and cdc5-2 mutants, which have defects in Swe1 degradation, show HU sensitivity because of their defects in Swe1 degradation. We further demonstrated that their HU sensitivity is not a result of DNA damage accumulation or incomplete DNA synthesis; instead the sensitivity is due to their dramatically delayed recovery from HU-induced S-phase arrest. Strikingly, our in vivo data indicate that Swe1 inhibits the kinase activity of Clb2-Cdk1, but not that of Clb5-Cdk1. Therefore, S-phase accumulated Swe1 prevents Clb2-Cdk1–mediated mitotic activities, but has little effect on Clb5-Cdk1–associated S-phase progression. These results explain a long-time puzzle regarding the question why the Cdk1 negative regulator Swe1, which is present in S-phase, does not affect DNA synthesis. The kinetochore is a protein complex that assembles on centromeric DNA to mediate chromosome-microtubule interactions. Most eukaryotic cells form the spindle and establish kinetochore-microtubule interactions during mitosis, but budding yeast cells finish these processes in S-phase. It has long been noticed that the S-phase spindle in budding yeast is shorter than that in metaphase, but the biological significance of this short S-phase spindle structure remains unclear. We addressed this issue by using ask1-3, a temperature sensitive kinetochore mutant that exhibits partially elongated spindles at permissive temperature in the presence of HU, a DNA synthesis inhibitor. After exposure to, and removal of, HU, ask1-3 cells show a delayed anaphase entry. This delay depends on the spindle checkpoint, which monitors kinetochore-microtubule interaction defects. Overproduction of microtubule-associated protein Ase1 or Cin8 also induces spindle elongation in HU-arrested cells. The spindle checkpoint-dependent anaphase entry delay is also observed after ASE1 or CIN8 overexpression in HU-arrested cells. Therefore, the shorter spindle in S-phase cells is likely to facilitate proper chromosome-microtubule interactions. In budding yeast, chromosomes are attached by spindle microtubules emanating from opposite spindle poles to establish bipolar attachment during S-phase. The bipolar attachment generates tension on chromosomes and defects in tension generation activate the tension checkpoint that blocks anaphase entry. The pulling force directed to the opposite spindle poles as well as the cohesin that holds sister chromatids together provides the mechanical mechanism for tension generation. Here we present evidence indicating that Cik1/Kar3, a minus-end-directed kinesin motor complex in budding yeast, binds to kinetochore protein and promotes generation of tension on sister kinetochores. Cells lacking Cik1/Kar3 exhibit tension defects as indicated by the decreased sister centromere separation in pre-anaphase cells. Moreover, cik1Δ mutant cells show reduced association of centromeric DNA with Ask1, a component of DASH complex that encircles a microtubule and mediates kinetochore-microtubule interactions. Since Kar3 has been shown to drive the poleward chromosome movement along microtubules, it is likely that Cik1/Kar3-dependent chromosome transport promotes tension generation and facilitates DASH-dependent kinetochore-microtubule interactions.
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Date Issued: 2009
Identifier: FSU_migr_etd-1142
Format: Thesis

Title: Mechanism of Replication Timing Program in Mammalian Systems.
Creator: Lu, Junjie, Gilbert, David M., Gunjan, Akash, Bass, Hank W., Davidson, Michael W., Epstein, Lloyd M., Department of Biological Science, Florida State University
Abstract/Description: DNA replication, the duplication of genetic and epigenetic information, is the basis of biological inheritance. In all eukaryotes, DNA replication follows a tightly regulated replication-timing program, which dynamically changes during development and has a lot of conserved characteristics across multiple species. However the molecular mechanism of replication timing program remains elusive. Using a Xenopus egg extract in vitro replication system, it was found that the replication timing...
Show moreDNA replication, the duplication of genetic and epigenetic information, is the basis of biological inheritance. In all eukaryotes, DNA replication follows a tightly regulated replication-timing program, which dynamically changes during development and has a lot of conserved characteristics across multiple species. However the molecular mechanism of replication timing program remains elusive. Using a Xenopus egg extract in vitro replication system, it was found that the replication timing program is established at a time point during early G1-phase of the cell cycle, termed the Timing Decision Point (TDP). The TDP is coincident with the re-positioning and anchorage of chromosome segments in the newly formed nucleus. Previous studies have found that many chromatin related factors were not able to determine replication timing. Here, I studied the roles of non-coding RNAs from mouse pericentric heterochromatin and histone methylatransferase G9a in replication timing determination and found that they were also not the main determinants. I further examined the replication timing property of chromatin in G2-phase and quiescent (G0) cells and demonstrated that G2-phase chromatin lacks determinants of replication timing but maintains the overall spatial organization of chromatin domains, using both in vitro system and in vivo cell culture. In contrast, chromatin from quiescent cells (G0) retains replication timing but exhibits disrupted spatial organization. These data support a model in which events at the TDP establish chromatin marks as replication timing determinants, potentially facilitated by spatial organization, which persist independent of such organization until the process of chromatin replication dilutes or disrupts those determinants. These results demonstrate that establishment and maintenance of replication timing program are mediated by different mechanisms, both of which potentially involve chromatin spatial organization. This work represents a major advance in our quest for the replication timing determinants and understanding of the mechanism of replication timing program.
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Date Issued: 2010
Identifier: FSU_migr_etd-1019
Format: Thesis

Title: Immunological Analysis of Allergenic Tree Nut Proteins.
Creator: Willison, Leanna N., Roux, Kenneth H., Sathe, Shridhar K., Keller, Thomas C. S., Department of Biological Science, Florida State University
Abstract/Description: Allergies are a growing problem in industrialized countries, with food allergies affecting 6% of children and 3-4% of adults. Tree nuts are a common cause of food-induced allergy and include walnut, cashew, almond, hazelnut, pistachio, pecan, chestnut, and Brazil nut. Tree nut allergy, in particular, affects 0.5% of the US population. Unlike other food allergies, tree nut allergy persists throughout life and is known to cause severe allergic reactions. Therefore, it is critical to identify...
Show moreAllergies are a growing problem in industrialized countries, with food allergies affecting 6% of children and 3-4% of adults. Tree nuts are a common cause of food-induced allergy and include walnut, cashew, almond, hazelnut, pistachio, pecan, chestnut, and Brazil nut. Tree nut allergy, in particular, affects 0.5% of the US population. Unlike other food allergies, tree nut allergy persists throughout life and is known to cause severe allergic reactions. Therefore, it is critical to identify allergenic proteins in tree nuts that are directly involved in the allergic response and study these proteins on both the biochemical and immunological level. To identify allergenic proteins in pistachio and almond, polymerase chain reaction (PCR) was preformed using degenerate or gene specific primers, designed to amplify likely tree nut allergens in complementary cDNA libraries. The coding genes for the 7S vicilin from pistachio and lipid transfer protein (LTP) from almond were identified and were ligated into an expression vector to be expressed as fusion proteins. The generated fusion proteins were purified and used to screen almond- or pistachio-allergic individuals for IgE reactivity using immunoblot, ELISA, and dot blot assays. IgE reactivity to the pistachio 7S was found in 7 of 19 (37%) patients tested, and the allergen was designated Pis v 3. Inhibition immunoblots using the 7S fusion protein lead to the identification of the native protein in the crude nut extract. It is not uncommon for food-allergic individuals to react to more than one allergen, and tree nut allergic individuals are often sensitized to multiple nuts. In particular, patients allergic to cashew frequently report allergy to pistachio as well, which is likely a result of cross-reactivity between the two closely related tree nuts. Analysis of the 7S vicilin pistachio allergen revealed that it was highly homologous to the 7S vicilin allergen, Ana o 1, from cashew. Cross-reactivity between these two allergens was investigated by inhibition dot- and immunoblot assays using serum IgE from pistachio and/or cashew-allergic individuals. The seven patients with IgE that recognized Pis v 3 also recognized Ana o 1. Similar results were obtained using a panel of murine anti-rAna o 1 monoclonal antibodies (MAbs), as six of nine (67%) MAbs tested showed reactivity to Pis v 3 on dot-blots. The data does not identify the primary sensitizing agent but suggests that IgE reactivity to Pis v 3 and Ana o 1 is focused on the most conserved regions of the proteins. The results demonstrate that Pis v 3 is a likely contributor to the observed co-sensitivity to pistachio and cashew in some patients. Plant LTPs have been identified as allergens in a variety of fruits, vegetables, and nuts, and several studies have demonstrated cross-reactivity among LTPs. Immunoblotting using a rabbit polyclonal antibody (pAb) raised against peach LTP identified native LTP in the crude nut extract. IgE reactivity to almond LTP was found in 5 of 25 (20%) patients tested and designated Pru du 3. LTP is found in low abundance in the seed, therefore, an enriched almond extract was used in immunoblotting assays with LTP-reactive patients. Of the five LTP-reactive patients, only two recognized native LTP in the enriched nut extract. The lack of IgE reactivity by immunoblot suggests that some patients recognize primarily conformational epitopes on LTP that are destroyed under the denaturing conditions of immunoblotting. Subsequent dot blot assays confirm this hypothesis as IgE reactivity was lost when LTP was treated with common reducing reagents used in immunoblotting. Overall, the results demonstrate that LTP is an allergenic protein in almond and IgE antibodies in allergic individuals are directed against both conformational and linear epitopes.
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Date Issued: 2009
Identifier: FSU_migr_etd-0941
Format: Thesis

Title: Multivariate Phenomics Approach to Inheritance of a Complex Trait.
Creator: Nye, Jessica, Houle, David, Hughes, Kimberly, Beerli, Peter, Department of Biological Science, Florida State University
Abstract/Description: Mortality in humans is primarily due to diseases with complex causes such as heart disease, cancer, and diabetes. Finding the genetic underpinnings of such diseases could lead to advances in medicine. Reviewing studies that try to locate causal variants in diseases has revealed several problems. With the utilization of the tools available in the model system Drosophila melanogaster and the complex phenotype of the wing, I attempt to improve mapping techniques of a complex phenotype in order...
Show moreMortality in humans is primarily due to diseases with complex causes such as heart disease, cancer, and diabetes. Finding the genetic underpinnings of such diseases could lead to advances in medicine. Reviewing studies that try to locate causal variants in diseases has revealed several problems. With the utilization of the tools available in the model system Drosophila melanogaster and the complex phenotype of the wing, I attempt to improve mapping techniques of a complex phenotype in order to investigate the genetic structure of a quantitative trait including number of causal loci, gene ontology, and gametic disequilibrium. This is done firstly by determining the number of dimensions of the phenotype containing significant additive genetic variation, and secondly by mapping the phenotype to the genotype using multivariate statistical tests and vector projections. The base population in this study is the Drosophila genetic reference panel (DGRP) which is a collection of inbred lines derived from the same natural population. The modeled genetic variance-covariance (G) matrix, from 185 of the DGRP lines, show that all measured traits (21) from the phenotype have significant genetic variation. The eigenvalues of each successive dimension decrease in importance by 26.8%, indicating a slow linear regression on a log scale. In an outbred population simulated by a series of diallel crosses, the modeled G matrix indicated 16 dimensions held significant genetic variation. The decrease in dimensionality is due to the loss of homozygozity due to dominance and epistasis. Mapping the inbred females from 165 of the DGRP lines to 85,000 SNPs resulted in 1,795 significant sites with a false discovery rate of 25%. The top mapped sites have a wide range of effect sizes, and most fall outside the coded protein. The mapping results suggest that there is a large amount of quantitative inputs from regulatory regions of the genome as well as protein coding regions.
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Date Issued: 2011
Identifier: FSU_migr_etd-2478
Format: Thesis

Title: The Effects of Population Size and Spatial Structure on Genetic Variation and Response to a Novel Environment in a Perennial Plant.
Creator: Oakley, Christopher G., Winn, Alice A., Stallins, J. Anthony, Beerli, Peter, Houle, David, Travis, Joseph, Department of Biological Science, Florida State University
Abstract/Description: While theory shows that the distribution of populations in space can have profound ecological and evolutionary consequences, the complexity of numerical and genetical dynamics in space has made evaluation of model assumptions and predictions in natural populations challenging. Evolutionary dynamics in a spatial context will involve complex interactions between natural selection, genetic drift, migration, and genetic architecture. These factors have seldom been evaluated for natural...
Show moreWhile theory shows that the distribution of populations in space can have profound ecological and evolutionary consequences, the complexity of numerical and genetical dynamics in space has made evaluation of model assumptions and predictions in natural populations challenging. Evolutionary dynamics in a spatial context will involve complex interactions between natural selection, genetic drift, migration, and genetic architecture. These factors have seldom been evaluated for natural populations. I explored evolution in a spatial context in the perennial plant Hypericum cumulicola, exploiting natural variation in population size and degree of isolation. This species has recently colonized a novel roadside habitat, and populations in the novel habitat show dramatic differences in life history traits suggesting divergent selection. In chapter 2, I quantified the pattern of population size and migration using molecular marker data and coalescent methods, and sought to determine the effects of population size on patterns of recessive or nearly recessive unconditionally deleterious mutations. I found that migration was overall very low but that it was difficult to reliably distinguish differences among populations based on estimates of effective size. Agreement of point estimates of effective size, Nei's gene diversity, and habitat patch area and census size indicate that in this system, differences in census size represent a reasonable proxy for difference in long term differences in relative population size; populations with more individuals have higher genetic diversity and occupy larger habitat patches than populations with fewer individuals. I performed controlled pollinations and quantified the fitness of the progeny in the greenhouse to examine the effects of population size on the distribution of recessive or nearly recessive deleterious mutations for 16 populations. I found strong (71%) heterosis in small populations only, indicating an accumulation of deleterious mutations fixed by genetic drift. Qualitatively lower inbreeding depression in small populations was also consistent the action of drift in small populations. In chapter 3, I conducted a field transplant experiment to examine if population size was positively associated with mean individual fitness in either the native scrub of novel roadside habitat. I also examined if there was local adaptation between populations in the two habitats, and quantified the pattern of phenotypic selection in each habitat. I found that population size is not associated with mean individual fitness in the native scrub habitat, but is in the roadside habit. A 200% increase in mean individual fitness in the road habitat, suggests that population size might influence the ability to respond to more beneficial growing conditions. I found no evidence of local adaptation between the native and novel habitat types. There was significant phenotypic selection for traits associated with faster growth, earlier reproduction, and larger adult size in both environments, with selection being stronger in the native scrub environment for some traits. In chapter 4, I asked if larger populations had greater quantitative genetic variation for potentially important traits. I quantified population differentiation and broad sense coefficients of genetic variation (CV) for phenotypic traits and examined the relationship of trait means and CV to population size for plants grown in their natural scrub habitat and in the greenhouse. I found limited evidence for reduced quantitative genetic variation in small populations, or depressed trait means for traits related to fitness. I did find consistently positive associations between estimates of Nei's gene diversity and CV for traits measured in their natural habitat. I suggest that reduced plasticity for relevant traits and increased frequency of deleterious mutations could limit the ability of small populations to respond to a novel environment. In sum, I detected an extreme degree of spatial subdivision in which populations of a dozen to a few hundred individuals are completely isolated on scales of just a few hundred meters. Such isolation and small population size appears to result in substantial fixation of recessive to nearly recessive deleterious mutations. Small populations also appear to have a reduced ability to capitalize on more beneficial environmental conditions. I find no effect of population size on levels of quantitative genetic variation, but suggest that the few cases in which populations are exchanging migrants may obscure the expected pattern. My results suggests that interactions among selection, drift, migration, and non-additive genetic architecture are unlikely to facilitate adaptive evolution in this system, but that there is an important role of genetic drift in how evolution proceeds.
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Date Issued: 2011
Identifier: FSU_migr_etd-2442
Format: Thesis

Title: Genetic Interactions in Evolutionary Processes.
Creator: Fierst, Janna L. (Janna Lynn), Houle, David, Bertram, Richard, Beerli, Peter, Hansen, Thomas F., Winn, Alice A., Department of Biological Science, Florida State University
Abstract/Description: Mutation is the origin of biological variation but how that variation is expressed and selected depends on the mapping from genetic variation to phenotypic variation, the effect on fitness, and selection. Until recently, there was little empirical knowl- edge about molecular mechanisms underlying these factors and to simplify theoretical and mathematical models, evolutionary genetic theory represented these processes through additive or average effects. New molecular and phenotypic...
Show moreMutation is the origin of biological variation but how that variation is expressed and selected depends on the mapping from genetic variation to phenotypic variation, the effect on fitness, and selection. Until recently, there was little empirical knowl- edge about molecular mechanisms underlying these factors and to simplify theoretical and mathematical models, evolutionary genetic theory represented these processes through additive or average effects. New molecular and phenotypic technologies have shown that the development and maintenance of most traits involves an underlying system of complex genetic interactions, and that the pattern of interactions influences the dynamics of mutations and trait evolution. In this dissertation, I analyzed the significance of genetic interactions in evolution- ary processes. In Chapter 2, I used analytical and computational modeling to explore the evolution of reproductive isolation through a polygenic trait with an underlying epistatic architecture. I quantified the relationship between epistatic interactions and the accumulation of hybrid incompatibilities, and found that simulated populations experienced suites of compensatory allelic changes that facilitated genetic divergence. Strong epistatic interactions prevented allelic divergence and reproductive isolation. These results indicate that genetic drift is a plausible hypothesis for the evolution of reproductive isolation. In Chapter 3, I modeled the evolution of a sexually dimorphic trait controlled by a genetic regulatory network to explore the relationship between network-level regulation, phenotypic identity and evolvability. Sexually dimorphic characters often change rapidly and while this is thought to be due to the strength of sexual selection xiv acting on the trait, a dimorphic character with an underlying pleiotropic architecture may also influence the evolution of the regulatory network that controls the charac- ter and result in higher evolvability. I tested this with a computational model of a genetic regulatory network and found that sexually dimorphic characters had higher robustness to mutation, evolvability, and conditional evolvability. These results indi- cate that producing two pleiotropically linked characters does not constrain either the production of a robust phenotype or adaptive potential. Instead, the genetic system evolves to maximize both quantities. In Chapter 4, I extended the sexually dimorphic regulatory network model to include sexual selection on the male trait. I studied four sexual selection scenarios- sensory bias, sexual conflict and two representations of the Fisher process- to deter- mine the evolutionary dynamics under each of these hypotheses when the genetic architecture is a complex regulatory network, and to assess the influence of the sex- ual selection scenario on the resulting robustness and evolvability of the underlying network. When the male trait and female preference were determined by a single network, all sexual selection scenarios decreased evolvability and robustness. When the male trait and female preference were determined by different networks, the type of sexual selection determined the link between evolvability and robustness. Under the sensory bias and sexual conflict scenarios, robustness and evolvability were not affected while the two Fisher scenarios resulted in evolved genotypes with reduced evolvability and robustness. These results indicate that sexual selection scenarios have implications for genetic as well as phenotypic evolution.
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Date Issued: 2010
Identifier: FSU_migr_etd-4463
Format: Thesis

Title: Skeletochronology of the American Alligator (Alligator Mississippiensis): Examination of the Utility of Elements for Histological Study.
Creator: Garcia, Bonnie Joan, Erickson, Gregory, Parker, William, Inouye, Brian, DuVal, Emily, Department of Biological Science, Florida State University
Abstract/Description: Neontological studies of reptilian growth are important as they provide a proxy allowing investigation of the life history of extinct relatives. As such, finding modern correlates for bone growth and histological types that can then be used in a skeletochronological capacity are important for unraveling prehistoric mysteries that involve the growth of the extinct relatives of modern taxa. Most skeletochronology studies have focused on the lines of arrested growth, or LAGs, generated in the...
Show moreNeontological studies of reptilian growth are important as they provide a proxy allowing investigation of the life history of extinct relatives. As such, finding modern correlates for bone growth and histological types that can then be used in a skeletochronological capacity are important for unraveling prehistoric mysteries that involve the growth of the extinct relatives of modern taxa. Most skeletochronology studies have focused on the lines of arrested growth, or LAGs, generated in the femur, as femoral size is large enough to study in even small reptiles and round in cross-sectional shape such that growth occurs evenly in all directions in the transverse plane of reference. No single study has yet to section every bone type in the body of an animal. This would allow for exploration of the extent to which other elements preserve the growth record and provide alternative elements to study growth, which may prove useful when the femur is not available. Modern archosaurs such as crocodilians, and in particular the American alligator (Alligator mississippiensis), not only allow for not only an interesting modern system but also provide a proxy for the past given their close evolutionary ties to fossil archosaurs such as dinosaurs. With the goal of generating a histological map of elements useful for aging in archosaurs, I conducted a histological analysis of an alligator previously in the care of the Florida Fish and Wildlife Commission was conducted. A representative of every type of bone was sectioned at multiple points and chemical label counts and tissue type characterizations were made. Aside from major long bone elements commonly used in histological studies, other skeletal structures such as ribs and phalanges exhibit areas of excellent LAG deposition that make them potentially useful in skeletochronologic analysis. From this data a "map" of the alligator skeleton was constructed regarding where along skeletal elements researchers are likely to find unobstructed deposition of LAGs. The results of this study elucidate which bones are best suited for analysis, as well as where along those bones information is preserved. Because histological analysis is a destructive technique, this information will allow researchers to make more informed decisions with regards to which skeletal elements to sample, thus reducing the potential for damaging more elements than is necessary. This will also open the possibility to age partial skeletons in which the femur is missing or unavailable for sampling.
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Date Issued: 2011
Identifier: FSU_migr_etd-4360
Format: Thesis

Title: The Role of Larval Dispersal in the Population Genetics and Ecology of Gag, Mycteroperca Microlepis, in the Gulf of Mexico.
Creator: Jue, Nathaniel Kenneth, Travis, Joseph, Landing, William, Beerli, Peter, Coleman, Felicia, Levitan, Don, Department of Biological Science, Florida State University
Abstract/Description: The role of larval dispersal in ecology and evolution is still largely undescribed in many marine organisms. Larval recruitment has long been viewed as being dominated by unpredictable, highly variable processes but also as a significant driver of population ecology and genetics. Without any knowledge about the directionality and magnitudes of larval recruitment, understanding marine species population dynamics remains a highly uncertain endeavor. Unfortunately, given the need for extensive...
Show moreThe role of larval dispersal in ecology and evolution is still largely undescribed in many marine organisms. Larval recruitment has long been viewed as being dominated by unpredictable, highly variable processes but also as a significant driver of population ecology and genetics. Without any knowledge about the directionality and magnitudes of larval recruitment, understanding marine species population dynamics remains a highly uncertain endeavor. Unfortunately, given the need for extensive management and conservation of many fishery species, this uncertainty can directly affect our ability to implement effective management policy. Gag, Mycteroperca microlepis, in the Gulf of Mexico are a perfect example of this conundrum where larval dispersal appears to be important, but we have a very limited understanding of how it is affecting populations. In this dissertation, I describe a series of studies pointed at describing the role of larval dispersal in patterns of population genetics of gag across the Gulf of Mexico as well as examining its relationship to the population ecology of the system. In Chapter Two, I examined patterns of genetic variation across life stages (adult and juvenile), space, and time to explore the role of larval dispersal in the effective population size of gag. Typically, marine systems are believed to exhibit low effective size to census size ratios due to large variation among individuals in reproductive success. While gag do exhibit this low ratio, there is little evidence to support "sweepstakes"-like reproductive success. I found little support for significant genetic differentiation among post-settlement juveniles both in space and time or for juvenile gag being a sample of a subset of adult genetic diversity. Also, adult gag did not exhibit any consistent spatial bias in contributions to juvenile cohorts. Overall, across the entire West Florida Shelf, juvenile gag appeared to be a well-mixed sample of adult gag offspring. The results suggest that larval dispersal has little effect on the population genetics of gag and the patterns of reproductive success among individuals. In Chapter Three, I examined the hypothesis that populations of gag on Campeche Bank off the Yucatan Peninsula are connected to populations of gag on the West Florida Shelf via larval dispersal. Additionally, I determined whether it was likely that this connectivity was due to on-going or historical gene flow. Ecological evidence suggests on-going connectivity. Genetic evidence showed significant, but low levels, of genetic differentiation between these two regions, consistent with a hypothesis of on-going gene flow among populations. Simulations of gag population genetics supported this hypothesis. Analysis of migration directionality indicated one-way migration from Campeche Bank to the West Florida Shelf, congruent with Gulf Loop Current flow. The timing of population divergences also suggested that current population genetic diversity could not be explained solely by historical population dynamics. These results indicate that population of reef fishes in the Gulf of Mexico may be connected across under-appreciated spatial scales on both ecological- and evolutionary-relevant time scales. In Chapter Four, I integrated population genetics data with population dynamics data to test for the support of data for different models of reproductive success and the role of demographic changes versus skewed reproductive success among individuals contributing to low effective size to census size ratios. Using an individual-based simulation model and Approximate Bayesian Computations, I evaluated different models and parameter sets for reproductive success and age-determined sex change using Bayes Factors to assess which factors are likely contributing to the observed patterns of genetic variation given the population ecology and biology of gag. Data generally supported models of skewed reproductive success among individuals, but the level of skew was not high enough to attribute "sweepstakes"-like variation among individuals in reproductive success to the pattern observed in the data. Instead, changes in demography had a much greater effect on support for models, indicating that documented changes in gag population sex ratio may have a large role in the observed patterns in the data.
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Date Issued: 2010
Identifier: FSU_migr_etd-3719
Format: Thesis

Title: The Role of Environment and Genetics in the Demography of Introduced and Natural Populations of the Endangered Shrub Conradina Glabra.
Creator: Bladow, Jason, Winn, Alice, Underwood, Nora, Mast, Austin, Department of Biological Science, Florida State University
Abstract/Description: Biodiversity continues to decline as many species face extinction. One way to mitigate possible extinction is to introduce new populations of a species to locations that the species does not currently occupy. Such introductions can sometimes fail due to environmental factors or to genetic problems that may arise from small population size. Monitoring introduced populations and collecting data can identify reasons for the success or failure of an introduction and provide valuable information...
Show moreBiodiversity continues to decline as many species face extinction. One way to mitigate possible extinction is to introduce new populations of a species to locations that the species does not currently occupy. Such introductions can sometimes fail due to environmental factors or to genetic problems that may arise from small population size. Monitoring introduced populations and collecting data can identify reasons for the success or failure of an introduction and provide valuable information for future management efforts. To evaluate the success of introductions, demographic models can be constructed and analyzed to determine if populations are growing and to identify parts of the life cycle that contribute most to the population growth rate. Conradina glabra is an endangered shrub that was introduced to three sites at the Apalachicola Bluffs and Ravines Preserve in Liberty County, Florida 1991. Three introduced populations and three natural populations were censused in 2009 and 2010 to construct stage-structured demographic models to project the current growth rates of the introduced populations and to compare them to natural populations. Sensitivity and elasticity analyses were performed to determine the importance of individual rates of growth, survival, and seed production to the overall success of each population. A Life Table Response Experiment analysis was conducted to determine how much differences in each of these vital rates contributed to the observed difference in the growth rate between introduced and natural populations. All of the populations examined were projected to grow or remain stable (λ ≥ 1) indicating that the introduced and natural populations are projected to be successful. As a whole, introduced populations grew faster (λ = 1.052) than natural populations (λ = 1.004). Stasis of large plants, or the proportion of large plants that survived and remained in the same stage class from one year to the next, was the most important vital rate in maintaining the population growth rate for both introduced and natural populations. Greater growth in early life stages and greater fecundity contributed to the greater population growth rate at introduced populations, while increased regression of larger plants to smaller plants and the stasis of young individuals contributed to the lower growth rate of natural populations. A second experiment looked for evidence of inbreeding depression in C. glabra. Flowers in one natural and one introduced population were hand selfed or outcrossed to estimate inbreeding depression in seed set, seed weight, and germination rate. Inbreeding depression in the number seeds produced per flower and percentage seed germination was significant. Outcrossed flowers produced 1.5 seeds on average, compared to 1.04 seeds per flower from selfed crosses. Seeds from outcrossed flowers germinated at a rate of 12% while seeds from selfed flowers germinated at a rate of 4.1%. There was no evidence of inbreeding depression in seed mass. The effects of inbreeding were incorporated into demographic models for the introduced and natural populations that assumed that all seed production was a result of selfing. This hypothetical scenario was compared to the models for unmanipulated populations to examine the potential effects of extreme inbreeding on the demography of C. glabra. When demographic models included the effects of inbreeding depression, the population growth rate became less than one, indicating that populations would decline if they become entirely selfing. At present, all populations are projected to remain stable or grow, although the timeframe over which data were collected was short relative to the lifetime of individuals, and the predicted success of populations is conditional upon the assumption that estimates of the vital rates accurately reflect long-term rates of growth, survival, and reproduction. Inbreeding depression can occur in these populations and is a threat that should be considered when introducing populations, even though at this time, C. glabra populations are growing despite the potential for inbreeding depression.
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Date Issued: 2010
Identifier: FSU_migr_etd-3682
Format: Thesis

Title: Control of Prolactin Secretion by Central Oxytocin in Cervically Stimulated Ovariectomized Rats.
Creator: McKee, De'Nise T., Freeman, Marc E., Logan, Timothy M., Bertram, Richard, Meredith, Michael, Overton, J Michael, Department of Biological Science, Florida State University
Abstract/Description: Prolactin is a protein hormone predominately synthesized in and secreted from cells in the anterior pituitary gland called lactotrophs. Although prolactin is elevated during other physiological states, mating induces a unique pattern of prolactin secretion. Prolactin is elevated twice a day for several days in response to a mating stimulus, suggesting a mnemonic. The neural pathway in which the mating stimulus initiates this unique prolactin secretion is unknown. However, hypothalamic...
Show moreProlactin is a protein hormone predominately synthesized in and secreted from cells in the anterior pituitary gland called lactotrophs. Although prolactin is elevated during other physiological states, mating induces a unique pattern of prolactin secretion. Prolactin is elevated twice a day for several days in response to a mating stimulus, suggesting a mnemonic. The neural pathway in which the mating stimulus initiates this unique prolactin secretion is unknown. However, hypothalamic inhibitory neuroendocrine dopamine neurons and stimulatory oxytocin neurons are demonstrated to be involved. Our laboratory has shown that a peripheral injection of oxytocin initiates prolactin surges like those induced by mating. This suggests that oxytocin may initiate the mating-induced prolactin surges. Whether the bolus injection of oxytocin initiated these surges by acting peripherally or centrally was investigated in this dissertation. Results showed that oxytocin does not initiate these surges by acting peripherally since a peripheral oxytocin antagonist did not block the initiation of the surges. However, peripheral oxytocin is important for maintaining the surges. Therefore, central oxytocin must initiate twice daily prolactin surges, likely from the hypothalamic paraventricular nucleus. Since the mating stimulus must convey its signal from the uterine cervix to the hypothalamus a pathway was investigated. A relay of the mating stimulus via brainstem noradrenergic input to the paraventricular nucleus was shown. Only brainstem noradrenergic neurons that projected to paraventricular nucleus were activated by the mating stimulus, suggesting a specific pathway for the initiation of mating-induced prolactin surges. In addition, norepinephrine was released in the paraventricular nucleus in response to a mating stimulus. Lastly, oxytocin neurons in the hypothalamic paraventricular nucleus were directly investigated. Surprisingly, hypothalamic oxytocin neurons were not acutely activated by a mating stimulus, suggesting that the paraventricular nucleus is not a source of oxytocin important in initiating the mating-induced prolactin surges.
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Date Issued: 2009
Identifier: FSU_migr_etd-2538
Format: Thesis

Title: Modeling Gag (Mycteroperca Microlepis) Population Dynamics to Predict Optimal Reserve Strategy.
Creator: Simmons, Erin Elizabeth, Miller, Thomas E., Coleman, Felicia, Travis, Joseph, Cogan, Nicholas, Department of Biological Science, Florida State University
Abstract/Description: Marine reserves are proposed not only as a management strategy but also as a proxy for un-fished conditions. As fishing pressures have risen over time, scientists have had no way to control for fishing in populations being studied. With the help of models, we can begin to understand the effects of heavy fishing on populations by comparing them with "un-fished reserves" that are simulated by the model. I have done this using the species Mycteroperca microlepis (commonly known as gag) to build...
Show moreMarine reserves are proposed not only as a management strategy but also as a proxy for un-fished conditions. As fishing pressures have risen over time, scientists have had no way to control for fishing in populations being studied. With the help of models, we can begin to understand the effects of heavy fishing on populations by comparing them with "un-fished reserves" that are simulated by the model. I have done this using the species Mycteroperca microlepis (commonly known as gag) to build an explicit, individual-based model that looks at the effects of fishing on population dynamics. In order to better understand how to manage this species, I have created a model that has incorporated factors that have, up to now, been largely ignored: (1) explicit dispersal and connectivity between spawning grounds and seagrass nursery habitat, (2) pre- and post-dispersal density dependence, (3) stage-structured population, (4) spatially explicit and stage-specific movement, (5) stochastic population dynamics, and (6) the exploration of percent, placement, and size of marine reserves both by themselves and coupled with other management techniques. A sensitivity analysis on all parameters was performed using Latin Hypercube Sampling to determine which parameters had the greatest effect as well as which output variables were affected most. I found that (1) fishing exaggerated the skewed population demography of this protogynous hermaphrodite, (2) the effects of fishing had a much greater significant effect on effective population size than total population size, (3) sex ratio in the form of the fertility function did not have a significant effect on population demography, total, or effective population size, (4) spatial distribution has a significant effect, usually as an interaction, on effective population size therefore increasing its susceptibility to fishing pressure, (5) output variables were sensitive to life history aspects including natural mortality, sex change, fecundity, and recruitment, which were all highly correlated with multiple output variables, (6) effective population size was the most sensitive to change as compared to other output variables, and (7) size limit (where age limit was used as a proxy), reserves, and spatial variation in fishing were found to be effective management strategies when used both alone and in suites. This model can be used as a basis to be modified for other species in the Gulf of Mexico by changing life history methods. Overall, this model will be beneficial in better understanding the effect of fishing on the population structure and genetic viability of the population, and for predicting proper management strategies in a manner that is both habitat and species specific.
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Date Issued: 2011
Identifier: FSU_migr_etd-1715
Format: Thesis

Title: Integration of Transpiration Rate, Photosynthesis, Abscisic-Acid Signaling, and Gaurd-Cell Gene Expression.
Creator: Sherdan, Danielle M. (Danielle Marie), Outlaw, William H., Gilmer, Penny J., Bates, George W., Bass, Hank W., Epstein, Lloyd M., Department of Biological Science, Florida State...
Show moreSherdan, Danielle M. (Danielle Marie), Outlaw, William H., Gilmer, Penny J., Bates, George W., Bass, Hank W., Epstein, Lloyd M., Department of Biological Science, Florida State University
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Abstract/Description: Guard cells in the leaf epidermis regulate transpiration rate (E) and carbon dioxide uptake by adjusting stomatal aperture size. Sucrose (suc) is a recent product of photosynthesis that accumulates around guard cells during transpiration. Similarly, abscisic acid (ABA) is a potent regulator of guard cell physiology that can be transported to guard cells via the transpiration stream. I tested the hypothesis that suc that accumulates around guard cells as a result of transpiration affects...
Show moreGuard cells in the leaf epidermis regulate transpiration rate (E) and carbon dioxide uptake by adjusting stomatal aperture size. Sucrose (suc) is a recent product of photosynthesis that accumulates around guard cells during transpiration. Similarly, abscisic acid (ABA) is a potent regulator of guard cell physiology that can be transported to guard cells via the transpiration stream. I tested the hypothesis that suc that accumulates around guard cells as a result of transpiration affects levels of messenger ribonucleic acids (mRNAs) in guard cells. I present preliminary investigations of a similar hypothesis for ABA.The hypothesis was tested with three approaches. One, sucrose was fed to leaves via the petiole. Two, guard cells were incubated in solutions of suc and/or ABA. Three, relative humidity (RH) was increased to decrease E. As expected, E and the rate of suc delivery to guard cells were around 4 fold and 3 7 fold lower, respectively, under high RH (90%) conditions than under control (60% RH) conditions. I measured mRNA levels in guard cells and leaves of Vicia faba of genes encoding protein products involved in sugar transport or metabolism and one mRNA induced in guard cells by ABA. Selected mRNAs encode a plasma membrane localized H+ suc symporter, a plasma membrane localized H+ monosaccharide symporter, a small subunit of ribulose 1, 5 bisphosphate carboxylase/oxygenase, a suc phosphate synthase, a suc synthase, a cell wall invertase, or a small subunit of adenosine diphosphate glucose pyrophosphorylase. I found that E linked accumulation of solutes in the guard cell apoplast affects mRNA levels of some sugar and ABA regulated genes. Our laboratory surmises that suc, ABA, and other signals found in the transpiration stream that are present at sub threshold levels in the transpiration stream, accumulate to threshold levels around guard cells, where they induce signal transduction. Signal transduction in these experiments was measured by changes in mRNA levels. Interestingly, E induced changes in mRNA levels measured during the first day following a switch to high RH growth conditions returned to control levels after several days in high humidity, possibly indicating acclimation, at the molecular level, to changes in E.
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Date Issued: 2009
Identifier: FSU_migr_etd-1760
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: Minichromosome Maintenance Protein Dynamics Throughout the Cell Cycle.
Creator: Kuipers, Marjorie A. (Marjorie Ann), Gilbert, David M., Gunjan, Akash, Bass, Hank W., Epstein, Lloyd M., Dennis, Jonathan H., Department of Biological Science, Florida State...
Show moreKuipers, Marjorie A. (Marjorie Ann), Gilbert, David M., Gunjan, Akash, Bass, Hank W., Epstein, Lloyd M., Dennis, Jonathan H., Department of Biological Science, Florida State University
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Abstract/Description: Replicative DNA polymerases require a ssDNA template, yet polymerases themselves lack dsDNA unwinding activity (Kawasaki and Sugino, 2001). Enter the helicase, a motor protein that uses NTP-dependent conformational changes to unwind duplex DNA by separating two annealed nucleic acid strands by breaking the hydrogen bonds between them. The heterohexameric complex known as the Mcm2-7 (minichromosome maintenance) complex has been presumed, for the last 15 years, to serve as this helicase in...
Show moreReplicative DNA polymerases require a ssDNA template, yet polymerases themselves lack dsDNA unwinding activity (Kawasaki and Sugino, 2001). Enter the helicase, a motor protein that uses NTP-dependent conformational changes to unwind duplex DNA by separating two annealed nucleic acid strands by breaking the hydrogen bonds between them. The heterohexameric complex known as the Mcm2-7 (minichromosome maintenance) complex has been presumed, for the last 15 years, to serve as this helicase in mammalian cells (Bochman and Schwacha, 2009), with recent recombinant in vitro experiments demonstrating that Mcm2-7 unwinds DNA, either alone or within the context of a larger complex (Bochman and Schwacha, 2008). To date, however, only data from studies done in non-physicolical settings exist concerning the mammalian Mcm2-7 proteins; they have never before been studied in a live cell mammalian setting, and so the true nature of Mcm2-7's interaction with chromatin has not been clear. Is it dyanamic like other replication associated proteins (Leonhardt et al., 2000; McNairn et al., 2005; Xouri et al., 2007), or stable like core histones (Kanda et al., 1998; Kimura and Cook, 2001)? Afterall, both classes of these proteins interact with DNA. Given that Mcm binding to chromatin marks the final step in the formation of the pre-replication complex (pre-RC) in the eukaryotic G1 phase of the cell cycle (a process referred to as "replication licensing" (Prasanth et al., 2004)), and that DNA replication is stringently controlled to ensure replication takes place just once during the cell cycle, some dyanamic shift should take place around the time of late mitosis/early G1, when Mcms load onto chromatin, through S phase, when Mcms clear from chromatin. To test this, we employed Fluoresence Recovery After Photobleaching (FRAP) as a tool to detect chromatin interactions in living cells, and found that the Mcms do show significant biophysical changes in their interactions with chromatin throughout the cell cycle, and that they do not exchange on and off chromatin during S phase.
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Date Issued: 2010
Identifier: FSU_migr_etd-2935
Format: Thesis

Title: Is Malate an Intermediate in the Signal-Transduction Network of Elevated Co2-Induced Stomatal Closure?.
Creator: Jiang, Tianran, Outlaw, William H., Bass, Hank W., Bates, George W., Keller, Laura R., Department of Biological Science, Florida State University
Abstract/Description: Stomata close when plants are exposed to elevated CO2 concentration and they open when plants are exposed to low CO2 concentration. However, molecular elements of the signal transduction network involved in CO2 sensing have not been identified. In addition to the intrinsic CO2 response that guard cells have, it has been proposed that high CO2 evoked malate release from mesophyll cells activates the guard cell anion channel, which initiates ion efflux. In our experimental system (Vicia faba, 3...
Show moreStomata close when plants are exposed to elevated CO2 concentration and they open when plants are exposed to low CO2 concentration. However, molecular elements of the signal transduction network involved in CO2 sensing have not been identified. In addition to the intrinsic CO2 response that guard cells have, it has been proposed that high CO2 evoked malate release from mesophyll cells activates the guard cell anion channel, which initiates ion efflux. In our experimental system (Vicia faba, 3 h from the onset of illumination when stomatal aperture size is increasing), guard cell K+ content tracked the increase in aperture and therefore activation of the anion channel was the cardinal event in stomatal closure. The time course and concentration of malate required to close stomata were studied using epidermal peels (to determine guard cell sensitivity to malate) and petiolar feeding (to infer apoplastic transport of malate to the guard cells). Isolation of the bulk leaf apoplastic malate pool was additionally studied using a novel sequential sampling method. The malate contents of the guard cell symplast and the guard cell apoplast were determined using quantitative histochemical techniques. During the photoperiod, at stomatal aperture size ≤5 μm, guard cell apoplast malate content (maximum = 0.13 pmol, ~ 31 mM) was negatively correlated with stomatal aperture size (R2 = 0.74), which indicates a regulatory role of malate at low apertures. As the essence of this study, the CO2 concentration around whole plants was elevated two fold from nominal pre industrial CO2 concentration (300 μL·L-1). Consistent with previous studies, the bulk leaf apoplastic malate concentration increased (by 2.6 fold) within 30 min of exposure to elevated CO2 concentration. However, stomata closed and the guard-cell K+ contents declined before the modest increase in the guard cell-apoplast malate content. These results do not support the hypothesis that elevated CO2 induced malate release from mesophyll cells initiates stomatal closure, however, an elevated guard cell apoplast malate content could be a contributing factor in the maintenance of stomatal closure under high CO2 concentration.
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Date Issued: 2004
Identifier: FSU_migr_etd-3517
Format: Thesis

Title: The Role of Tadpole Predation in the Habitat Distribution of the Green Treefrog (Hyla Cinerea).
Creator: Gunzburger, Margaret Sarah, Travis, Joseph, Smith, James, James, Frances, Miller, Thomas, Houpt, Thomas, Means, Bruce, Department of Biological Science, Florida State University
Abstract/Description: Understanding the factors that contribute to the distribution of species across habitats allows predictions of the effects habitat alterations on species distributions. Amphibians are important components of wetland ecosystems and are especially susceptible to changes in aquatic and terrestrial habitats. My research focused on the role of predation in the habitat distribution of tadpoles of the green treefrog, Hyla cinerea. Predation pressure on H. cinerea tadpoles varies across a gradient of...
Show moreUnderstanding the factors that contribute to the distribution of species across habitats allows predictions of the effects habitat alterations on species distributions. Amphibians are important components of wetland ecosystems and are especially susceptible to changes in aquatic and terrestrial habitats. My research focused on the role of predation in the habitat distribution of tadpoles of the green treefrog, Hyla cinerea. Predation pressure on H. cinerea tadpoles varies across a gradient of 23 localities in northern Florida. The main division in the predator community is between ponds with and without fish; H. cinerea occurs primarily in permanent ponds with fish. The most effective predators are bass (Micropterus salmoides), odonates (Aeshnidae, Libellulidade), and newts (Notophthalmus viridescens). A predation pressure index constructed from predation rates and predator abundances varies strongly across habitats, but odonates are the most important predators in most habitats and fishless habitats generally have higher predation pressure index values than permanent habitats with fish. Combinations of multiple predator species tend to have similar effects on H. cinerea tadpoles. Tadpole survival with combinations of three predators (M. salmoides, aeshnid and libellulid odonates) was similar in five predator treatments, and slightly higher in the libellulid and aeshnid + libellulid treatments. Comparisons of behavior and predation rates revealed that H. cinerea tend to hide more in response to warmouth (Lepomis gulosis) predation than their sister taxon H. gratiosa. There were not strong differences in survival between these species with warmouth or odonate predators. My research demonstrates that H. cinerea tadpoles are subject to strong variation in predation pressure across the range of habitats in which they breed, and an adaptation to cope with this variation may be the ability to have similar survival with various combinations of multiple predator species. Predation probably plays a role in the habitat distribution of H. cinerea tadpoles because tadpole predation pressure tends to be lower in the habitats in which H cinerea typically choose for breeding. Other factors which may result in H. cinerea choosing permanent habitats include adult habitat selection and other factors influencing tadpole survival, such as competition and abiotic effects.
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Date Issued: 2004
Identifier: FSU_migr_etd-3913
Format: Thesis

Title: Structure Variations in Lipoxygenase: Mutagenesis of Soybean Lipoxygenase-1.
Creator: Imber, Ann Nicole, Gaffney, Betty J., Taylor, Kenneth A., Bass, Hank W., Department of Biological Science, Florida State University
Abstract/Description: The focus of this study is a prototypical member of the lipoxygenase protein family (acc. PF00305), soybean lipoxygenase-1 (LOX-1). I am interested in two aspects of this enzyme: a comparison of its structure and sequence with lipoxygenase enzymes and the overall shape of the active site cavity with respect to catalysis. The first topic is primarily focused on an unusual member of the lipoxygenase protein family that binds manganese instead of iron, manganese lipoxygenase (MnLO). In order to...
Show moreThe focus of this study is a prototypical member of the lipoxygenase protein family (acc. PF00305), soybean lipoxygenase-1 (LOX-1). I am interested in two aspects of this enzyme: a comparison of its structure and sequence with lipoxygenase enzymes and the overall shape of the active site cavity with respect to catalysis. The first topic is primarily focused on an unusual member of the lipoxygenase protein family that binds manganese instead of iron, manganese lipoxygenase (MnLO). In order to perform mutational studies on structural regions of importance in LOX-1, the sequence of LOX-1 will be compared to the sequence of MnLO. In addition, the known structure of LOX-1 will be used as a platform to model features suggested by MnLO. This is possible because of the considerable sequence identity within the conserved motifs of lipoxygenase family members. The results of this approach include implications for the metal selectivity of lipoxygenase enzymes and the structural significance of the highly conserved ð-helix found in the metal binding region. The second topic centers on the origin of the positional and stereochemical specificity of the lipoxygenase catalytic products. Members of the lipoxygenase family catalyze the insertion of molecular oxygen to their long-chain fatty acid substrates in a highly specific manner. The structural rationale for this chemical specificity is implicated in the overall shape of the active site cavity. A stereodeterminant influencing the cavity shape was recently identified as a single residue conserved as a Gly in R- specific lipoxygenases and an Ala in S- specific enzymes. By performing mutagenesis on this residue in the LOX-1 enzyme and evaluating its EPR spectra, it is possible to monitor a catalytically relevant change in the lipoxygenase active site cavity that is propagated to the metal ion. Thus, this study proposes to examine key variations in the lipoxygenase amino acid sequence, especially in terms of enzyme catalysis and the details of the active site cavity and its metal binding site.
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Date Issued: 2006
Identifier: FSU_migr_etd-3882
Format: Thesis

Title: Gene Expression in Leaves, Mesophyll Tissues and Guard Cells of Arabidopsis Thaliana and the Effect of Sucrose.
Creator: Chattopadhyay, Maitreyi, Bates, George W., Bass, Hank W., Jr., William H. Outlaw, Department of Biological Science, Florida State University
Abstract/Description: Sugar, the end product of photosynthesis, not only plays an important role in carbon and energy metabolism and in polymer biosynthesis, it also functions as a hormone like signaling molecule that regulates the expression of many genes involved in growth, development and resource allocation in plants. This study focuses on the gene expression profiles in leaves and guard cells of the model plant Arabidopsis thaliana and the response of these genes to sugar. Guard cells are specialized cells...
Show moreSugar, the end product of photosynthesis, not only plays an important role in carbon and energy metabolism and in polymer biosynthesis, it also functions as a hormone like signaling molecule that regulates the expression of many genes involved in growth, development and resource allocation in plants. This study focuses on the gene expression profiles in leaves and guard cells of the model plant Arabidopsis thaliana and the response of these genes to sugar. Guard cells are specialized cells that flank each stoma on the surface of the leaf epidermis. Guard cells regulate stomatal aperture thereby controlling the rate of carbon dioxide uptake during photosynthesis and the rate of water loss during transpiration. During stomatal opening, solute content within guard cells builds up due to the uptake of ions and other solutes from outside of the cell. Solute content also builds up by intercellular solute production within guard cells. As a result water is driven into guard cells osmotically, turgor pressure within the cells increases, the guard cells swell, and stomatal aperture increases in size. Stomatal closure is initiated with the dissipation of these solute gradients. Guard cell chloroplasts cannot perform significant amounts of photosynthetic carbon fixation due to a low activity of the enzyme rubisco. Guard cells also lack functional plasmodesmata. Therefore, guard cells depend on extracellular sugars (specifically sucrose). Under the conditions of high light intensity and high transpiration rates, sucrose is swept by the transpiration stream from the mesophyll cells and is deposited in the cell wall of guard cells. Delivery of sucrose into guard cells via sucrose transporters, or breakdown of the sucrose by different sucrose cleaving enzymes on the cell wall and transport of those hexoses into cell, provides the energy needed for guard cell metabolism and may also have a signaling role in the swelling and shrinking of guard cells. This study examines gene expression profiles in leaves, mesophyll cells, and in guard cells, and the transcriptional responses of these genes to sucrose. The ultimate goal of the study was to demonstrate the feasibility of analyzing gene expression in small samples of individually dissected guard cells (about 40-50 guard cells per sample) of Arabidopsis. The panel of genes studied here included several genes preferentially expressed in guard cells (monosaccharide symporter 1 (STP1), trehalose phosphate synthase 1 (TPS1), protein phosphatase 2C (HAB1), potassium inward channel (KAT1) and ADP-glucose pyrophosphorylase (ADPGase)), a gene that should be preferentially expressed in mesophyll cells (ribulose bisphosphate carboxylase, RBCS), two genes inovled in sucrose transport (sucrose transporters 1 and 2, SUC1 and SUC2), and three control genes (Actin-2, ACT2, elongation factor 1a, EF1, and Cyclophilin, CYP). Gene expression was assayed by quantitative real-time PCR (Q-PCR). In an initial experiment gene expression was analyzed in Arabidopsis leaf strips that were incubated in either 150 mM sucrose or mannitol for 5 hours. This work established the gene expression profile in leaves and showed that the expression of ACT2, HAB1, and KAT1 were up regulated by sucrose, and RBCS was down regulated. RBCS was the most highly expressed of all the genes assayed in leaf strips, followed by SUC1, then STP1, HAB1, and SUC2. CYP, ADPGase, KAT1, and TPS1 were all expressed at low levels in leaf strips. Next, small samples (approximately 10 μg) of mesophyll tissues were microdissected from freeze-dried Arabidopsis leaf strips that had been treated with sucrose or mannitol. RNA was isolated and analyzed by Q-PCR. The mesophyll cells gave a similar profile of gene expression to that observed in the leaf strips. Because of inter sample variation, and because this experiment was done in duplicate rather than triplicate, it was not possible to document sucrose induced changes in gene expression in the mesophyll tissues. Next, the mesophyll RNA samples were diluted 100 and 1000 fold, the RNA was amplified using T7 RNA polymerase, and transcript levels were determined by Q-PCR. The amplified RNA samples gave gene expression profiles that were very similar to that in the unamplified RNA from mesophyll cells. This result indicates the reliability of RNA amplification using T7 RNA polymerase. Finally, samples of guard cells were microdissected from freeze-dried Arabidopsis leaves (50 guard cells per sample), RNA was isolated, amplified using T7 RNA polymerase, and transcript levels were determined by Q-PCR. The gene expression profile observed in the guard cells was quite different from what was found in leaf strips and in mesophyll cells. In guard cells STP1 and KAT1 had the highest level of expression relative to ACT2. TPS1, HAB1, ADPGase, and CYP were all expressed at higher levels in guard cells than in mesophyll cells. SUC2 expression was very low in guard cells, but surprisingly RBCS transcripts levels were moderately high in the guard cell samples. To date very little work has examined changes in gene expression in guard cells because of the difficulty of isolating sufficient numbers of these cells. This study documents the feasibility of studying gene expression in guard cells by manually microdissecting them out of free dried leaves and then amplifiying the RNA with T7 RNA polymerase to give sufficient transcript levels for Q-PCR analysis. This approach opens the possibility of studying the role of gene expression in stomatal movements.
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Date Issued: 2007
Identifier: FSU_migr_etd-3872
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: Smooth Muscle Titin Interactions with Alpha-Actinin.
Creator: Chi, Richard Jue-Hsien, Keller, Thomas C. S., Blaber, Michael, Taylor, Kenneth A., Fajer, Piotr G., Chase, P. Bryant, Department of Biological Science, Florida State University
Abstract/Description: The striated muscle Z-line is a complex network of proteins in which N-terminal domains of titin interact with the actin filament cross-linker alpha-actinin, and other proteins to establish and maintain the structural integrity of the sarcomere. Actin-myosin II filament-based contractile structures in smooth muscle and nonmuscle cells also contain the actin filament-crosslinking protein alpha-actinin. We previously discovered a titin isoform - originally named smitin, hereafter called sm...
Show moreThe striated muscle Z-line is a complex network of proteins in which N-terminal domains of titin interact with the actin filament cross-linker alpha-actinin, and other proteins to establish and maintain the structural integrity of the sarcomere. Actin-myosin II filament-based contractile structures in smooth muscle and nonmuscle cells also contain the actin filament-crosslinking protein alpha-actinin. We previously discovered a titin isoform - originally named smitin, hereafter called sm-titin - in smooth muscle cells suggesting that similar interactions may exist in the alpha-actinin rich dense bodies and dense plaques that act as the smooth muscle equivalent of the Z-line. I have found that purified native smooth muscle alpha-actinin binds with nanomolar affinity to sm-titin in sm-titin-myosin coassemblies in vitro. Smooth muscle alpha-actinin also interacts with striated muscle titin. In contrast to striated muscle alpha-actinin interaction with titin and sm-titin, which is significantly enhanced by PIP2, smooth muscle alpha-actinin interacts with sm-titin and titin equally well in the presence and absence of PIP2. Using expressed regions of smooth muscle alpha-actinin, I have demonstrated sm-titin-binding sites in the smooth muscle alpha-actinin R2-R3 spectrin-like repeat rod domain and a C-terminal domain formed by cryptic EF hand structures. These sm-titin-binding sites are highly homologous to the titin-binding sites of striated muscle alpha-actinin. This suggests that sm-titin contains at least some of the domains found in titin that are known to bind to the alpha-actinin sites. In striated muscle Z-disks, titin N-terminal Z-repeat domains interact with the alpha-actinin EF hand region and the titin Zq domain interacts with the alpha-actinin central rod. RT-PCR analysis of RNA from various smooth muscle sources and western blot analysis with an N-terminal region-specific antibody presented here reveals that sm-titin contains the alpha-actinin-binding Z repeats Zr1, Zr2, Zr3, and Zr7 and the Zq domain encoded by the titin gene. I investigated whether the sm-titin Zq domain interacts with R2 and R3 spectrin repeat-like domain loops that lie in proximity on the surface of the smooth muscle alpha-actinin central rod. I found alanine and phosphomimetic mutations of alpha-actinin R2 and R3 loop residues decreased binding to expressed sm-titin Zq domain in GST-pull-down and solid phase binding assays. Likewise, surface plasmon resonance experiments revealed that alanine mutagenesis of a Zq domain region with high propensity to form alpha-helix decreased binding to the alpha-actinin R2-R3 region. Additionally, the Zq peptide migrates on FPLC size exclusion chromatography as an apparent dimer. I present a model of how the sm-titin Zq domain, which may form an anti-parallel homodimer, could interact with mirror image sites formed by R2 and R3 loops in the smooth muscle alpha-actinin central rod domain. Taken together, our results suggest that direct interaction between alpha-actinin and titin or titin isoforms is a common feature of actin-myosin II contractile structures in striated muscle and smooth muscle cells and that the molecular bases for alpha-actinin interaction with these proteins are similar, although regulation of these interactions may differ according to tissue.
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Date Issued: 2007
Identifier: FSU_migr_etd-3821
Format: Thesis

Title: Female Rats Show Greater Sensitivity to High-Strength Magnetic Fields: Role of Vestibular System and Estrogen.
Creator: Cason, Angela Marie, Houpt, Thomas A., Eckel, Lisa A., Freeman, Marc E., Gaffney, Betty J., Smith, James C., Department of Biological Science, Florida State University
Abstract/Description: This dissertation discusses the investigation of sex differences in the response to magnetic field (MF) exposure and the role of the vestibular system and estrogen in the effects of MFs. Advances in magnetic resonance imaging are driving the development of higher resolution machines equipped with high-strength static MFs. The behavioral effects of high-strength MFs are largely uncharacterized. Our laboratory has previously shown that following high-strength static magnetic field (MF) exposure...
Show moreThis dissertation discusses the investigation of sex differences in the response to magnetic field (MF) exposure and the role of the vestibular system and estrogen in the effects of MFs. Advances in magnetic resonance imaging are driving the development of higher resolution machines equipped with high-strength static MFs. The behavioral effects of high-strength MFs are largely uncharacterized. Our laboratory has previously shown that following high-strength static magnetic field (MF) exposure, male rats suppress normal rearing behavior, walk in circles, acquire a conditioned taste aversion (CTA), and express c-Fos in vestibular and visceral relays of the brainstem. These results suggest that the MF may interact with the vestibular system producing effects similar to rotation or motion sickness. Here, I provide evidence for sex differences in the response to MF-exposure by examining how fluctuations in endogenous estrogen across the female rat's estrous cycle and removal of the ovaries and subsequent hormone replacement influence the behavioral response to MF-exposure compared to male rats. I then provide evidence that the inner ear is required for both the behavioral and neural response to MF-exposure by showing that bilateral removal of the inner ear by chemical labyrinthectomy eliminates MF-induced CTA and MF-induced c-Fos expression in vestibular and visceral relays. I also demonstrate that estrogen influences the amount of c-Fos expression in the vestibular and visceral relays. Compared to ovariectomized rats, chronic estrogen replacement decreased c-Fos expression in the LC and SuV and reversed significant hemispheric asymmetries in other vestibular and visceral relays suggesting potential neural sites where estrogen could interact to influence the behavioral response to MF exposure. In addition, I provide evidence that the estrogen receptor subunits are present within specific areas of the vestibular and visceral relays that are responsive to MF-exposure and could potentially mediate the effects of estrogen.
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Date Issued: 2006
Identifier: FSU_migr_etd-4079
Format: Thesis

Title: Development of a DNA Probe Assay for Rare Transfer RNAs and Its Use in Measuring the Expression of the ArgX Gene in Escherichia Coli.
Creator: Cavnar, Katie, Reeves, Robert H., Keller, Thomas, Epstein, Lloyd, Department of Biological Science, Florida State University
Abstract/Description: In Escherichia coli, the argX gene is the first gene in a cluster with three other tRNA genes. The tRNA products of the three downstream genes, hisR, leuT and proM are abundant in E. coli, and all four of the tRNAs are processed from the same precursor RNA transcript. The argX promotor appears to be a strong promotor and is preceded by an Up element. By all accounts, the gene product of argX, tRNAarg3 should be a major tRNA. Like the three downstream genes in this cluster, argX must be...
Show moreIn Escherichia coli, the argX gene is the first gene in a cluster with three other tRNA genes. The tRNA products of the three downstream genes, hisR, leuT and proM are abundant in E. coli, and all four of the tRNAs are processed from the same precursor RNA transcript. The argX promotor appears to be a strong promotor and is preceded by an Up element. By all accounts, the gene product of argX, tRNAarg3 should be a major tRNA. Like the three downstream genes in this cluster, argX must be transcribed at a high level because of its location at the beginning of the gene cluster. However, tRNAarg3 is considered a rare tRNA in E. coli. Its low level is correlated with the low codon usage of its corresponding codon, CGG. It appears that the nucleotide sequence just upstream of argX can base pair with the 5'- end of the tRNA. This alternative secondary structure in the precursor RNA transcript may prevent the maturation of the tRNAarg3 by disrupting the base pairing of the aminoacyl-tRNA stem. This structure is required by ribonuclease P for maturation of the 5'-ends of all tRNAs and also prevents the 3'-exonucleases from digesting into the tRNA from the CCA (3'-) end. Different constructs of the argX gene have been cloned into high copy number plasmids. These constructs differ in the nucleotides just upstream of the argX gene. Deletion of nucleotides results in higher expression of tRNAarg3 and the loss of an alternative secondary structure involving the 5'–end of the tRNA. The data suggests alternative secondary structure formation during transcription may in part be responsible for tRNAarg3 abundance in E. coli. A tRNA assay has been developed to measure the levels of rare tRNAs from extracts. This assay is both sensitive and specific and demonstrates its ability to detect low levels of tRNAs.
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Date Issued: 2004
Identifier: FSU_migr_etd-4015
Format: Thesis

Title: Troponin Complex Conformation Changes Upon Calcium Binding and Phosphorylation.
Creator: Chamoun, Jean Elias, Fajer, Peter G., Logan, Timothy M., Chase, P. Bryant, Keller, Thomas C. S, Hurt, Myra M., Department of Biological Science, Florida State University
Abstract/Description: Striated muscle contraction is regulated by troponin complex which consist of three subunits: troponin C (TnC) binds to calcium, troponin I (TnI) inhibits actomyosin ATPase activity and troponin T (TnT) anchors the complex to the thin filament. An increase in calcium concentration induces conformational changes in TnI and TnC. These changes are believed to result in the repositioning of tropomyosin (Tm), exposing myosin binding sites on actin and eventually leading to force production. The N...
Show moreStriated muscle contraction is regulated by troponin complex which consist of three subunits: troponin C (TnC) binds to calcium, troponin I (TnI) inhibits actomyosin ATPase activity and troponin T (TnT) anchors the complex to the thin filament. An increase in calcium concentration induces conformational changes in TnI and TnC. These changes are believed to result in the repositioning of tropomyosin (Tm), exposing myosin binding sites on actin and eventually leading to force production. The N-terminal extension (30-33 a.a) of the cardiac TnI subunit contains two PKA phosphorylation sites (serine 23/24). In the ON-state (+Ca2+), the position of the cTnI switch peptide (150-159) is in close proximity to the N-lobe of TnC whereas in the OFF-state (-Ca2+) its location is unknown. One of many possibilities is that the peptide is close to the coiled-coil region of cTnT (226-275) and cTnI (90-136). We designed mutants with labels on the N-lobe of TnC, the TnI/TnT coiled coil and at either end of the switch peptide to probe the ON/OFF states conformational changes of the switch peptide within the troponin complex. Conventional dipolar EPR and Double Electron-Electron Resonance (DEER) were used for distance measurements between the different mutants in the reconstituted troponin complex. For the ON-state the measured distance is in agreement with the crystal structure (Takeda et al. 2003). In the OFF-state we concluded that the switch peptide is found at two different positions a) close to the N-lobe of TnC and b) close to the TnI/TnT coiled-coil. Cardiac muscle regulation is modulated by the phosphorylation of serines (23/24) in the N-terminal extension. A model of the bisphosphorylated N-terminal extension suggests that phosphorylation induces the movement of the N-terminal extension toward the inhibitory region. We measured the relative distance in both unphosphorylated and bisphosphorylated states between N-terminal extension and the inhibitory region (TnI9/142) in the presence of calcium. Our results showed no distance change between un-&-bisphosphorylated states of TnI9/142.
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Date Issued: 2008
Identifier: FSU_migr_etd-3924
Format: Thesis

Title: Octamer Formation and Stability in a Mitochondrial Creatine Kinase from a Protostome Invertebrate.
Creator: Hoffman, Gregg G., Ellington, W. Ross, Chapman, Michael S., Chase, P. Bryant, Moerland, Timothy S., Roux, Kenneth H., Department of Biological Science, Florida State University
Abstract/Description: The free energy yield from ATP hydrolysis is directly related to the [ATP]/ ([ADP] [Pi]) ratio within any given cell, and it has long been clear that cells can not maintain functionality when this ratio undergoes a significant change. This energetic challenge is most pronounced within the cells of excitable tissues with high and variable energy demands, cells such as cardiomyocytes, neurons, transport epithelia, and primitive free swimming spermatozoa. It is within these very cells that the...
Show moreThe free energy yield from ATP hydrolysis is directly related to the [ATP]/ ([ADP] [Pi]) ratio within any given cell, and it has long been clear that cells can not maintain functionality when this ratio undergoes a significant change. This energetic challenge is most pronounced within the cells of excitable tissues with high and variable energy demands, cells such as cardiomyocytes, neurons, transport epithelia, and primitive free swimming spermatozoa. It is within these very cells that the creatine kinase (CK) system comes into play. CK catalyzes the reversible transfer of the ã-phosphoryl group from MgATP to the guanidine group of creatine, thereby maintaining ÄGATP by buffering the [ATP]/([ADP] [Pi]) ratio when there are temporal and spatial mismatches of ATP supply and ATP demand. Three distinct CK gene families exist – mitochondrial, cytoplasmic and flagellar – each targeted to different intracellular compartments. These genes appear to have evolved at the dawn of the radiation of multicellular animals. As part of on-going efforts to probe the evolutionary physiology in the CK gene family, the cDNAs for mitochondrial CKs from the protostome polychaete Chaetopterus variopedatus (CVMtCK) and chicken cardiac tissue (SarMtCK) have been cloned, inserted into an expression vector and recombinant protein expressed, purified and characterized. Recombinant CVMtCK was primarily octameric as was the well-characterized chicken SarMtCK. Using two independent methods (size-exclusion chromatography and dynamic light scattering, or DLS), studies of oligomerization dynamics showed that CVMtCK exhibited the same reversible transition between octamers and dimers as has been reported for MtCKs from higher organisms, and that these ancient octamers displayed the same dissociation and reassociation profile as that seen in the MtCKs from birds and man under various thermal and concentration regimes. However, the rate of change in both directions is much more rapid for CVMtCK. Interestingly, and perhaps importantly, when CVMtCK was converted to the transition state analog complex (TSAC) in the presence of NO3-, MgADP, and creatine, both size exclusion chromatography and DLS showed that there was minimal dissociation of octamers into dimers while SarMtCK octamers were highly unstable as the TSAC. To evaluate the potential structural correlates of the observed differences in octamer stability, a homology model was developed using the octameric crystal structures of SarMtCK and human ubiquitous UbiMtCK as templates. The resulting model was validated by a variety of on-line tools. Comparison of the structures showed some differences in the interactions occurring across the dimer - dimer interface which are likely to impact the stability of the octameric structure. In all structures, a key and absolutely conserved tryptophan residue is present in this interface. Site-directed mutagenesis procedures were employed to mutate this Trp residue to Cys, Phe, Leu and Tyr. In all cases, the specific activity was unaffected but the recombinant protein was dimeric; no octameric protein was detected using chromatography or DLS. The overall results of this effort show that octamer formation is a primitive character of MtCKs and that there has been some fine-tuning – in an evolutionary sense – of the nature of the interactions promoting and stabilizing the octameric state.
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Date Issued: 2005
Identifier: FSU_migr_etd-3992
Format: Thesis

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