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(1 - 27 of 27)
- Title
- The Afferent Circuitry of the Ventromedial Hypothalamus and Its Activation in Paternal Behavior of the Socially Monogamous Prairie Vole.
- Creator
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Rogers, Richard S., Department of Chemistry and Biochemistry
- Abstract/Description
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Paternal behavior is an interesting and important research topic due to its integral contribution to the fitness and well-being of multiple species, including humans. Although paternal behavior is well described in literature, attempts at neurobiological characterization have yielded conflicting results that fail to address brain region interconnectivity. This study was designed to evaluate the relationship between afferent VMH circuitry and the onset of paternal behavior, using the prairie...
Show morePaternal behavior is an interesting and important research topic due to its integral contribution to the fitness and well-being of multiple species, including humans. Although paternal behavior is well described in literature, attempts at neurobiological characterization have yielded conflicting results that fail to address brain region interconnectivity. This study was designed to evaluate the relationship between afferent VMH circuitry and the onset of paternal behavior, using the prairie vole (Microtus ochrogaster) model. Sexually naïve male prairie voles received injections of the retrograde neurotracer Fluoro-Gold (FG), into the VMH. Two weeks later, subjects were exposed to either conspecific pups, contained within a tea-ball, or an empty tea-ball (control) for 1 hr. Immunohistochemical labeling was conducted for both FG and the neuronal activity marker Egr-1, in order to evaluate neuronal and afferent pathway activation between the ventromedial hypothalamus (VMH) and the amygdala (AMYG), bed nucleus of the stria terminalis (BNST), lateral septum (LS) and ventral tegmental area (VTA). Similar to the pathway implicated in the onset of maternal behavior, the results of this study showed pup exposure-induced neuronal activation in the AMYG and BNST, particularly in the efferent pathways from these two brain areas to the VMH. This effect was not found in the LS and VTA projection neurons to the VMH. Together, the data suggests a brain region-specific neuronal activation by pup exposure in particular brain circuitry, implicating its possible involvement in paternal behavior.
Show less - Date Issued
- 2015
- Identifier
- FSU_migr_uhm-0545
- Format
- Thesis
- Title
- Assessment of Synthetic Matrix Metalloproteinase Inhibitors by Fluorogenic Substrate Assay.
- Creator
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Lively, Ty J., Department of Chemistry and Biochemistry
- Abstract/Description
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Matrix metalloproteinases (MMPs) are a family of metzincin enzymes that act as the principle regulators and remodelers of the extracellular matrix (ECM). While MMPs are involved in many normal biological processes, unregulated MMP activity has been linked to many detrimental diseases, including cancer, neurodegenerative diseases, stroke, and cardiovascular disease. To develop tools to investigate MMP functions and potential new therapeutics, matrix metalloproteinase inhibitors (MMPIs) have...
Show moreMatrix metalloproteinases (MMPs) are a family of metzincin enzymes that act as the principle regulators and remodelers of the extracellular matrix (ECM). While MMPs are involved in many normal biological processes, unregulated MMP activity has been linked to many detrimental diseases, including cancer, neurodegenerative diseases, stroke, and cardiovascular disease. To develop tools to investigate MMP functions and potential new therapeutics, matrix metalloproteinase inhibitors (MMPIs) have been designed, synthesized, and tested to regulate MMP activity. Inhibitor potencies were evaluated in terms of half maximal inhibitory concentrations (IC50 point) and apparent inhibition constants (Kiapp) for a series of YHJ cyclopentane and pyrolidine-based mercaptosulfonamide inhibitors using collagenase (MMPs-1), gelatinase A (MMP-2), matrilysin (MMP-7), and gelatinase B (MMP-9). MMPs with a shallow S1' binding pocket (MMP-1 and -7) were unable to distinguish between inhibitors showing low potency for nearly all synthetic analogs, the exception being GM6001. Conversely, potency levels of inhibitors tested with MMPs with an intermediate S1' pocket (MMP-2 and -9) varied among inhibitor. The most interesting variation occurred with YHJ-6-286 which was more than 30-fold more selective for MMP-2 than MMP-9, despite belonging to the same gelatinase class. To investigate the role stereoselectivity plays in enzyme inhibition, a dye-conjugate of inhibitor YHJ-7-52, YHJ-7-207, was tested for MMP-9. Results gathered suggest that the dye component of YHJ-7-207 produces a significant amount of steric hindrance as inhibition assays against MMP-9 revealed YHJ-7-207 having a larger IC50 point and Kiapp value than YHJ-7-52.
Show less - Date Issued
- 2015
- Identifier
- FSU_migr_uhm-0536
- Format
- Thesis
- Title
- Automated Analysis of Protein Side Chain Spectra.
- Creator
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Hart, Andrew, Department of Chemistry and Biochemistry
- Abstract/Description
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Manual Nuclear Magnetic Resonance (NMR) spectral analysis of proteins is a time intensive effort with methods often specific to each analysis. The method described in this thesis automates the resonance assignment of protein side chains using a TOCSY (Totally Correlated Spectroscopy) NMR experiment. The system under study is Ubiquitin (8.6 kDa). 54 of the 70 available amino acid side chains were identified by a single TOCSY spectrum in less than 5 min of local computer runtime using the...
Show moreManual Nuclear Magnetic Resonance (NMR) spectral analysis of proteins is a time intensive effort with methods often specific to each analysis. The method described in this thesis automates the resonance assignment of protein side chains using a TOCSY (Totally Correlated Spectroscopy) NMR experiment. The system under study is Ubiquitin (8.6 kDa). 54 of the 70 available amino acid side chains were identified by a single TOCSY spectrum in less than 5 min of local computer runtime using the algorithms described. Automation of spectral analysis can enhance reproducibility and create standards of spectral analysis.
Show less - Date Issued
- 2013
- Identifier
- FSU_migr_uhm-0244
- Format
- Thesis
- Title
- Biophysical Characterization of a ssDNA Virus.
- Creator
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Morrison, Anneliese J., Department of Chemistry and Biochemistry
- Abstract/Description
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Viral capsids must assemble into stable structures that resist dissociation in extreme environments between hosts yet they simultaneously must be unstable enough to release their genome upon infection. The conflicting functions that the viral capsid must fulfill suggests that they exhibit an evolutionarily fine-tuned structure/function relationship that is not apparent in many other systems. Biophysical characterization of viral assembly and disassembly processes can aid in developing an...
Show moreViral capsids must assemble into stable structures that resist dissociation in extreme environments between hosts yet they simultaneously must be unstable enough to release their genome upon infection. The conflicting functions that the viral capsid must fulfill suggests that they exhibit an evolutionarily fine-tuned structure/function relationship that is not apparent in many other systems. Biophysical characterization of viral assembly and disassembly processes can aid in developing an understanding of the physical mechanisms that underlie the relationship between tightly linked phenotypes in complex protein systems. In this honor's thesis project, the dissociation process was biophysically characterized in an ssDNA bacteriophage. After the development of a PEG precipitation based purification method, intrinsic fluorescence spectroscopy, static light scattering, and plaque assays were used to develop a two-step model that describes the molecular events that occur during Microvirid bacteriophage capsid dissociation. At 57˚C using a scan rate of 1˚C/min, loss of 99% of viral activity is observed corresponding to loss of the major spike protein. At 69˚C transitions are seen in fluorescence and light scattering spectra that indicate a structural change is occurring. Plaque assays confirm that immediately after the structural transition occurs all viral activity is lost, indicating that this second step represents global capsid dissociation.
Show less - Date Issued
- 2014
- Identifier
- FSU_migr_uhm-0428
- Format
- Thesis
- Title
- The Characterization of the Conformational Changes of Sar1 upon Activation.
- Creator
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CreveCoeur, Travis, Stagg, Scott, Bhattacharya, Nilakshee, Wood-Cohan, Nathaniel, Department of Chemistry and Biochemistry
- Abstract/Description
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Vesicle transport is an essential function for eukaryotes in the transfer of molecular cargo throughout the cell. Studying the formation of vesicles is key in understanding eukaryotic cell biology. Coat Protein Complex II facilitates exocytic vesicle formation from the endoplasmic reticulum to the Golgi apparatus, where cargo is further modified. Sar1, a subunit of the COPII coat and GTPase, is involved in the budding and fission of vesicles through interactions between its amphipathic N...
Show moreVesicle transport is an essential function for eukaryotes in the transfer of molecular cargo throughout the cell. Studying the formation of vesicles is key in understanding eukaryotic cell biology. Coat Protein Complex II facilitates exocytic vesicle formation from the endoplasmic reticulum to the Golgi apparatus, where cargo is further modified. Sar1, a subunit of the COPII coat and GTPase, is involved in the budding and fission of vesicles through interactions between its amphipathic N-terminal α-helix and the ER. My project aims to characterize how Sar1 physically changes its conformation upon activation. This is observed through Site-Directed Spin Labeling analyses and Electron Paramagnetic Resonance. This will aid in the determination of the physical distance the N-terminal α-helix moves, which will ultimately shed light on the mechanism of Sar1 insertion into lipid membranes. Since spin labels exclusively attach to cysteine residues, mutations to Sar1 were either inserted or removed at selected residues. Manipulations to Leu181, Ser14, and Cys102 residues provided insight to the conformational change of Sar1 upon activation. The majority of my work focused on determining optimal conditions to obtain soluble Sar1 protein. Although different conditions were tested, using M9 minimal media to grow bacterial cells and inducing protein expression at cold temperatures proved optimal for some mutants.
Show less - Date Issued
- 2013
- Identifier
- FSU_migr_uhm-0250
- Format
- Thesis
- Title
- CRISPR-Cas9 Utility in Genome Engineering.
- Creator
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McCullers, Michelle R., Department of Biological Science
- Abstract/Description
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The field of genomic engineering and manipulation has made great strides in recent years with the development of genome-altering techniques to alleviate disease by flexing control on an epigenetic scale. Facioscapulohumeral muscular dystrophy (FSHD) poses a series of points within its pathophysiology where it is possible to examine the utility of these manipulation techniques. This paper specifically focuses on how three approaches can be applied to ultimately stop the expression of the full...
Show moreThe field of genomic engineering and manipulation has made great strides in recent years with the development of genome-altering techniques to alleviate disease by flexing control on an epigenetic scale. Facioscapulohumeral muscular dystrophy (FSHD) poses a series of points within its pathophysiology where it is possible to examine the utility of these manipulation techniques. This paper specifically focuses on how three approaches can be applied to ultimately stop the expression of the full length double homeobox 4 DUX4 gene transcript which is thought to be responsible for the upper body muscular atrophy exhibited in most FSHD cases. With this information, we can surmise what the future holds for epigenetics, including the purpose of repetitive DNA, the role of epigenetics in disease manifestation, and how to apply new genetic engineering techniques in creative ways.
Show less - Date Issued
- 2015
- Identifier
- FSU_migr_uhm-0450
- Format
- Thesis
- Title
- The Effects of Nicotine on Neurotrophic Factor Expression in the Adult Male Zebra Finch.
- Creator
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Peoples, Jessica, Chemistry and Biochemistry
- Abstract/Description
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This thesis examines the effects of nicotine on the expression of Brain- Derived Neurotrophic Factor (BDNF) in the brain of adult male zebra finches (Taeniopygia guttata) under isolated and social housing conditions. BDNF, which is essential for survival, growth, and neuroplasticity of neurons, is significantly decreased in patients with affective disorders, such as depression and schizophrenia. A significant number of these patients use tobacco products, which induce an increase in plasma...
Show moreThis thesis examines the effects of nicotine on the expression of Brain- Derived Neurotrophic Factor (BDNF) in the brain of adult male zebra finches (Taeniopygia guttata) under isolated and social housing conditions. BDNF, which is essential for survival, growth, and neuroplasticity of neurons, is significantly decreased in patients with affective disorders, such as depression and schizophrenia. A significant number of these patients use tobacco products, which induce an increase in plasma BDNF levels. Isolated rodents exposed to nicotine showed an increase in central BDNF levels. The zebra finch is an established model to study cognitive functioning, and as such we examined how nicotine interacts with BDNF expression in zebra finch brain areas involved in cognitive functioning such as the song nuclei and the hippocampal area. Adult male zebra finches were exposed to nicotine and the expression of BDNF was examined between isolated and social housed male zebra finches, using immunocytochemistry. The results show that the housing conditions did not have an effect on the expression of BDNF in the examined song nuclei or the hippocampal area. However, nicotine induced an increased expression of BDNF in the song nuclei HVC, RA (Robust Nucleus of the Archistriatum), and Area X. The HVC and RA contain nicotinic acetylcholinergic receptors, which could explain our finding. Area X is involved in song learning, which is not applicable to our animals, as adult animals have a crystallized pattern. The results could be explained by the fact that Area X might be under direct control of the HVC.
Show less - Date Issued
- 2012
- Identifier
- FSU_migr_uhm-0069
- Format
- Thesis
- Title
- The Evaluation of Industrial Methods of Size-Exclusion Chromatography (SEC) of Difficult-to-Dissolve Polymers.
- Creator
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McNeel, Kelsey, Chemistry
- Abstract/Description
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In experiments presented here, well-characterized polystyrene and poly(methyl methacrylate) standards were analyzed using size-exclusion chromatography and an approach in which the mobile phase differed from the solvent in which the polymer was dissolved. Solvent combinations included a mobile phase in which the polymer was insoluble and a mobile phase that was immiscible with the solvent. It is important to determine the accuracy of molar mass averages and distributions obtained from such...
Show moreIn experiments presented here, well-characterized polystyrene and poly(methyl methacrylate) standards were analyzed using size-exclusion chromatography and an approach in which the mobile phase differed from the solvent in which the polymer was dissolved. Solvent combinations included a mobile phase in which the polymer was insoluble and a mobile phase that was immiscible with the solvent. It is important to determine the accuracy of molar mass averages and distributions obtained from such experiments because the averages and distributions are frequently used in industry to give information about physical properties of the analyte. It is often expensive and time consuming to determine the ideal solvent in which to analyze a polymer, and even more so to purge the system of the previous solvent and condition it with the new one. To circumnavigate these problems, polymers are sometimes dissolved in a known solvent and injected into an instrument containing a chemically different mobile phase, a mobile phase that is already in the instrument. We found that performing experiments using this industrial approach needs to be done with caution. The molar mass averages and distributions can be determined accurately when the mobile phase is a solvent for the polymer and miscible with the solvent, but other cases were less conclusive. Using a mobile phase that is not a solvent for the polymer appears to yield accurate results for low molar mass polymers (<20,000 g/mol) but, in some solvent combinations, yields exclusively solvent peaks. It is likely that the larger polymers precipitate when the miscible solvent and mobile phase mix and the polymers are adsorbed onto the column. Experiment set 5, in which the mobile phase was a solvent for the polymer but was immiscible with the solvent in which the polymer was dissolved, yielded no peaks in these experiments. It is hypothesized that this lack of peaks is a result of enthalpic interactions between the stationary phase, sample solution, and mobile phase. It appears that the elution of the polymer may not be driven by entropic interactions, as is the case in a size-exclusion mechanism, resulting in the co-elution of the polymer and the solvent.
Show less - Date Issued
- 2011
- Identifier
- FSU_migr_uhm-0026
- Format
- Thesis
- Title
- Expression and purification of notch protein and its ligand delta.
- Creator
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Vanderpool, Christopher D.
- Abstract/Description
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The Notch signaling pathway is a cell-to-cell communication system involved in cell fate decisions of multicellular organisms. The Notch receptor is a transmembrane protein embedded in the plasma membrane of the cell. This receptor consists of extracellular and intracellular domains, each with distinct functions. Our lab is interested in the extracellular domain, as its function is to regulate ligand binding through glycosylation. Ligands, Delta and Serrate (commonly referred to as DSL family...
Show moreThe Notch signaling pathway is a cell-to-cell communication system involved in cell fate decisions of multicellular organisms. The Notch receptor is a transmembrane protein embedded in the plasma membrane of the cell. This receptor consists of extracellular and intracellular domains, each with distinct functions. Our lab is interested in the extracellular domain, as its function is to regulate ligand binding through glycosylation. Ligands, Delta and Serrate (commonly referred to as DSL family), bind to the Notch receptor in Drosophila. The binding of two specific regions of receptor and ligand activates the cell-to-cell communication. In Drosophila, epidermal growth factor like-repeats (ELRs) 11 and 12 of the extracellular domain of Notch are required for interaction with ligands Delta and Serrate. These ligands contain an epidermal growth factor like-repeat motif binding domain (EBD) region, which binds to the Notch receptor. The goal of research in the Logan lab is to characterize the biophysical interactions between the Notch receptor and ligands. The focus of my research was to express and purify the ELRs 10-13 of the Notch receptor and EBD region of Delta. The resulting work will complement future research goals of the Logan lab.
Show less - Date Issued
- 2006-05-24
- Identifier
- 160485, FSDT160485, fsu:18933
- Format
- Document (PDF)
- Title
- In Vivo Analysis of Phenotypes Arising from Mutations in Cardiac Troponin C.
- Creator
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Gonzalez-Martinez, David A., Department of Chemistry and Biochemistry
- Abstract/Description
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Mutations in cardiac troponin C (TnC) linked to hypertrophic cardiomyopathies (HCM) and dilated cardiomyopathies (DCM) have predominantly been studied in vitro. Using two genetically engineered mice, one bearing the mutation Ala8Val (KI-TnC-A8V) and the other bearing the mutation Glu40Ala (KI-TnC-E40A) in Troponin C (TnC), this study aims to further our understanding of the changes in Ca2+ sensitivity of the myofilament and the molecular remodeling of Ca2+ handling proteins that results upon...
Show moreMutations in cardiac troponin C (TnC) linked to hypertrophic cardiomyopathies (HCM) and dilated cardiomyopathies (DCM) have predominantly been studied in vitro. Using two genetically engineered mice, one bearing the mutation Ala8Val (KI-TnC-A8V) and the other bearing the mutation Glu40Ala (KI-TnC-E40A) in Troponin C (TnC), this study aims to further our understanding of the changes in Ca2+ sensitivity of the myofilament and the molecular remodeling of Ca2+ handling proteins that results upon the in vivo expression of mutant cardiac TnC. Echocardiogram studies showed a reduced end diastolic volume and end systolic volume in 9 month old KI-TnC-A8V+/+ mice (34.7 ± 3.7µl, 6.5 ± 1.5 µl) when compared to WT mice (53.2 ± 5.5 µl, 1.50 ± 2.6 µl), a characteristic phenotype in HCM while a greater end diastolic and end systolic volume was seen in 6 month old KI-TnC-E40A+/+ mice (97.4 ± 5.0 µl, 61.0 ± 5.6 µl) when compared to WT mice (61.9 ± 2.8 µl, 21.0 ± 2.3 µl), a characteristic phenotype in DCM. After validating these genetically engineered mice as viable models for HCM and DCM, the changes in Ca2+ of the myofilament and maximum force were studied using skinned fiber bundles and the changes in protein expression of Ca2+ handling proteins were characterized using western blot methods thus furthering our understanding of the link between mutations in TnC and the development of HCM and DCM.
Show less - Date Issued
- 2016
- Identifier
- FSU_migr_uhm-0586
- Format
- Thesis
- Title
- Investigating the role of the e(y)1 gene in the Notch signaling pathway.
- Creator
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Alvarado, Francisco, Department of Chemistry and Biochemistry
- Abstract/Description
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Drosophila melanogaster serve as an excellent model to study the highly conserved notch signaling pathway, which is involved in a broad array of developmental events. The Notch signal transduction pathway in Drosophila is vastly complex and involves a large number of proteins and genes that positively or negatively influence Notch signaling. In this study we analyze a gene that may be affecting the Notch pathway in follicle cell development. We provide not only new information about e(y)1,...
Show moreDrosophila melanogaster serve as an excellent model to study the highly conserved notch signaling pathway, which is involved in a broad array of developmental events. The Notch signal transduction pathway in Drosophila is vastly complex and involves a large number of proteins and genes that positively or negatively influence Notch signaling. In this study we analyze a gene that may be affecting the Notch pathway in follicle cell development. We provide not only new information about e(y)1, but also the notch signaling pathway itself and add onto the understanding of germline-follicle cell signaling during oogenesis.
Show less - Date Issued
- 2013
- Identifier
- FSU_migr_uhm-0284
- Format
- Thesis
- Title
- Isolating and Crystallizing the Permuted HD Domain of CRISPR.
- Creator
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Hubert, Joshua, Li, Hong, Ramia, Nancy, Department of Chemistry and Biochemistry
- Abstract/Description
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CRISPR-cas systems have been found to confer RNA guided immunity in prokaryotes comparable to the eukaryotic RNA interference. These Clustered Regularly Interspaced Short Palindromic Repeats, as their name entails, are repeated sequences varying from 25 to 45 nucleotides in length separated by variable spacers. The CRISPR system has been found in many different bacteria and Archaea. Associated with the CRISPR locus are cas genes, which are thought to encode for nucleases, helicases, and...
Show moreCRISPR-cas systems have been found to confer RNA guided immunity in prokaryotes comparable to the eukaryotic RNA interference. These Clustered Regularly Interspaced Short Palindromic Repeats, as their name entails, are repeated sequences varying from 25 to 45 nucleotides in length separated by variable spacers. The CRISPR system has been found in many different bacteria and Archaea. Associated with the CRISPR locus are cas genes, which are thought to encode for nucleases, helicases, and polymerases involved in the CRISPR defense mechanism. The mechanism involved with the defense occurs when the CRISPR locus is transcribed into a long RNA that will be processed into short sequences used as a guide to target and cleave the invader genome sequences through base pairing. Together, the CRISPR-cas systems are able to protect the bacteria or archaea from invading DNA or RNA. Cas 10 is the signature protein of type III CRISPR systems and is characterized by a permuted Histidine-Aspartic acid (HD) domain predicted to possess a nuclease activity. The presence of the permuted HD and the presence of a nucleotidyl cylcase-like have guided the belief that the permuted HD domain may be the activity site. After several attempts, it was found that the HD domain protein from both PF1129 and TTHB147 is insoluble in water and is consistently lost in the pellet during centrifugation. However, there is still a small concentration of the domain collected in the elution, showing that some of the HD domain can be purified to the last step. The max concentration collected of the permuted HD domain from the TTHB147 was found to be 2.45 mg/mL.
Show less - Date Issued
- 2014
- Identifier
- FSU_migr_uhm-0317
- Format
- Thesis
- Title
- Lipid Profiling of Algae Biofuel Feedstock Grown in Wastewater.
- Creator
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Baxter, Alexis, Chemistry and Biochemistry
- Abstract/Description
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Algae represent a carbon neutral feedstock for biofuel production. However, to fully realize the benefits of coupling algae production with nutrient reduction of wastewater, maximum usable biofuel output must be achieved. Indeed, identification of appropriate algal strains, understanding biochemical pathways, and optimization of production of fuel precursors (i.e. lipids) have been identified as some of the most important challenges that need to be addressed before this technology becomes...
Show moreAlgae represent a carbon neutral feedstock for biofuel production. However, to fully realize the benefits of coupling algae production with nutrient reduction of wastewater, maximum usable biofuel output must be achieved. Indeed, identification of appropriate algal strains, understanding biochemical pathways, and optimization of production of fuel precursors (i.e. lipids) have been identified as some of the most important challenges that need to be addressed before this technology becomes economically feasible. Prospective biofuels originate from the medium-chain fatty acids encased in algal cell membranes. Under ideal conditions, algae synthesize primarily proteins and carbohydrates that are necessary for cell growth. However, when stressed, algae will synthesize large quantities of triacylglycerols (TAGs) that are comprised of mid- to long-chain fatty acids (FAs) bound to glycerol, and these FAs are the primary source of biodiesel. By manipulating growth conditions (e.g. temperature, light, nutrients, pH etc.) the concentration and composition of the FAs can be altered. In this work, various species of freshwater algae grown on domestic wastewater were harvested to determine their potential as a source of combustible biofuels. The goal was to determine which algal strains and stress conditions optimize lipid composition. Total lipid content in dried algal pellets was screened with UV-Vis spectroscopy after derivatization of free fatty acids. Since the quality of the lipids produced is also important in determining biofuel conversion efficiency, the composition of individual lipids (e.g. carbon chain length, number and position of double bonds) was characterized by capillary gas chromatography.
Show less - Date Issued
- 2012
- Identifier
- FSU_migr_uhm-0102
- Format
- Thesis
- Title
- Metal ion-dependent folding of the U2-U6 snRNA spliceosomal complex.
- Creator
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Griffin, Laura
- Date Issued
- 2006-05-22
- Identifier
- 160681, FSDT160681, fsu:19025
- Format
- Document (PDF)
- Title
- Mutational Analysis Reveals The Mechanism Of Proton-Coupled Electron Transfer In Sulfite Reductase Hemoprotein.
- Creator
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Smith, Kyle, Department of Chemistry and Biochemistry
- Abstract/Description
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An essential step in the biogeochemical cycling of sulfur is the six electron reduction of sulfite (SO32-) to sulfide (S2-) catalyzed by the enzyme sulfite reductase (SiR). SiR performs the largest single atom reduction in any biological pathway with the exception of the analogous six electron reduction of nitrite to ammonia. The reduction of SO32- to S2- is critical to the dissimilatory anaerobic respiration pathway in sulfate-reducing bacteria and assimilatory pathway responsible for...
Show moreAn essential step in the biogeochemical cycling of sulfur is the six electron reduction of sulfite (SO32-) to sulfide (S2-) catalyzed by the enzyme sulfite reductase (SiR). SiR performs the largest single atom reduction in any biological pathway with the exception of the analogous six electron reduction of nitrite to ammonia. The reduction of SO32- to S2- is critical to the dissimilatory anaerobic respiration pathway in sulfate-reducing bacteria and assimilatory pathway responsible for incorporation of sulfur into biomolecules in plants, bacteria, and archaea. This project has successfully used mutational analysis of assimilatory sulfite reductase hemoprotein (SiRHP) to reveal a proton coupled electron transfer mechanism with nonredundant proton donors at several step of catalysis. Four hypothesized proton donors (R83, R153, K215, and K217) were independently mutated to serine, resulting in changes in substrate binding, the dynamics of an active site loop, and the number of electrons transferred per sulfur reduced. A fifth mutation was made (N149W) in an attempt to mimic an inactive siroheme site observed in the dissimilatory pathway SiR. This mutation resulted in changes in active site loop dynamics and protease sensitivity, but enhanced, rather than inhibited, the activity of the enzyme. Crystal structures were determined of the R153S variant in the oxidized state and of the N149W variant in the oxidized and reduced substrate bound state. Finally, newer work exploring an active heterodimeric form of SiR (SiRHF) is discussed. A proposal for future experiments with SiRHF is included.
Show less - Date Issued
- 2013
- Identifier
- FSU_migr_uhm-0221
- Format
- Thesis
- Title
- Novel Carbon Nanotube Structures and Microcellular Foams.
- Creator
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Amrhein, Christina, Department of Biological Science
- Abstract/Description
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Structural foams reinforced with carbon nanotubes could prove to be beneficial in numerous industrial applications. High internal phase emulsion foams are emerging as new and important forms of microcellular foams. Microcellular foams, foams with a pores size of 0.1-10 μm, have numerous advantages, such as high impact strength. The dispersal of carbon nanofibers, as wall pore reinforcements, within microcellular foams promises to enhance the foams' mechanical properties. This research has...
Show moreStructural foams reinforced with carbon nanotubes could prove to be beneficial in numerous industrial applications. High internal phase emulsion foams are emerging as new and important forms of microcellular foams. Microcellular foams, foams with a pores size of 0.1-10 μm, have numerous advantages, such as high impact strength. The dispersal of carbon nanofibers, as wall pore reinforcements, within microcellular foams promises to enhance the foams' mechanical properties. This research has developed a novel method for creating such foam.
Show less - Date Issued
- 2014
- Identifier
- FSU_migr_uhm-0290
- Format
- Thesis
- Title
- Posttranslational Modifications Are Crucial for the Recruitment of Histone Variant H3.3 to DNA Damage Sites.
- Creator
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Canzani, Daniele, Department of Chemistry and Biochemistry
- Abstract/Description
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Histones are essential proteins that package DNA inside the cell nucleus and regulate access to the genetic information contained within it. Histone variant H3.3 is frequently mutated in children and young adults with high-grade glioblastomas, chondroblastomas, and giant cell tumors of the bone. Live cell imaging shows that H3.3 is rapidly recruited to sites of laser induced DNA damage, where it appears to be playing an important role in facilitating DNA repair. Since many proteins involved...
Show moreHistones are essential proteins that package DNA inside the cell nucleus and regulate access to the genetic information contained within it. Histone variant H3.3 is frequently mutated in children and young adults with high-grade glioblastomas, chondroblastomas, and giant cell tumors of the bone. Live cell imaging shows that H3.3 is rapidly recruited to sites of laser induced DNA damage, where it appears to be playing an important role in facilitating DNA repair. Since many proteins involved in DNA repair undergo posttranslational modifications (PTMs) such as phosphorylation and acetylation, this study was designed to determine if H3.3 PTMs affect its recruitment to damaged DNA. For this, acetylation at specific sites was prevented through the generation of lysine to arginine (K to R) mutations at positions 14, 18, 23, 36 and 37 in H3.3, which were chosen based on our preliminary mass spectrometry data and acetylation sites reported in the literature. In addition, histone acetyltransferase (HAT) inhibitors such as anacardic acid and curcumin were used to prevent acetylation. Similarly, phosphorylation at specific residues was prevented through the generation of serine to alanine (S to A) mutations at 28, 31, 86 and 96 positions on H3.3 and a tyrosine to phenylalanine mutation at position 99 (Y99F). These potential phosphorylation sites were chosen primarily based on their proximity to amino acid residues that are unique to histone H3.3, thereby placing them in a unique sequence context in this protein. My findings reveal the importance of acetylation and phosphorylation of H3.3 for recruitment to DNA damage sites, with major reductions in recruitment for all H3.3 mutants generated in an additive manner. Recruitment of mutant H3.3 was down to 17% for K14, 18R acetylation defective mutant and 15% for phosphorylation defective S28, 31, 86, 96A Y99F quintuple mutant compared to the wild type H3.3. A preliminary investigation of several DNA repair factors belonging to different DNA repair pathways suggests a specific role for H3.3 in homologous recombination mediated double strand break repair.
Show less - Date Issued
- 2015
- Identifier
- FSU_migr_uhm-0558
- Format
- Thesis
- Title
- Protein biochemistry.
- Creator
-
Tellechea, Laura M.
- Date Issued
- 2006-05-24
- Identifier
- 160461, FSDT160461, fsu:18921
- Format
- Document (PDF)
- Title
- Quantitative Detection of Protein Electrostatic Environment via Intrinsic pKa Calculations.
- Creator
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Stribling, Dan, Department of Chemistry and Biochemistry
- Abstract/Description
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One of the primary contributors to protein structure and functioning in biological systems is the electrostatic environment experienced by the protein. This environment is caused by charged and polar chemical interactions, with the acidic protein residues ASP and GLU often acting as a significant source of charged interactions in protein systems. Knowledge of the charged states of these residues is given by the determination of their pKa values and provides a significant source of information...
Show moreOne of the primary contributors to protein structure and functioning in biological systems is the electrostatic environment experienced by the protein. This environment is caused by charged and polar chemical interactions, with the acidic protein residues ASP and GLU often acting as a significant source of charged interactions in protein systems. Knowledge of the charged states of these residues is given by the determination of their pKa values and provides a significant source of information on the electrostatic character of protein environments. This knowledge provides insight into a diverse array of biological processes such as enzymatic function, membrane transport, and immunological activity. Simulation study has recently arisen as a cost-effective method to determine the pKa value of acidic protein residues. This study has successfully used molecular dynamics simulation with the Orthogonal Space Tempering method to determine the pKa values of the acidic ASP and GLU residues on the reduced form of the Human Thioredoxin Protein, an enzyme responsible for combatting oxidative stress in the human body. The study achieved an unprecedented accuracy in the computational determination of the pKa values of these residues with an overall RSMD of 0.71 units. Notably, the pKa value of the buried ASP residue was obtained to a before-unseen accuracy with a deviation of the pKa value from the experimental values by 0.6 units. This study demonstrates the utility of the OST computational method and its major potential for use in the prediction of pKa values of acidic residues in protein systems.
Show less - Date Issued
- 2014
- Identifier
- FSU_migr_uhm-0344
- Format
- Thesis
- Title
- The Role of Insulin/Insulin-like Signaling Pathway in Regulating Female Drosophila Melanogaster Remating Behaviors.
- Creator
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Dudek, Karrie, Department of Biological Science
- Abstract/Description
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Mating incurs costs to lifespan across species. Given that mating is a necessity for survival of most species, but yet has health costs, a method of modulating reproductive strategies under various environmental conditions to give optimal outcomes is observed in different species. Since remating is thought to be energy dependent, nutrition is thought to play a critical role in the remating rates of female Drosophila melanogaster. The mechanisms underlying these regulatory responses, however,...
Show moreMating incurs costs to lifespan across species. Given that mating is a necessity for survival of most species, but yet has health costs, a method of modulating reproductive strategies under various environmental conditions to give optimal outcomes is observed in different species. Since remating is thought to be energy dependent, nutrition is thought to play a critical role in the remating rates of female Drosophila melanogaster. The mechanisms underlying these regulatory responses, however, are unknown and are of great interest. It is thought that the Insulin/IGF (Insulin-like growth factor)-like signaling (IIS) pathway is a key factor in regulating post-mating responses, including remating rates. This study addresses the effects of the IIS pathway on post-mating behaviors, specifically the effects of Drosophila insulin-like peptides (DILPs) on remating rates of female D. melanogaster. The conservation of genes that encode the DILPs and other IIS pathway signaling components between Drosophila and vertebrates suggests that insights gained from Drosophila studies could give insight into how the IIS pathway could affect humans and their behaviors. By employing a Drosophila insulin-like peptide knockdown system, I was able to demonstrate that knockdown of the IIS pathway, by reduction of dilp2, dilp3, or dilp5, results in reduced remating rates. The IIS is nutrient sensing and the females' remating response is thought to be energy dependent, therefore, I also investigated the effects of different caloric intake values. The results, however, show no significant differences between remating rates of females housed on high, medium, or low calorie foods.
Show less - Date Issued
- 2013
- Identifier
- FSU_migr_uhm-0246
- Format
- Thesis
- Title
- A Simplified Protein Folding Simulation Using a Metropolis Monte Carlo Algorithm.
- Creator
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Franklin, Robert D., Department of Chemical and Biomedical Engineering
- Abstract/Description
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The major outcome of this project is "Protein Folding Toy," an iPad program (app) which is designed to allow children (ages 10-18) to interact with a system that behaves like a single polymer chain on the molecular level. The user can design a single chain, specifying a sequence of beads that each could be neutral (black), hydrophobic (green), or charged (red/blue). The app can then perform a Metropolis Monte Carlo simulation and allows the user to vary temperature in real time. Observed...
Show moreThe major outcome of this project is "Protein Folding Toy," an iPad program (app) which is designed to allow children (ages 10-18) to interact with a system that behaves like a single polymer chain on the molecular level. The user can design a single chain, specifying a sequence of beads that each could be neutral (black), hydrophobic (green), or charged (red/blue). The app can then perform a Metropolis Monte Carlo simulation and allows the user to vary temperature in real time. Observed chain behavior depends on inter-bead interactions as well as temperature-dependent stochastic motions.
Show less - Date Issued
- 2015
- Identifier
- FSU_migr_uhm-0521
- Format
- Set of related objects
- Title
- The Structural Characterization of AfXPB Bound to G-Quadruplex DNA.
- Creator
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Jackson, Trevia M., Department of Chemistry and Biochemistry
- Abstract/Description
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Xeroderma Pigmentosum (XP) is an autosomal recessive disorder characterized by extreme sensitivity to sunlight, higher incidence of skin cancers, pigmented alterations in the skin, and in some cases neurological abnormalities. XP is caused by a mutation in one of eight genes designated XP groups A-G and V. Recent studies show that helicases encoded by XP-group B (XPB) and XP group D (XPD) genes bind specifically to G-quadruplex (G4) DNA. G4 DNA is a form of DNA rich in guanine nucleotides...
Show moreXeroderma Pigmentosum (XP) is an autosomal recessive disorder characterized by extreme sensitivity to sunlight, higher incidence of skin cancers, pigmented alterations in the skin, and in some cases neurological abnormalities. XP is caused by a mutation in one of eight genes designated XP groups A-G and V. Recent studies show that helicases encoded by XP-group B (XPB) and XP group D (XPD) genes bind specifically to G-quadruplex (G4) DNA. G4 DNA is a form of DNA rich in guanine nucleotides that form planar tetrads using Hoogsteen hydrogen base pairing. These tetrads then stack upon each other yielding a quadruplex structure. The structure of free XPB has been determined previously, but the structure of XPB bound to G4 DNA has not yet been obtained. The protein-DNA complex structure is valuable in understanding how this helicase binds on the atomic level as this could potentially answer additional questions about its function in vivo. Furthermore, due to similar characteristics of XPB and XPD, we hypothesize that XPB acts a regulatory enzyme competitively inhibiting enzyme activity of the robust helicase, XPD. The goal of this study is to determine the mechanism by which these proteins bind by addressing two specific aims. First, determine the structure of XPB bound to G4 DNA in order to structurally assess the mechanism through which this protein binds using X-ray crystallography. Second, test the competitive inhibition of XPD by XPB in the presence of G4 DNA using biochemical assays. By optimizing preparative protocols in gene expression and protein purification, we enhanced the recovery of homogenous samples of XPB essential for carrying out structural studies. However, to improve the XPB:G4 DNA complex stability in solution, studies shifted to optimizing the buffer system for this complex; as this is the next essential step in moving all structural and biochemical studies forward. Understanding the interactions between XPB and G4 DNA will not only provide insight into the interactions between enzymes and G-quadruplex DNA, but also further insight into the Xeroderma Pigmentosum disorder with hopes of providing more knowledge for enhanced gene therapies and treatments.
Show less - Date Issued
- 2015
- Identifier
- FSU_migr_uhm-0580
- Format
- Thesis
- Title
- Use of Liquid Chromatography-Mass Spectrometry to Detect Potential Metabolite Biomarkers for Alzheimer's Disease.
- Creator
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Ogunrinde, Elizabeth, Department of Chemistry and Biochemistry
- Abstract/Description
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Alzheimer's disease (AD) is a neurodegenerative disease currently affecting about five million Americans. The devastating impact of Alzheimer's disease and the growing cost of the disease make it an emerging public health problem causing a significant societal burden. Lack of completely specific AD biomarkers necessitates the investigation of biomarkers that are stable, specific, and can serve as viable diagnostics for AD. This work focuses on using liquid chromatography-mass spectrometry (LC...
Show moreAlzheimer's disease (AD) is a neurodegenerative disease currently affecting about five million Americans. The devastating impact of Alzheimer's disease and the growing cost of the disease make it an emerging public health problem causing a significant societal burden. Lack of completely specific AD biomarkers necessitates the investigation of biomarkers that are stable, specific, and can serve as viable diagnostics for AD. This work focuses on using liquid chromatography-mass spectrometry (LC-MS) for the identification of metabolite biomarkers in Alzheimer's disease. The six metabolites examined in this study were alanine, glutamine, glutamate, tryptophan, γ-Aminobutyric acid (GABA), and N-acetyl-L-aspartate (NAA). Hippocampal tissue was obtained from two male wild type and two male Alzheimer's disease mice. The Alzheimer's mice were double transgenic APPswe, PSEN1dE9 mice expressing a chimeric mouse/human amyloid precursor protein (Mo/HuAPP695swe) and a mutant human presenilin 1(PS1-dE9). Controls were noncarriers of the mutation. Metabolites were extracted from the hippocampal tissue and subsequently analyzed using a triple quadrupole mass spectrometer. There were no significant differences in metabolite levels between the wild type and Alzheimer's disease mice in either the left or right hippocampus.
Show less - Date Issued
- 2014
- Identifier
- FSU_migr_uhm-0336
- Format
- Thesis
- Title
- Zinc Regulation of Bone Marrow-Derived Mesenchymal Stem Cell Neuronal Differentiation.
- Creator
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Faye, Sari, Department of Chemistry and Biochemistry
- Abstract/Description
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The multipotent ability of mesenchymal stem cells (MSC) to differentiate into a large variety of mature cell types gives them a high potential for use in a variety of therapeutic purposes. Recently, it was discovered that bone marrow derived MSC could be induced to take on a neuronal phenotype through the addition of cobalt chloride (CoCl2) to the growth media. It is also well known that the trace element zinc is vital for both neuronal proliferation and differentiation from neuronal...
Show moreThe multipotent ability of mesenchymal stem cells (MSC) to differentiate into a large variety of mature cell types gives them a high potential for use in a variety of therapeutic purposes. Recently, it was discovered that bone marrow derived MSC could be induced to take on a neuronal phenotype through the addition of cobalt chloride (CoCl2) to the growth media. It is also well known that the trace element zinc is vital for both neuronal proliferation and differentiation from neuronal precursor cells. Thus, this work tested the hypothesis that zinc plays a role in the differentiation of MSC into neurons. Secondly, because zinc is unable to enter or exit cells without the assistance of zinc transport proteins (ZnT), this work tested the hypothesis that two transport proteins, ZnT-1 and ZnT-4, would be regulated both by zinc and by treatment with cobalt. This work used both cell morphology and markers of neuronal differentiation (TuJ1 and neuronal specific enolase) to show that zinc deficiency (ZD) combined with CoCl2 treatment appeared to induce differentiation of rat MSC. Furthermore, the zinc transporters were differentially regulated such that ZnT-4 was increased on the cell membrane by zinc deficiency, while ZnT-1 levels at the membrane were highest in the combined zinc deficiency-cobalt treatment group. These data implicate zinc in the mechanisms associated with MSC function.
Show less - Date Issued
- 2013
- Identifier
- FSU_migr_uhm-0235
- Format
- Thesis
- Title
- Zinc Regulation of Mesenchymal Stem Cell Proliferation and Survival.
- Creator
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Hagler, Shaye, Department of Chemistry and Biochemistry
- Abstract/Description
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Mesenchymal stem cells (MSC) have a wide variety of promising clinical applications including the treatment of brain disorders and injury, cardiovascular disease, and cancer. To fully exploit their potential, we need a better understanding of the cellular and molecular mechanisms that govern stem cell division and survival. We have hypothesized that the essential trace element zinc regulates the proliferation and survival of rat and human bone marrow-derived MSC. Proliferation of MSC is...
Show moreMesenchymal stem cells (MSC) have a wide variety of promising clinical applications including the treatment of brain disorders and injury, cardiovascular disease, and cancer. To fully exploit their potential, we need a better understanding of the cellular and molecular mechanisms that govern stem cell division and survival. We have hypothesized that the essential trace element zinc regulates the proliferation and survival of rat and human bone marrow-derived MSC. Proliferation of MSC is impaired by zinc deficiency. For example, after 48h of zinc deficiency, proliferation was reduced by 50% (p
Show less - Date Issued
- 2013
- Identifier
- FSU_migr_uhm-0181
- Format
- Thesis
- Title
- βTRCP: Linking Circadian Rhythms and Metabolism.
- Creator
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Sweeney, Megan C., Department of Biomedical Sciences
- Abstract/Description
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Shifts in circadian rhythms, like in shift work or jet lag, have been shown to increase the risk of many metabolic disorders. Therefore, it is not surprising that many genes involved in the circadian clock mechanism have demonstrated a regulatory role in metabolism. It has been shown that E3 ubiquitin ligases can influence metabolism as well. In initial studies, my lab created a knockout of two E3 ubiquitin ligases thought to be essential to the clock, βTRCP1/2, in a mouse model in order to...
Show moreShifts in circadian rhythms, like in shift work or jet lag, have been shown to increase the risk of many metabolic disorders. Therefore, it is not surprising that many genes involved in the circadian clock mechanism have demonstrated a regulatory role in metabolism. It has been shown that E3 ubiquitin ligases can influence metabolism as well. In initial studies, my lab created a knockout of two E3 ubiquitin ligases thought to be essential to the clock, βTRCP1/2, in a mouse model in order to study the proteasomal degradation machinery in mammals. Upon characterizing the circadian phenotype of this mouse, we noticed an unprecedented, metabolic phenotype after deletion of these vital ligases. These novel mutant mice lose over 30% of their body weight within 5 days while still maintaining an eating and drinking regime similar to wild-type mice. In this project, in vivo and sequence analysis studies aimed to look further into the causes of this phenomenon and the molecular mechanisms underlying them.
Show less - Date Issued
- 2015
- Identifier
- FSU_migr_uhm-0455
- Format
- Thesis
- Title
- Φ-Value Analysis of Symfoil-4T.
- Creator
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Sutherland, Mason A., Department of Biological Science
- Abstract/Description
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A critical consideration in the process of de novo protein architecture design and protein evolution is the folding pathway and behavior a protein undertakes in transitioning to its functional tertiary structure. Of particular interest is a cryptic element within protein primary structure that enables an efficient folding pathway, and is postulated to be a heritable element in the evolution of protein architecture, the "folding nucleus" (FN). However, almost nothing is known regarding how the...
Show moreA critical consideration in the process of de novo protein architecture design and protein evolution is the folding pathway and behavior a protein undertakes in transitioning to its functional tertiary structure. Of particular interest is a cryptic element within protein primary structure that enables an efficient folding pathway, and is postulated to be a heritable element in the evolution of protein architecture, the "folding nucleus" (FN). However, almost nothing is known regarding how the FN changes as simpler peptide motifs join to form more complex polypeptides. To this effect, the structure and folding properties of foldable intermediates along the evolutionary trajectory of the β-trefoil protein type were tested. This study specifically used and compared data from Symfoil-4T (an engineered β-trefoil protein) to several mutants to show that the FN is acquired during gene fusion events, incorporating novel turn structure generated by gene fusion. Furthermore, the FN of β-trefoils are adjusted by circular permutation in response to destabilizing functional mutations to allow the survival of FN (which is made possible by the intrinsic C3 cyclic symmetry of β-trefoil architecture) identifying a selective advantage that helps explain extant cyclic structural symmetry in the proteome.
Show less - Date Issued
- 2015
- Identifier
- FSU_migr_uhm-0453
- Format
- Thesis
