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Title: Biophysical Characterization of a ssDNA Virus.
Creator: Morrison, Anneliese J., Department of Chemistry and Biochemistry
Abstract/Description: 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.
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Date Issued: 2014
Identifier: FSU_migr_uhm-0428
Format: Thesis

Title: Isolating and Crystallizing the Permuted HD Domain of CRISPR.
Creator: Hubert, Joshua, Li, Hong, Ramia, Nancy, Department of Chemistry and Biochemistry
Abstract/Description: 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.
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Date Issued: 2014
Identifier: FSU_migr_uhm-0317
Format: Thesis

Title: Automated Analysis of Protein Side Chain Spectra.
Creator: Hart, Andrew, Department of Chemistry and Biochemistry
Abstract/Description: 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.
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Date Issued: 2013
Identifier: FSU_migr_uhm-0244
Format: Thesis

Title: The Role of Insulin/Insulin-like Signaling Pathway in Regulating Female Drosophila Melanogaster Remating Behaviors.
Creator: Dudek, Karrie, Department of Biological Science
Abstract/Description: 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.
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Date Issued: 2013
Identifier: FSU_migr_uhm-0246
Format: Thesis

Title: The Characterization of the Conformational Changes of Sar1 upon Activation.
Creator: CreveCoeur, Travis, Stagg, Scott, Bhattacharya, Nilakshee, Wood-Cohan, Nathaniel, Department of Chemistry and Biochemistry
Abstract/Description: 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.
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Date Issued: 2013
Identifier: FSU_migr_uhm-0250
Format: Thesis

Title: Use of Liquid Chromatography-Mass Spectrometry to Detect Potential Metabolite Biomarkers for Alzheimer's Disease.
Creator: Ogunrinde, Elizabeth, Department of Chemistry and Biochemistry
Abstract/Description: 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.
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Date Issued: 2014
Identifier: FSU_migr_uhm-0336
Format: Thesis

Title: Mutational Analysis Reveals The Mechanism Of Proton-Coupled Electron Transfer In Sulfite Reductase Hemoprotein.
Creator: Smith, Kyle, Department of Chemistry and Biochemistry
Abstract/Description: 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.
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Date Issued: 2013
Identifier: FSU_migr_uhm-0221
Format: Thesis

Title: Novel Carbon Nanotube Structures and Microcellular Foams.
Creator: Amrhein, Christina, Department of Biological Science
Abstract/Description: 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.
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Date Issued: 2014
Identifier: FSU_migr_uhm-0290
Format: Thesis

Title: Lipid Profiling of Algae Biofuel Feedstock Grown in Wastewater.
Creator: Baxter, Alexis, Chemistry and Biochemistry
Abstract/Description: 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.
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Date Issued: 2012
Identifier: FSU_migr_uhm-0102
Format: Thesis

Title: The Effects of Nicotine on Neurotrophic Factor Expression in the Adult Male Zebra Finch.
Creator: Peoples, Jessica, Chemistry and Biochemistry
Abstract/Description: 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.
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Date Issued: 2012
Identifier: FSU_migr_uhm-0069
Format: Thesis

Title: Assessment of Synthetic Matrix Metalloproteinase Inhibitors by Fluorogenic Substrate Assay.
Creator: Lively, Ty J., Department of Chemistry and Biochemistry
Abstract/Description: 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.
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Date Issued: 2015
Identifier: FSU_migr_uhm-0536
Format: Thesis

Title: Posttranslational Modifications Are Crucial for the Recruitment of Histone Variant H3.3 to DNA Damage Sites.
Creator: Canzani, Daniele, Department of Chemistry and Biochemistry
Abstract/Description: 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.
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Date Issued: 2015
Identifier: FSU_migr_uhm-0558
Format: Thesis

Title: In Vivo Analysis of Phenotypes Arising from Mutations in Cardiac Troponin C.
Creator: Gonzalez-Martinez, David A., Department of Chemistry and Biochemistry
Abstract/Description: 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.
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Date Issued: 2016
Identifier: FSU_migr_uhm-0586
Format: Thesis

Title: βTRCP: Linking Circadian Rhythms and Metabolism.
Creator: Sweeney, Megan C., Department of Biomedical Sciences
Abstract/Description: 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.
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Date Issued: 2015
Identifier: FSU_migr_uhm-0455
Format: Thesis

Title: Φ-Value Analysis of Symfoil-4T.
Creator: Sutherland, Mason A., Department of Biological Science
Abstract/Description: 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.
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Date Issued: 2015
Identifier: FSU_migr_uhm-0453
Format: Thesis

Title: The Structural Characterization of AfXPB Bound to G-Quadruplex DNA.
Creator: Jackson, Trevia M., Department of Chemistry and Biochemistry
Abstract/Description: 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.
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Date Issued: 2015
Identifier: FSU_migr_uhm-0580
Format: Thesis

Title: The Afferent Circuitry of the Ventromedial Hypothalamus and Its Activation in Paternal Behavior of the Socially Monogamous Prairie Vole.
Creator: Rogers, Richard S., Department of Chemistry and Biochemistry
Abstract/Description: 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.
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Date Issued: 2015
Identifier: FSU_migr_uhm-0545
Format: Thesis

Title: Zinc Regulation of Bone Marrow-Derived Mesenchymal Stem Cell Neuronal Differentiation.
Creator: Faye, Sari, Department of Chemistry and Biochemistry
Abstract/Description: 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.
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Date Issued: 2013
Identifier: FSU_migr_uhm-0235
Format: Thesis

Title: CRISPR-Cas9 Utility in Genome Engineering.
Creator: McCullers, Michelle R., Department of Biological Science
Abstract/Description: 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.
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Date Issued: 2015
Identifier: FSU_migr_uhm-0450
Format: Thesis

Title: Investigating the role of the e(y)1 gene in the Notch signaling pathway.
Creator: Alvarado, Francisco, Department of Chemistry and Biochemistry
Abstract/Description: 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.
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Date Issued: 2013
Identifier: FSU_migr_uhm-0284
Format: Thesis

Title: Zinc Regulation of Mesenchymal Stem Cell Proliferation and Survival.
Creator: Hagler, Shaye, Department of Chemistry and Biochemistry
Abstract/Description: 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
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Date Issued: 2013
Identifier: FSU_migr_uhm-0181
Format: Thesis

Title: The Evaluation of Industrial Methods of Size-Exclusion Chromatography (SEC) of Difficult-to-Dissolve Polymers.
Creator: McNeel, Kelsey, Chemistry
Abstract/Description: 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.
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Date Issued: 2011
Identifier: FSU_migr_uhm-0026
Format: Thesis

Title: Quantitative Detection of Protein Electrostatic Environment via Intrinsic pKa Calculations.
Creator: Stribling, Dan, Department of Chemistry and Biochemistry
Abstract/Description: 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.
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Date Issued: 2014
Identifier: FSU_migr_uhm-0344
Format: Thesis

Title: Characterization of a Novel CRISPR Type II-C Cas9 Endonuclease from Bacterial Thermophile Acidothermus Cellulolyticus 11B.
Creator: Tsui, Tsz Kin Martin, Li, Hong (Chemistry and Biochemistry), Alabugin, Igor V. (Professor), Yang, Wei, Miller, Brian G., Taylor, Kenneth A., Florida State University, College of...
Show moreTsui, Tsz Kin Martin, Li, Hong (Chemistry and Biochemistry), Alabugin, Igor V. (Professor), Yang, Wei, Miller, Brian G., Taylor, Kenneth A., Florida State University, College of Arts and Sciences, Program in Molecular Biophysics
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Abstract/Description: The discovery of Cas9, a large protein that involves in all processes the type II system of Clustered Regularly Interspaced Short Palindromic Repeat, an adaptive immune system from bacteria and archaea, is a paradigm shifting molecular tool in the world of cell biology research due to its potential for various biotechnology applications. As an RNA-guided DNA targeting endonuclease, Cas9 can essentially cleave any DNA sequence of interest based on Watson-Crick base pairs using the guide region...
Show moreThe discovery of Cas9, a large protein that involves in all processes the type II system of Clustered Regularly Interspaced Short Palindromic Repeat, an adaptive immune system from bacteria and archaea, is a paradigm shifting molecular tool in the world of cell biology research due to its potential for various biotechnology applications. As an RNA-guided DNA targeting endonuclease, Cas9 can essentially cleave any DNA sequence of interest based on Watson-Crick base pairs using the guide region of a single guide RNA (sgRNA), which is comprised of the processed sequence of CRISPR RNA (crRNA) that carries the spacer sequence that requires searching for the targeting DNA, covalently linked to a trans-activating crRNA (tracrRNA) that provides a scaffold for Cas9 protein to bind to the crRNA. While Cas9 has tremendous application in any DNA-associated problems, which includes eradicating genetic diseases or gene mutations, Cas9 has an inherent off-target DNA cleavage – that is cleaving a potential DNA target that may have a similar but incorrect sequence compared to the on-target DNA sequence – due to one or multiple mismatches between the gRNA and the targeting DNA. As a result, it poses a concern regarding its use in gene therapy in human or other animal systems. We chose to address this DNA target specificity and efficiency issue by establishing and studying through a novel Cas9 system from a subtype different than the established systems – a type II-C Cas9 from thermophile Acidothermus Cellulolyticus 11B (AceCas9). Further information of the classification of Cas9, molecular processes that involved Cas9, the significance of Cas9 and this research project are addressed in Chapter 1. Without any previous studies on AceCas9, we established the in vitro biochemical functions of AceCas9 through combinatorial methods to determine 1) the sgRNA sequence that yields a Cas9 RNP, 2) a functional PAM sequence that permits AceCas9 to cleave dsDNA through an in vitro DNA library assay, 3) determine the target specificity of PAM and target DNA in vitro through mutational analysis on oligo DNA substrates, and 4) environmental conditions that influence target efficiency of AceCas9 in chapter 2. The results showed that AceCas9 recognized a novel, cytosine-specific PAM sequence (5’–NNNCC–3’) and proves to be functional in vitro. AceCas9 depends on limited divalent cations for DNA cleavage, yet it proves to be functional at a wider range of temperature, from 37 °C to 60 °C. To further establish DNA cleavage specificity and efficiency, we performed cleavage assays as well as a series of single-turnover kinetics assays to determine how specific and efficient AceCas9 cleaves plasmid DNA substrates with various mutation and/or DNA topologies under in vitro condition in chapter 3. Results showed that substrates with higher helicity permit AceCas9 to cleave those substrates quicker, yet decrease AceCas9 target specificity. Finally, with the hypothesis that the sgRNA may influence target efficiency and specificity, we performed both in vitro and a bacterial-based in vivo assays to determine how elongation on the guide length may influence DNA interference by AceCas9. We demonstrated that an elongated guide length from 20-nt to 24-nt significantly improves AceCas9 DNA targeting efficiency both in vitro and in vivo, but it does not contribute significantly in target specificity. Intriguingly, AceCas9 can only be functional in vivo with either a 24-nt or 26-nt guide in sgRNA, suggesting that AceCas9 is selective to the length of its spacer, contrary to its counterparts.
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Date Issued: 2017
Identifier: FSU_SUMMER2017_Tsui_fsu_0071E_13952
Format: Thesis

Title: Development of a Simple Microfluidic Device for Characterizing Chemotaxis of Macrophage in Response to Myelin Basic Protein.
Creator: Jia, Xiaolin, Chung, Hoyong, Mohammadigoushki, Hadi, Florida State University, College of Engineering, Department of Chemical and Biomedical Engineering
Abstract/Description: Microfluidic devices are widely used for cell-based analysis. There are always needs to develop simpler, more effective and/or less costly devices than the existing ones for this application. A simple microfluidic device has been fabricated and tested for studying chemotaxis of macrophages in this study. The device was made of polydimethylsiloxane bound to a cell culture dish. It consisted of a millimeter-sized cavum and two arrays of straight channels of 5 um in width and 6um height and...
Show moreMicrofluidic devices are widely used for cell-based analysis. There are always needs to develop simpler, more effective and/or less costly devices than the existing ones for this application. A simple microfluidic device has been fabricated and tested for studying chemotaxis of macrophages in this study. The device was made of polydimethylsiloxane bound to a cell culture dish. It consisted of a millimeter-sized cavum and two arrays of straight channels of 5 um in width and 6um height and about two millimeters in length. The channels connected the cavum, in which a chemoattractant was loaded, with the surrounding environment, in which the macrophages were cultured. The device was first tested with a known chemoattractant - fetal bovine serum and the chemoattractive property of myelin basic protein (MBP) was then studied using the device. The macrophages were found to migrate towards to the MBP-loaded cavum in larger quantity and greater distance than those in the control samples. The results prove the usefulness of the microfluidic device for chemotaxis assay and indicate that MBP is a chemoattractant for the macrophages.
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Date Issued: 2017
Identifier: FSU_SUMMER2017_Jia_fsu_0071N_13978
Format: Thesis

Title: Studies Towards the Total Synthesis of 1-Deoxy-8-Demethyl Taxol.
Creator: Koo, Ki Chul, Holton, Robert A., Reeves, Robert H., Krafft, Marie E., Dudley, Gregory B., Logan, Timothy M., Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: The total synthesis towards 1-deoxytaxol analogs is described. The synthesis relies on new methods to install the C3-C4 bond and to achieve the tricyclic taxane ring skeleton. The first section describes the formation of the C3-C4 bond via a highly stereoselective one pot tandem enone formation and Michael addition reaction. The precursor for the tandem reaction was prepared from a taxane AB-ring intermediate which was developed for the synthesis of 1-deoxytaxol analogs in our laboratory. An...
Show moreThe total synthesis towards 1-deoxytaxol analogs is described. The synthesis relies on new methods to install the C3-C4 bond and to achieve the tricyclic taxane ring skeleton. The first section describes the formation of the C3-C4 bond via a highly stereoselective one pot tandem enone formation and Michael addition reaction. The precursor for the tandem reaction was prepared from a taxane AB-ring intermediate which was developed for the synthesis of 1-deoxytaxol analogs in our laboratory. An elimination reaction provided a transient α,β-unsaturated ketone and the following intramolecular Michael addition reaction produced the desired C3-C4 bond-linked lactone as a single diastereomer. Section two describes the highly efficient C-ring closure by consecutive Michael addition and aldol condensation reaction of the previously obtained lactone. Indeed, Michael addition with acrolein led to the corresponding aldehyde adduct. The aldehyde was then employed for the intramolecular aldol condensation reaction to give the crucial tricyclic taxane intermediate. In the third section, preliminary studies describing the reactivity of the taxane intermediate towards the synthesis of 1,7-dideoxy-8-demethyltaxol are reported.
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Date Issued: 2009
Identifier: FSU_migr_etd-2842
Format: Thesis

Title: Multi-Detector Hydrodynamic Chromatography: Particle Characterization and Ultra-High Molar Mass Polymer Analysis.
Creator: Brewer, Amandaa Kimberly, Striegel, André M., Collier, John R., Dorsey, John G., Steinbock, Oliver, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Particle size, shape, and their distributions directly influence a variety of processing and end-use properties related to packing, mixing, and transport of powders, solutions, and suspensions. Many of the techniques currently employed for particle size characterization have found limited applicability for broadly polydisperse and/or non-spherical particles. We have attempted to overcome many of the limitations of these techniques through the systematic development of multi-detector...
Show moreParticle size, shape, and their distributions directly influence a variety of processing and end-use properties related to packing, mixing, and transport of powders, solutions, and suspensions. Many of the techniques currently employed for particle size characterization have found limited applicability for broadly polydisperse and/or non-spherical particles. We have attempted to overcome many of the limitations of these techniques through the systematic development of multi-detector hydrodynamic chromatography (HDC) methodology for the characterization of mono- and polydisperse spherical and non-spherical particles and ultra-high molar mass polymers. Initially, HDC with multi-angle static light scattering (MALS), quasi-elastic light scattering(QELS), differential viscometry (VISC), and differential refractometry (DRI) detection, was used to characterize the size, shape, and compactness of monodisperse spherical polystyrene and poly(methyl methacrylate) particle standards. From these analyses, we were able to measure three colloidal radii, and also to determine the shape and structure or compactness of the particles. Once the HDC/MALS/QELS/VISC/DRI technique was proven successful for characterizing monodisperse spherical particles, our analytical approach was refined to include spherical particles polydisperse in size, compactness, and chemistry. Analysis of these polydisperse particles demonstrated the robustness of the HDC method, in which the multiplicity of detection methods employed helps compensate for the inherently low chromatographic resolution of the separation method. The next step in the sequential development of multi-detector HDC methodology was to examine non-spherical particles. A polydisperse prolate ellipsoidal silica particle was successfully characterized based on molar mass, three colloidal radii, and their distributions. The synergetic combination of size information obtained through the use of multiple physical detectors provided information on particle shape and compactness across the HDC elution profile, through the application of various dimensionless size ratios. A collaboration with Inovatia Laboratories LLC., then allowed us to extend the HDC method to the study of nanoparticles that had been shown to be polydisperse in both size and shape by transmission electron microscopy (TEM). These analyses confirmed the accuracy and precision of the HDC technique, which provided a more complete structural picture of the nanoparticles than did TEM, in a fraction of the time needed for the latter. We then evaluated the ability of multi-detector HDC to analyze irregular-shaped objects by analyzing a colloidal assembly of silica particles, with a shape similar to that of a string-of-pearls. Two different separation techniques, HDC and size-exclusion chromatography (SEC), were used to characterize this sample. The string-of-pearls was shown to degrade during SEC analysis, but not during HDC analysis. The multiple detection methods employed allowed us to expand the type of information obtained in previous studies to include the observation that the sample contained "strings" varying in degree of polymerization, as well as individual "pearls." Lastly we used, HDC to study ultra-high molar mass polymers which had been shown to experience on-column, flow-induced degradation by SEC. Multi-detector HDC was able to determine the molar mass and size of the ultra-high molar mass polysaccharide alternan more accurately than SEC, in a fraction of the time. In conclusion, multi-detector HDC has been successfully and systematically developed into a method for characterizing complex particles and ultra-high molar mass polymers based on their size, shape, and compactness. Because the multi-detector HDC technique employed instrumentation common to most macromolecular separations laboratories, it shows great promise in the areas of particle and polymer characterization.
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Date Issued: 2010
Identifier: FSU_migr_etd-3045
Format: Thesis

Title: Structure and Biophysical Characterization of b-Turn Regions of Human Acidic Fibroblast Growth Factor.
Creator: Kim, Jaewon, Blaber, Michael, Keller, Thomas C. S., Logan, Timothy M., Safron, Sanford A., Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: The structure of human acidic fibroblast growth factor (FGF-1) is classified a b-trefoil structure, one of the fundamental protein superfolds. The x-ray crystal structures of wild type and various mutants of FGF-1 have been solved in five different space groups: C2, C2221, P21 (4 molecules/asu), P21 (3 molecules/asu) and P212121. These structures reveal two characteristically different conformations for the b8/b9 b-hairpin comprising residue positions 90-94. This region in the wild-type FGF-1...
Show moreThe structure of human acidic fibroblast growth factor (FGF-1) is classified a b-trefoil structure, one of the fundamental protein superfolds. The x-ray crystal structures of wild type and various mutants of FGF-1 have been solved in five different space groups: C2, C2221, P21 (4 molecules/asu), P21 (3 molecules/asu) and P212121. These structures reveal two characteristically different conformations for the b8/b9 b-hairpin comprising residue positions 90-94. This region in the wild-type FGF-1 structure (P21, four molecules/asu), a His-tagged His93®Gly mutant (P21, three molecules/asu) and a His-tagged Asn106®Gly mutant (P212121) adopts a 3:5 b-hairpin known as a type I (1-4) G1 b-bulge (containing a type I turn). However, a His-tagged form of wild-type FGF-1 (C2221) and a His-tagged Leu44®Phe mutant (C2) adopt a 3:3 b-hairpin (containing a type I' turn) for this same region. A structural feature that distinguishes these two types of b-hairpin structures is the number and location of side chain positions with eclipsed Cb and main chain carbonyl oxygen groups (Y @ +60°). The effects of glycine mutations upon stability, at positions within the hairpin, have been used to identify the most likely b-hairpin structure in solution. The results indicate that the 3:3 b-hairpin containing a type I' turn in some FGF-1 crystal forms is adopted due to the effects of crystal packing interactions. Type I' turns in the structural data bank are quite rare, and a survey of these turns reveals that a large percentage exhibit crystal contacts within 3.0Å. The results suggest that many of the type I' turns in x-ray structures may be adopted due to crystal packing effects. However, the results also suggest that type I' tur ns may identify structurally dynamic regions within proteins. Specific residues in a polypeptide may be key contributors to the stability and foldability of the unique native structure. Identification and prediction of such residues is, therefore, an important area of investigation in solving the protein-folding problem. Atypical main chain conformations can help identify strain within a folded protein, and by inference, positions where unique amino acids may have a naturally high frequency of occurrence due to favorable contributions to stability and folding. Non-Gly residues located near the left-handed a-helical region (L-a) of the Ramachandran plot are a potential indicator of structural strain. Although many investigators have studied mutations at such positions, no consistent energetic or kinetic contributions to stability or folding have been elucidated. Here we report a study of the effects of Gly, Ala and Asn substitutions found within the L-a region at a characteristic position in defined b-hairpin turns within FGF-1, and demonstrate consistent effects upon stability and folding kinetics. The thermodynamic and kinetic data are compared to available data for similar mutations in other proteins, with excellent agreement. The results have identified that Gly at the i+3 position within a subset of b-hairpin turns is a key contributor towards increasing the rate of folding to the native state of the polypeptide while leaving the rate of unfolding largely unchanged. Turn 4 and turn 8 regions are two turns related by the b-trefoil 3- fold axis of symmetry within FGF-1. These two turns have different turn conformations, even though there are no insertions or deletions within turn regions. By using Nuclear Magnetic Resonance (NMR) and Gly substitutions, we prove that the double conformation of turn 4 is not from the crystal-packing artifact. We also prove that the turn 4 region is a lowenergy conformation with little associated structural strain by using Gly screening and turn sequence swapping using the turn 8 sequence into the turn 4 sequence. We also show that the turn 8 region has a structural strain based on the significant effects upon both the protein stability and rate of folding or unfolding. By using three turn 4 mutant crystal structures and one turn 8 mutant analyzed by NMR, we show that the variable turn sequences within turn regions do not affect the structure. Rather we find the adjacent residue to turn region 8 (Leu89) is the key residue for determining the turn structure. Previously, we demonstrated that by adding one more criterion (requiring the position to be the i+3 residue within a type I b-hairpin), we could identify a consistent stability effect for Gly and Ala substitutions within the L-a of the Ramachandran plot. We extended our target turn region to three additional 3:5 type I turn in FGF-1 to see the consistent results of previous chapter. We substituted each i+3 residues (all Gly) of three turn regions with Ala. The stability data of two-turn regions GlyÃ Ala substitution was consistent with what we expected (~10 kJ/mol destabilization effect). One mutant (Gly71Ã Ala), however, showed an extra destabilization effect (~15 kJ/mol). We found this additional effect came from a bad contact of non-Gly residue with neighboring atoms. Nonetheless, we could identify a single structural property, independent of local sequence or neighboring effects, with a predictable effect upon protein stability.
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Date Issued: 2003
Identifier: FSU_migr_etd-3117
Format: Thesis

Title: Interactions and Conformations of Polyelectrolytes in Solutions, Thin Films, and Complexes.
Creator: Markarian, Marie Z., Schlenoff, Joseph B., Van Winkle, David H., Cooper, William T., Mattoussi, Hedi, Roper, Michael G., Department of Chemistry and Biochemistry, Florida State...
Show moreMarkarian, Marie Z., Schlenoff, Joseph B., Van Winkle, David H., Cooper, William T., Mattoussi, Hedi, Roper, Michael G., Department of Chemistry and Biochemistry, Florida State University
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Abstract/Description: Polyelectrolyte complexes have gained great attention over the past few decades for their applications in materials science, engineering, and medicine. They can be assembled into thin films via the layer-by-layer deposition of oppositely charged polyelectrolytes or transformed into moldable complexes, or hydrogels, through their controlled mixing. The properties of the resulting materials are highly tunable depending on the nature and composition of the system at hand; of great importance are...
Show morePolyelectrolyte complexes have gained great attention over the past few decades for their applications in materials science, engineering, and medicine. They can be assembled into thin films via the layer-by-layer deposition of oppositely charged polyelectrolytes or transformed into moldable complexes, or hydrogels, through their controlled mixing. The properties of the resulting materials are highly tunable depending on the nature and composition of the system at hand; of great importance are the polyelectrolytes' interactions and their conformations. In this dissertation, we walk through several chemiscapes revealing the properties of synthetic, biocompatible, and biological polyelectrolytes and their complexes. Polynucleotides are the focus herein; they have been used as materials in biocompatible polyelectrolyte composites for the many controllable properties they offer: hydrogen bonding, electrostatic interactions, and conformational changes depending on pH, ionic strength and temperature. DNAs have also caught the attention of theoretical investigators; they are used as model polyelectrolytes due to their defined structure and charge density. On the other hand most of the studies have been conducted under conditions far from physiological while the applications are targeted for medicinal purposes as implants or for drug and/or gene delivery. The first section of the manuscript describes the studies conducted on the isothermal pairing of oligonucleotides in solutions under molecular crowding conditions and different ionic strengths mimicking physiological conditions where polyethylene glycol is used as crowder. The osmotic properties of polyethylene glycol and sodium chloride solutions were investigated to establish the thermodynamics of the DNA pairing while spectroscopy was used to monitor the conformational changes and the binding properties of the strands under these conditions. In the following section, the discussion extends to the hybridization of polynucleotides incorporated into polyelectrolyte multilayers. Biodegradable capsules of polynucleotides based on the layer-by-layer technique are candidates for gene delivery. It is therefore important to understand the properties and stability of polynucleotides in polyelectrolyte matrices. We employed surface characterization and spectroscopic methods to establish the polynucleotides' hydrogen bonding efficacy under varying ionic and temperature conditions. Polyelectrolyte thin films are also promising coatings as anti-fouling, anti-corrosion, and anti-sticking agents. To improve these characteristics it is necessary to have a clear vision of the composition of the thin films to better control their chemical, physical and mechanical properties. In this dissertation, scintillation counting using radiolabeled ions is described for the determination of the ionic content of the polyelectrolyte films and the extent of polyelectrolyte interpenetration and charge overcompensation, the driving forces of the layer-by-layer technique. Finally, we present the synthesis and analysis of polyelectrolyte complexes which have gained attention as materials for bio-implants and cell culture scaffolds. This section details the methods used for the synthesis of stoichiometric complexes followed by their characterization by neutron scattering methods where the conformation of individual polyelectrolyte chains is explored under different ionic conditions presenting a first study of the structural properties of the charged polymers in these compact matrices.
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Date Issued: 2011
Identifier: FSU_migr_etd-7189
Format: Thesis

Title: Radical Fragmentations: From Conformational Control of Enediyne Reactivity to 1,2-C,O Transposition and Metal-Free Synthesis of Benzoates and Benzamides from Phenols.
Creator: Baroudi, Abdulkader, Alabugin, Igor V., Locke, Bruce R., Dudley, Gregory B., Steinbock, Oliver, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: The work described in this thesis is a demonstration of how theory and experiment can be used side by side in developing research in organic chemistry, from mechanistic studies to the design of new reactions. In chapter one we describe a variety of fragmentations and rearrangements following Bergman cyclization in enediynes equipped with acetal rings mimicking the carbohydrate moiety of natural enediyne antibiotics of the esperamicine and calchiamicine families. In the first step of all these...
Show moreThe work described in this thesis is a demonstration of how theory and experiment can be used side by side in developing research in organic chemistry, from mechanistic studies to the design of new reactions. In chapter one we describe a variety of fragmentations and rearrangements following Bergman cyclization in enediynes equipped with acetal rings mimicking the carbohydrate moiety of natural enediyne antibiotics of the esperamicine and calchiamicine families. In the first step of all these processes, intramolecular H-atom abstraction efficiently intercepts the p-benzyne product of the Bergman cyclization through a six-membered transition state (TS) and transforms the p-benzyne into a new more stable radical. Depending on the substitution pattern and reaction conditions, this radical follows four alternative paths: (a) abstraction of an external hydrogen atom, (b) O-neophyl rearrangement which transposes O- and C-atoms of the substituent, (c) fragmentation of the O─C bond in the acetal ring, or (d) fragmentation with elimination of the appended acetal moiety as a whole. Experiments with varying concentrations of external H-atom donor (1,4-cyclohexadiene) were performed in order to gain further insight into the competition between intermolecular H-abstraction and the fragmentations. The Thorpe-Ingold effect in gem-dimethyl substituted enediynes enhances the efficiency of fragmentation to the extent where it cannot be prevented even by a large excess of external H-atom donor. These processes provide insight into a possible mechanism of unusual fragmentation of esperamicin A1 upon its Bergman cycloaromatization and lay foundation for a new approach for the conformational control of reactivity of these natural antitumor antibiotics. Such an approach, in conjunction with supramolecular constraints, may provide a plausible mechanism for resistance to enediyne antibiotics by the enediyne-producing microorganisms. In chapter two we demonstrate our new reaction that can transform phenol derivatives directly into esters and amides of benzoic acids via a new radical cascade. This newly designed radical transformation was inspired by the radical rearrangement discovered for one of the THP-equipped enediynes described in chapter one. We found that this process can be rendered practically useful for C,O-transposition in diaryl thiocarbonates and thiocarbamates. These compounds are available in a single high-yielding step from phenols, and can be transformed into benzoates derivatives through a radical cascade inspired by selective addition of silyl radicals at the sulfur atom of the C=S moiety. This addition step, analogous to the first step of the Barton-McCombie reaction, affords an anomerically stabilized carbon radical which can undergo a reversible 1,2 O→C transposition through O-neophyl rearrangement. The usually unfavorable equilibrium in the rearrangement step is shifted via a highly exothermic C─S bond scission in the O-centered radical which furnishes the final benzoic ester or benzamide product. This sequence provides a metal-free approach for the preparation of benzoate derivatives from phenols, a potentially useful alternative to metal catalyzed carbonylation of aryl triflates.
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Date Issued: 2010
Identifier: FSU_migr_etd-4623
Format: Thesis

Title: Functional and Structural Study of Crispr Processing Endonuclease Cas6.
Creator: Wang, Ruiying, Li, Hong, Epstein, Lloyd M., Miller, Brian G., Roper, Michael, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) interference is a newly discovered small-RNA based immune system used by bacteria and archaea. One of the essential steps in this interference is to process long CRISPR RNAs into small CRISPR RNAs (crRNAs). Cas6 has been found to perform the CRISPR RNA processing endonuclease activity in archaea, and our goal is to study the molecular basis of Cas6's function as the endonuclease to bind and cleave the repeat RNAs. The work...
Show moreCRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) interference is a newly discovered small-RNA based immune system used by bacteria and archaea. One of the essential steps in this interference is to process long CRISPR RNAs into small CRISPR RNAs (crRNAs). Cas6 has been found to perform the CRISPR RNA processing endonuclease activity in archaea, and our goal is to study the molecular basis of Cas6's function as the endonuclease to bind and cleave the repeat RNAs. The work described in this manuscript mostly focuses on study of the structures of Cas6 protein and Cas6-RNA complexes. The main objectives of this dissertation have been outlined. In chapter 2, our goal is to understand the CRISPR processing and the enzyme involved in this small RNA generating process. A high resolution crystal structure of free Cas6 from Pyrococcus furiosus has been obtained. This structure, together with the cleavage and binding activity assays, reveals the characteristics of this endoribonuclease. In chapter 3, we want to understand the molecular mechanism of the binding and cleavage activity of Cas6, the complex structure of Cas6 and its substrate RNA was solved. Our findings suggest that Cas6 processes CRISPR RNA by a beads-on-a-string model. And Cas6 is a single-stranded RNA specific endonuclease. In chapter 4, we apply the crystallization on various repeat RNAs and Cas6 protein's non-catalytic homologue (Cas6nc) to understand how RAMP proteins, the most abundant family of proteins involved in CRISPR interference, interact with various CRISPR repeat RNAs. The structure and the complementary biochemical analysis suggest a wrap-around model of RAMP-RNA interactions, and it also sheds light on how RAMP proteins are able to specifically interact with different repeat RNA substrates.
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Date Issued: 2010
Identifier: FSU_migr_etd-4631
Format: Thesis

Title: Polyelectrolytes in Optoelectronics and Biomaterials.
Creator: Al-Hariri, Lara A., Schlenoff, Joseph B., Alamo, Rufina G., Marshall, Alan, Steinbock, Oliver, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Polyelectrolyte multilayers are built by the alternating deposition of oppositely charged polymers from solutions onto a substrate. The physical and chemical properties of the multilayers can be controlled by the polyelectrolyte combination, the build up, and the post-build up conditions. The demands of novel material coatings that could be used at the surface of biomedical devices and nano-assemblies that can be used in optoelectronic devices are the trigger for material scientist to explore...
Show morePolyelectrolyte multilayers are built by the alternating deposition of oppositely charged polymers from solutions onto a substrate. The physical and chemical properties of the multilayers can be controlled by the polyelectrolyte combination, the build up, and the post-build up conditions. The demands of novel material coatings that could be used at the surface of biomedical devices and nano-assemblies that can be used in optoelectronic devices are the trigger for material scientist to explore the new research areas. Polyelectrolyte multilayers are investigated in an aim to fulfill the spectrum of needs ranging from biomaterial to optoelectronic field. In this dissertation, the thermal elimination of a water soluble poly(xylylidene) precursor to poly(phenylene vinylene) (PPV) is investigated using small and macro-counterion. The aqueous route to PPV is investigated to yield high photoluminescence efficiency at low elimination temperature. The polyelectrolyte multilayers terminated with different heteroatom polycations interaction with cells is investigated for application as a coating for stent. Fluorescence, UV-vis and Fourier transform infra red spectroscopy were used in order to investigate the optical properties of PPV. PPV elimination was accomplished in air at 80 ºC using the macro-counterion poly(ethylene glycol)-4-nonylphenyl-3-sulfoproply ether (PEGNOPS). The route to PPV using PEGNOPS was low on carbonyl content, with a possible contribution from PPV chain separation. The PEGNOPS precursor showed no carbonyl formation on long term storage in contrast to other counterions. Orthogonal Space Random Walk (OSRW) was performed to understand the exceptionally low temperature (80 ºC) required for thermal conversion of PPV precursor containing PEGNOPS. Simulations of the solvent free-system converged for starting points where the backbone was expanded or compressed. The simulation revealed the ability of PEGNOPS to partially pre-order the precursor chain in a conformation that favors the E2 elimination. The elimination mechanism of poly(xylydiene tetrahydrothiophene) (PXT), precursor to PPV, in assembly in polyelectrolyte multilayers was investigated using FT-IR. PXT assembly with poly(styrene sulfonate) (PSS) and poly(acrylic acid) (PAA) as a polycation were investigated. The heteroatom effect on polyelectrolyte multilayer assembly was explored. Polycations having the same charge density, molecular weight, and molecular weight distribution were employed for multilayer assembly. The polycations differed only in the heteroatom on which the positive charge resided. The three multilayers terminated with the three different polycations poly(vinyl benzyl trimethyl ammonium) (PVBTMA), poly(vinyl benzyl trimethyl phosphonium) (PVBTMP) and poly(vinyl benzyl dimethyl sulfonium) (PVBDMS) had similar build up characteristics, hydration and wettability. The interaction of the smooth muscle cells (A7r5) with the PVBTMA, PVBTMP and PVBDMS terminated multilayers was investigated. The PVBTMA and PVBTMP are cytophobic to A7r5 cells whereas the PVBDMS multilayer is cytophilic. The effect of UV sterilization of the PVBDMS multilayer on cell interaction was explored.
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Date Issued: 2011
Identifier: FSU_migr_etd-4596
Format: Thesis

Title: Proteomics of Putative Protein Biomarkers in Human Breast Cancer Tissues.
Creator: Semaan, Suzan M., Sang, Qing-Xiang Amy, Keller, Thomas C. S., Marshall, Alan, Stagg, Scott, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Breast cancer is the second leading death-causing cancer in women, after lung cancer. Breast cells are normally responsive to the estrogen hormone as they have the receptor for it. Breast cancer types that express the estrogen receptor have been targeted by anti-hormonal drugs yet treatment is not always successful due to the resistance of some breast cancer cells. Studies have shown that some breast cancer cells overlying broken breast-duct walls have lost their estrogen receptor (ER) and...
Show moreBreast cancer is the second leading death-causing cancer in women, after lung cancer. Breast cells are normally responsive to the estrogen hormone as they have the receptor for it. Breast cancer types that express the estrogen receptor have been targeted by anti-hormonal drugs yet treatment is not always successful due to the resistance of some breast cancer cells. Studies have shown that some breast cancer cells overlying broken breast-duct walls have lost their estrogen receptor (ER) and became more aggressive favoring metastasis more than the ER positive (ER+) cancer cells lying beside them in the duct. Those ER negative (ER-) cells are not affected by anti-hormonal treatments. Therefore more prognostic biomarkers are needed to increase the life expectancy in breast cancer affected women. Post translational modifications, such as glycosylation and phosphorylation, are essential to correct functioning of proteins within the cell. The addition of sugar moieties to a glycoprotein changes its structure and thus its function, which has been frequently correlated with cancerous transformation. Similarly, phosphorylation or dephosphorylation of a protein directly affects the protein's function. Identifying such post translational differences at different stages of breast cancer is essential for recognizing possible targets or specific biomarkers. In this work, proteins extracted from ER+ and ER- human breast cancer tissues were separated by two dimensional gel electrophoresis (2-DE), and stained for glycoproteins and total proteins. Breast cancer tissue lysates were N-deglycosylated in order to identify the N-linked glycoproteins. The 2-DE scans were analyzed and the proteins with significant differential expression among the ER+, ER-, and their N-deglycosylated proteins were identified by means of a reversed-phase nano-liquid chromatography coupled with a hybrid linear quadrupole ion trap/Fourier transform ion cyclotron resonance mass spectrometer (LC-LTQ/FTICR-MS). Spot count results showed that 30% of the total lysate proteins are glycoproteins. Alpha-1-acid, alpha-1- antitrypsin, calmodulin, and superoxide dismutase mitochondrial precursor were among the identified glycosylated proteins, further verified by Western blotting in both ER+ and ER- human breast tissues. A possible glycosylation difference in alpha-1-antitrypsin was found to be expressed highest in the ER- samples. This glycosylation difference is a potential tumor-derived biomarker for breast cancer progression.
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Date Issued: 2011
Identifier: FSU_migr_etd-7228
Format: Thesis

Title: Supramolecular Multichromophoric Dye Sensitized Solar Cells.
Creator: Goodson, Flynt, Saha, Sourav, Siegrist, Theo, Zhu, Lei, Mattoussi, Hedi, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Global demand for clean sustainable energy is growing with the planet's population. While fossil fuels are the current standard, many are looking to the nearly endless energy supplied by the Sun. Because of the potential cost benefit over conventional silicon solar cells, dye sensitized solar cells (DSSCs) are a popular research topic. In Chapter One, current fuel sources and cost as well as the present DSSC research are discussed. A description and explanation of DSSC device principles are...
Show moreGlobal demand for clean sustainable energy is growing with the planet's population. While fossil fuels are the current standard, many are looking to the nearly endless energy supplied by the Sun. Because of the potential cost benefit over conventional silicon solar cells, dye sensitized solar cells (DSSCs) are a popular research topic. In Chapter One, current fuel sources and cost as well as the present DSSC research are discussed. A description and explanation of DSSC device principles are also provided. Chapter Two provides an overview of the challenges for DSSC and ways these might be overcome: multichromophoric absorption, better charge injection, donor-acceptor charge separation. In Chapter Three the synthetic schemes and methods for the dye molecules are discussed. Characterization of these dyes by 1H and 13C NMR and mass spectrometry are also presented. In Chapter Four, individual dyes are assembled into Zinc-porphyrin***peryleneimide and zinc phthalocyanine***peryleneimide dyads in solution. The dyads as well as individual dyes will be characterized in solution by UV-vis, NMR, and electrochemical techniques to measure the absorption, binding constants, and redox potentials. Chapter Five describes how DSSC are constructed from a dye coated working electrode, platinized indium tin oxide (Pt/ITO) counter electrode, and I−/I3− redox mediator. The TiO2-coated F: SnO2 (FTO), working electrode was functionalized with supramolecular dyad systems in a two-step process. Then Current/Voltage relationship and Incident Photon to Current Conversion Efficiency of the DSSC devices were measured. These measurements found that multichromphoric dye-sensitized solar cells (DSSCs) based on self-assembled zinc-porphyrin***peryleneimide and zinc phthalocyanine***peryleneimide dyads exhibit higher device efficiency than individual dye DSSCs. The novel ZnPc***peryleneimide system has the highest reported efficiency (η = 2.3%) among DSSCs made of supramolecular dyad chromophores. Increased efficiency of dyad systems can be attributed to co-sensitization as well as vectorial electron transfer that lead to better electron-hole separation in the device. Chapter Six describes how the electron deficient 3,4,9,10-perylenediimide (PDI-12) selectively undergoes thermal ET from strong Lewis basic OH− and F− anions, but remains electronically and optically silent to poor Lewis basic anions, as ET and CT events are turned OFF. These interactions have been fully characterized by UV/Vis, NMR, and EPR spectroscopies. These results demonstrate the generality of anion-induced ET events in aprotic solvents, as long as the electronic requirements are satisfied.
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Date Issued: 2014
Identifier: FSU_migr_etd-8991
Format: Thesis

Title: Structural and Mechanistic Studies of Ribonucleoprotein Particle Pseudouridine Synthase.
Creator: Zhou, Jing, Li, Hong, Schlenoff, Joseph B., Striegel, Andre, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Box H/ACA ribonucleoprotein particle (RNP), an RNA-guided RNA modification enzyme, is comprised of four conserved proteins (Cbf5, Nop10, Gar1 and L7Ae) and one guide RNA. This complex recognizes the substrates by the guide RNA and catalyzes the chemical modification by the protein partners. In archaea and eukaryotes, box H/ACA RNP is responsible for pseudouridylation in most stable RNAs, using more than 100 guide RNAs. In this thesis, we present the structural studies of this most complex...
Show moreBox H/ACA ribonucleoprotein particle (RNP), an RNA-guided RNA modification enzyme, is comprised of four conserved proteins (Cbf5, Nop10, Gar1 and L7Ae) and one guide RNA. This complex recognizes the substrates by the guide RNA and catalyzes the chemical modification by the protein partners. In archaea and eukaryotes, box H/ACA RNP is responsible for pseudouridylation in most stable RNAs, using more than 100 guide RNAs. In this thesis, we present the structural studies of this most complex pseudouridine synthase. Based on this complicated system, we extend our understanding of the catalytic mechanism of pseudouridylation. In Chapter 2, we present a crystal structure of the substrate binding archaeal box H/ACA RNP complex which shows the detailed information of the active site. The 5-fluorouridine on the target position of substrate RNA is fully docked and rearranged into (5S,6R)-5-fluoro-6-hydroxy-pseudouridine (f5oh6Ψ) similar to other known structures of non RNA-guide pseudouridine synthases. This structure also reveals the substrate RNA binding details and the functions of conserved protein partners. In Chapter 3, we apply the crystallization on various substrate analogs with the functional H/ACA RNP complex. We also combine the activity assays and structures, provide a complete view of the enzyme active site along the reaction pathway, and suggest pre-organization as an important catalytic strategy. In Chapter 4, we focus on the specificity of PUA domain of Cbf5 for the absolutely conserved ACA sequence on guide RNA. We discovered that the high specificity enables the Cbf5 to catch the short RNA substrate containing ACA at the 3'end during crystallization. The activity assay shows that ACA trinucleotides block the Cbf5 activity on the small hairpin RNA substrate in a non RNA-guide manner.
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Date Issued: 2010
Identifier: FSU_migr_etd-7263
Format: Thesis

Title: Selective Additions to Unsaturated Carbon-Carbon Bonds by the Use of N-Heterocyclic Carbene-Copper(I) Catalysts.
Creator: Ondrusek, Brian A., McQuade, D. Tyler, Locke, Bruce, Roper, Michael, Dudley, Gregory, Alabugin, Igor, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: ABSTRACT The development of easily-accessible and densely-functionalized synthetic intermediates is a longstanding goal of the organic chemistry community. The use of transition metal catalysts has allowed many synthetic targets to be produced with minimal waste. Copper(I) has distinct advantages to its use as a catalyst for organic transformations, in that it is relatively cheap and non-toxic by comparison to other metals which see frequent use. Hydroboration chemistry, in particular, has...
Show moreABSTRACT The development of easily-accessible and densely-functionalized synthetic intermediates is a longstanding goal of the organic chemistry community. The use of transition metal catalysts has allowed many synthetic targets to be produced with minimal waste. Copper(I) has distinct advantages to its use as a catalyst for organic transformations, in that it is relatively cheap and non-toxic by comparison to other metals which see frequent use. Hydroboration chemistry, in particular, has seen some of these benefits, as copper(I) catalyst systems for hydroboration have been the source of much research in the past 15 years, enabling the production of a wide variety of complex boron-containing synthetic intermediates via addition reactions to unsaturated bonds. The research contained herein describes our efforts to improve the methods for functionalization of unsaturated carbon-carbon bonds using N-heterocyclic carbene-copper(I) catalysts. The formation of chiral allylboronates via allylic substitution is an important class of reactions owing to the versatile products that result, which can be used subsequently to produce allylic alcohols, homoallylic alcohols as well as to form C-C bonds via coupling reactions. Previously, only a few examples of chiral allylboronates have been described by the asymmetric addition of boron, therefore this was designated a good starting point for our research into this area. Through the use of a copper(I) catalyst with a 6-membered N-heterocyclic carbene (6-NHC) ligand, we have demonstrated the ability to produce chiral allylboronates with a high degree of selectivity. Additionally, this system was found to be stereoconvergant, meaning that both the E and Z-isomeric starting materials resulted in products with the same absolute stereochemistry. In addition to polarized allylic alkenes, we also investigated the hydroboration of alkenes which are strained but otherwise unactivated, a class of substrates understudied for copper-catalyzed hydroboration. Through the use of a 5-NHC ligand pioneered by Glorius, we were able to achieve the selective hydroboration of unfunctionalized strained alkenes in the presence of non-strained alkenes for a variety of norbornene and styrenic substrates. In addition to the hydroboration of C-C double bonds, we then became interested in the addition of boron to alkynyl species to produce functionalized vinylboronates. Regioselective hydroborations utilizing copper(I) catalysis have been reported for terminal alkynes and for some internal alkyne systems, however propargylic substrates have been understudied and the ability to access any desired regioiosmer remains elusive. Our efforts have shown that boron addition to propargylic alcohols can be controlled by the careful selection of catalyst and O-protecting group. Specifically, we have demonstrated that the use of a 6-NHC-Cu(I) complex with a m-nitrophenyl propargyl ether results in selectivity for the alpha position with a high degree of selectivity. The use of a 5-NHC-Cu(I) complex to an unprotected propargylic alcohol produced the desired beta-borylated material, also with good selectivity. In this way we have been able to produce a variety of functionalized vinylboronates to which we then turned our attention. In an effort to showcase the utility of these vinylboronate species, we have also developed an ate-mediated allylic substitution, or AMAS, which can produce tertiary allylboronates by the simple addition of organomagnesium reagent. A variety of Grignard reagents were employed, and it was demonstrated that the AMAS procedure is compatible with most functional groups that are stable under Grignard conditions. To showcase the synthetic viability of this procedure we performed a total synthesis of the sugar molecule methyl axenoside from entirely achiral starting materials. This natural product was synthesized in 9 steps with an overall yield of 10%.
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Date Issued: 2014
Identifier: FSU_migr_etd-9066
Format: Thesis

Title: The Effects of Apples and Exercise on Clinical Parameters of Metabolic Syndrome in Women with Polycystic Ovary Syndrome.
Creator: Booth, Brandon Kody, Spicer, Maria, Figueroa, Arturo, McGee, Daniel, Department of Nutrition, Food, and Exercise Science, Florida State University
Abstract/Description: Purpose: The purpose of this study was to examine the effect of the consumption of apples and moderate intensity resistance training, twice weekly, on blood glucose, abdominal circumference, blood lipids, blood pressure, BMI and insulin resistance in overweight/obese college age women with polycystic ovary syndrome. Methods: Premenopausal women (ages 18-27) were randomly assigned to four treatment groups: apple supplement without exercise (Apple) (n=5), apple supplement with exercise (ExApp) ...
Show morePurpose: The purpose of this study was to examine the effect of the consumption of apples and moderate intensity resistance training, twice weekly, on blood glucose, abdominal circumference, blood lipids, blood pressure, BMI and insulin resistance in overweight/obese college age women with polycystic ovary syndrome. Methods: Premenopausal women (ages 18-27) were randomly assigned to four treatment groups: apple supplement without exercise (Apple) (n=5), apple supplement with exercise (ExApp) (n=6), cereal supplement (placebo) without exercise (Cereal) (n=3), or cereal supplement with exercise (ExCer) (n=3) for eight weeks. Participants in the apple groups received 75 grams of dried apples. The control supplement was 65 grams of cereal, matched in calories and fiber content with the apple supplement. Participants were provided with 28 days worth of supplements to be eaten in the first 4 weeks, returned to the lab for midpoint assessments, and given another 4 weeks worth of supplements. The exercise protocol consisted of resistance training twice per week with each session allowing 48-72 hours for recovery. At each training session participants performed 3 sets of 12 repetitions at the 12RM. During each visit a total of 10 exercises were performed; one for each major muscle group. The non-exercise groups did not perform any physical activity outside of their usual activities. Anthropometric measurements and blood were collected at the beginning (0wk) and at the end (8wk) of the study. Results: Fasting blood glucose significantly increased (p<0.05) in the Cereal and ExCereal groups, but did not change in the Apple and ExApp groups. The Cereal groups, with and without exercise, showed significant increases (p≤0.05) in fasting insulin and HOMA-IR. Both the Cereal and ExApp groups exhibited significant increases (p≤0.05) in total cholesterol. The Cereal group had significantly elevated (p<0.05) LDL cholesterol levels while the ExApp group had significantly elevated (p<0.05) TG levels. Conclusions: This study found that consuming 75 grams of dried apples every day for 2 months can prevent increases in insulin resistance and prevent glucose, insulin, and LDLc from significantly increasing in young, sedentary women with PCOS.
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Date Issued: 2010
Identifier: FSU_migr_etd-3525
Format: Thesis

Title: Determining the Solution Conformational Entropy of Oligosaccharides: Isolating the Effects of Anomeric Configuration, Glycosidic Linkage, Degree of Polymerization, Linearity versus Cyclicity and Hydrogen Bonding.
Creator: Boone, Marcus Alexander, Striegel, André M., Collins, Emmanuel, Dorsey, John, Nymeyer, Hugh, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Oligosaccharides have various fundamental roles, ranging from providing nutritional and flavoring support to being involved in fertilization and parasite infection processes. Maltooligosaccharides are used heavily in the food and pharmaceutical industries, cellooligosaccharides have shown promise as prebiotic candidates, and cyclodextrins are potential candidates for drug delivery. Factors such as degree of polymerization and hydrogen bonding play key roles in the aforementioned processes and...
Show moreOligosaccharides have various fundamental roles, ranging from providing nutritional and flavoring support to being involved in fertilization and parasite infection processes. Maltooligosaccharides are used heavily in the food and pharmaceutical industries, cellooligosaccharides have shown promise as prebiotic candidates, and cyclodextrins are potential candidates for drug delivery. Factors such as degree of polymerization and hydrogen bonding play key roles in the aforementioned processes and phenomena. Other factors such as anomeric configuration have been shown to influence bacterial binding and docking, while the glycosidic linkage is known to be a factor in binding processes such as those related to the interspecies spreading of avian influenza virus. Our group has pioneered the use of size-exclusion chromatography (SEC) in investigating solution conformational entropy (∆S) of select O-linked disaccharides and their monosaccharide constituents. I recently expanded the scope of these investigations to quantitate the ∆S of several homologous series of oligosaccharides, malto- and cellooligosaccharides and cyclodextrins. By using SEC and appropriate selection of hydrogen-bond accepting or non hydrogen-bond accepting solvents, I was able to isolate and quantitate the individual contributions of degree of polymerization, anomeric configuration, linearity versus cyclicity, and hydrogen bonding to the solution conformational entropy of the oligosaccharides. Due to the importance of oligosaccharides in biomolecular recognition phenomena, the next step was to study oligosaccharides at quasi-physiological conditions, i.e., in an aqueous solvent, at 37° C, and at a pH of 7.39. In the final step of the research I isolated the effects of glycosidic linkage on solution conformational entropy. To this effect, I compared homologous series of α-(1→6) and β-(1→3) oligomers, individually, to their α-(1→ 4) and β-(1→4) counterparts, respectively. The α-(1→6) oligomers were the isomaltosides with DP 2-7, which were compared to the maltooligosaccharides. The β-(1→3) series examined were the laminarbiosides with DP 2-7, compared to the previously studied cellooligosaccharides.
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Date Issued: 2009
Identifier: FSU_migr_etd-3533
Format: Thesis

Title: Computational and Experimental Exploration of Conformational Changes in Nucleic Acids Associated Biomolecular Functions.
Creator: Nelson, Joycelynn D., Yang, Wei, Logan, Timothy, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: The works discussed within this dissertation explore two major considerations. The first of which explores key structural conformations within the branch site helix that are critical for ribonucleic acid (RNA) splicing. The branch site helix is formed by pairing U2 snRNA and intron from S. cerevisiae including a phylogenetically conserved pseudouridine (ψ) in U2 snRNA indicates that the branch site adenosine (A) of the intron strand is extruded from the helix and participates in a base triple...
Show moreThe works discussed within this dissertation explore two major considerations. The first of which explores key structural conformations within the branch site helix that are critical for ribonucleic acid (RNA) splicing. The branch site helix is formed by pairing U2 snRNA and intron from S. cerevisiae including a phylogenetically conserved pseudouridine (ψ) in U2 snRNA indicates that the branch site adenosine (A) of the intron strand is extruded from the helix and participates in a base triple (Newby and Greenbaum 2002a). Conformational changes experienced by the branch site adenosine(A), which is responsible for the first step in splicing, were observed by altering base triple formation. The function of the base triple was unknown , yet was thought to be a critical component for proper positioning of the branch site A, given its proximity within the branch site helix. 2-aminopurine fluorescence spectroscopy and nuclear magnetic resonance (NMR) were used to establish qualitative and quantitative structural data, respectively, of the branch site A and the branch site helix (Nelson and Greenbaum 2006). These data show that the introduction of an AU → UA mutation to the base triple were consistent with a stacked intrahelical conformation of the branch site A (Nelson and Greenbaum 2006). In order to obtain atomistic details of these solution structures, molecular dynamics simulations were used. A novel and innovative sampling strategy known as orthogonal space random walk (OSRW) (Zheng, Chen, Yang 2008; Zheng, Chen, Yang 2009) was utilized to efficiently calculate the free energy differences between conformational states of interest by using alchemical transitions to flipping uridine or ψ 180º at the phylogenetically conserved position. These data show that uridine significantly prefers it canonical conformation, where ψ may assume either conformation. Base flipping of the branch site A was quantified by using a new algorithm known as multi-time scale technique (MTST) (Lv, Nelson, Yang 2010). Information about this type of phenomenon are of significant importance, given this is common strategy of enzymes and other binding protein to acquired access to chemical relevant nucleic acids. Data obtained in these studies indicated that ψ encourage base flipping through the major groove and is energetically more favored as opposed to uridine. Uridine favors flipping of the branch site A toward the minor groove and energetically less favored. These data identify an energetically favored structural conformation, in the presence of ψ, that is highly probable to interact with other branch site proteins which affect the efficiency of splicing. In the second half of these works, an evaluation of metal binding in iron-dependent repressors found in Tuberculosis (TB) and Diphtheria Toxin (DT) was conducted. Interestingly, crystal structures for these repressors have been solved under several divalent metal ionic conditions with similar overall structure and metal ion coordination (Wang and others 1999; White and others 1998; Wisedchaisri, Holmes, Hol 2004). Semavina et al. and Stapleton et al. were able to detect cooperative and non-cooperative behaviors between various divalent metal ions using equilibration dialysis and fluorescence (Semavina, Beckett, Logan 2006; Stapleton and Logan 2010). OSRW was use to calculate the free energy difference upon divalent metal binding in various site in the repressors in order to understand cooperatively and the order of metal binding. These data aid in corroboration of data established in experimental approaches. The repressor found in TB has two possible pathways to bind nickel. The second of which seems to be the most favorable.
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Date Issued: 2010
Identifier: FSU_migr_etd-2660
Format: Thesis

Title: Development and Implementation of an NMR-Compatible 3-D Perfusion Bioreactor to Study HMSC Microenvironment.
Creator: Crowe, Jason J., Ma, Teng, Goldsby, Kenneth, Logan, Timothy, Grant, Samuel, Li, Hong, Program in Molecular Biophysics, Florida State University
Abstract/Description: Tissue engineering is one of the fastest growing biotechnology industries. The overarching research goals of the tissue engineering industry are highly intertwined with the field of medicine. The tissue engineering field seeks to create a variety of technologies that can be used in a clinical setting for tissue repair therapy. These technologies include everything from cell-based trophic factor therapies to engineered tissue grafts, and potentially engineered tissues. At the forefront of...
Show moreTissue engineering is one of the fastest growing biotechnology industries. The overarching research goals of the tissue engineering industry are highly intertwined with the field of medicine. The tissue engineering field seeks to create a variety of technologies that can be used in a clinical setting for tissue repair therapy. These technologies include everything from cell-based trophic factor therapies to engineered tissue grafts, and potentially engineered tissues. At the forefront of tissue engineering research is the extensive use of adult stem cells. Adult stem cells are located throughout the body and have the ability to differentiate into a variety of different tissue types. This plasticity enables adult stem cells to be used in a wide range of tissue engineering research. Despite all of the advancements made in the tissue engineering field, some obstacles still need to be overcome before tissue engineering can be used extensively as a clinical therapy for tissue damage and repair. One of the largest obstacles is the creation of tissue engineering technologies that mimic the existing human body conditions as close as possible. The creation of fully biomimetic tissue engineering technologies will increase the quality and functionality of the engineered grafts and therapies. The research presented will discuss the development and implementation of a biomimetic perfusion bioreactor. The reactor was designed to incorporate a number of environmental factors that play a large role in controlling cellular behaviors into a culture system capable of supporting the growth of a human mesenchymal stem cell construct. Furthermore, the reactor was constructed to allow the tissue construct to be studied in a non-invasive manner. This characteristic provides a way to keep the integrity of the cell construct throughout the experimentation process so it remains functional and viable until it is potentially needed for tissue therapy.
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Date Issued: 2009
Identifier: FSU_migr_etd-2986
Format: Thesis

Title: Increasing the Yield of Thy-1 EGFP Expression in Insect and Mammalian Cells.
Creator: Kasprzak, Agnieszka, Logan, Timothy M., Li, Hong, Dorsey, John, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Thy-1 is being pursued as a model system for NMR studies of structure and dynamics of intact glycoproteins. However, the low expression yield of the Thy-1 EGFP glycoprotein in insect and mammalian cells has been an obstacle for these studies. The objective of the following studies is to increase the yield of purified Thy-1 EGFP with correct post translational modifications and homogeneity in the three glycosylation sites. To improve the yield in the baculovirus based insect cell expression...
Show moreThy-1 is being pursued as a model system for NMR studies of structure and dynamics of intact glycoproteins. However, the low expression yield of the Thy-1 EGFP glycoprotein in insect and mammalian cells has been an obstacle for these studies. The objective of the following studies is to increase the yield of purified Thy-1 EGFP with correct post translational modifications and homogeneity in the three glycosylation sites. To improve the yield in the baculovirus based insect cell expression systems we investigated the effect of viral titer, multiplicity of infection (MOI), cell density, and length of expression. The MOI and cell density need to be optimized to give the highest yield of recombinant protein. It was found that the titer of a virus needs to be at least 1X108 pfu/ml and that more than two amplifications decrease both titer and expression levels. The optimal MOI used in for virus amplification was found to be between 0.01 to 0.05, while for expression, the optimal MOI is between 1-5. Optimal cell density for amplification of virus is 2.5 X106 cells/ml, while expression cell density is optimal at 3.0 X106 cells/ml. Optimized time of expressing Thy-1 EGFP is 72 hours post infection. Using these optimized conditions, the maximum yield of Thy-1 was 500 μg per liter of culture. Mammalian cell expression in Lec-1 cells provides a more homogenous glycosylation pattern on Thy-1 EGFP than observed from wild type CHO cells. In lec-1 cells the factors investigated to optimize the expression yield were media volume, time of expression and cell confluency. The optimal conditions for expression were found to be 48 hours in 50 mls of serum free media with 2mM butyric acid and 20mM HEPES. The cell confluency should be 100% before initiating expression. Using these optimized conditions the maximum protein yield was 2 mg of Thy-1 per liter of expression media. In a final set of experiments we sought to inhibit the activity of proteases that co-purify with Thy-1 through most of the purification steps. We screened for the effect of protease inhibitors such as E-64, leupeptin, aprotinin, and PMSF to minimize the degradation during purification. Protease activity was analyzed via zymogram assay and PMSF yielded the highest inhibition at a 0.5 mM concentration. The other protease inhibitors were less or completely ineffective.
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Date Issued: 2005
Identifier: FSU_migr_etd-3327
Format: Thesis

Title: Addition / C-C Bond Cleavage Reactions of Vinylogous Acyl Triflates and Their Application to Natural Product Synthesis.
Creator: Jones, David Mack, Dudley, Gregory B., Taylor, Kenneth, Saltiel, Jack, McQuade, D. Tyler, Goldsby, Kenneth, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: This dissertation describes the synthetic utility of tandem addition / C-C bond cleavage reactions of vinylogous acyl triflates. The first chapter provides background into carbon-carbon bond breaking reactions that have been applied in organic synthesis and the preliminary data that allowed for the original work presented here. Chapter 2 explains the significance as well as the prior syntheses of a commercially important moth pheromone, (Z)-6-heneicosen-11-one. The second chapter culminates...
Show moreThis dissertation describes the synthetic utility of tandem addition / C-C bond cleavage reactions of vinylogous acyl triflates. The first chapter provides background into carbon-carbon bond breaking reactions that have been applied in organic synthesis and the preliminary data that allowed for the original work presented here. Chapter 2 explains the significance as well as the prior syntheses of a commercially important moth pheromone, (Z)-6-heneicosen-11-one. The second chapter culminates in the synthesis of the sex attractant through a fragmentation reaction made possible by the direct extension of the initial nucleophile-triggered fragmentation studies to include the use of Grignard reagents. Chapter 3 describes the application of the fragmentation method, coupled to a benzannulation reaction, to afford penta- and hexasubstituted indanes. This two step sequence provides the basis for future work directed toward the synthesis of alcyopterosin A, a known cytotoxic agent with possible biological applications. The current difficulties pertaining to the treatment of melanoma are discussed in Chapter 4. Recently, an exciting natural product that provides promising activity against this horrible cancer was discovered. Palmerolide A has the ability to kill melanoma cells selectively at low concentrations. The fragmentation method developed in these laboratories provides entry into a key fragment. The Claisen-type condensation reaction of vinylogous acyl triflates was expanded to the synthesis of a novel beta-ketophosphine oxide olefinating reagent, which allowed for the rapid synthesis of the eastern hemisphere (C1-C15) of this exciting natural product. Optimization of the Claisen-type condensation reaction to provide the beta-ketophosphine oxide reagent, led to the optimal reduction of the number of equivalents of the nucleophile. Intrigued by this, these reactions were explored in more detail. The results of this investigation are described in Chapter 5. The reduction in the number of equivalents of nucleophile, a key feature in these reactions, may be attributed to the ability of the phosphorus atom to form of an oxaphosphetane-like intermediate. As a result, new, potentially useful, beta-ketophosphonates were synthesized.
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Date Issued: 2010
Identifier: FSU_migr_etd-3451
Format: Thesis

Title: Nanomaterials: Synthesis, Characterization, and Their Bio-Integration.
Creator: Jennings, Travis L., Strouse, Geoﬀrey, Chase, Bryant, Kasha, Michael, Kroto, Harry W., Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: The purpose of this dissertation is to utilize the changing optical and electronic properties of metallic and semiconductor nanomaterials for applications to biotechnology. The dynamic optical properties of metals and semiconductors with size is discussed in regard to the ability of these materials to accept electronic excitation energy from classical molecular fluorescent dyes (Chap. 2). Absorption, photoluminescence, and time-resolved photoluminescence experiments are performed on metal...
Show moreThe purpose of this dissertation is to utilize the changing optical and electronic properties of metallic and semiconductor nanomaterials for applications to biotechnology. The dynamic optical properties of metals and semiconductors with size is discussed in regard to the ability of these materials to accept electronic excitation energy from classical molecular fluorescent dyes (Chap. 2). Absorption, photoluminescence, and time-resolved photoluminescence experiments are performed on metal nanoparticle-dye pairs at separation distances controlled via synthetic DNA spacers where the distance, dye, and nanoparticle size are varied (Chaps. 3 and 4). It is found that the efficiency of energy transfer to small metal nanoparticles is greater than expected for a 1/R^6 Förster mechanism of energy transfer and the measurable separation distance is increased, following a 1/R^4 dependence. The 1/R^4 distance dependence is the theoretically established relationship of an excited molecule to a metallic surface. This tool, termed "Nanometal Surface Energy Transfer" (NSET) is then used in Chapters 5 and 6 to measure the kinetics and conformational changes associated with a hammerhead ribozyme as a model test subject for NSET methodology. Finally, ZnS-overcoated CdSe semiconductor quantum dots, (Chap. 7) are synthesized controllably and characterized in terms of their potential for biological incorporation for detection or in vitro studies.
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Date Issued: 2006
Identifier: FSU_migr_etd-3532
Format: Thesis

Title: Comparative Analysis of Human Milk Peptides: Gestational and Lactation Stage Effects.
Creator: Jeffries‐Grant, Kirsten, Rill, Randolph, Roux, Kenneth, Logan, Timothy, Cooper, William, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: In this study methods were developed for the analysis of endogenous human milk peptides with molecular weights
Date Issued: 2008
Identifier: FSU_migr_etd-3540
Format: Thesis

Title: Engineering Human Fibroblast Growth Factor-1 (Fgf-1) as a Human Therapeutic.
Creator: Xia, Xue, Blaber, Michael, Miller, Brian G., Stefanovic, Branko, Megraw, Timothy L., Florida State University, College of Medicine, Department of Biomedical Sciences
Abstract/Description: Fibroblast growth factor-1 (FGF-1) is an angiogenic factor with therapeutic potential for the treatment of ischemic disease. FGF-1 has low intrinsic thermostability and is characteristically formulated with heparin as a stabilizing agent. Heparin, however, adds a number of undesirable properties that negatively impact safety and cost. Mutations that increase the thermostability of FGF-1 may obviate the need for heparin in formulation and may prove to be useful "2nd-generation" forms for...
Show moreFibroblast growth factor-1 (FGF-1) is an angiogenic factor with therapeutic potential for the treatment of ischemic disease. FGF-1 has low intrinsic thermostability and is characteristically formulated with heparin as a stabilizing agent. Heparin, however, adds a number of undesirable properties that negatively impact safety and cost. Mutations that increase the thermostability of FGF-1 may obviate the need for heparin in formulation and may prove to be useful "2nd-generation" forms for therapeutic use. Chapter one is a report on a pharmacokinetic (PK) study in rabbits of human FGF-1 in the presence and absence of heparin, as well as three mutant forms having differential effects upon thermostability, buried reactive thiols, and heparin affinity. The results support the hypothesis that heparan sulfate proteoglycan (HSPG) in the vasculature of liver, kidney and spleen serves as the principle peripheral compartment in the distribution kinetics. The addition of heparin to FGF-1 is shown to increase endocrine-like properties of distribution. Mutant forms of FGF-1 that enhance thermostability or eliminate buried reactive thiols demonstrate a shorter distribution half-life, a longer elimination half-life, and a longer mean residence time (MRT) in comparison to wild-type FGF-1. The results show how such mutations can produce useful 2nd-generation forms with tailored PK profiles for specific therapeutic application. Chapter two is a systematic mutational analysis to eliminate buried free cysteines in FGF-1. Buried free Cys residues can contribute to an irreversible unfolding pathway that promotes protein aggregation, increases immunogenic potential, and significantly reduces protein functional half-life. Consequently, mutation of buried free Cys residues can result in significant improvement in the storage, reconstitution, and pharmacokinetic properties of protein-based therapeutics. Mutational design to eliminate buried free Cys residues typically follows one of two common heuristics: either substitution by Ser (polar and isosteric), or substitution by Ala or Val (hydrophobic); however, a detailed structural and thermodynamic understanding of Cys mutations is lacking. We report a comprehensive structure and stability study of Ala, Ser, Thr and Val mutations at each of the three buried free Cys positions (Cys16, Cys83, and Cys117) in fibroblast growth factor-1 (FGF-1). Mutation was almost universally destabilizing, indicating a general optimization for the wild-type Cys, including van der Waals and H-bond interactions. Structural response to Cys mutation characteristically involved changes to maintain, or effectively substitute, local H-bond interactions -- by either structural collapse to accommodate the smaller oxygen radius of Ser/Thr, or conversely, expansion to enable inclusion of novel H-bonding solvent. Despite the diverse structural effects, the least destabilizing average substitution at each position was Ala, and not isosteric Ser. Chapter three is a report on a gain-of-function mutation FGF-1 S116R and its potential interaction with the FGFR-1c. The 18 signaling members of the mammalian fibroblast growth factor (FGF) family have a broad range of functions in development, tissue homeostasis, and metabolism. This family includes the paracrine and endocrine FGFs - distinguished by the high heparan sulfate (HS) affinity of the former. HS plays a key role in cell signaling of the paracrine FGFs through formation of a ternary complex of FGF\FGF receptor (FGFR)\HS. Binding to HS on the surface of vascular endothelial cells also defines the peripheral compartment in the pharmacokinetics of the paracrine FGFs, and modulation of FGF HS affinity by specific mutation has the potential to tailor paracrine/endocrine properties. A designed S116R mutation in FGF-1 as constructed with the goal of increasing HS affinity. X-ray structure and biophysical analyses indicate that the S116R mutation has no discernable structural perturbation or change in protein stability or folding cooperativity in comparison to wild-type (WT) FGF-1. Isothermal titration calorimetry (ITC) analysis of complexation with sucrose octasulfate (SOS; a disaccharide HS analogue) indicates a statistically significant, albeit minor, increase in affinity. Surprisingly, assays of mitogenic activity show an order of magnitude gain of function. Several X-ray structures for distinctly different FGF\FGFR\HS ternary complexes have been reported. These structures predict substantially different environments for position 116 - varying from solvent exposure to participating in specific intermolecular FGFR contacts. The pronounced gain of function for the S116R FGF-1 mutation is most consistent with a recently reported novel FGF-1\FGFR1c\HS complex structure, which predicts the establishment of unique FGF\FGFR heterodimerization contacts involving S116R, and is distinct from either of the prevailing "2:2:2 symmetric" or "2:2:1 asymmetric" models.
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Date Issued: 2015
Identifier: FSU_2015fall_Xia_fsu_0071E_12832
Format: Thesis

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

Title: Influencing Human Mesenchymal Stem Cell Behavior with Small Molecular Compounds.
Creator: Bosco, Dale Braxton, Sang, Qing-Xiang Amy, Dudley, Gregory B., Roper, Michael Gabriel, Logan, Timothy M., Li, Yan, Florida State University, College of Arts and Sciences,...
Show moreBosco, Dale Braxton, Sang, Qing-Xiang Amy, Dudley, Gregory B., Roper, Michael Gabriel, Logan, Timothy M., Li, Yan, Florida State University, College of Arts and Sciences, Program in Molecular Biophysics
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Abstract/Description: Human mesenchymal stem cells are a class of adult multipotent cells that are of interest to researchers for their clinical potential. While this cell type has been intensely investigated, there is still a significant amount to be learned about how these cells function. The presented studies utilized small molecular compounds to investigate the abilities of hMSCs. Project one investigated the roles matrix metalloproteinases (MMPs) play in adipogenic differentiation of hMSCs. MMPs are a family...
Show moreHuman mesenchymal stem cells are a class of adult multipotent cells that are of interest to researchers for their clinical potential. While this cell type has been intensely investigated, there is still a significant amount to be learned about how these cells function. The presented studies utilized small molecular compounds to investigate the abilities of hMSCs. Project one investigated the roles matrix metalloproteinases (MMPs) play in adipogenic differentiation of hMSCs. MMPs are a family of metzincin proteinases that cleave the extracellular matrix (ECM), an activity that is critical to the differentiation process. These studies utilized the novel YHJ series of mercaptosulfonamide-based MMP inhibitors, which display high selectivity and potency for intermediate and deep pocket MMPs. Differentiation assays, kinetic assays, and qPCR were employed to characterize these inhibitors and determine their affect upon adipogenesis. Even though the YHJ MMPIs are not as stable as the hydroxamate-based MMPI GM6001, they produced comparable effects. Reduction in Lipid accumulation was also comparable to inhibition of peroxisome-proliferator activated receptor gamma (PPARγ) with the antagonist T0070907. Moreover, MMP inhibition was able to suppress lipid accumulation in cells co-treated with Troglitazone, a potent agonist of PPARγ. This is important as MMP inhibition is a potential avenue for obesity treatment research. Finally, the selectivity provided by the YHJ MMPIs allowed for the identification of MMP-3 (stromelysin-1) as a possible regulator of adipogenesis. Project two investigated hMSC resistance to the apoptotic effects of chemotherapeutic drugs. This has been of major interest, as hMSCs can confer resistance to tumor microenvironments. However, the effects of internalized chemotherapeutics upon hMSCs remain largely unexplored. Cellular viability and proliferation assays, combined with different biochemical approaches, were used to investigate the effects of Paclitaxel exposure upon hMSCs. The results indicate that hMSCs are highly resistant to the cytotoxic effects of Paclitaxel treatment, even though there was no detectable expression of the efflux pump P-glycoprotein, the usual means by which a cell resists Paclitaxel treatment. Moreover, Paclitaxel treatment induces hMSCs to adopt a non-proliferative fibroblastic state, as evidenced by changes to morphology, cellular markers, and a reduction in differentiation potential that is not directly coupled to the cytoskeletal effects of Paclitaxel. Taken together, these results show that Paclitaxel treatment does not induce apoptosis in hMSCs, but does induce quiescence and phenotypic changes.
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Date Issued: 2015
Identifier: FSU_migr_etd-9555
Format: Thesis

Title: Selective Heating of Polar Solutes in a Homogeneous Solution: Evidence of Microwave-Specific Effects and a Method to Quantify These Effects.
Creator: Rosana, Michael R., Dudley, Gregory B., Gunjan, Akash, Saha, Sourav, Alabugin, Igor, Stiegman, Albert, Dorsey, John, Department of Chemistry and Biochemistry, Florida State...
Show moreRosana, Michael R., Dudley, Gregory B., Gunjan, Akash, Saha, Sourav, Alabugin, Igor, Stiegman, Albert, Dorsey, John, Department of Chemistry and Biochemistry, Florida State University
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Abstract/Description: Microwave reactors offer convenience in sealed tube, high temperature reactions that can lower reaction time, increasing the value of certain synthetic procedures. Benzylation of alcohols, carboxylic acids and arenes using 2-benzyloxy-1-methylpyridinium triflate (BnOPT) translates smoothly from conventional heating to a microwave-heated method in trifluorotoluene in at 120 °C. Use of BnOPT avoids typical highly acidic and highly basic conditions of Benzylation, and compared to the analogous...
Show moreMicrowave reactors offer convenience in sealed tube, high temperature reactions that can lower reaction time, increasing the value of certain synthetic procedures. Benzylation of alcohols, carboxylic acids and arenes using 2-benzyloxy-1-methylpyridinium triflate (BnOPT) translates smoothly from conventional heating to a microwave-heated method in trifluorotoluene in at 120 °C. Use of BnOPT avoids typical highly acidic and highly basic conditions of Benzylation, and compared to the analogous reaction in an oil bath is completed in 20 minutes as opposed to 24 hours. In addition to shortening reaction times, 2-benzyloxy-1-methylpyridinium tetrakis(3, 5-bistrifluoromethylphenyl) borate (BnOPB), an analog of BnOPT, strongly supports the existence of microwave effects in homogeneous solutions despite widespread skepticism. Inspired by several unexplained observations in the area of microwave chemistry, we hypothesized that microwave effects would most likely be observed with a thermally-activated, highly polar (microwave-absorbing) reagent dissolved in a largely nonpolar (microwave transparent) solvent. Applied microwave energy is selectively absorbed by BnOPB leading to Friedel-Crafts benzylation of the solvent (toluene or xylene) that is not dependent solely on observed bulk temperature and which cannot be reproduced in an oil bath. A reflux-controlled reaction circumvents the fallibility of temperature probes which demonstrated an enhanced reactivity in a microwave reactor versus an oil bath (36% versus 67% after 30 minutes). We have coined the term microwave-actuated organic reactions: reactions for which there is clear benefit under microwave-heated conditions as compared to an oil bath reaction of the same temperature. Though using a solvent's physical property of boiling point, our data and conclusions are still met skeptically. In a debate style series of essays in Angewandte Chemie International Edition, we defended our work providing further data to support our conclusions and pointing out fallacies in the opposing essay denouncing our work. Three main points were addressed in our rebuttal essay: 1) our conclusions were misstated by the opposing author, 2) our experiments were inappropriately replicated and 3) the text of the authors essay and their supporting information are inconsistent with one another. Each of these points is addressed in full detail below, but the biggest issue raised by Kappe, et al. was that our refluxing toluene temperature was actually higher than literature-reported boiling point under microwave-heating, which could certainly undermine our conclusions. A quick analysis of the boiling point using a NIST-calibrated fiber-optic probe under several conditions proved to liberate us from these claims. It is clear to us that microwave effects exist, yet our desire to distance our work from the controversy associated with the nebulous terms of "microwave effects", "athermal microwave effects" and "specific microwave effects" has led us to develop a new term: microwave-specific thermal effects (MSTE). We define MSTEs as reactivity enhancements that are thermal in nature, but cannot be replicated outside of a microwave reactor. It is our belief that selective absorption of microwave energy by BnOPB in nonpolar solutions creates a temperature differential between the bulk solvent and the solvent cage surrounding the reagent. Reactivity should thus be based on the temperature of the microenvironment surrounding the reagent, not the bulk solvent temperature. Having shown that MSTEs exist, it became our goal to begin quantifying the extent of their effect. First order kinetic plots were created from conversion data of xylene to benzylxylene in the presence of BnOPB at varying temperatures from which Arrhenius plots were derived. An activation energy of 30.2 kcal/mol and a pre-exponential value of 9.64 x 1013 sec-1 were calculated. In conjunction with the Arrhenius equation (k = Ae(-Ea/RT)), kinetic data allows us to relate how observed changes can affect different varaiables of the Arrhenius equation that may approximate the extent of observed MSTEs. Best results were obtained using external cooling of a reaction vessel heated at high microwave power where we observed about a seven-fold increase in reaction rate under microwave heating. Success was shown in demonstrating MSTEs in another reaction: the conversion of allyl p-nitrophenyl ether to 2-allyl-4-nitrophenol by Claisen rearrangement. The system was examined under constant temperature, constant power and pulsed microwave heating. Constant temperature conditions nearly completely hid MSTEs. MSTEs were clearly observed under constant power conditions showing about a 4-fold rate enhancement. Best results were observed under pulsed microwave heating which showed about a 9-fold rate enhancement.
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Date Issued: 2014
Identifier: FSU_migr_etd-9081
Format: Thesis

Title: Wild-Type and Drug Resistant M2 Proton Channel from Influenza A: Structural Influences of Cholesterol, Drug and Proton Binding.
Creator: Ekanayake, Ekanayake Vindana, Cross, Timothy A., Fadool, Debra Ann, Cooper, William T., Marshall, Alan G., Florida State University, College of Arts and Sciences, Department of...
Show moreEkanayake, Ekanayake Vindana, Cross, Timothy A., Fadool, Debra Ann, Cooper, William T., Marshall, Alan G., Florida State University, College of Arts and Sciences, Department of Chemistry and Biochemistry
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Abstract/Description: Membrane proteins contribute about 30% of the proteome and about 60% of the drugs in the market target membrane proteins. But only about 1.5% of structures in Protein Data Bank are membrane proteins which reflect the challenges in membrane protein studies. In this dissertation work M2 full-length protein from Influenza A, a drug target to treat Influenza, is studied in lipid bilayer environment. ssNMR, the best technique to date to study membrane proteins at atomic resolution, has been...
Show moreMembrane proteins contribute about 30% of the proteome and about 60% of the drugs in the market target membrane proteins. But only about 1.5% of structures in Protein Data Bank are membrane proteins which reflect the challenges in membrane protein studies. In this dissertation work M2 full-length protein from Influenza A, a drug target to treat Influenza, is studied in lipid bilayer environment. ssNMR, the best technique to date to study membrane proteins at atomic resolution, has been utilized and intermonomer distance measurement is carried out throughout the study. Differently isotopically labeled protein mixtures were utilized for experiments to obtain unambiguous measurements, even though this method significantly reduces the sensitivity. Influence of amantadine, an antiviral drug, towards distinctive residues at different locations of the channel, such as N-terminus channel pore opening, channel interior and C-terminus of the channel has been characterized. M2 is known to be sensitive to its environment and here the influence of factors such as drugs, pH and membrane constituents such as cholesterol is studied. Distance information obtained for M2 full-length is compared with some of the existing M2 structures of truncated constructs and the importance of studying M2 protein as much as its native-like environment is emphasized. Among multiple drug resistant mutations of M2, S31N mutation contribute almost 100% of drug resistant strains observed in flu infected humans. Therefore, studies involving S31N mutation is an essential part of M2 proton channel studies. Hence, multiple experiments has been performed to obtain structural insights of this oligomeric protein and to compare them with that of M2 wild-type protein, in addition to study of its effect to environmental conditions such as pH.
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Date Issued: 2015
Identifier: FSU_migr_etd-9593
Format: Thesis

Title: Asymmetric Michael Additions of Lithium Propionate Enolates to α,β-Unsaturated Esters: A Study Towards the Total Synthesis of Lonomycin A.
Creator: Jo, Sunjin, Holton, Robert A., Reeves, Robert H., Krafft, Marie E., Zakarian, Armen, Blaber, Michael, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: The Michael addition of lithium enolates to α,β unsaturated esters is considered to be one of the powerful and widely used C-C bond forming methods in organic synthesis. We investigated the Michael addition of lithium propionate enolates using the various chiral auxiliaries to α,β unsaturated esters, methyl 2-bromo-3-methoxy acrylate and dioxinones. Various chiral propionates were prepared from the optically pure and commercially available terpenes in several steps. Michael additions using...
Show moreThe Michael addition of lithium enolates to α,β unsaturated esters is considered to be one of the powerful and widely used C-C bond forming methods in organic synthesis. We investigated the Michael addition of lithium propionate enolates using the various chiral auxiliaries to α,β unsaturated esters, methyl 2-bromo-3-methoxy acrylate and dioxinones. Various chiral propionates were prepared from the optically pure and commercially available terpenes in several steps. Michael additions using the chiral auxiliary introduced moderate to high stereoselectivities, and the factors that influence the stereoselection were examined. These results are consistent with a chelated transition state. The stereochemistry of Michael adducts was determined by the Mosher's esterification and NMR experiments. Lonomycin A, which was isolated from Streptomyces ribosidificus in 1975, is a polycyclic ether constituted of six highly functionalized rings and 23 stereocenters. Our synthetic strategy relying on the asymmetric Michael addition of lithium propionate enolate to α,β unsaturated esters introduced stereoselectively the contiguous and alternating methyl and methoxy moieties. This attractive methodology gave an easy access to the crucial intermediates in the synthetic approach towards the right-half fragment of Lonomycin A.
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Date Issued: 2008
Identifier: FSU_migr_etd-3505
Format: Thesis

Theses and Dissertations (188)	+ -
Undergraduate Honors Theses (23)	+ -

Chemistry (148)	+ -
Biophysics (82)	+ -
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