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Title: Petroleum Analysis by Atmospheric Pressure Photoionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.
Creator: Purcell, Jeremiah Michael, Marshall, Alan, Salters, Vincent, Cooper, William, Logan, Timothy, Rodgers, Ryan, Hendrickson, Christopher, Department of Chemistry and Biochemistry,...
Show morePurcell, Jeremiah Michael, Marshall, Alan, Salters, Vincent, Cooper, William, Logan, Timothy, Rodgers, Ryan, Hendrickson, Christopher, Department of Chemistry and Biochemistry, Florida State University
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Abstract/Description: Petroleum and petroleum products are an integral part of today's society. Although petroleum is projected to be the dominant energy source for the next fifty years, the depletion of light sweet crude oil reserves has led to the refinement of heavier feedstocks. Heavier petroleum feedstocks contain higher weight percent sulfur-, nitrogen- and oxygen-containing species. Not only is the combustion of these species harmful to the environment, they can also poison catalytic and hydrotreatment...
Show morePetroleum and petroleum products are an integral part of today's society. Although petroleum is projected to be the dominant energy source for the next fifty years, the depletion of light sweet crude oil reserves has led to the refinement of heavier feedstocks. Heavier petroleum feedstocks contain higher weight percent sulfur-, nitrogen- and oxygen-containing species. Not only is the combustion of these species harmful to the environment, they can also poison catalytic and hydrotreatment refining equipment. The United States Environmental Protection agency has limited allowable heteroatom weight percents in petroleum products. Moreover, sulfur is the third most abundant element in petroleum and has been regulated to parts-per-million levels and further reduction slated for the year 2010. To meet the more stringent environmental regulations, refineries are facing major challenges. Mass spectrometry has proven to be a valuable tool for the molecular speciation of petroleum. Notably, electrospray ionization Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry has proven invaluable for the speciation of the polar compounds in crude oil. This analysis has added to the understanding of specific refinery problems, e.g., solid deposition and flocculation. However, hydrocarbons and non-polar sulfur species are not accessible by ESI mass spectrometry. Atmospheric Pressure PhotoIonization (APPI) can produce ions from non-polar (and polar) species. Chapter 1 is a brief discussion of basic ICR principles, APPI pathways, instrumentation and data analysis. In Chapter 2, I describe an APPI source coupled to the in house built 9.4 Tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer at the National High Magnetic Field Laboratory (NHMFL) in Tallahassee, Florida. This chapter highlights the complexity of crude oil analysis with an APPI source. The possibility of forming two ion types (protonated compounds and radical molecular ions) from one compound complicates an already complex spectrum. Model compound spectra demonstrate the necessity of ultra-high resolution mass spectrometry to resolve common mass doublets (3.4 mDa, the mass difference between C3 vs. SH4; 4.5 mDa, the mass difference between 12CH and 13C) found in petroleum spectra. Also, this report establishes the highest number of resolved (and assigned elemental formulas) spectral peaks (>12,000 peaks in a single mass spectrum and up to 63 peaks of the same nominal mass) in one mass spectrum.12,000 peaks in a single mass spectrum and up to 63 peaks of the same nominal mass) in one mass spectrum. Although APPI is considered to be a soft ionization technique, the analyte is nebulized and heated before ion formation. On the other hand, ESI is a well established soft ionization process. Therefore, in Chapter 3, I compare ESI and APPI data from the same crude oil and also pyridinic and pyrrolic nitrogen model compounds. The chapter defines instrument parameters which can cause fragmentation (loss of H2) and parameters which do not. ESI and APPI crude oil spectra yield the same elemental species, providing evidence that APPI can produce an ion population without fragmentation. A dopant (proton donor) is advantageous for APPI mass spectrometry because proton transfer reactions are enhanced. For simple mixture analysis, the proton donor is predominantly the dopant. However, for complex mixture analysis (crude oil), the solution matrix can contain species which could also participate in proton transfer reactions. In Chapter 4, I investigate the proton transfer reaction for a Canadian bitumen petroleum in deuterated toluene (C7D8). Nitrogen class compounds are also analyzed in deuterated toluene. The dopant percent contribution to the even-electron ions (protonated and deuterated compounds) of the petroleum is ~5 %. The nitrogen model compounds exhibited a similar trend. Petrochemical analysis commonly employs the saturates-aromatic-resins-asphaltenes (SARA) separation method. In Chapter 5, the sulfur containing compounds of a Middle East crude oil are speciated. The crude oil is additionally fractionated by the SARA method and its fractions are analyzed by APPI FT-ICR mass spectrometry. Molecular species from the whole crude oil and its fractions are compared to ascertain differences and similarities between sulfur species in the fractions. Non-polar sulfur species are not efficiently ionized by ESI. However, derivatization chemistry can methylate polycyclic aromatic sulfur species and form cations in solution with subsequent analysis by ESI mass spectrometry. In Chapter 6, the derivatized and non-derivatized samples of a petroleum vacuum bottom residue (the highest boiling point fraction of petroleum and hence, the most complex heteroatom content) are analyzed by ESI and APPI. Significant differences in the double bond equivalent values (DBE, value equal to the number of rings plus double bonds in the molecular structure calculated from the elemental formula) between the ESI and APPI analyzed sulfur species are identified. Furthermore, this report provides data that probes APPI ionization efficiency. Chapter 7 is a synopsis of the APPI technology applied to petroleum analysis. The chapter also includes a real world application of APPI FT-ICR mass spectrometry. The Institute of Petroleum at France (IFP) is interested in the development of new hydroconversion processes to upgrade vacuum bottom residue to more useful petroleum products. A substantial fraction of vacuum bottom residue is the asphaltenes; the most heteroatom rich fraction in petroleum. The chapter presents molecular speciation from intermediate stages of a hydroconversion process; a first step in hydroconversion catalytic technology improvement. A Ph.D. thesis may also include research outside the scope of the primary dissertation research to achieve a broader understanding of the sciences. Appendix A describes the ongoing construction and adaptation of an ion cluster source to an existing FT-ICR mass spectrometer. The primary investigator is Professor Harry Kroto, Nobel prize laureate for the discovery of fullerenes. Fullerenes are closed cage molecules consisting of 12 pentagonal and several hexagonal rings. Fullerenes with 60 carbon atoms or larger follow the isolated pentagon rule (IPR). Smaller fullerenes ( In appendix B, the reaction products of C60 and hydrogen at high temperature and pressure are resolved and identified. The product species formed at elevated temperature and hydrogen pressure are characterized by APPI FT-ICR mass spectrometry. Only the APPI analysis (and Field Desorption, FD) were accomplished at Florida State University and the first report (of three published reports) is presented.
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Date Issued: 2007
Identifier: FSU_migr_etd-0477
Format: Thesis

Title: Integrin αIibβ3 Conformational Change Visualized in a Membrane Environment by Cryoelectron Tomography.
Creator: Ye, Feng, Taylor, Kenneth A., Chapman, Michael S., Roberts, Thomas M., III, Thomas C. S. Keller, Roux, Kenneth H., Program in Molecular Biophysics, Florida State University
Abstract/Description: Integrin signaling is critical for many biological functions including cell survival, cell migration, development, immunity and wound healing. Integrins perform their function through a structural change that is propagated from the cytoplasm to the ligand binding domain in inside-out signaling and from the ligand binding domain to the cytoplasm during outside-in signaling events. However, the structural basis for the signal transduction in a native-like lipid bilayer environment is poorly...
Show moreIntegrin signaling is critical for many biological functions including cell survival, cell migration, development, immunity and wound healing. Integrins perform their function through a structural change that is propagated from the cytoplasm to the ligand binding domain in inside-out signaling and from the ligand binding domain to the cytoplasm during outside-in signaling events. However, the structural basis for the signal transduction in a native-like lipid bilayer environment is poorly understood. We investigated the inactive and active conformations of integrin alpha IIb beta 3 in a membrane environment to understand the structural basis of integrin signaling. We used reconstituted small unilamellar vesicles to mimic the native membrane environment and used cryo-electron tomography of ice embedded specimens and 3-D averaging to obtain the structures. Our results showed that, in this membrane environment, both active and inactive integrins are in an upright conformation. They differ in the separation of the leg regions of the alpha and beta chains. Inactive integrins have the legs together, similar to the 3-D structure of detergent solubilized alpha IIb beta 3 observed in ice but with a more upright orientation with respect to the membrane. The active integrins have the legs separated by about 5.6 nm at the membrane surface. These results support a model in which integrin signaling is achieved by the relative movement of the leg regions of the two subunits.
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Date Issued: 2005
Identifier: FSU_migr_etd-0421
Format: Thesis

Title: Activation Profiles and Regulatory Cascades of the Human Kallikrein-Related Peptidases.
Creator: Yoon, Hyesook, Blaber, Michael, Tang, Hengli, Miller, Brian, Steinbock, Oliver, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: This work describes the activation profiles and regulatory cascades of the human kallikrein-related peptidases. The human kallikrein (KLK)-related peptidases are the largest family of serine peptidases, comprising 15 members (KLK1–15) and with the majority (KLK4–15) being identified only within the last decade. Members of this family are associated with important diseased states (including cancer, inflammation, and neurodegeneration) and have been utilized or proposed as clinically important...
Show moreThis work describes the activation profiles and regulatory cascades of the human kallikrein-related peptidases. The human kallikrein (KLK)-related peptidases are the largest family of serine peptidases, comprising 15 members (KLK1–15) and with the majority (KLK4–15) being identified only within the last decade. Members of this family are associated with important diseased states (including cancer, inflammation, and neurodegeneration) and have been utilized or proposed as clinically important biomarkers or therapeutic targets of interest. All human KLKs are synthesized as prepro-forms that are proteolytically processed to secreted pro-forms via the removal of an amino-terminal secretion signal peptide. The secreted inactive pro-KLKs are then activated extracellularly to mature peptidases by specific proteolytic release of their amino-terminal propeptide. Although a key step in the regulation of KLK function, details regarding the activation of the human pro-KLKs are unknown, to a significant extent, but have been postulated to involve "activation cascades" with other KLKs and endopeptidases. To characterize more completely the KLK activation cascades, an individual KLK propeptides fused to the amino terminus of a soluble carrier protein have been expressed from Escherichia coli. The ability of 14 different mature KLKs to process the 15 different pro-KLK peptide sequences has been determined. Various autolytic and cross activation relationships identified using this system have subsequently been characterized using recombinant pro-KLK proteins. The results demonstrate the potential for extensive KLK activation cascades and, when combined with available data for the tissue-specific expression of the KLK family, permit the construction of specific regulatory cascades. One such tissue-specific cascade is proposed for the central nervous system. Much recent work has been devoted to elucidating the potential for activation cascades between members of the KLK family, with physiologically relevant KLK regulatory cascades now described in skin desquamation and semen liquefaction. Despite this expanding knowledge of KLK regulation, details regarding the potential for functional intersection of KLKs with other regulatory proteases are essentially unknown. To elucidate such interaction potential, the ability of proteases associated with thromostasis to hydrolyze the pro-peptide sequences of the KLK family have been characterized, using the previously described (chapter 2) pro-KLK fusion protein system. A subset of positive hydrolysis results were subsequently quantified with proteolytic assays using intact recombinant pro-KLK proteins. The results show that proteases of the thrombostasis family can efficiently activate specific pro-KLKs, uncovering for the first time the potential for important regulatory interactions between these two major protease families. Proteases of the thrombostasis system are activated under conditions of injury, inflammation, and tissue remodeling, such as occurs in various disease states, including cancer. Activation of KLKs by thrombostasis proteases is therefore relevant for KLK activity in such diseases states.
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Date Issued: 2008
Identifier: FSU_migr_etd-0712
Format: Thesis

Title: Preparing Packed Capillaries for Use in Capillary Electrochromatography: Studies of Mobility, Dispersion, and Reproducibility.
Creator: Piraino, Stephanie M., Dorsey, John G., Dorsey, Jodee, Vickers, Thomas, Stiegman, Albert, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Packing capillaries for use in Capillary Electrochromatography (CEC) is described in detail. Qualitative observations regarding the general packing procedure are elucidated. Because forming frits to retain the stationary phase is one of the greatest challenges regarding the fabrication of packed capillaries, the performance of several types of frits including sintered frits, photopolymerized frits, and frits made by sol- gel technologies is evaluated. The frits are formed in open capillaries,...
Show morePacking capillaries for use in Capillary Electrochromatography (CEC) is described in detail. Qualitative observations regarding the general packing procedure are elucidated. Because forming frits to retain the stationary phase is one of the greatest challenges regarding the fabrication of packed capillaries, the performance of several types of frits including sintered frits, photopolymerized frits, and frits made by sol- gel technologies is evaluated. The frits are formed in open capillaries, and the electroosmotic mobilities are calculated and compared to those obtained in an open capillary. Run-to-run, day-to-day, and column- to-column reproducibilities are evaluated. In all cases, the run-to-run reproducibility varied by less than 4%. The greatest mobility was through the capillaries with the sol- gel frits (1.29 x 10-3 cm2/V·s) which also exhibited the best day-to-day reproducibility. The capillaries with the photopolymerized frits had the best column-tocolumn reproducibility yet the slowest mobility (1.00 x 10-3 cm2/V·s). The second central moment of benzene as a solute is calculated and plotted as a function of time to estimate the differences in peak dispersion among the various frits studied. Although the mobilities through the capillaries with the sintered frits were the most irreproducible, these frits are associated with the least amount of band broadening. As the sintered frits are the most common type of frit used for CEC, modifications to the equipment used to sinter the frits were made in an effort to make more reproducible and reliable sintered frits. The revisions made to the frit heater include the use of a transformer to vary the voltage, a nichrome ribbon to generate smaller frits, and a thermocouple to estimate the temperature while heating the capillary. The band broadening of an uncharged solute was measured as a function of sintering times and temperatures to calculate the amount of heat used to sinter the frits. It was determined that more homogeneous frits were made as more heat was applied to sinter the frit, resulting in less dispersion.
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Date Issued: 2003
Identifier: FSU_migr_etd-0674
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: 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: Early Secretory Mechanism and Progress Toward Structural Determination of the Voltage-Gated Potassium Channel Kv1.3.
Creator: Spear, John Michael, Stagg, Scott, Hyson, Richard L., Fadool, Debra Ann, Cross, Timothy A., Stroupe, Margaret Elizabeth, Florida State University, College of Arts and Sciences,...
Show moreSpear, John Michael, Stagg, Scott, Hyson, Richard L., Fadool, Debra Ann, Cross, Timothy A., Stroupe, Margaret Elizabeth, Florida State University, College of Arts and Sciences, Program in Molecular Biophysics
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Abstract/Description: Voltage-gated potassium channels (Kv channels) play a vital roll in cellular excitability and neuronal communication by catalyzing the translocation of potassium ions across cellular membranes. After synthesis occurs in the endoplasmic reticulum (ER), Kv channels must reach the cellular periphery. The coat protein complex II (COPII) mediates this trafficking process. However, the mechanism used by COPII to associate with Kv channels remains controversial. I show that trafficking of the of the...
Show moreVoltage-gated potassium channels (Kv channels) play a vital roll in cellular excitability and neuronal communication by catalyzing the translocation of potassium ions across cellular membranes. After synthesis occurs in the endoplasmic reticulum (ER), Kv channels must reach the cellular periphery. The coat protein complex II (COPII) mediates this trafficking process. However, the mechanism used by COPII to associate with Kv channels remains controversial. I show that trafficking of the of the Kv channel subfamily member 3, Kv1.3, partially relies upon the tandem di-acidic motif E443-E445-E447, and is a bona fide client of the Sec24a cargo recruiter of the COPII coat. These results expand upon previous studies aimed at deciphering the disputed Kv1.3 secretory trafficking mechanism. Biophysical properties and surface localization of Kv channels can be modulated by their association with auxiliary subunits termed beta subunits (Kvβ). The subfamily member 2 (Kvβ2.1) protein has been shown crystallographically to exist in more than one plausible biological assembly, but its oligomeric state in solution has not been well characterized. I show that Kvβ2.1 in solution exists as an octamer, but upon increased reduction potential Kvβ2.1 octamers dissociate into tetramers in vitro. Furthermore, I use cryogenic electron microscopy (cryoEM) to structurally characterize the octameric complex and find a previously unidentified density in the N-termini, which could play a role in the formation of Kvβ2.1 octamers or Kv channel-Kvβ2.1 complexes. These results demonstrate that there could be a physiological cue that couples the dissociation of Kvβ2.1 octamers to tetramers followed by the association of Kvβ2.1 tetramers with Kv channels. With technological advances in electron detection devices used in cryoEM, I examine some of the influences that potentially hinder achieving atomic resolution using cryoEM single particle reconstructions. I tested previous metrics shown to greatly enhance the ability to achieve atomic resolution using cryoEM, and further expanded on those metrics by showing the effects of rigorous dose compensation and image alignment. To this extent I was able to determine a 2.8 Å structure of the adeno-associated virus using our new metrics. These results demonstrate how cryoEM is ushering in a new era in structural biology. Determining the full-length, high-resolution structure of a Kv channel in a native-like membrane in a non-crystalline environment is a long sought after goal in structural biology. Moreover, structural characterization of the interactions that occur between COPII and a membrane-embedded cargo molecule has not been completed. I present an innovative method and advances towards determining the high-resolution cryoEM structure of membrane reconstituted Kv1.3 encapsulated within a COPII coat. This work lays a foundation to solve the high-resolution structure of membrane-embedded Kv1.3 and examine the COPII-Kv1.3 interactions that occur within a coated vesicle.
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Date Issued: 2015
Identifier: FSU_2015fall_Spear_fsu_0071E_12928
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: 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: 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: Insights into the Complex Formation between Nucleoside Diphosphate Kinase and a Highly Polymorphic DNA G-Quadruplex.
Creator: Kopylov, Mykhailo, Stroupe, M. Elizabeth (Margaret Elizabeth), McGinnis, Karen M., Bass, Hank W., Yang, Wei, Li, Hong, Florida State University, College of Arts and Sciences,...
Show moreKopylov, Mykhailo, Stroupe, M. Elizabeth (Margaret Elizabeth), McGinnis, Karen M., Bass, Hank W., Yang, Wei, Li, Hong, Florida State University, College of Arts and Sciences, Program in Molecular Biophysics
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Abstract/Description: Non-canonical forms of DNA like the guanine quadruplex (G4) play important roles in regulating transcription and translation through interactions with their protein partners. G4s comprise a class of nucleic acid structures formed by stacking of guanine base quartets in a quadruple helix. This G4 DNA can form within or across single stranded DNA molecules and is mutually exclusive with duplex B-form DNA. The core of a G4 is formed in G-rich stretches of DNA by Hoogsteen base-paired guanines...
Show moreNon-canonical forms of DNA like the guanine quadruplex (G4) play important roles in regulating transcription and translation through interactions with their protein partners. G4s comprise a class of nucleic acid structures formed by stacking of guanine base quartets in a quadruple helix. This G4 DNA can form within or across single stranded DNA molecules and is mutually exclusive with duplex B-form DNA. The core of a G4 is formed in G-rich stretches of DNA by Hoogsteen base-paired guanines that assemble as planar stacks, stabilized by a central cation like K+. These structures are reversible and structurally diverse, which makes them highly versatile genetic structures, as demonstrated by their roles in various functions including DNA replication, transcription, translation and telomere metabolism. The structural information on protein-G4 complexes remains scarce, especially little is known about G4-interacting proteins in the plant kingdom. In the present study, we addressed the following aims to tackle this deficiency: 1. Bioinformatically determine the abundance and localization of putative G4s in maize, a model organism for plant species; 2. Identify plant G4-binding proteins by expression library screening. 3. Analyze the structural heterogeneity of a polymorphic G4-forming oligonucleotide hex4_A5U. 4. Structurally characterize complex formation between hex4_A5U G4 and a G4-binding protein ZmNDPK1 using cryo-electron microscopy (cryoEM). G4 forming sequences were first identified in telomeres and then recognized in other genomic loci. To investigate their potential roles in a large-genome model plant species, we computationally identified 149,988 canonical non-telomeric putative G4s in maize, 29 percent of which were in non-repetitive genomic regions. Putative G4 hotspots exhibited non-random enrichment in genes at three locations: one on the antisense strand in the 5‘UTR (A5U class); second one also on the antisense strand at the 5’ end of the first intron (A5I class); and third one on the sense strand adjacent to transcription start site (ATG class). Maize hexokinase4 gene has one G4 from each class (hex4_A5U, hex4_A5I and hex4_AUG) which we shown to form G4s in vitro. Overall the G4 motifs were prevalent in key regulatory genes associated with hypoxia, oxidative stress, and energy status pathways. Putative G4 elements have been identified in, or near, genes from species as diverse as bacteria, mammals, and plants, but little is known about how they might function as cis-regulatory elements or as binding sites for trans-acting protein partners. In fact, until now, no G4 binding partners have been identified in the plant kingdom. Here, we report on the identification, cloning and characterization of the first plant-kingdom gene known to encode a G4-binding protein, maize (Zea mays L.) Nucleoside Diphosphate Kinase1 (ZmNDPK1). Structural characterization by X-ray crystallography reveals that it is a homohexamer, akin to other known NDPKs like the human homolog NM23-H2. Further probing into the G4-binding properties of both NDPK homologs shows that ZmNDPK1 possesses properties distinct from that of NM23-H2, which is known to interact with a G-rich sequence element upstream of the c-myc gene and, in doing so, modulate its expression. We also demonstrate that the G4-binding activity of ZmNDPK1 is independent of nucleotide binding and kinase activity, suggesting that the G4-binding region and the enzyme active site are separate. Together, these findings establish a broad evolutionary conservation of some NDPKs as G4-DNA binding enzymes, but with potentially distinct biochemical properties that may reflect divergent evolution or species-specific deployment of these elements in gene regulatory processes. A single G4-forming sequence can adopt a variety of 3D structures depending on: strand order and orientation (parallel, antiparallel), number of tetrads in a core (two, three, four), identity of the central cation (K+, Na+) and presence of bulges in G-tracts. Here I investigate the conformational heterogeneity of a hex4_A5U. This sequence adopts extensive polymorphic G4 conformations including non-canonical bulged G4 folds that co-existed in solution. The nature of this polymorphism depends, in part, on the incorporation of different sets of adjacent guanines into a G4 core that allowed formation of the different conformations. Additionally, I show that the ZmNDPK1 specifically recognizes and promotes formation of a subset of these conformations.
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Date Issued: 2017
Identifier: FSU_FALL2017_Kopylov_fsu_0071E_14171
Format: Thesis

Title: Siroheme Biosynthesis and Regulation of Siroheme Deficiency.
Creator: Pennington, Joseph Masters, Li, Hong, Jones, Kathryn M., Stroupe, M. Elizabeth (Margaret Elizabeth), Blaber, Michael, Yang, Wei, Florida State University, College of Arts and...
Show morePennington, Joseph Masters, Li, Hong, Jones, Kathryn M., Stroupe, M. Elizabeth (Margaret Elizabeth), Blaber, Michael, Yang, Wei, Florida State University, College of Arts and Sciences, Institute of Molecular Biophysics
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Abstract/Description: This thesis analyzes the structure and function of enzymes involved in the biosynthesis of the tetrapyrrole cofactor, siroheme, a critical cofactor used in sulfur and nitrogen metabolism in plants, bacteria, and some archaea. The multifunctional enzyme, siroheme synthase, from Salmonella typhimurium and two newly identified enzymes from Mycobacterium tuberculosis are used to understand how these ancient enzymes function. Additionally, the effects of siroheme deficiency in Escherichia coli are...
Show moreThis thesis analyzes the structure and function of enzymes involved in the biosynthesis of the tetrapyrrole cofactor, siroheme, a critical cofactor used in sulfur and nitrogen metabolism in plants, bacteria, and some archaea. The multifunctional enzyme, siroheme synthase, from Salmonella typhimurium and two newly identified enzymes from Mycobacterium tuberculosis are used to understand how these ancient enzymes function. Additionally, the effects of siroheme deficiency in Escherichia coli are studied in the context of sulfite reductase, an enzyme central to sulfur assimilation. Siroheme synthase (CysG) is a trifunctional enzyme responsible for the three terminal steps of siroheme biosynthesis in Salmonella typhimurium. The enzyme is composed of two functional modules, CysGA that accomplishes the first reaction, and CysGB that accomplishes the final two reactions. Interestingly, the same active site in CysGB is responsible for two very distinct chemistries where in other structural homologs, this is not observed. The work here shows how CysG distinguishes between these reactions to produce siroheme. Point mutagenesis, in vivo complementation assays, spectroscopic activity assays, and X-ray diffraction studies were used to piece together how CysG binds and orients the substrates and intermediates needed to catalyze siroheme. The co-crystal structures of precorrin-2-, sirohydrochlorin-, and cobalt-sirohydrochlorin-bound CysG were solved allowing characterization of the residues involved in binding and how their orientations change throughout catalysis. In Mycobacterium tuberculosis, the enzyme (or enzymes) responsible for siroheme production are unknown even though the siroheme cofactor is present in the bacteria’s sulfur metabolic pathway. This work reports the identification and characterization of two enzymes, MtCysG and MtChe1, that work together to produce siroheme. Molecular cloning teachniques, in vivo complementation assays, spectroscopic activity assays, and X-ray diffraction were used to isolate and identify MtCysG and MtChe1 as the enzymes necessary and sufficient for siroheme production. Interestingly, MtCysG is structurally homologous to Salmonella typhimurium CysG but is not a functional chelatase. Instead, MtChe1 fulfills this function to catalyze siroheme. Assimilatory NADPH-sulfite reductase (SiR) from Escherichia coli catalyzes the six-electron reduction of sulfite to sulfide. Two subunits, one a flavin-binding flavoprotein (SiRFP) and the other an iron-containing hemoprotein (SiRHP), assemble to make a holoenzyme ~800 kDa. How the two subunits assemble is not known. The iron-rich cofactors in SiRHP are unique because they are a covalent arrangement of a Fe4S4 cluster attached through a cysteine ligand to an iron-containing porphyrinoid called siroheme. The link between cofactor biogenesis and SiR stability is also ill-defined. Through hydrogen/deuterium exhchange, biochemical analysis and small-angle X-ray scattering (SAXS) we explore how the holoenzyme assembles and the structure of the N-terminal oligomerization domain of SiRHP. Apo-SiRHP forms a homotetramer, also dependent on its N-terminus, that is unable to assemble with SiRFP. From these results, we propose that homo-tetramerization of apo-SiRHP serves as a quality control mechanism to prevent formation of inactive holoenzyme in the case of limiting cellular siroheme.
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Date Issued: 2019
Identifier: 2019_Spring_Pennington_fsu_0071E_15083
Format: Thesis

Title: Metabolism and Redox Cycle in Human Mesenchymal Stem Cell with Culture Induced Senescence: Homeostasis and Rejuvenation.
Creator: Yuan, Xuegang, Ma, Teng, Logan, Timothy M., Grant, Samuel C., Li, Yan, Florida State University, FAMU-FSU College of Engineering, Department of Chemical and Biomedical Engineering
Abstract/Description: Human mesenchymal stem cells (hMSCs) isolated from various adult tissues are primary candidates in cell therapy and being tested in clinical trials for a wide range of diseases. The pro-regenerative and therapeutic properties of hMSCs are largely attributed to their trophic effects that coordinately modulate the progression of inflammation and enhance the endogenous tissue repair by host progenitor cells. However, immediately after isolation and upon in vitro culture expansion, hMSCs lose...
Show moreHuman mesenchymal stem cells (hMSCs) isolated from various adult tissues are primary candidates in cell therapy and being tested in clinical trials for a wide range of diseases. The pro-regenerative and therapeutic properties of hMSCs are largely attributed to their trophic effects that coordinately modulate the progression of inflammation and enhance the endogenous tissue repair by host progenitor cells. However, immediately after isolation and upon in vitro culture expansion, hMSCs lose their in vivo quiescent state and start to accumulate genetic and phenotypic changes that significantly alter their phenotypic properties, with increased heterogeneity and reduced therapeutic potential. The proliferation of hMSCs is limited and long-term culture-induced changes lead to cellular senescence and metabolic alteration, resulting in reduced therapeutic outcome. Since clinical application requires large-scale production of hMSCs with defined cellular properties, preserving cellular homeostasis during hMSCs in vitro expansion is a major barrier for hMSCs based industrial production. Once viewed as a mere consequence of the state of a cell, metabolism is now acknowledged to play regulatory roles in cellular events and signaling pathways that govern stem cell phenotype and functional properties. Regulation of hMSC metabolism via preconditioning strategies have been proposed to enhance hMSC stem cell properties. However, the mechanistic details of metabolic and redox alterations in hMSC replicative senescence are not well understood. The current study is to understand the role of energy metabolism in regulating hMSC cell fate during in vitro culture expansion to develop metabolic strategies to augment hMSCs therapeutic outcome. We studied therapeutic relevant properties of hMSC such as immune modulation with regards to the energy metabolism and cellular signaling networks in Chapter 2. Moreover, preconditioning of hMSCs via 3D aggregation regulated energy metabolism and redox cycle, further activated PI3K/Akt survival pathways. The therapeutic potentials of 3D aggregate-derived hMSCs were studied in a rat MCAO stroke model in Chapter 3. To address the scale-up production of hMSC aggregates for potential pre-clinical applications, a novel microcarrier-based bioreactor was developed with thermal-response materials. Non-invasive and non-enzymatic procedures can be achieved for hMSC expansion and 3D aggregates production as demonstrated in Chapter 4. At last, we reported the breakdown of cellular homeostasis in hMSCs with culture-induced senescence. Basic cellular characteristics including proliferation, regenerative potential, cell cycle, and mitochondrial function were disrupted during culture expansion of hMSCs. Culture-induced senescence of hMSCs also induced impairment of migratory ability and immunomodulation. Decrease of basal autophagy and mitophagy indicated the breakdown of cellular homeostasis in hMSCs with replicative senescence. GC-MS metabolomics and proteomics revealed the loss of glycolytic phenotype and energy homeostasis with replicative expansion of hMSCs, which reconfigured hMSCs to an insufficient energy production state from glycolysis towards OXPHOS following senescence. Rapid production of energy required for maintaining cellular properties of hMSCs induced mitochondrial dysfunction and redox imbalance. We also found that nicotinamide adenine dinucleotide (NAD+) plays a central role in regulating senescent response along with hMSC expansion. It has been shown that NAD+ repletion restored mitochondrial and stemness in improving longevity of rodent. Our results show a significant decline of NAD+ during rapid expansion of hMSCs. NAD+/Sirtuin axis plays a crucial role in restoring mitochondrial function, including mitochondrial biogenesis, membrane potential and electron transport ability. By repletion of NAD+ to senescent hMSCs, various stem cell properties were recovered. Together, the results revealed the mechanistic connection between metabolic regulation and hMSC fate and therapeutic potentials, and provided metabolic and redox target to maintain hMSC cellular homeostasis for cell therapy applications in manufacturing.
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Date Issued: 2019
Identifier: 2019_Spring_Yuan_fsu_0071E_15098
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: Predicting the Thermodynamic Properties of Proteins Using Computer Simulations.
Creator: Nguemaha, Valery Marcel, Piekarewicz, Jorge, Stagg, Scott, Zhou, Huan-Xiang, Xiong, Peng, Florida State University, College of Arts and Sciences, Department of Physics
Abstract/Description: Protein molecules, sometimes referred to as the molecules of life, are the drivers of virtually every biological function. In this dissertation, we describe a series of computational studies to dissect the mystery of complex protein molecules. We consider a large collection of protein systems, ranging from globular proteins to Intrinsically Disordered Proteins (IDPs) with a focus on predicting thermodynamic observables that can be quantitatively compared with experimental data. In the first...
Show moreProtein molecules, sometimes referred to as the molecules of life, are the drivers of virtually every biological function. In this dissertation, we describe a series of computational studies to dissect the mystery of complex protein molecules. We consider a large collection of protein systems, ranging from globular proteins to Intrinsically Disordered Proteins (IDPs) with a focus on predicting thermodynamic observables that can be quantitatively compared with experimental data. In the first part of this dissertation, we study the effects of the phenomenon of macromolecular crowding and how it affects the properties of two different groups of proteins. First, we investigate the effects of crowding on globular proteins by calculating the free energy of all-atom proteins in crowded environments. Second, we study how crowding affect the conformational ensembles of disordered proteins with a focus on comparing computations with experiments. In the second part of this dissertation, we apply Monte Carlo simulation techniques to study protein droplet formation and Liquid Liquid Phase Separation in protein systems.
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Date Issued: 2018
Identifier: 2018_Fall_Nguemaha_fsu_0071E_14858
Format: Thesis

Title: Investigating Oligomerization as a Form of Enzyme Regulation in Human Glucokinase.
Creator: McCray, Malcolm M. R. K. (Malcolm Marvin Rufus Kenyatta), Miller, Brian G., Zhu, Lei, Roper, Michael Gabriel, Stagg, Scott, Florida State University, College of Arts and...
Show moreMcCray, Malcolm M. R. K. (Malcolm Marvin Rufus Kenyatta), Miller, Brian G., Zhu, Lei, Roper, Michael Gabriel, Stagg, Scott, Florida State University, College of Arts and Sciences, Department of Chemistry and Biochemistry
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Abstract/Description: Recent studies have shown the importance of enzyme regulation through the formation of higher order structures. This is exemplified by Acetyl coenzyme A Carboxylase, and Cytidine Triphosphate synthase, two enzymes that form long chain filaments in the presence of specific activators [5-7,14,22,26]. Recently, yeast glucokinase (Glk1p) was found to form filaments in the presence of increasing concentrations of Glucose-6-phosphate (G6P), the product of the reaction that it catalyzes [18]. Due to...
Show moreRecent studies have shown the importance of enzyme regulation through the formation of higher order structures. This is exemplified by Acetyl coenzyme A Carboxylase, and Cytidine Triphosphate synthase, two enzymes that form long chain filaments in the presence of specific activators [5-7,14,22,26]. Recently, yeast glucokinase (Glk1p) was found to form filaments in the presence of increasing concentrations of Glucose-6-phosphate (G6P), the product of the reaction that it catalyzes [18]. Due to this fact, and the consistent appearance of what seem to be higher order and oligomer like peaks in size exclusion chromatography (SEC) chromatograms of human glucokinase (GK) expressed from Escherichia coli, an investigation of the nature of these peaks was conducted. Through the use of SEC, a method described in Tayyab at al 1992 was used to determine the molecular weight [38]. This resulted in the discovery that only the oligomeric peak was formed over time, was the approximate size of a trimeric glucokinase structure. SEC also discovered that factors, including time and ligand presence, affected the formation of the oligomeric complex, while spectrophotometric assays of the oligomeric protein indicated a decrease in overall activity. The oligomeric form of GK produced little to no activity in the presence of increasing concentrations of its substrate, glucose. This finding is consistent with the fact that the oligomer is an inactive form of GK that is not affected by glucose concentrations.
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Date Issued: 2019
Identifier: 2019_Spring_McCray_fsu_0071N_15200
Format: Thesis

Title: Effects of Obesity-Related Inflammation on Breast Cancer Progression.
Creator: Vallega, Karin Andrea, Sang, Qing-Xiang, Ren, Yi, Fadool, Debra Ann, Stagg, Scott, Steinbock, Oliver, Florida State University, College of Arts and Sciences, Department of...
Show moreVallega, Karin Andrea, Sang, Qing-Xiang, Ren, Yi, Fadool, Debra Ann, Stagg, Scott, Steinbock, Oliver, Florida State University, College of Arts and Sciences, Department of Chemistry and Biochemistry
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Abstract/Description: In the United States, breast cancer is the most commonly diagnosed cancer in women, except for skin cancers. The American Cancer Society estimates there will be 268,600 new cases in 2019 alone. It is also the second leading cause of cancer mortality in U.S. women, after lung cancer, with an estimated 41,760 deaths set to occur in 2019. However, worldwide, breast cancer is the most frequently diagnosed cancer in women and the leading cause of female cancer deaths. There are many disparities in...
Show moreIn the United States, breast cancer is the most commonly diagnosed cancer in women, except for skin cancers. The American Cancer Society estimates there will be 268,600 new cases in 2019 alone. It is also the second leading cause of cancer mortality in U.S. women, after lung cancer, with an estimated 41,760 deaths set to occur in 2019. However, worldwide, breast cancer is the most frequently diagnosed cancer in women and the leading cause of female cancer deaths. There are many disparities in breast cancer incidence and prognosis. Among the starkest of these disparities is the difference between African American and Caucasian American women. African American women present with higher rates of aggressive triple-negative subtype, earlier age at diagnosis, and have a 39% higher mortality rate than Caucasian American women. African American women also have higher rates of obesity than Caucasian American women. Obesity and inflammation are also very closely linked to breast cancer. Obesity causes chronic inflammation, and is a risk factor for post-menopausal breast cancer and worse prognosis. This work looks at several effects of obesity-related inflammation in the progression of breast cancer. Bioinformatics was used to explore the differential expression of resistin, a pro-inflammatory cytokine that has 4-fold higher expression in African American women. Due to the pro-inflammatory behavior, and role in adipose tissue, resistin may be a link between obesity, inflammation, and cancer. Resistin was differentially expressed in African American early-stage receptor negative subtypes. It was especially linked to estrogen receptor negative breast cancer subtype. Resistin expression was also higher in triple-negative subtype compared to luminal A subtype, which is hormone receptor positive. The high levels of resistin could contribute to African American breast cancer phenotype and high mortality rates. It may also serve as an early detection biomarker, since it is linked to early stages. Another effect of obesity that was analyzed in this work was the role of macrophages in breast cancer cell-adipocyte crosstalk. Obesity triggers increased infiltration of macrophages into adipose tissue. An innovative cell co-culture system was used to study the paracrine interactions between adipocytes, macrophages, and breast cancer cells, and how they can benefit tumor progression. Macrophage conditioned media intensified the effects of breast cancer cell-adipocyte crosstalk. In this crosstalk, adipocytes become cancer-associated, meaning they become delipidated and increase production of pro-inflammatory cytokines. Breast cancer cells then benefit from this increased inflammation and become more aggressive. Macrophage conditioned media in breast cancer cell and adipocyte co-culture increased tumor cell proliferation and migration compared to co-culture with nonconditioned media. Macrophage conditioned media also increased the expression of pro-inflammatory cytokines by adipocytes, even in the absence of breast cancer cells, although the expression levels were highest with all three cell components. Additionally, in co-culture of adipocytes and breast cancer cells with macrophage conditioned media, adipocytes showed signs of delipidation. Therefore, macrophages contribute to adipocyte inflammation and cancer-association, and help drive tumorigenesis.
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Date Issued: 2019
Identifier: 2019_Summer_Vallega_fsu_0071E_15362
Format: Thesis

Title: Microfluidic-Enabled Quantitative Measurements of Insulin Release Dynamics from Single Pancreatic Islets of Langerhans.
Creator: Bandak, Basel, Roper, Michael Gabriel, Fajer, Piotr G., Bleiholder, Christian, Stagg, Scott, Florida State University, College of Arts and Sciences, Department of Chemistry and...
Show moreBandak, Basel, Roper, Michael Gabriel, Fajer, Piotr G., Bleiholder, Christian, Stagg, Scott, Florida State University, College of Arts and Sciences, Department of Chemistry and Biochemistry
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Abstract/Description: The work in this dissertation presents a microfluidic method for the quantitative measurement of insulin secretion rates and patterns from single pancreatic islets of Langerhans. Proper release of insulin from islets is essential for maintaining glucose homeostasis. For full efficacy, both the pattern and the amount of hormone release are critical. It is therefore important to understand how insulin levels are secreted from single islets in both a quantitative fashion and in a manner that...
Show moreThe work in this dissertation presents a microfluidic method for the quantitative measurement of insulin secretion rates and patterns from single pancreatic islets of Langerhans. Proper release of insulin from islets is essential for maintaining glucose homeostasis. For full efficacy, both the pattern and the amount of hormone release are critical. It is therefore important to understand how insulin levels are secreted from single islets in both a quantitative fashion and in a manner that resolves temporal dynamics. Although several systems have been described for high time resolution measurements, many are limited in their ability to quantify release. Previous microfluidic systems for single islet hormone secretion measurements used pressure-driven perfusion systems to deliver glucose solutions to an islet chamber and sampled secretions by electroosmotic flow (EOF). Because of a discrepancy in these flow rates, only a small fraction of the secretions was sampled. Experimental variables, such as islet proximity to the sampling channel, can alter that percentage, hindering islet-to-islet comparisons of insulin measurements. Using finite element analysis, a microfluidic system was designed that ensured cellular secretions were homogenized (RSDs < 3%) prior to sampling, permitting quantitative monitoring of insulin and examination of inter-islet biological variability. Using the new design, the system was tested with standard insulin solutions and demonstrated RSDs of < 2% as well as a detection limit of 10 nM insulin, low enough for single islet sampling. The application of this system to monitor insulin release from murine islets demonstrated biphasic secretory rates and dynamics that were in good agreement with other reports. Single islets from healthy and T2DM human donors were also sampled, and with this system, blunted phase 1 peaks and lower secretion rates were quantified in the diseased samples compared with the healthy donor samples. Chronically elevated levels of lipids have been associated with insulin resistance and impaired insulin secretion. Using this quantitative microfluidic system, the acute and chronic effects of two classes of lipids were investigated: palmitic acid, a free fatty acid (FFA), and 5-palmitic acid hydroxy stearic acid (5-PAHSA), which is a member of the novel fatty acid hydroxy fatty acid (FAHFA) class of lipids that are upregulated in non-diabetic individuals. Acute exposure of these two classes of lipids to islets induced elevated secretion rates, consistent with published reports. Chronic incubation (48-h) with 5-PAHSA significantly augmented glucose-stimulated insulin secretion (GSIS) rates and dynamics at the single islet level compared to chronic incubation without the lipid. Incubation in the presence of palmitic acid (PA) resulted in impaired insulin release, as characterized by lower release rates and the loss of pulsatility. The studies were continued in human islets from both healthy and type 2 diabetes mellitus (T2DM)-diagnosed donors. Total amounts of GSIS were not only augmented in islets that were chronically incubated with 5-PAHSA, but the dynamic insulin release profiles also improved as noted by more pronounced insulin oscillations. With this quantitative microfluidic system, the anti-diabetic effects of 5-PAHSA were corroborated by demonstrating improved islet function after chronic incubation with this lipid via improved oscillatory dynamics along with higher basal and peak release rates. It has been shown that cellular stress derived from reactive oxygen species (ROS) plays a critical role in the impairment and apoptosis of insulin secreting cells. A microfluidic analytical method has been developed that permits the simultaneous measurements of real-time oxidative stress dynamics with insulin release patterns from single murine islets in vitro. A redox-sensitive biosensor (Grx1-roGFP2) was virally delivered to islets of Langerhans and selectively expressed in β-cells. The ratiometric fluorescence output of the biosensor was utilized to image intracellular ROS dynamics in response to extracellular stimuli, simultaneously with insulin release patterns using a microfluidic dual microscopy system. Single islets were loaded on the microfluidic device and stimulated with 11 mM glucose while ROS and insulin levels were measured simultaneously. The resulting secretory profile of insulin was biphasic, in which the first phase response was observed with a duration of 5-10 min, followed by second phase oscillations with periods of 3-5 min. The biosensor fluorescence also exhibited similar dynamic profiles, with the fluorescence ratio rapidly increasing during first phase insulin release and showing pulsatility that was synchronized with insulin oscillations in second phase release. Dynamic stimulations of infected islets with 20 mM glucose from 11 mM levels also showed a dose-dependent response in the redox state of islet β-cells. These results suggest that ROS generation is associated with insulin release dynamics and highlight the potential role of ROS in insulin release signaling. The experimental method presented here is amenable to the quantitative examination of acute changes of other intracellular metabolites simultaneously with the release of other hormones.
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Date Issued: 2019
Identifier: 2019_Summer_Bandak_fsu_0071E_15312
Format: Thesis

Title: Structural and Dynamic Characterization of CrgA; a Small Helical Membrane Protein in a Lipid Bilayer Using Solid-State NMR.
Creator: Shin, Yiseul, Cross, Timothy A., Alamo, Rufina G., Hu, Yan-yan, Stagg, Scott, Fajer, Piotr G., Florida State University, College of Arts and Sciences, Department of Chemistry...
Show moreShin, Yiseul, Cross, Timothy A., Alamo, Rufina G., Hu, Yan-yan, Stagg, Scott, Fajer, Piotr G., Florida State University, College of Arts and Sciences, Department of Chemistry and Biochemistry
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Abstract/Description: CrgA is a small helical membrane protein from Mycobacterium tuberculosis (Mtb) and known to recruit other cell division proteins to the divisome, a substantial protein complex for Mtb cell division. There is a lack of information about the formation of the divisome, because most of the Mtb cell division proteins, including CrgA, do not have homologs in the traditional model bacteria, such as E. coli. Thus, structural information is needed to understand how these proteins interact and function...
Show moreCrgA is a small helical membrane protein from Mycobacterium tuberculosis (Mtb) and known to recruit other cell division proteins to the divisome, a substantial protein complex for Mtb cell division. There is a lack of information about the formation of the divisome, because most of the Mtb cell division proteins, including CrgA, do not have homologs in the traditional model bacteria, such as E. coli. Thus, structural information is needed to understand how these proteins interact and function in the divisome. However, small helical membrane proteins are such a challenging target for structural biology, because they are surrounded by the complex membrane environment, which has varying biophysical properties in terms of the dielectric constants, water concentration, fluidity, and a lateral pressure profile. The entire membrane protein does not experience the same environment, and therefore it is critical to understand the dynamics of each domain in order to characterize its structure. CrgA was characterized as having a cytoplasmic N-terminus (30 residues long) consisting of two dynamically different regions: an intrinsically disordered region (IDR) and a β-strand. Even though most Mtb divisome proteins were predicted to have at least one IDR, the roles of IDRs in membrane proteins has not been fully explored. In this dissertation, different solid-state NMR techniques were used to characterize the water soluble regions such as IDRs of membrane proteins. Identifying a small portion of the protein in the lipid interfacial region is not feasible without providing a native-like lipid bilayer environment. The observation of the β-strand addressed the importance of the appropriate membrane mimetic to characterize the interfacial region of CrgA. Also, unambiguous interhelical distance restraints are essential for determining the correct tertiary and quaternary structure of a helical membrane protein. However, high contents of hydrophobic residues in the transmembrane domain make sequence specific assignments a difficult task, so various methods, such as specific isotope labeling schemes and mutagenesis were implemented to obtain the distance restraints. The sequence specific assignment strategies presented here for CrgA can be applied to other membrane protein systems. Combining the structural restraints from both magic angle spinning and oriented sample NMR led to the dimeric structure of CrgA using molecular dynamics simulations. Additionally, the mutagenesis data helped to define the dimeric interface for CrgA. In the case of CrgA, it is necessary to sort out any residues at the helix-helix interfacial region in the same monomer versus those located in the monomer-monomer interface of the dimer. Excluding those residues involved in the dimer interface will facilitate defining the lipid-facing residues available for CrgA's binding partners. Since CrgA is a pivotal component of the divisome, the structure of CrgA will provide great insight into the formation of the divisome to advance knowledge about Mtb cell division.
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Date Issued: 2019
Identifier: 2019_Summer_Shin_fsu_0071E_15392
Format: Thesis

Title: Mechanistic Enzymological Studies of Human Glucokinase and the Development of a Biocatalyst for Chemical Synthesis.
Creator: Sternisha, Shawn M., Miller, Brian G., Zhu, Fanxiu, Hoekman, Timothy, Yang, Wei, Frederich, James H., Florida State University, College of Arts and Sciences, Department of...
Show moreSternisha, Shawn M., Miller, Brian G., Zhu, Fanxiu, Hoekman, Timothy, Yang, Wei, Frederich, James H., Florida State University, College of Arts and Sciences, Department of Chemistry and Biochemistry
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Abstract/Description: The hexokinase (HK) family of enzymes catalyze the conversion of glucose to glucose 6-phosphate in a process that is utilized for energy production in most organisms. Hexokinase IV, commonly known as glucokinase (GCK), is functionally distinct from the rest of the HK isozymes. It is characterized by a high substrate concentration at half-maximal velocity and is not inhibited by an abundance of its product. GCK also displays positive kinetic cooperativity, despite functioning as a monomer and...
Show moreThe hexokinase (HK) family of enzymes catalyze the conversion of glucose to glucose 6-phosphate in a process that is utilized for energy production in most organisms. Hexokinase IV, commonly known as glucokinase (GCK), is functionally distinct from the rest of the HK isozymes. It is characterized by a high substrate concentration at half-maximal velocity and is not inhibited by an abundance of its product. GCK also displays positive kinetic cooperativity, despite functioning as a monomer and containing only one glucose binding site. This responsiveness is such that the inflection point occurs in the range of physiological blood glucose levels, providing the enzyme with exceptional sensitivity in this region. GCK's unique functional properties allow it to control the rates of insulin release and glycogen synthesis. The significance of proper GCK function is emphasized by various pathological conditions that arise from mutations in the gck gene. These discoveries are expanded upon in Chapter 1 and have led to GCK being considered the glucose sensor of the body. Millisecond timescale fluctuations of the small domain have been shown to be essential for cooperativity in GCK. However, a detailed picture of GCK's dynamic conformational landscape, including the number of accessible states, their relative populations, and the timescales on which they interconvert is absent in the literature. In Chapter 2, we map the intrinsic dynamics and structural heterogeneity of GCK on the nanosecond timescale using a combination of unnatural amino acid incorporation, time-resolved fluorescence spectroscopy and 19F nuclear magnetic resonance spectroscopy. Based on these results, we propose a catalytic model in which cooperativity originates from correlation between nanosecond and millisecond timescale motions. Activating GCK mutations abolish cooperativity and manifest themselves in the clinic as congenital hyperinsulinism. In Chapter 3, we use steady- and transient-state kinetics, and hydrogen-deuterium exchange mass spectrometry, to demonstrate that mutational activation of GCK occurs via two distinct mechanisms: α and β. Our data reveal that α-activation results from a shift in the conformational ensemble of unliganded GCK toward a state resembling the glucose-bound, closed conformation. β-type activation is instead caused by increased mobile loop dynamics, which accelerate the product release rate. This work elucidates the molecular basis of naturally occurring, activated GCK disease variants. Due to its essential role in maintaining whole-body glucose homeostasis, GCK activity is extensively regulated at virtually every level in the cell. The hormonal, metabolic, and transcriptional regulation of GCK have been described in great detail by other laboratories.1,2 Protein-protein interactions and post-translational modifications involving GCK elicit an array of physiological consequences and intrinsic conformational dynamics provide GCK with an additional layer of functional control. In Chapter 4, we offer insights into how these regulatory strategies are integrated and coordinated within the broader context of the cell. Of these regulatory mechanisms, the post-translational conjugation of the small ubiquitin-like modifier (SUMO1) protein to GCK remains one of the most poorly understood. Recently, it was reported that SUMOylation increases GCK's activity and stability, and mediates nuclear translocation of the enzyme.3,4 However, the inability to isolate homogenous, SUMOylated proteins often inhibits full characterization of the modification. In Chapter 5 we describe our efforts to generate SUMOylated GCK using semi-synthetic and coexpression approaches. We conclude with a look to the future, emphasizing the need for continued investigation and describing future experiments. In Chapter 6, we deviate from investigations of GCK and describe our efforts to characterize CyrI, a unique iron-dependent, nonheme oxygenase. This enzyme is expressed in cyanobacteria, where it catalyzes the final step in the biosynthesis of the toxic drinking water contaminant cylindrospermopsin. CyrI catalyzes a challenging C-H oxidation step with exquisite selectivity and appears to be depend on a sulfate group as a substrate recognition motif. CyrI is intriguing to develop from a chemical synthesis perspective as the selective functionalization of C-H bonds among numerous similarly reactive C-H bonds is a considerable challenge in organic synthesis. We detail our analysis of CyrI stability and crystallization and provide insights into future experimentation.
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Date Issued: 2019
Identifier: 2019_Summer_Sternisha_fsu_0071E_15294
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: 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

Title: Biomimetic Cascade Reactions Towards the Synthesis of Ciguatoxin.
Creator: Bou Hamdan, Farhan Ramez, Holton, Robert A., Keller, Laura L., Schwartz, Martin A., Krafft, Marie E., Logan, Timothy M., Department of Chemistry and Biochemistry, Florida State...
Show moreBou Hamdan, Farhan Ramez, Holton, Robert A., Keller, Laura L., Schwartz, Martin A., Krafft, Marie E., Logan, Timothy M., Department of Chemistry and Biochemistry, Florida State University
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Abstract/Description: Considerable attention has recently been given to the preparation of the ciguatoxins, the major group of toxins implicated with the onset of ciguatera fish poisoning (CFP) – a seafood-borne illness associated with the consumption of reef fish in tropical and subtropical areas. Ciguatoxins are characterized by a very complex polycyclic framework of 13 ether rings ranging from five to nine members. Previous studies directed towards the synthesis of the ciguatoxin skeleton have illustrated the...
Show moreConsiderable attention has recently been given to the preparation of the ciguatoxins, the major group of toxins implicated with the onset of ciguatera fish poisoning (CFP) – a seafood-borne illness associated with the consumption of reef fish in tropical and subtropical areas. Ciguatoxins are characterized by a very complex polycyclic framework of 13 ether rings ranging from five to nine members. Previous studies directed towards the synthesis of the ciguatoxin skeleton have illustrated the difficulty associated with the construction of medium rings, particularly eight and nine-membered. We envisaged that an electrophile-induced epoxy-alcohol cascade cyclization, previously developed in our laboratory and utilized in the synthesis of hemibrevetoxin-B, could be used for the construction of medium-ring ethers. As little was known about this cyclization cascade, an investigation has been launched to test the potential of this biomimetic approach in the construction of [6,8]-trans-fused bicyclic ethers and its applicability towards the synthesis of the HIJ rings of the ciguatoxins. This study has revealed that the construction of oxocanes via this epoxy-alcohol cascade cyclization methodology is feasible, and that molecular tethers facilitate the reaction. Several tethers including a benzene ring, a cis or trans-fused dioxolane were tested. Interestingly, the use of a dioxolane tether has demonstrated that a considerable level of diastereocontrol can be obtained. Moreover, the incorporation of the nucleophilic alcohol group within a cyclic structure has proven to have a large effect on the Baldwin vs. anti-Baldwin selectivity. A theoretical study regarding that aspect has further supported our findings. Finally, this work has resulted in the synthesis of an advanced tricylic intermediate of the ciguatoxin skeleton. It is one of the most efficient pathways reported so far, as it provides this advanced intermediate in 23 linear steps and 11% overall yield.
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Date Issued: 2007
Identifier: FSU_migr_etd-3506
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: Development of Copper(II)-Mediated Azide-Alkyne Cycloaddition Reactions Using Chelating Azides.
Creator: Brotherton, Wendy S., Zhu, Lei, Chase, P. Bryant, Dudley, Gregory B., Alabugin, Igor V., Roper, Michael G., Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: This dissertation describes the development of copper(II)-mediated azide-alkyne cycloaddition reactions using chelating azides. The first chapter provides an introduction to the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) through describing the history of the CuAAC reaction, its current mechanistic understanding, and selected applications of this widely used reaction. Within the second chapter, the development of copper(II)-accelerated azide-alkyne cycloaddition (AAC) is presented....
Show moreThis dissertation describes the development of copper(II)-mediated azide-alkyne cycloaddition reactions using chelating azides. The first chapter provides an introduction to the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) through describing the history of the CuAAC reaction, its current mechanistic understanding, and selected applications of this widely used reaction. Within the second chapter, the development of copper(II)-accelerated azide-alkyne cycloaddition (AAC) is presented. Therein, 1,4-disubstituted-1,2,3-triazoles were synthesized via the azide-alkyne cycloaddition in good to excellent yields using copper(II) salts in alcoholic solvents. The developed procedures avoided the need for an added reducing agent. Two pathways could be operational to generate copper(I) under these conditions: oxidation of alcoholic solvent or oxidative homocoupling of the alkyne. 2-Picolylazide behaved as a superior substrate under these conditions due to its chelating ability to copper that facilitates the cycloaddition. Preliminary spectroscopic results (EPR, UV-Vis spectroscopy) provided evidence that copper(I) was generated after an induction period. Within the third chapter, the reactivity of other chelating azides and their products as assisting ligands regarding the Cu(OAc)2-accelerated AAC reaction are briefly discussed. This data, combined with the results from the initial study, allowed us to study the mechanism. Results pertaining to solvent screening and alkyne screening are discussed in detail. In regards to solvent, the reaction can proceed in aprotic organic solvents but required longer reaction times than reactions in protic solvents. The Cu(OAc)2-accelerated AAC reactions were also observed to proceed fairly rapidly in aqueous solvents. The alkyne screening results under preparative, heterogeneous conditions show no clear trend between the structure of the alkyne and efficiency of the reaction. However, under homogeneous conditions used for the kinetics studies, a clear trend was observed where electron-withdrawing substituents on the para-position of phenylacetylene show shorter induction periods and react very rapidly. During the solvent and alkyne screening, a discontinuous reaction profile was observed suggesting the structure evolution of the catalyst, therefore lowering the catalytic activity. Application of the developed Cu(OAc)2-accelerated AAC reaction for the facile and rapid synthesis of tridentate 2,6-bis(1,2,3-triazol-1-ylmethyl)pyridine ligands is described in Chapter 4. Upon coordination with transition metal ions, the pyridyl nitrogen as well as the less Lewis basic N2 nitrogen of the 1,2,3-triazole ring were found to participate in binding. The ligands created in this study complement other well-studied tridentate ligands such as the triazolyl-based terpy motif and the 2,6-bis(pyrazol-1-ylmethyl)pyridine systems. Additionally, a ligand was designed to include two bidentate binding sites at both the N3 and N2 positions forming a five- and six-membered chelation ring, respectively. Its coordination was studied and showed that metals prefer the 5-membered planar chelation pocket over the puckered 6-membered pocket that contains the N2 nitrogen of the 1,2,3-triazole. Stable copper(II)/organic azide complexes from chelating azides were also observed and their features described. All copper(II)/azide complexes exhibit the alkylated nitrogen atom (Nα) of the azido group coordinating to the copper(II) ion. Analysis of the bond lengths show that copper(II) coordination at Nα enhances the electrophilicity of the terminal Nγ, accelerating the CuAAC reaction. The information gained from this study enhances our knowledge and understanding of the coordination chemistry of 1,4-substituted-1,2,3-triazole molecules, particularly in regards to the N2 atom of the 1,2,3-triazole. Chapter five describes the synthesis of 5-iodo-1,2,3-triazoles. These compounds were synthesized with the intention of functionalizing the 5-position with an electron donating group to enhance the electron density at the N2 position and enhance binding affinity. Unlike previous reports, 5-iodo-1,4-disubstituted-1,2,3-triazoles were generated from 2-picolylazide and iodoalkynes without the need of an assisting ligand. Moderate to good yields of the 5-iodotriazole products were obtained from a small set of iodoalkynes screened. Wanting to circumvent the synthesis of iodoalkynes, a one-pot method was created where copper(II) salts are reduced by NaI. This in situ method generates the necessary copper(I) catalyst as well as the iodinating source. The reaction requires an equivalent of base and was able to proceed in a variety of solvents. The reaction performed well with a variety of alkynes and azides, however, the reaction is sensitive to excess base and those with tertiary amines exhibited lower yields due to the formation of protonated triazole. The in situ generating conditions are more reactive than that of the direct addition of CuI and I2. Use of another electrophile, allyl iodide, under these conditions gave the 5-allyl-1,4-disubstituted-1,2,3-triazole in a multicomponent, one-pot reaction. 1,4,5-trisubstituted-1,2,3-triazoles were obtained through palladium cross-coupling reactions, such as the Sonogashira or the Suzuki reactions. However, minor amounts of the dehalogenated triazole by-product complicates purification of these compounds. Further study of 1,4,5-trisubstituted-1,2,3-triazoles is underway in our laboratory.
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Date Issued: 2012
Identifier: FSU_migr_etd-5533
Format: Thesis

Title: NMR Near Ferroelectric, Magnetic, and Quantum Phase Transitions.
Creator: Besara, Tiglet, Dalal, Naresh S., Alabugin, Igor, Chiorescu, Irinel, Knappenberger, Ken L., Kroto, Sir Harold W., Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: This dissertation details the use of solid state 1H nuclear magnetic resonance (NMR) to investigate three compounds exhibiting phase transitions. The metal-organic framework (MOF) [(CH3)2NH2]Zn(HCOO)3 has a ferroelectric (FE) transition at T=156 K. Spin-lattice relaxation time (T1) measurements are used to probe the local environment of the DMA+ cations. It is shown that the FE transition involves a smooth slowing-down of the three-fold hopping motion of the nitrogen in DMA+, rather than a...
Show moreThis dissertation details the use of solid state 1H nuclear magnetic resonance (NMR) to investigate three compounds exhibiting phase transitions. The metal-organic framework (MOF) [(CH3)2NH2]Zn(HCOO)3 has a ferroelectric (FE) transition at T=156 K. Spin-lattice relaxation time (T1) measurements are used to probe the local environment of the DMA+ cations. It is shown that the FE transition involves a smooth slowing-down of the three-fold hopping motion of the nitrogen in DMA+, rather than a complete freezing. The T1 data reveal that this compound exhibits several different local structures with close-lying ground states, which is a manifestation of glassy behavior. This is observed through the appearance of new and distinct metastable relaxation paths as the sample is thermally cycled. The MOF [(CH3)2NH2]Mn(HCOO)3 is a multiferroic, exhibiting both a FE (T=190 K) and an antiferromagnetic transition (AFM, TN=8.5 K). Spectra, T1, and spin-spin relaxation time (T2) measurements reveal that the electron-nucleus magnetic interactions play a dominant role at temperatures as high as the FE transition. The AFM transition is observed in all measurements at higher temperature than reported, indicating that the local magnetic structure around the hydrogens is already an almost fully ordered magnetic lattice at the NMR time scale. The transition occurs at different temperatures as the sample is thermally cycled, and the spectral shape in the ordered state is different each time, indicating that this material exhibits different magnetic structures, a sign of glassy behavior. The AFM Cr(C4H13N3)(O2)2·H2O exhibits a quantum phase transition. T1 data reveal that the critical field appears to be Hc=12.50 T, in good agreement with previously reported value. Near the quantum critical point, the 2-D antiferromagnet behaves as a 3-D Ising ferromagnet.
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Date Issued: 2011
Identifier: FSU_migr_etd-5511
Format: Thesis

Title: Low Temperature Microwave Driven C1 Reactions: The Catalytic Partial Oxidation of Methanol to Formaldehyde and the Gasification of Coal.
Creator: Crosswhite, Mark, Stiegman, Albert, Chanton, Jeff, Dorsey, John, Marshall, Alan G., Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: The selective heating observed by Spencer et al. in 1946 by microwave irradiation is the same observation that is showing up in recent articles published on microwave driven reactions. The unique way in which microwaves heat some materials selectively (reagents and catalysts) depending on their physical properties facilitates lower observed reaction temperatures, lower energy consumption, and higher reaction rates when compared to traditional thermal heating. Because of these advantages of...
Show moreThe selective heating observed by Spencer et al. in 1946 by microwave irradiation is the same observation that is showing up in recent articles published on microwave driven reactions. The unique way in which microwaves heat some materials selectively (reagents and catalysts) depending on their physical properties facilitates lower observed reaction temperatures, lower energy consumption, and higher reaction rates when compared to traditional thermal heating. Because of these advantages of microwave heating there has been an increase in publications on the topic over the last 25 years, from 3 publications in 1986 to 4000 in 2011. In early literature the observed microwave efficiencies were explained by the reality that microwaves instantaneously heat systems whereas convective heating requires time to reach the desired temperature. The literature debate now hinges around if there is a "special microwave" (non thermal) effect. In the studies presented here, two microwave driven chemical reaction systems are investigated. A heterogeneously catalyzed one in which methanol is oxidized over microwave absorbing solid catalyst. In the second, the direct absoption of microwaves by carbon is used to drive the highly energy intensive carbon-steam reaction for the production of synthesis gas. Our initial catalytic studies focused on oxidation reactions that fit two criteria. One is that they are of interest as pathways to industrially important commodity or fine chemicals. The second is that there is some literature precedent that suggests that oxides such as spinels will catalyze these transformations. In the former system we have developed microwave specific catalysts materials based on magnetic spinel oxides to accomplish difficult oxidation processes. For the particular system of chromite spinels of the general form MCr2O4 (M=Fe2+, Co2+, Cu2+) we were able to oxidize methanol to formaldehyde in aqueous solutions under very mild conditions. In our studies of the carbon-steam reaction, which is the primary reaction in coal gasification, we identified a reaction that is driven thermally at high temperatures whose reactants are strongly microwave absorbing. In this study our objective was to heat the reagents using microwave irradiation in order to drive the reaction at a lower temperature than traditional heating methods. We are able to directly heat the carbon to temperatures at which it reacts with steam (also generated using the microwaves) to produce synthesis gas (CO + H2). In our work we observe that microwave heating contributes to increased reaction rates, which in one case is attributed to efficient heating and in the other case a "microwave effect". Microwave heating is underutilized by the scientific community. With the possibility of realizing a microwave effect and the assurance of highly efficient heating, researchers should consider using microwave heating as a possible substitute to the traditional convective heating that is used in many labs.
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Date Issued: 2012
Identifier: FSU_migr_etd-5580
Format: Thesis

Title: Metabolic and Functional Plasticity in Bacteria Revealed with Genetic Selections for Triosephosphate Isomerase Activity and Bromoacetate Resistance.
Creator: Desai, Kevin, Miller, Brian, Bass, Hank, Li, Hong, Zhu, Lei, Stroupe, M. Elizabeth, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Modern protein catalysts are often viewed as possessing exquisite specificities for their cognate physiological substrates. In contrast, primordial catalysts are thought to have possessed much broader substrate specificities, a characteristic that likely afforded the survival of their host organisms under a plethora of diverse environmental conditions. Recent experimental work suggests that present day enzymes often retain the ability to recognize and transform a variety of natural and...
Show moreModern protein catalysts are often viewed as possessing exquisite specificities for their cognate physiological substrates. In contrast, primordial catalysts are thought to have possessed much broader substrate specificities, a characteristic that likely afforded the survival of their host organisms under a plethora of diverse environmental conditions. Recent experimental work suggests that present day enzymes often retain the ability to recognize and transform a variety of natural and unnatural compounds that are structurally distinct from their target substrate. The widespread existence of such promiscuity could prove generally useful both in the natural and directed evolution of new proteins. To probe the persistence of enzyme promiscuity in modern proteomes we studied the model organism Escherichia coli due to its rapid growth, ease of genetic manipulation and many years of prior research on this organism which have generated abundant knowledge on its metabolism. The first exploration into uncovering enzyme promiscuity, described in chapter two, examines the proton transfer reaction catalyzed by triosephosphate isomerase (TIM). Triosephosphate isomerase catalyzes the interconversion of D-glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, an essential step in glycolytic and gluconeogenic metabolism. To uncover promiscuous isomerases embedded within the E. coli genome, we searched for genes capable of restoring growth of a TIM-deficient bacterium under gluconeogenic conditions. Rather than discovering an isomerase, we selected yghZ, a gene encoding for a member of the aldo-keto reductase superfamily. Here we show that YghZ catalyzes the stereospecific, NADPH-dependent reduction of L-glyceraldehyde 3-phosphate, the enantiomer of the TIM substrate. This transformation provides an alternate pathway to the formation of dihydroxyacetone phosphate. In chapter three we show that Gpr co-purifies with a b-type heme cofactor. Gpr associates with heme in a 1:1 stoichiometry to form a complex that is characterized by a Kd value of 5.8 ± 0.2 µM in the absence of NADPH and a Kd value of 11 ± 1.3 µM in the presence of saturating NADPH. The absorbance spectrum of reconstituted Gpr indicates that heme is bound in a hexacoordinate low-spin state under both oxidizing and reducing conditions. The physiological function of heme association with Gpr is unclear, as the L-glyceraldehyde 3-phosphate reductase activity of Gpr does not require the presence of the cofactor. Bioinformatics analysis reveals that Gpr clusters with a family of putative monooxygenases in several organisms, suggesting that Gpr may act as a heme-dependent monooxygenase. The discovery that Gpr associates with heme is interesting because Gpr shares 35% amino acid identity with the mammalian voltage-gated K+ channel β-subunit, an NADPH-dependent oxidoreductase that endows certain voltage-gated K+ channels with hemoprotein-like, O2-sensing properties. To date the molecular origin of O2 sensing by voltage-gated K+ channels is unknown and the results presented herein suggest a role for heme in this process. In chapter four we probe the network of genes within E. coli that can provide resistance to the nonnatural toxin bromoacetate. Microbial niches contain toxic chemicals that are capable of forcing organisms into periods of intense natural selection to afford survival. Elucidating the mechanisms by which microbes evade environmental threats has direct relevance for understanding and combating the rise of antibiotic resistance. In this study we used a toxic small-molecule, bromoacetate, to model the selective pressures imposed by antibiotics and anthropogenic toxins. We report the results of genetic selection experiments that identify nine genes from Escherichia coli whose overexpression affords survival following exposure to a lethal concentration of bromoacetate. Eight of these genes encode putative transporters or transmembrane proteins, while one encodes the essential peptidoglycan biosynthetic enzyme, UDP-N-acetylglucosamine enolpyruvoyl transferase (MurA). Biochemical studies demonstrate that the primary physiological target of bromoacetate is MurA, which becomes irreversibly inactivated via alkylation of a critical active-site cysteine. Genetic experiments also identify 63 single-gene mutants of E. coli that display increased susceptibility to bromoacetate. One hypersensitive bacterium lacks yliJ, a gene that encodes a glutathione transferase capable of catalyzing the detoxification of bromoacetate with a kcat/Km value of 5.4 × 103 M-1 s-1. The catalytic proficiency of YliJ, which exceeds 5 orders of magnitude, is particularly noteworthy considering the enzyme is unlikely to have previously encountered bromoacetate. In total, our results indicate that nearly 2% of the E. coli proteome contributes to, or can be recruited to provide, bromoacetate resistance. This illustrates the wealth of intrinsic survival mechanisms that can be exploited by bacteria when they are challenged with toxins. The work described here illuminates the vast metabolic and functional plasticity of protein function harbored within bacteria. Their ability to recruit latent and weakly active proteins for novel functions enables survival under diverse nutritional and environmental challenges.
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Date Issued: 2010
Identifier: FSU_migr_etd-4608
Format: Thesis

Title: Synthesis of Cerium Rich Intermetallics Using Molten Metal Eutectics.
Creator: Tucker, Patricia Christine, Latturner, Susan, Dalal, Naresh, Shatruk, Michael, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Metal eutectic fluxes are useful for exploratory synthesis of new intermetallic phases. In this work the use of cerium/transition metal eutectics such as: Ce/Co, Ce/Ni, and Ce/Fe have yielded many new synthetically and magnetically complex phases. Structural units that were previously observed in phases grown in La/Ni eutectic reactions have also been observed in new structures and analogs grown from cerium/transition metal eutectics. These structural units include a main group element...
Show moreMetal eutectic fluxes are useful for exploratory synthesis of new intermetallic phases. In this work the use of cerium/transition metal eutectics such as: Ce/Co, Ce/Ni, and Ce/Fe have yielded many new synthetically and magnetically complex phases. Structural units that were previously observed in phases grown in La/Ni eutectic reactions have also been observed in new structures and analogs grown from cerium/transition metal eutectics. These structural units include a main group element coordinated by 9 rare-earth atoms (such as the Al@Ce9 clusters seen in Ce31.0(2)Fe11.8(5)Al6.5(6)B13C4), trigonal planar FeC3 units (also seen in Ce31.0(2)Fe11.8(5)Al6.5(6)B13C4), iron clusters capped by light elements (Fe46 frustrated tetrahedral in Ce21Fe8M7C14, and larger Fe clusters in Ce33Fe14B25C34). Variants of these building blocks were observed in Ce10Co2B7C16 with square Co units and chains of B and C connected to them, Fe2C8 units observed in Ce7Fe2C9, and FeC4 observed in Ce4FeGa0.85Al0.15C4 and Ce4FeAlC4. Two new phases were grown from Ce/Fe eutectic, Ce33Fe14B25C34 and Ce33Fe13B18C34 which exhibits very similar structures, but significantly different magnetic behavior. Structurally these two phases are similar. Both crystallize in the Im-3m space group, but differ by the centering of the Fe clusters. Ce33Fe14B25C34 contains Fe clusters centered by B atoms and Al doped on the Fe2 site. In Ce33Fe13B18C34 , the Fe cluster is a perfect cuboctahedron. Ce33Fe14B25C34 exhibits mixed valent behavior of cerium at 75K and no magnetic moment on iron, where-as Ce33Fe13B18C34 exhibits tetravalent cerium and its iron clusters undergo a ferromagnetic transition at 180K. Another borocarbide, Ce10Co2B7C16 was synthesized from Ce/Co eutectic flux. This structure features squares of Co surrounded by chains of C and B and a sea of cerium atoms. Temperature dependent magnetic susceptibility measurements at 1 Tesla were fit to a modified Curie-Weiss law and a moment per Ce was calculated to be 2.70µB. Field dependent data were collected at 200K and 2K. Paramagnetic behavior dominated at 200K and at 2K, ferromagnetic behavior was observed. XPS measurements were used to confirm that Ce is in the 3+ oxidation state. Intermetallics containing different Fe clusters (Y5Mg5Fe4AlSi, La6Fe10Al3Si, Ce21Fe8Al7-xSixC12, and Ce33Fe13.1Al1.1B24.8C34) were explored as potential catalysts for conversion of methane to Carbon Nanotubes (CNT). Different growth temperatures were explored. At 690ºC, Ce33Fe14B25C34 catalyzed the growth of single walled carbon nanotubes, Ce21Fe8Al7-xSixC12 multiwalled carbon nanotubes, and all other structures did not catalyze the growth of CNT.
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Date Issued: 2012
Identifier: FSU_migr_etd-5450
Format: Thesis

Title: The Fabrication of Self-Healing Cellulose Triacetate Polymer Composites and Dicyclopentadine Polymeric Foam.
Creator: Spann, Artrease, Kroto, Harold, Collins, Emmanuel, Knappenberger, Ken, Zhu, Lei, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Damage continues to plague the structural integrity of polymer composites. Continued stress leads to cracking and weakening of a composite system. Engineering polymer composites with carbon nanotube reinforcements and self-healing capabilities can potentially enhance the composites properties and extend the service lifetime. Carbon nanotubes (CNTs) promise to improve the electrical and mechanical properties of composite materials into which they are incorporated. These hollow tubes comprised...
Show moreDamage continues to plague the structural integrity of polymer composites. Continued stress leads to cracking and weakening of a composite system. Engineering polymer composites with carbon nanotube reinforcements and self-healing capabilities can potentially enhance the composites properties and extend the service lifetime. Carbon nanotubes (CNTs) promise to improve the electrical and mechanical properties of composite materials into which they are incorporated. These hollow tubes comprised completely of carbon, which have nanometer scale diameters, as well as low density, possess unique paradigm-shifting mechanical and electrical properties. The major problem prohibiting the widespread use of enhanced polymer composites has been the difficulty fabricating these composites. Research in developing methods capable of fully integrating the components is essential. This study sought to develop fabrication strategies to overcome the problem of integration and create polymer composites with advanced behavior useful in multiple applications. Engineering polymer composites with CNT and self-healing capabilities could enhance the composites' properties and extend the lifetime. Polymer composites are prone to degradation and damage. The damage leads to major system failure. The strategy developed here was inspired by living systems that evolved to mend damage and regain function. Engineering "smart" synthetic composites with infrastructures which can respond to damage is necessary for improving polymer usefulness. The molecular processes that alleviates damage aids in avoiding total mechanical failure. Such self-healing materials repair damage and continue function post repair. There several methods for creating composites with self-healing capability. The one studied here involves embedded microcapsules whose envelopes burst open releasing their contents in response to the material cracking. Microcapsules containing a reactive liquid "healing" core are embedded in the polymer during fabrication. A mechanical defect in the polymer breaks the shells of the microcapsules releasing the "healing" core. The core material polymerizes, thus healing the crack. As defects are repaired, functionality should be restored and the serviceable lifetime extended. The results obtained in this study indicate microcapsules can be modified to incorporate CNT. Also self-healing capabilities can be extended to new polymer systems in a cost-effective manner. In addition to enhancing polymers for sustainability, CNT reinforced structural foams could also prove to be beneficial in numerous industrial applications, where mechanically strong foams are needed. CNTs are already being investigated as structural enhancements for other foams, such as polyurethane - the most common and widely used polymer. High internal phase emulsion foams, highly porous emulsion template foams, are emerging as new and important forms of microcellular foams with numerous advantages, such as high impact strength and high stiffness-to-weight ratio. This research developed a novel method for creating carbon nanotube reinforced microcellular foam.
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Date Issued: 2012
Identifier: FSU_migr_etd-5440
Format: Thesis

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