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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: Activation Profiles of Human Kallikrein-Related Peptidases by Matrix Metalloproteinases.
Creator: Yoon, Hyesook, Blaber, Sachiko, Li, Wu, Scarisbrick, Isobel, Blaber, Michael
Abstract/Description: Abstract The 15 human kallikrein-related peptidases (KLKs) are clinically important biomarkers and therapeutic targets of interest in inflammation, cancer, and neurodegenerative disease. KLKs are secreted as inactive pro-forms (pro-KLKs) that are activated extracellularly by specific proteolytic release of their amino-terminal pro-peptide, and this is a key step in their functional regulation. Physiologically relevant KLK regulatory cascades of activation have been described in skin...
Show moreAbstract The 15 human kallikrein-related peptidases (KLKs) are clinically important biomarkers and therapeutic targets of interest in inflammation, cancer, and neurodegenerative disease. KLKs are secreted as inactive pro-forms (pro-KLKs) that are activated extracellularly by specific proteolytic release of their amino-terminal pro-peptide, and this is a key step in their functional regulation. Physiologically relevant KLK regulatory cascades of activation have been described in skin desquamation and semen liquefaction, and work by a large number of investigators has elucidated pairwise and autolytic activation relationships among the KLKs with the potential for more extensive activation cascades. More recent work has asked whether functional intersection of KLKs with other types of regulatory proteases exists. Such studies show a capacity for members of the thrombostasis axis to act as broad activators of pro-KLKs. In the present report, we ask whether such functional intersection is possible between the KLKs and the members of the matrix metalloproteinase (MMP) family by evaluating the ability of the MMPs to activate pro-KLKs. The results identify MMP-20 as a broad activator of pro-KLKs, suggesting the potential for intersection of the KLK and MMP axes under pathological dysregulation of MMP-20 expression.
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Date Issued: 2013
Identifier: FSU_migr_biomed_faculty_publications-0042, 10.1515/hsz-2012-0249, PMC3709557
Format: Citation

Title: Activation Profiles of Human Kallikrein-Related Peptidases by Proteases of the Thrombostasis Axis.
Creator: Yoon, Hyesook, Blaber, Sachiko, Evans, D., Trim, Julie, Juliano, Maria, Scarisbrick, Isobel, Blaber, Michael
Abstract/Description: The human kallikrein-related peptidases (KLKs) comprise 15 members (KLK1-15) and are the single largest family of serine proteases. The KLKs are utilized, or proposed, as clinically important biomarkers and therapeutic targets of interest in cancer and neurodegenerative disease. All KLKs appear to be secreted as inactive pro-forms (pro-KLKs) that are activated extracellularly by specific proteolytic release of their N-terminal pro-peptide. This processing is a key step in the regulation of...
Show moreThe human kallikrein-related peptidases (KLKs) comprise 15 members (KLK1-15) and are the single largest family of serine proteases. The KLKs are utilized, or proposed, as clinically important biomarkers and therapeutic targets of interest in cancer and neurodegenerative disease. All KLKs appear to be secreted as inactive pro-forms (pro-KLKs) that are activated extracellularly by specific proteolytic release of their N-terminal pro-peptide. This processing is a key step in the regulation of KLK function. 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, we have characterized the ability of proteases associated with thrombostasis to hydrolyze the pro-peptide sequences of the KLK family using a previously described pro-KLK fusion protein system. A subset of positive hydrolysis results were subsequently quantified with proteolytic assays using intact recombinant pro-KLK proteins. Pro-KLK6 and 14 can be activated by both plasmin and uPA, with plasmin being the best activator of pro-KLK6 identified to date. Pro-KLK11 and 12 can be activated by a broad-spectrum of thrombostasis proteases, with thrombin exhibiting a high degree of selectivity for pro-KLK12. The results show that proteases of the thrombostasis family can efficiently activate specific pro-KLKs, demonstrating the potential for important regulatory interactions between these two major protease families.
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Date Issued: 2008
Identifier: FSU_migr_biomed_faculty_publications-0009
Format: Citation

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

Title: Adeno-Associated Virus-2 and Its Primary Cellular Receptor-Cryo-EM Structure of a Heparin Complex.
Creator: O’Donnell, Jason, Li, Hong, Chapman, Michael, Taylor, Kenneth, Safron, Sanford, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Adeno-associated virus serotype 2 (AAV-2) is a leading candidate vector for gene therapy. Cell entry starts with attachment to a primary receptor, Heparan Sulfate Proteoglycan (HSPG) before binding to a co-receptor. Here, cryo-electron microscopy provides direct visualization of the virus–HSPG interactions. Single particle analysis was performed on AAV-2 complexed with a 17 kDa heparin fragment at 8.3 Å resolution. Heparin density covers the shoulder of spikes surrounding viral 3-fold...
Show moreAdeno-associated virus serotype 2 (AAV-2) is a leading candidate vector for gene therapy. Cell entry starts with attachment to a primary receptor, Heparan Sulfate Proteoglycan (HSPG) before binding to a co-receptor. Here, cryo-electron microscopy provides direct visualization of the virus–HSPG interactions. Single particle analysis was performed on AAV-2 complexed with a 17 kDa heparin fragment at 8.3 Å resolution. Heparin density covers the shoulder of spikes surrounding viral 3-fold symmetry axes. Previously implicated, positively charged residues R448/585, R451/588 and R350/487 from another subunit cluster at the center of the heparin footprint. The footprint is much more extensive than apparent through mutagenesis, including R347/484, K395/532 and K390/527 that are more conserved, but whose roles have been controversial. It also includes much of a region proposed as a co-receptor site, because prior studies had not revealed heparin interactions. Heparin density bridges over the viral 3-fold axes, indicating multi-valent attachment to symmetry-related binding sites.
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Date Issued: 2009
Identifier: FSU_migr_etd-2459
Format: Thesis

Title: Advanced Analytical Characterization of Molecular Changes Occurring in the Dissolved Organic Matter of Oxidized Landfill Leachate.
Creator: Osborne, Daniel M., Cooper, William T., Locke, Bruce R., Dorsey, John G., Safron, Sanford A., Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: The industrialization and growth of the world population has consequently led to the generation of larger quantities of both industrial and municipal wastes. Modern landfills are designed to sequester municipal solid wastes into a compact area and protect the surrounding environment from any hazardous materials. Precipitation, irrigated water, and the inherent moisture in the waste filter through the landfill absorbing inorganic and organic matter, and this resulting liquid waste is known as...
Show moreThe industrialization and growth of the world population has consequently led to the generation of larger quantities of both industrial and municipal wastes. Modern landfills are designed to sequester municipal solid wastes into a compact area and protect the surrounding environment from any hazardous materials. Precipitation, irrigated water, and the inherent moisture in the waste filter through the landfill absorbing inorganic and organic matter, and this resulting liquid waste is known as landfill leachate. As the water filters through the various layers of a waste material, pollutants are transferred to the leachate through a variety of different processes. Four groups of pollutants are present in varying compositions in all leachates, and they are: inorganic macrocomponents, heavy metals, xenobiotic organic carbons (XOCs), and dissolved organic matter (DOM). DOM is typically quantified by chemical oxygen demand (COD) or total organic carbon (TOC) measurements. As a landfill matures, the leachate that is produced becomes increasingly stabilized and biodegradability decreases, and the ratio of the biological oxygen demand over 5 days (BOD5) to the COD is used to quantify the biodegradability of a sample. Biological, physical and chemical methods can all be used to remove COD and increase BOD5/COD ratios, making the effluent more suitable for domestic wastewater treatment plants. Chemical oxidation of DOM with ozone can occur through direct molecular reactions or indirect pathways that result in the formation of hydroxyl radicals. Techniques where large concentrations of hydroxyl radicals are generated are known as advanced oxidation processes (AOPs), and one such AOP involves the photolysis of ozone with ultraviolet (UV) light. While COD and BOD5/COD are suitable methods for monitoring the effectiveness of landfill leachate treatment, they do not yield any molecular information regarding changes that occur to the complex DOM mixture during ozone or ozone/UV treatments. This work describes the use of ultraviolet/visible absorption spectroscopy (UV/Vis), excitation-emission matrix fluorescence spectroscopy (EEM), and size exclusion chromatography (SEC). These techniques were utilized to determine bulk properties of the DOM found in untreated landfill leachate and leachate treated using the following four reaction modes: aeration, aeration/UV, ozonation, and ozonation/UV. These reactions were carried out in a home-built bubble column reactor. The UV/Vis data yielded information about the relative size of the DOM components and the SEC experiments made it possible to calculate and compared average molecular weights and distribution. The EEM plots generated data about the types of fluorophores found in the DOM and how these fluorophores were affected by the given treatments. Ultrahigh resolution negative ion electrospray ionization Fourier transform-ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) was used to determine molecular level information about the DOM components of both the raw and treated leachates. Spectral slope and absorbance ratios from the UV spectra of the treated leachates indicated that the ozone and ozone/UV treatments both reduced the relative size of the DOM components. The EEM plots showed that over 90% of DOM fluorescence was removed over the course of the two hour ozone and ozone/UV treatments. Also, integrated areas of fluorophores with the EEM plot suggested that aromatic, humic-like fluorophores may be preferentially oxidized during these treatments. Weight average molecular weights and polydispersities were lowered following both of the ozone methods, confirming that the DOM molecules were smaller after treatment and indicating that the size was also becoming more uniform. Molecular level information from the ESI FT-ICR MS experiments supported the findings from the other bulk property measurements. The average number of carbons in each molecule and the nominal mass were shown to decrease after oxidation. Double bond equivalence (DBE) and DBE-oxygen (DBE-O) distributions were lower for the treated samples. However, the shift in DBE-O was larger, suggesting that the oxidized DOM contained more oxygen, and elemental ratios supported these findings. Molecules that were less saturated and had lower O/C ratios were preferentially "removed" and DOM molecules with higher O/C ratios were "formed" during the both ozone reactions. Sulfur-containing molecules were also preferentially removed which may suggest that the sulfur moieties are on the edges of the DOM and not part of the backbone of the molecule.
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Date Issued: 2011
Identifier: FSU_migr_etd-5877
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: Allosteric Activation of Human Glucokinase.
Creator: Bowler, Joseph M., Miller, Brian G., Stefanovic, Branko, Logan, Timothy M., Yang, Wei, Zhu, Lei, Florida State University, College of Arts and Sciences, Department of Chemistry...
Show moreBowler, Joseph M., Miller, Brian G., Stefanovic, Branko, Logan, Timothy M., Yang, Wei, Zhu, Lei, Florida State University, College of Arts and Sciences, Department of Chemistry and Biochemistry
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Abstract/Description: The hexokinase family of enzymes catalyzes the ATP-dependent phosphorylation of glucose to generate glucose 6-phosphate and ADP. Consistent with highly evolved catalysts, hexokinases I-III possess high apparent substrate affinities (low Km) as a result of specificity optimization. Hexokinase IV, commonly known as glucokinase (GCK), has a low apparent affinity for its physiological substrate glucose and is not significantly inhibited by the reaction product. Interestingly, it displays...
Show moreThe hexokinase family of enzymes catalyzes the ATP-dependent phosphorylation of glucose to generate glucose 6-phosphate and ADP. Consistent with highly evolved catalysts, hexokinases I-III possess high apparent substrate affinities (low Km) as a result of specificity optimization. Hexokinase IV, commonly known as glucokinase (GCK), has a low apparent affinity for its physiological substrate glucose and is not significantly inhibited by the reaction product. Interestingly, it displays positively cooperative kinetic behavior while functioning exclusively as a monomer with a single glucose binding site. A lag in steady-state saturation curves places the inflection point in the range of physiological blood glucose levels, allowing activity to be highly tunable in this region. Fasting and fed states can therefore dictate the rate of glucose phosphorylation in the reaction that represents the rate-limiting factor of insulin release and glycogen synthesis. The most compelling evidence for the pivotal role of GCK in glucose homeostasis was the discovery of mutations in the glk gene that result in long-term glycemic complications. Established as a central regulator of blood glucose, efforts to target GCK in the treatment of diabetic disorders resulted in the development of glucokinase activators. Small-molecule activators of GCK bind to an allosteric site and increase the enzyme's apparent affinity for glucose (K0.5). Effects on turnover (kcat) and cooperativity (Hill coefficient) can vary widely, and these differences have remained largely uninvestigated. Conflicting kinetic models have been proposed in the literature that disagree as to whether glucose binding is pre-requisite to activator association. Our kinetic activity assays, obtained via stopped-flow, suggest that activators can indeed associate with some population of GCK in the absence of glucose. To investigate the differing effects on turnover by activators, we performed viscosity variation assays. We found that the kcat of GCK is partly dependent on some diffusion-limited process, such as product release. In the presence of different activators, whose effects on turnover varied from unchanged to augmented 30%, we observed a significant change in dependence for just one of the three compounds tested. Compound A increased kcat by 10% and showed a nearly complete dependence upon solvent viscosity, suggesting that the activator functions by accelerating some preceding event that makes product release the determinant for turnover. The precise in vivo regulation of GCK and the presence of its highly effective allosteric activation site have led some to speculate the existence of an endogenous activator. Such a molecule would likely provide a safe scaffold in the design of future GCK therapeutics. We tested a variety of biogenic compounds using linked-enzyme assays for activating properties. When these tests proved unsuccessful, we developed a methodology that would provide the ability to genetically select for activating cyclic peptides among a library of 106 unique members. We combined a proven genetic selection system devised in-house with a uniquely engineered plasmid construct that generates randomized cyclic peptides in vivo. The methodology was optimized to be sensitive to the presence of GCK activation. In addition, we describe alternative approaches and ongoing efforts on this front.
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Date Issued: 2014
Identifier: FSU_migr_etd-9147
Format: Thesis

Title: Analysis of the Inter- and Intraparticle Obstruction Factors in Size-Exclusion Chromatography.
Creator: Richard, Dustin Joseph, Striegel, Andre, Chella, Ravi, Logan, Timothy, Roper, Michael, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Size-exclusion chromatography (SEC) is a separation technique commonly used for size and molar mass analysis. The primary mechanism of separation by SEC is not by enthalpic interactions, like in such techniques as gas chromatography (GC) or reversed-phase liquid chromatography (RPLC), but instead by an entropic mechanism. This entropic mechanism of separation can be beneficial when used to determine and understand diffusion, or obstructed diffusion, during a chromatographic separation. The...
Show moreSize-exclusion chromatography (SEC) is a separation technique commonly used for size and molar mass analysis. The primary mechanism of separation by SEC is not by enthalpic interactions, like in such techniques as gas chromatography (GC) or reversed-phase liquid chromatography (RPLC), but instead by an entropic mechanism. This entropic mechanism of separation can be beneficial when used to determine and understand diffusion, or obstructed diffusion, during a chromatographic separation. The lack of enthalpic interactions allows for easier isolation of individual contributions of chromatographic peak broadening, which is primarily caused by analyte diffusion. Measuring changes in certain band broadening contributions under various conditions allows for the determination of the effective diffusion coefficient, or hindered diffusion due to the chromatographic media, of the analyte. It's these effective diffusion coefficients that allow for the measurement of how different the measured effective diffusion coefficient is to the diffusion of the analyte in an open or unobstructed environment. This difference in diffusion is known as the obstruction factor, γ. The capability to quantify and understand obstructed diffusion, and how it relates to band broadening, has its advantages. Currently, the most common method of determining molar mass distributions of large polydisperse analytes is done using a molar mass calibration curve. This type of calibration curve, as well as other types of calibrations, is vulnerable to small changes in band broadening due to the logarithmic dependence of molar mass to retention volume. Measuring or knowing an analyte's obstructed diffusion gives the system operator the ability to predict how much band broadening will be present, depending on system conditions or operator-controlled variables. This study investigates several types of obstruction factors. First, the intraparticle obstruction factor, γp, takes into account the obstruction that occurs as an analyte partitions in and out of the pores of chromatographic media. Second, the interparticle obstruction factor, γe, accounts for obstruction that occurs solely around the spherical particles present within most chromatographic media. And last, the "total" or combined obstruction factor, γt, represents the combined contribution to obstruction of both, γp and γe, as an analyte travels around and within a column's particles. These three types of obstruction are measured in different ways. The intraparticle obstruction factor was determined via a variable-flow-rate method that measures how much hindrance of diffusion occurs as it travels in and out of the particle's pores. By measuring the increase in peak broadening as the volumetric flow rate of the system was increased, we were able to determine γp. Several operator-controlled variables were changed to see what effect, if any, they had on γp, such as molar mass, analyte, and solvent, as well as several system parameters such as column particle size, pore size, and temperature. The results of the experiments show that γp is very small and in each case, except with change in temperature, that the intraparticle obstruction factors change depending on the variable of the system. The other two obstruction factors, interparticle and combined obstruction, are determined using a stop-flow method, which measures the change in longitudinal diffusion of an analyte over time. This measurement was performed by having a sample sit on a column for various lengths of time (from 1 hour to 72 hours). The resulting series of broadening peaks was then used to determine the effective diffusion coefficient. Depending on the column used, either γe or γt was determined. If an analyte is measured on a column that has pores too small for the analyte to enter, then only γe is measured, as it cannot enter the column's pores. Conversely, if the analyte is small enough to enter the column's pores, then the γt is measured due to the analyte's ability to diffuse not only within particles, but also around them. The results for γe were constant and comparable to other published results. Values for γt were also obtained and used to relate both the γe and γp to γt. This study provides not only information pertinent to SEC, but also the obstruction factor measurements provides a greater fundamental understanding of the diffusion and band broadening processes that happen in other forms of chromatography, such as GC or RPLC. Other scientific areas where diffusion plays an important role, such as biochemistry or the oil industry, can also benefit from the results of these experiments.
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Date Issued: 2011
Identifier: FSU_migr_etd-5930
Format: Thesis

Title: Anionic Rearrangement of 2-Benzyloxypyridine Derivatives and a Synthetic Approach to Aldingenin B.
Creator: Yang, Jingyue, Dudley, Gregory B., Keller, Thomas C. S., Alabugin, Igor, Zhu, Lei, Shatruk, Michael, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: [1,2]-Anionic rearrangements are important tools for altering the complexity of molecules at hand. In Part I of this dissertation, an anionic rearrangement of 2-benzyloxypyridine is described. Pyridine-directed metallation of the benzylic carbon leads to 1,2 migration of pyridine via a postulated associative mechanism (addition / elimination). Several aryl pyridyl carbinols were obtained in high yields. A formal synthesis of carbinoxamine, an antihistamine drug used for the treatment of...
Show more[1,2]-Anionic rearrangements are important tools for altering the complexity of molecules at hand. In Part I of this dissertation, an anionic rearrangement of 2-benzyloxypyridine is described. Pyridine-directed metallation of the benzylic carbon leads to 1,2 migration of pyridine via a postulated associative mechanism (addition / elimination). Several aryl pyridyl carbinols were obtained in high yields. A formal synthesis of carbinoxamine, an antihistamine drug used for the treatment of seasonal allergies and hay fever, emerges from this methodology. As an update, the [1,2]-anionic rearrangement of benzyl 2-pyridyl ethers can also be accessed by a distinct and unusual mechanism: addition of alkyllithium reagents to α-(2-pyridyloxy)-styrene triggers anionic rearrangement to teriary pyridyl carbinols. This will be presented in Chapter 4 and the process is explained by invoking contraelectronic, pyridine-directed carbolithiation of the enol ether π-system. Part II of this dissertation is focused on a synthetic approach to aldingenin B. The synthesis of the tricyclic core of aldingenin B from a key internal alkyne was completed. Synthesis of alkynes by fragmentation is an on-going interest of the Dudley lab. One current goal is to apply our methodology in conjunction with an innovative oxidative alkyne ketalization to achieve a short and efficient synthesis of aldingenin B. The specific goal for this dissertation was to prepare a model alkyne by conventional methods and establish the feasibility of the oxidative alkyne ketalization. The preparation and selenium-mediated cyclo-ketalization of an alkyne-diol will be described as a model study for the synthesis of aldingenin B in Chapter 8. The oxidative cyclization is a simplifying transformation for aldingenin B, as it provides a convenient method for generating the tricyclic core of the natural product from a functionalized carbocycle. Some preliminary experiments to guide future efforts for completing the synthesis of aldingenin B will be presented in Chapter 9.
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Date Issued: 2011
Identifier: FSU_migr_etd-1017
Format: Thesis

Title: Application of FT-ICR Mass Spectrometry in Study of Proteomics, Petroleomics and Fragmentomics.
Creator: Mao, Yuan, Marshall, Alan G., Blaber, Michael, Dalal, Naresh S., Roper, Michael G., Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: With the advent of two "soft" ionization techniques in the late 1980s—electrospray ionization (ESI) and matrix assisted laser desorption/ionization (MALDI) for the routine and general formation of intact molecular ions—and continuing effort in the instrumentation development directed toward improving two key parameters of mass spectrometric performance—mass resolving power and mass accuracy, mass spectrometry has been an indispensable analytical technique for chemical and biochemical sample...
Show moreWith the advent of two "soft" ionization techniques in the late 1980s—electrospray ionization (ESI) and matrix assisted laser desorption/ionization (MALDI) for the routine and general formation of intact molecular ions—and continuing effort in the instrumentation development directed toward improving two key parameters of mass spectrometric performance—mass resolving power and mass accuracy, mass spectrometry has been an indispensable analytical technique for chemical and biochemical sample analysis, especially for highly complex mixture systems, e.g., proteomics, petroleomics, lipidomics, metabolomics, etc. The introduction of ESI and MALDI ionization techniques has extended the accessibility of mass spectrometry-based analysis from small volatile molecules to large non-volatile molecules, whereas mass accuracy and mass resolving power directly determine the usefulness of mass spectrometric experiments. Among the high resolution mass analyzers such as reflectron/multipass time of flight (TOF), orbitrap, and Fourier transform ion cyclotron resonance (FT-ICR), FT-ICR MS provides ten-fold higher mass resolution and mass measurement accuracy than other mass analyzers and has become the most powerful techniques that can deal with the complexity of various samples, e.g., it is possible to routinely achieve high mass resolving power of 400,000 (m/∆m50% ≈ 400,000, in which m is molecular mass and ∆m50% is the mass spectral peak width at half-maximum peak height) and mass accuracy (~ 100 ppb) up to 800 Da from high-field (≥9.4 T) FT-ICR MS, thus resolving > 40,000 different elemental compositions in a single mass spectrum and providing their unambiguous molecular formulas. Chapter 1 introduces the concept of mass resolving power, mass resolution and mass measurement accuracy, the principle of the FT-ICR instrument for mass measurement, ionization methods, factors that control the mass measurement accuracy of FT-ICR MS and utility of high mass accuracy for analysis of biological samples and complex mixtures. Chapter 2 describes the application of high mass accuracy for distinction of N-terminal and C-terminal electron capture dissociation/electron transfer dissociation (ECD/ETD) product ions of c and z⋅ based on their number of hydrogen plus nitrogen atoms determined by accurate mass measurement, and forms a basis for de novo peptide sequencing. The effect of mass accuracy (0.1-1 ppm error) on c/z⋅ overlap and unique elemental composition overlap is evaluated for a database of c/z⋅ product ions each based on all possible amino acid combinations and four subset databases containing the same c ions but with z⋅ ions determined by in silico digestion with trypsin, Glu-C, Lys-C, or chymotrypsin. High mass accuracy reduces both c/z⋅ overlap and unique elemental composition overlap. Of the four proteases, trypsin offers slightly better discrimination between N- and C-terminal ECD/ETD peptides. Interestingly, unique elemental composition overlap curves for c/c and z⋅/z⋅ peptide ions exhibit discontinuities at certain nominal masses for 0.1-1.0 ppm mass error. Also, the number of ECD/ETD product ion amino acid compositions as a function of nominal mass increases exponentially with mass, but with a superimposed modulation due to higher prevalence of certain elemental compositions. Chapter 3 presents an idea that at sufficiently high mass accuracy, it is possible to distinguish phosphorylated from unmodified peptides by mass measurement alone. We examine the feasibility of that idea, tested against a library of all possible in silico tryptic digest peptides from the human proteome database. The overlaps between in silico tryptic digest phosphopeptides generated from known phosphorylated proteins (1-12 sites) and all possible unmodified human peptides are considered for assumed mass error ranges of ±10, ±50, ±100, ±1,000, and ±10,000 ppb. We find that for mass error ±50 ppb, 95% of all phosphorylated human tryptic peptides can be distinguished from nonmodified peptides by accurate mass alone through the entire nominal mass range. We discuss the prospect of on-line LC MS/MS to identify phosphopeptide precursor ions in MS1 for selected dissociation in MS2 to identify the peptide and site(s) of phosphorylation. ETD and ECD are two similar fragmentation approaches, producing extensive and nonspecific fragmentation (c/z⋅ ions formed by cleavage of N-Cα backbone bond) while retaining thermally labile post-translational modifications. In chapter 4, we implemented dual electrospray ionization ETD on a custom-built 9.4 T FT-ICR MS. Two separate electrospray emitters are automatically switched for injection of positive (analyte) and negative (reagent) ions. Decarboxylated 2-(fluoranthene-8-carbonyl) benzoic acid is the ETD reagent anion. A linear octopole ion trap is the ETD ion/ion reaction chamber, and an RF voltage is applied to the front and back ion trap electrodes to confine both cations and reagent anions for ETD, after which the c- and z- type product ions are passed to the ICR cell for high resolution and mass accuracy analysis. Comparison of ETD and ECD spectra of standard peptides shows that ETD provides similar sequence coverage and fragmentation pattern to ECD. Chapter 5 describes a calibration procedure in which accurate masses of spacings from any two same type neighboring fragment ions differing by one amino acid residue are used to calibrate ECD and collision activated dissociation (CAD) MS/MS spectra of standard peptides with different molecular weights and charge states. High mass accuracy of tandem mass spectra is crucial for confident extraction and identification of spacings. FT-ICR mass spectrometry provides ultrahigh mass accuracy and resolving power and acquired MS/MS spectra with ppm mass accuracy level are routinely obtained when combined with external calibration by substance P fragments. Calibration by accurate masses of extracted spacings shows up to ~ 30% further reduction of rms mass error of MS/MS spectra on average compared with substance P MS/MS external calibration. ~ 25% improvement of c/z⋅ ion unambiguous distinction from ECD spectra based on valence parity rule increases the confidence of peptide sequencing. FT-ICR MS with ultrahigh resolving power and mass accuracy is essential to resolve and uniquely identify elemental compositions of thousands of components in complex organic mixtures, e.g., petroleum crude oils. To study how much resolving power and mass accuracy is necessary, in chapter 6 all possible closest mass doublets (0<∆m<45 mDa, ∆m is the mass difference of mass doublet) were counted for both electrospray ionization (ESI) and atmospheric pressure photoionization (APPI) absorption-mode spectra automatically by use of the algorithm written in LabWindows/CVI. As many as thousands of mass doublets with the mass difference less than 10 mDa (as low as ~ 0.70 mDa) were observed in APPI and ESI absorption-mode broadband mass spectra. Histograms of mass doublet distribution for APPI and ESI were plotted. In chapter 7 the effect of mass error (10 - 500 ppb) on elemental composition overlap is evaluated for ESI and APPI databases containing all possible elemental compositions with proper constraints, CcHhNnOoSs13Ccc34Sss, c, h unlimited, 0≤n<5, 0≤o<10, 0≤s≤3, 0≤cc<3 and 0≤ss<2 for even-electron ions (M+H)+ (or (M-H)-) in ESI and for both M+⋅ and (M+H)+ (or M-⋅ and (M-H)-) in APPI with nominal mass of 200-1200 Da. Number of element compositions of all possible components in complex mixtures is reduced by ~ 1100 on average for each class after applying 90% rule. High mass accuracy reduces elemental composition overlap and facilitates the unique identification of elemental compositions for components up to 1200 Da at mass errors of 200 ppb in ESI and 100 ppb in APPI. All possible theoretical mass doublets which may occur in petroleum crude oils are calculated based on elemental compositions of all possible components from ESI and APPI databases. Mass doublets with mass difference as low as 0.20 mDa, even smaller than mass of electron (0.548 mDa) and NO213C vs. C2H3S (0.71 mDa) (the smallest one currently observed in 9.4 T broadband absorption-mode ESI/APPI FT-ICR MS) are calculated and can only be resolved with higher-field FT-ICR MS (e.g., absorption-mode 21T). FTICR mass spectrometer coupled with ECD/ETD offers ultrahigh broadband mass resolving power (>105) and mass accuracy (<1 ppm) for detection of accurate precursor mass as well as the vast amount of isotopically resolved fragment ions required for protein identification and has become an increasing useful tool for top-down analysis. In chapter 8 we perform top-down ECD FT-ICR MS for structural analysis of an intact monoclonal antibody (IgG1-kappa (κ) isotype, ~148 kDa). Simultaneous ECD for all charge states (42+ to 58+) generates more extensive cleavages than ECD for an isolated single charge state. The cleavages are mainly localized in the variable domains of both heavy and light chains, the respective regions between the variable and constant domains in both chains, the region between heavy chain constant domains CH2 and CH3, and the disulfide bond (S-S) linked heavy chain constant domain CH3. The light chain yields mainly N-terminal fragment ions due to the protection of the inter-chain disulfide bond between light and heavy chain, and limited cleavage sites are observed in the variable domains for each chain where the S-S spans the polypeptide backbone. Only a few cleavages in the S-S linked light chain constant domain, hinge region, and heavy chain constant domains CH1 and CH2 are observed, leaving glycosylation uncharacterized. Top-down ECD with a custom-built 9.4 T FT-ICR MS for structural characterization of IgG1κ provides more extensive sequence coverage than top-down collision induced dissociation (CID) and ETD with time-of-flight but comparable sequence coverage with top-down ETD with orbitrap MS.
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Date Issued: 2013
Identifier: FSU_migr_etd-7489
Format: Thesis

Title: Application of Solid State Nuclear Magnetic Resonance in Drug Discovery.
Creator: Wright, Anna Kozlova, Cross, Timothy A., Dudley, Gregory B., Blaber, Michael, Marshall, Alan G. (Alan George), Keller, Thomas C. S., Florida State University, College of Arts...
Show moreWright, Anna Kozlova, Cross, Timothy A., Dudley, Gregory B., Blaber, Michael, Marshall, Alan G. (Alan George), Keller, Thomas C. S., Florida State University, College of Arts and Sciences, Program in Molecular Biophysics
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Abstract/Description: The M2 proton channel from Influenza A is an established drug target, with multiple functions during the viral lifecycle. Amino acid mutations, in the residues lining the channel pore, have rendered M2 resistant to previously licensed inhibitors. Given the propensity for genetic reassortment of Influenza A and history of pandemics due to emergence of novel human strains, M2 has been subject of numerous structural characterization efforts. Attempts at rational drug design targeting M2 proton...
Show moreThe M2 proton channel from Influenza A is an established drug target, with multiple functions during the viral lifecycle. Amino acid mutations, in the residues lining the channel pore, have rendered M2 resistant to previously licensed inhibitors. Given the propensity for genetic reassortment of Influenza A and history of pandemics due to emergence of novel human strains, M2 has been subject of numerous structural characterization efforts. Attempts at rational drug design targeting M2 proton channel have been impeded by the limited number of experimental techniques having capabilities for elucidating atomic level interactions of the protein-ligand complexes in the native-like membrane mimetic environment. Solid state Nuclear Magnetic Resonance (ssNMR) is a technique that has all of these capabilities for structural characterization of membrane protein drug targets in lipid bilayers. Coupling ssNMR with Computer Aided Drug Discovery (CADD) is the precise approach needed to decrease time and resources required to generate novel therapeutics. Here, we present a first structural characterization of the full length wild type M2 channel in complex with inhibitor, and of the S31N mutant in the apo and drug-bound state. Through the use of diverse ssNMR experiments we tested stereoselectivity of drug binding in the channel pore, structural changes due to mutation, and characterized novel inhibitor interactions. Molecular dynamic simulations were performed by our collaborators and were in good agreement with our experimental findings. Together these results deepen our understanding of the atomic level interactions stabilizing wild type inhibitors in the channel pore, and structural changes in the mutant leading to loss of compound efficacy. Most importantly, specific interaction described herein are essential for successful outcomes from structure based CADD and be used in future computational efforts.
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Date Issued: 2015
Identifier: FSU_2016SP_Wright_fsu_0071E_13033
Format: Thesis

Title: Applications of High Frequency Electron Paramagnetic Resonance on Materials from Quantum to Classical Regime.
Creator: Wang, Zhenxing (EPR Specialist), Dalal, Naresh S., Brooks, James, Kroto, Harold, Strouse, Geoffrey F., Shatruk, Michael, Department of Chemistry and Biochemistry, Florida State...
Show moreWang, Zhenxing (EPR Specialist), Dalal, Naresh S., Brooks, James, Kroto, Harold, Strouse, Geoffrey F., Shatruk, Michael, Department of Chemistry and Biochemistry, Florida State University
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Abstract/Description: This dissertation has focused on studying the electron spin dynamics in the quantum and classical limit and, most importantly, at the quantum-classical boundary. We have successfully used high frequency electron paramagnetic resonance (HF-EPR) techniques to characterize six paramagnetic materials with increasing number of unpaired electrons and molecular sizes. The samples studied are Mn2+-doped CdSe Quantum Dots (Mn:CdSe QDs), Na20[Cu2Pd22PV12O60(OH)8] (Cu2), Na12[X2W18Cu3O66(H2O)3].32H2O (X...
Show moreThis dissertation has focused on studying the electron spin dynamics in the quantum and classical limit and, most importantly, at the quantum-classical boundary. We have successfully used high frequency electron paramagnetic resonance (HF-EPR) techniques to characterize six paramagnetic materials with increasing number of unpaired electrons and molecular sizes. The samples studied are Mn2+-doped CdSe Quantum Dots (Mn:CdSe QDs), Na20[Cu2Pd22PV12O60(OH)8] (Cu2), Na12[X2W18Cu3O66(H2O)3].32H2O (X = As, Sb) (Cu3), [Fe7O4(O2CPh)11(dmem)2].4MeCN (Fe7), [Mn7O4(pdpm)6(N3)4](ClO4)2 (Mn7) and Na34[Mn19(OH)12(SiW10O37)6].115H2O (Mn19). Our results have illustrated that four samples including Mn:CdSe QDs, Cu2, Cu3 and Fe7 can be perfectly described with quantum mechanics while sample Mn19 behaves like a typical classical system. Most interestingly, sample Mn7 (S = 29/2) straddles the interface between the classical and quantum mechanical spin descriptions. Chapter 1 gives the motivation, overview and organization of this dissertation. Chapter 2 describes synthetic details of the materials studied, introduction of two HF-EPR spectrometers, as well as the computer simulation programs employed in this undertaking. Chapter 3 summarizes the HF-EPR studies of Mn:CdSe QDs, the first application of HF-EPR to magnetic QDs. Chapter 4 presents the structure and magnetic characterization of an octahedrally coordinated Cu(II) pair, a very rare bonding for Cu(II) ions. Chapter 5 reports the coherent manipulation of electron spins in an antiferromagnetically coupled spin triangle {Cu3} impregnated in free standing nanoporous silicon (NS) by using 240 GHz microwave pulses. Chapter 6 discusses continuous wave (cw) and pulsed HF-EPR measurements on an Fe-based magnetic cluster: Fe7. Chapter 7 describes the HF-EPR characterization of a high spin (S) compound, Mn7, whose properties straddle the interface between the classical and quantum mechanical spin descriptions. Chapter 8 reports the structure and magnetic properties of a novel, unique, discrete polyanion comprising a cationic, planar Mn19 assembly incorporated in a 60-tungsto-6-silicate. Finally, chapter 9 summarizes the major results and conclusions of this dissertation.
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Date Issued: 2012
Identifier: FSU_migr_etd-5258
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: 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: The Autolytic Regulation of Human Kallikrein-Related Peptidase 6.
Creator: Blaber, Sachiko, Yoon, Hyesook, Scarisbrick, Isobel, Juliano, Maria, Blaber, Michael
Abstract/Description: Human kallikrein-related peptidase 6 (KLK6) is a member of the kallikrein family of serine-type proteases, characterized as an arginine-specific digestive-type protease capable of degrading a wide-variety of extracellular matrix proteins. KLK6 has been proposed to be a useful biomarker for breast and ovarian cancer prognosis, is abundantly expressed in the CNS and cerebrospinal fluid, and is intimately associated with regions of active inflammatory demyelination in multiple sclerosis (MS)...
Show moreHuman kallikrein-related peptidase 6 (KLK6) is a member of the kallikrein family of serine-type proteases, characterized as an arginine-specific digestive-type protease capable of degrading a wide-variety of extracellular matrix proteins. KLK6 has been proposed to be a useful biomarker for breast and ovarian cancer prognosis, is abundantly expressed in the CNS and cerebrospinal fluid, and is intimately associated with regions of active inflammatory demyelination in multiple sclerosis (MS) lesions. Inhibition of KLK6 results in delayed onset and reduced severity of symptoms associated with experimental autoimmune encephalomyelitis, suggesting a key effector role for this protease in CNS inflammatory disease. KLK6 has been shown to autolytically cleave internally, leading to inactivation and suggesting a negative feedback inhibition control mechanism. Alternatively, the ability of KLK6 to self-activate has also been reported, suggesting a positive feedback activation loop control mechanism. Activation of pro-KLK6 requires hydrolysis after a Lys residue; however, KLK6 exhibits 2 order of magnitude reduced affinity for hydrolysis after Lys versus Arg residues; therefore, the ability to autolytically activate has been called into question. In the present study the catalytic activity of KLK6 toward its pro-sequence and internal autolytic sequence is characterized. The results show that the ability of KLK6 to activate pro-KLK6 is essentially negligible when compared to the rate of the internal autolytic inactivation or to the ability of other proteases to activate pro-KLK6. The results thus show that the primary autolytic regulatory mechanism of KLK6 is negative feedback inhibition, and activation is likely achieved through the action of a separate protease.
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Date Issued: 2007
Identifier: FSU_migr_biomed_faculty_publications-0002, 10.1021/bi6025006, PMC2517904
Format: Citation

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: Backbone Dynamics in an Intramolecular Prolylpeptide SH3 Complex from Diphtheria Toxin Repressor, DtxR.
Creator: Bhattacharya, Nilakshee, Logan, Timothy M., Zhou, Huan-Xian, Li, Hong, Steinbock, Oliver, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Diphtheria toxin repressor is a regulatory protein from Corynebacterium diphtheriae, the causal agent of Diphtheria. The diphtheria toxin repressor (DtxR) contains an SH3-like domain that forms an intramolecular complex with a proline-rich (Pr) peptide segment that serves to stabilize the inactive state of the repressor. During activation of DtxR by transition metals, this intramolecular complex must dissociate as the SH3 domain and Pr segment form different interactions in the active...
Show moreDiphtheria toxin repressor is a regulatory protein from Corynebacterium diphtheriae, the causal agent of Diphtheria. The diphtheria toxin repressor (DtxR) contains an SH3-like domain that forms an intramolecular complex with a proline-rich (Pr) peptide segment that serves to stabilize the inactive state of the repressor. During activation of DtxR by transition metals, this intramolecular complex must dissociate as the SH3 domain and Pr segment form different interactions in the active repressor. In this study we investigate the dynamics of this intramolecular complex using backbone amide nuclear spin relaxation rates determined experimentally using NMR spectroscopy and computed from molecular dynamics trajectories. The SH3 domain in the unbound and bound states showed typical dynamics in that the secondary structures were fairly ordered with high generalized order parameters and low effective correlation times while residues in the loops connecting b-strands exhibited reduced generalized order parameters and required additional motional terms to adequately model the relaxation rates. Residues forming the Pr segment also exhibited low order parameters with internal rotational correlation times on the order of 0.6 â 1 ns. Further analysis showed that the SH3 domain was rich in ms motions while the Pr segment was rich in motions on the 100s ms timescale. Molecular dynamics trajectories of PrSH3 and SH3 indicated structural rearrangements that might contribute to the observed relaxation rates and, together with the observed relaxation rate data, suggested that the Pr segment exhibits a binding â unbinding equilibrium. The intramolecular complex resisted any significant change in the binding affinity between the Pr segment and the SH3 domain due to mutations in the Pr segment. The results of this study provide key insights into the nature of the intramolecular complex and provide a better understanding of the biological role of the SH3 domain in regulating DtxR activity.
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Date Issued: 2007
Identifier: FSU_migr_etd-1408
Format: Thesis

Title: Batch Anion Exchange Separation: A Prefractionation Technique for Proteome Research and Its Applications on In Vivo Cancer Samples.
Creator: Sahab, Ziad Joseph, Sang, Qing-Xiang A., Deng, Wu-Min, Cooper, William T., Marshall, Alan G., Schlenoff, Joseph B., Department of Chemistry and Biochemistry, Florida State...
Show moreSahab, Ziad Joseph, Sang, Qing-Xiang A., Deng, Wu-Min, Cooper, William T., Marshall, Alan G., Schlenoff, Joseph B., Department of Chemistry and Biochemistry, Florida State University
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Abstract/Description: Two-dimensional gel electrophoresis (2-DE) is used to compare the protein profiles of different crude biological samples. Narrow pH range Immobilized pH Gradient (IPG) strips were designed to increase the resolution of these separations. To take full advantage of IPG strips, the ideal sample should be composed primarily of proteins that have isoelectric point (pI) values within the pH range of the IPG strip. Prefractionation of cell lysates from a human prostate cancer cell line cultured in...
Show moreTwo-dimensional gel electrophoresis (2-DE) is used to compare the protein profiles of different crude biological samples. Narrow pH range Immobilized pH Gradient (IPG) strips were designed to increase the resolution of these separations. To take full advantage of IPG strips, the ideal sample should be composed primarily of proteins that have isoelectric point (pI) values within the pH range of the IPG strip. Prefractionation of cell lysates from a human prostate cancer cell line cultured in the presence or absence of epigallocatechin-3-gallate was achieved in fewer than 30 minutes using an anion exchange resin and two expressly-designed buffers. The procedure was carried out in a centrifuge tube and standard instrumentation was used. The cell lysates were prefractionated into two fractions: proteins with pI values above 7 and between 4 and 7, respectively. The fractions were then analyzed by 2-DE, selecting appropriate pH ranges for the IPG strips, and the gels were compared with those of unprefractionated cell lysates. Protein loading capacity was optimized and resolution and visualization of the less abundant and differentially-expressed proteins were greatly improved. Furthermore, this technique was applied successfully to eliminate albumin from serum samples. Elimination of albumin, constituting more than 50% of total serum proteins, allowed for increasing protein loads on polyacrylamide gels. Visualization and resolution of lower-abundance proteins that were previously masked by albumin was achieved as well.
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Date Issued: 2005
Identifier: FSU_migr_etd-2111
Format: Thesis

Title: Biochemical Characterization of Human Matrix Metalloproteinases and Their Newly Designed Inhibitors Related to Stroke.
Creator: Cao, Qiang, Sang, Qing-Xiang Amy, Wang, Yan-Chang, Li, Hong, Alabugin, Igor, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Matrix metalloproteinases (MMPs), a family of enzymes known for their proteolytic activities on processing extracellular matrix substrates, may play an integral role in blood-brain barrier opening following an ischemic stroke. Several matrix metalloproteinases are proposed to play vital roles in early or late stages of blood brain barrier opening. Matrix metalloproteinase inhibitor (MMPI) has been showing beneficial effect in the treatment of the blood brain opening related to stroke. As old...
Show moreMatrix metalloproteinases (MMPs), a family of enzymes known for their proteolytic activities on processing extracellular matrix substrates, may play an integral role in blood-brain barrier opening following an ischemic stroke. Several matrix metalloproteinases are proposed to play vital roles in early or late stages of blood brain barrier opening. Matrix metalloproteinase inhibitor (MMPI) has been showing beneficial effect in the treatment of the blood brain opening related to stroke. As old generation matrix metalloproteinase inhibitor failed in oncology clinical trials, our collaborators, Drs. Martin A. Schwartz and Yonghao Jin, have designed and synthesized new biologically friendly mercaptosulfonamide inhibitors. Characterization and selection of effective matrix metalloproteinase inhibitors were performed by evaluating their stability, potency, and selectivity by enzymatic kinetics. According to dissociation constant related to enzyme and inhibitor binding, our data indicates that those inhibitors are capable of inhibiting MMP-2, -9, and membrane-type 1 MMP (MT1-MMP) effectively and selectively. Selected inhibitors were studied with cell wound healing assays in human microvascular endothelial cell model to investigate selected MMPI activities and impact on cell behavior. By blocking MMP activities in cell culture, our inhibitors were able to reduce human brain microvascular endothelial cell wound healing process. Protein expression patterns in cell culture were investigated with proteomics after inhibitor treatment. The reduced expressions of several proteins, which are related to cell division, cell adhesion and cell death, have been discovered. It is also verified the blocking function of our inhibitor in human brain microvascular endothelial cell wound healing assay. Overall, our newly designed matrix metalloproteinase inhibitor efficiently inhibits matrix metalloproteinase which carries intermediate or deep S1' pocket at protein and cellular level. Upon application of matrix metalloproteinase inhibitor, it has been implicated that blocking of matrix metalloproteinase activities are involved in the decreasing of other cell function modulators. The most potent and specific inhibitors have been selected as promising compounds, which have been further tested in animal models to evaluate their efficacy in the prevention of blood brain barrier opening associated with stroke by our collaborator Dr. Gary A. Rosenberg. This study is the first enzymological and cellular analysis of mercaptosulfonamide inhibitors.
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Date Issued: 2010
Identifier: FSU_migr_etd-4624
Format: Thesis

Title: Biochemical Characterization of the RNA Splicing Endonuclease.
Creator: Calvin, Kate, Li, Hong, Hurt, Myra, Cross, Timothy, Logan, Timothy, Miller, Brian, Program in Molecular Biophysics, Florida State University
Abstract/Description: In eukaryotes and archaea 5-25% of transfer RNA (tRNA) precursors contain intervening sequences, or introns, that interrupt the molecules' functional regions. Because functional tRNA molecules are necessary for protein synthesis, removing these introns is essential to sustain life. tRNA introns are removed in a two-to-three step process mediated by three different proteins. The RNA splicing endonuclease acts first to cleave two phosphodiester bonds at the intron boundaries within the folded...
Show moreIn eukaryotes and archaea 5-25% of transfer RNA (tRNA) precursors contain intervening sequences, or introns, that interrupt the molecules' functional regions. Because functional tRNA molecules are necessary for protein synthesis, removing these introns is essential to sustain life. tRNA introns are removed in a two-to-three step process mediated by three different proteins. The RNA splicing endonuclease acts first to cleave two phosphodiester bonds at the intron boundaries within the folded precursor RNAs. The endonuclease performs this function upon nuclear tRNA introns and all archaeal introns. It is well-established that in all organisms the endonuclease step in the splicing pathway is completely conserved, with evidence for the conservation of cleavage chemistry being provided by biochemical studies. However, no detailed information was previously available regarding the endonuclease's specific mechanisms. This research addresses two key aspects of the splicing endonuclease mechanism, namely, substrate recognition and catalysis. Chapter 2 explores the structural elements in a phenotypical archaeal splicing endonuclease and its RNA substrate required for recognition and catalysis. These assays explicitly demonstrate the enzyme and substrate elements involved in recognition and binding. They also support previous findings regarding a conserved triad hypothesized to be catalytic and lay the foundation for the more in-depth studies in Chapter 3. Chapter 3 presents a series of kinetics experiments investigating this conserved triad in which kinetic parameters KM and k2 are obtained. The primary substrate recognition elements in the endonuclease are strictly conserved. However, splicing endonucleases in different organisms are found to have different subunit compositions and substrate specificities. No biochemical studies to date have shed light on how this occurs. Chapter 4 presents studies exploring how the enzyme's quaternary structure affects substrate recognition and cleavage. These studies are continued in Chapter 5, where it is demonstrated that enzyme assembly alone can dictate both substrate specificity and activity. Taken in total, the work presented in this Dissertation provides significant insight regarding how the endonuclease precisely recognizes intron-exon junctions and accelerates the cleavage reaction. It also sheds considerable light on how enzyme subunit composition and quaternary structure relate to the mechanism of RNA recognition.
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Date Issued: 2007
Identifier: FSU_migr_etd-4514
Format: Thesis

Title: Biochemistry Students.
Creator: Florida State University
Date Issued: 1947, 1960
Identifier: FSU_HPUA_2016003_B7_F2_008
Format: Image (JPEG2000)

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: 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: Broadband Phase Correction of Fourier Transform Ion Cyclotron Resanonce Mass Spectra.
Creator: Xian, Feng, Marshall, Alan G., Hendrickson, Christopher L., Hill, Stephen, Dalal, Naresh S., Roper, Michael, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: It has been known for 35 years that phase correction of Fourier transform ion cyclotron resonance (FT-ICR) mass spectral data can in principle produce an absorption-mode spectrum with mass resolving power as much as a factor of 2 higher than conventional magnitude-mode display, an improvement otherwise requiring a (much more expensive) increase in magnetic field strength. However, temporally dispersed excitation followed by time-delayed detection results in steep quadratic variation of signal...
Show moreIt has been known for 35 years that phase correction of Fourier transform ion cyclotron resonance (FT-ICR) mass spectral data can in principle produce an absorption-mode spectrum with mass resolving power as much as a factor of 2 higher than conventional magnitude-mode display, an improvement otherwise requiring a (much more expensive) increase in magnetic field strength. However, temporally dispersed excitation followed by time-delayed detection results in steep quadratic variation of signal phase with frequency. We developed a robust, rapid, automated method to enable accurate broadband phase correction for all peaks in the mass spectrum. Low-pass digital filtering effectively eliminates the accompanying baseline roll. Experimental FT-ICR absorption-mode mass spectra exhibit at least 40% higher resolving power (and thus an increased number of resolved peaks) as well as higher mass accuracy relative to magnitude mode spectra, for more complete and more reliable elemental composition assignments for mixtures as complex as petroleum. Absorption-mode FT-ICR mass spectrum, which is produced by automatic broadband phase correction algorithm, demonstrates baseline distortion even with low-pass filter baseline correction. Significant baseline roll affects peaks picking algorithm and results in incorrect peak height measurement. Isotopic distribution in spectra presenting large baseline roll couldn't display correct information. Thus, identification and characterization of biomolecule become much more difficult. In Chapter 2, we designed a fast, robust and automated baseline correction process. Each minimum data point of reversed peak in absorption-mode spectrum has been collected as bases of baseline model, and then further linear interpolation and boxcar smoothing technique help to complete the baseline model. Finally, the baseline model is subtracted from original spectrum to produce a flat baseline. This algorithm has been experimentally proven to automatically flatten baseline of crude oil, environmental sample and biomolecule FT-ICR mass spectra. More peaks have been identified from absorption-mode spectrum with flat baseline without loss of mass accuracy. Isotopic distribution also demonstrates very accurate profile. Apodization function and zero-filling are two basic steps in data processing of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry and their effect on the conventional Fourier transform ion cyclotron resonance (FT-ICR) experimental and simulated magnitude-mode mass spectra and single-peak absorption-mode spectra are well known. In Chapter 4, we examine the effects of each of twelve apodization (window) functions and 0, 1, and 2 zero-fills for absorption-mode Fourier transform mass spectra peak height-to-noise ratio, mass measurement accuracy, and mass resolving power for dense FT-ICR mass spectra of petroleum. Half function windowing is best for resolving close absorption-mode doublets, whereas full function windowing is best for resolving magnitude-mode doublets. Absorption-mode offers significantly higher mass resolving power than magnitude-mode for any given windowing function. Half apodization increases absorption-mode mass accuracy, irrespective of the choice of window function. One (but not more than one) zero-fill improves mass accuracy for absorption-mode mass accuracy but not for magnitude-mode. Peak height-to-noise ratio for both absorption and magnitude spectra is improved by zero-filling. Although we have successfully demonstrated the automated phase correction method for complex Fourier transform ion cyclotron resonance (FT-ICR) mass spectrum, we can't express the exact quadratic phase function of frequency from calculated phase for discrete data point. In Chapter 5, we applied stationary phase method to excitation and detection signal and derived the accurate phase spectra for both the linear chirp excitation and detected FT-ICR signals analytically. Because phase spectrum of detected signal represents correct variation of accumulated phase with frequency, it could be directly used to recover the absorption-mode FT-ICR mass spectra. Also, the phase correction of FT-ICR mass spectra from stored waveform inverse Fourier transform (SWIFT) by phase spectrum has been experimentally described. The analytically phase correction results are compared to the previous results produced by automated phase correction method in terms of resolving power and mass measurement accuracy Except for phase correction based on mathematical calculation of accurate phase for different frequencies. Scientists have demonstrated that simultaneous excitation and detection (SED) enable Fourier deconvolution to provide broadband phase correction with no user interaction. However, the capacitive nulling technique which is applied in SED method for removing the saturated excitation signal in front of detected signal is not practical due to unstable capacitors. In Chapter 6, we describe a new data processing procedure to enable broadband phase correction of FT-ICR mass spectra by SED without any hardware modification. The resulting absorption-mode spectra yield improvement in resolving power as well as reduction in assignment errors relative to conventional magnitude-mode spectra. The Fourier deconvolution procedure has the additional benefit of correcting for spectral variation resulting from nonuniform power distribution over the excitation bandwidth and phasing spectra from different excitation waveforms (e.g., SWIFT with different magnitude modulations). Fourier transform mass spectrometry (FTMS) of the isolated isotopic distribution for a highly charged biomolecule produces time-domain signal containing large amplitude signal "beats" separated by extended periods of much lower signal magnitude. Signal-to-noise ratio for data sampled between beats is low, due to destructive interference of the signals induced by members of the isotopic distribution. Selective blanking of the data between beats has been used to increase spectral signal-to-noise ratio. However, blanking also eliminates signal components, and thus can potentially distort the resulting FT spectrum. In Chapter 7, we simulate the time-domain signal from a truncated isotopic distribution for a single charge state of an antibody. Comparison of the FT spectra produced with or without blanking and with or without added noise clearly show that blanking does not improve mass accuracy and introduces spurious peaks at both ends of the isotopic distribution (thereby making it more difficult to identify posttranslational modifications and/or adducts). Ergo, blanking should never be employed: it has no advantages and major disadvantages.
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Date Issued: 2012
Identifier: FSU_migr_etd-6074
Format: Thesis

Title: Cascade Reactions for the Synthesis of Polycyclic Aromatic Hydrocarbons and Carbon Nanoribbons.
Creator: Byers, Philip M., Alabugin, Igor V., Alamo, Rufina, Strouse, Geoffrey, Saha, Sourav, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Radical and electrophile-promoted nucleophilic closure cascade reactions have been used for the synthesis of polycyclic aromatic hydrocarbons (PAHs) and carbon nanoribbons. Chapter 1 provides a discussion on radical cascade reactions, with a brief history and examples of well documented radical cascade reactions. Investigations into radical chain processes as well as redox processes are discussed. There is much development in these cascades currently due to the complex products that can be...
Show moreRadical and electrophile-promoted nucleophilic closure cascade reactions have been used for the synthesis of polycyclic aromatic hydrocarbons (PAHs) and carbon nanoribbons. Chapter 1 provides a discussion on radical cascade reactions, with a brief history and examples of well documented radical cascade reactions. Investigations into radical chain processes as well as redox processes are discussed. There is much development in these cascades currently due to the complex products that can be formed in a minimal amount of synthetic steps. Chapter 2 discusses the radical cascade that we developed, using a radical chain process. Selective radical generation in conjugated oligomeric o-aryleneethynylenes initiates a cascade which involves five fast radical cyclizations followed by aromatization via a 1,5-H shift to yield PAHs. Computation and 2D NMR studies were performed to determine the final cascade products. Chapter 3 discusses the growing trend in which gold catalysts are used to initiate cascade reactions through an electrophile-promoted nucleophilic closure mechanism. A number of current examples are also provided which demonstrate how gold catalyzed cascade reactions can efficiently synthesize complex PAH molecules. Chapeter 4 demonstrates our approach for the synthesis of fused benzofuran structures through gold catalysis. Through a gold catalyzed electrophile-promoted nucleophilic closure, PAHs containing benzofuran units with a polycyclic aromatic backbone were synthesized through a Sonogashira/5-endo-dig/6-endo-dig cascade. Depending on which mechanism is used, all exo cyclizations can be achieved (radical), or all endo cyclizations (electrophile-promoted nucleophilic closure). Starting materials for either cascade mechanism can be synthesized in a modular fashion, allowing for the systematic and controlled preparation of functionalized PAHs and carbon nanoribbons where, potentially, each of the peripheral aromatic rings can be different. The materials synthesized have future applications in the nano-materials and -electronics industries. Chapter 5 discusses future work in the field of gold catalyzed cascade reactions and provides conclusions on the work presented here within.
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Date Issued: 2013
Identifier: FSU_migr_etd-7737
Format: Thesis

Title: Challenges in Characterizing Membrane Proteins and Intrinsically Disordered Regions Involved in Mycobacterium Tuberculosis Cell Division.
Creator: Escobar Bravo, Cristian Andres, Cross, Timothy A., Jones, Kathryn M. (Kathryn Marjorie), Fajer, Peter G., Logan, Timothy M., Mattoussi, Hedi, Stagg, Scott M., Florida State...
Show moreEscobar Bravo, Cristian Andres, Cross, Timothy A., Jones, Kathryn M. (Kathryn Marjorie), Fajer, Peter G., Logan, Timothy M., Mattoussi, Hedi, Stagg, Scott M., Florida State University, College of Arts and Sciences, Institute of Molecular Biophysics
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Abstract/Description: Structural biology has been successful in the characterization of soluble protein, which has given insight in the inner working of several thousands of proteins. However, structural characterization of proteins has been limited in two areas. One area corresponds to the rise on interest on intrinsically disorder proteins. This kind of proteins has changed the structure-function paradigm since they can perform important cellular function without a defined three dimensional structure. The second...
Show moreStructural biology has been successful in the characterization of soluble protein, which has given insight in the inner working of several thousands of proteins. However, structural characterization of proteins has been limited in two areas. One area corresponds to the rise on interest on intrinsically disorder proteins. This kind of proteins has changed the structure-function paradigm since they can perform important cellular function without a defined three dimensional structure. The second are is the characterization of membrane proteins, which contrary to soluble proteins, they are embedded in an anisotropic environment that makes them difficult to characterize. This dissertation work was focused in the characterization of two important membrane proteins involved in cell division of Mycobacterium tuberculosis, ChiZ and FtsX. The first part of this work is related to the characterization of ChiZ, a cell division protein possibly involved in peptidoglycan remodeling and control of cell division progression. Structural characterization of ChiZ was mainly focused on the N-terminal soluble region that is intrinsically disordered. This region was previously identified to have peptidoglycan hydrolysis activity. Thus, a combination of solution and solid state NMR to characterize the structure of ChiZ N-terminal region in solution and in the full length protein reconstituted in liposomes. In addition to solution NMR, ChiZ N-terminal intrinsically disordered region was characterized with other method including circular dichroism, dynamic light scattering, size exclusion chromatography and small angle X-ray scattering. Thus, the objective was to obtain a clear picture of the behavior of the intrinsically disordered region in solution. Furthermore, characterization of ChiZ peptidoglycan hydrolysis activity was attempted with the goal of correlating structural properties of the N-terminal region and the protein function. The second part of this study focuses on the challenges of working with membrane proteins. The subject of study was FtsX, a membrane protein is one of the components of the cell division machinery or divisome. This part of the work attempts to give a general guideline for optimizing membrane protein purification and reconstitution with large emphasis on detergent selection. Detergent selection can affect each step of FtsX work, from protein purification to membrane protein reconstitution and sample preparation for solid state NMR experiments.
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Date Issued: 2017
Identifier: FSU_2017SP_EscobarBravo_fsu_0071E_13765
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: Characterization of Hepatitis C Virus Subgenomic Replicon Resistance to Cyclosporine in Vitro.
Creator: Robida, John, Tang, Hengli, Keller, Thomas C. S., Zhu, Fanxiu, Department of Biological Science, Florida State University
Abstract/Description: The current treatment for hepatitis C virus (HCV) consists of a combination therapy of alpha interferon (IFN-alpha) and ribivirin (RBV). Due to IFN resistance and side effects, new classes of drugs are needed to combat HCV infection. Cyclosporine A (CsA), an immunosuppressive and anti-inflammatory drug, has been shown to suppress HCV via a mechanism independent of the IFN pathway. In order to study the mechanism of CsA action on HCV, CsA resistant strains of HCV subgenomic replicon were...
Show moreThe current treatment for hepatitis C virus (HCV) consists of a combination therapy of alpha interferon (IFN-alpha) and ribivirin (RBV). Due to IFN resistance and side effects, new classes of drugs are needed to combat HCV infection. Cyclosporine A (CsA), an immunosuppressive and anti-inflammatory drug, has been shown to suppress HCV via a mechanism independent of the IFN pathway. In order to study the mechanism of CsA action on HCV, CsA resistant strains of HCV subgenomic replicon were selected and characterized. Here we report that different levels of resistance can be seen in different replicons and that different sets of mutations are associated with the different levels of resistance. Several different single cell clones with varying levels of CsA resistance contained mutations in the nonstructural protein 5B (NS5B), the HCV-encoded polymerase. When engineered into wildtype replicon these mutations were sufficient to confer a certain degree of resistance, but not to the original levels of selected replicons. Furthermore, these mutations, both individually and in groups, were able to rescue the lethal phenotype of a point mutation in NS5B (P540A) that has been previously implicated in the blockade of cyclophilins binding. These results demonstrate that CsA exerts selective pressure on the HCV genome despite being known to act on a cellular protein and identify a major target of CsA-mediated inhibition of HCV replication.
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Date Issued: 2009
Identifier: FSU_migr_etd-1814
Format: Thesis

Title: Characterization of Metal Linked Activation of the Iron-Dependent Regulator from Mycobacterium Tuberculosis.
Creator: Stapleton, Brian L., Logan, Timothy, Keller, Thomas C. S., Miller, Brian, Alabugin, Igor, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: The Iron-dependent regulator (IdeR) protein is a member of the diphtheria toxin repressor (DtxR) family of metal responsive proteins that regulates genes involved in iron homeostasis, oxidative stress response, and virulence in Mycobacterium tuberculosis. The roles of metals involved in activating IdeR are not completely understood. A better understanding of the metal binding process could possibly provide insight to a metal's role in virulence and other cell functions. The biological...
Show moreThe Iron-dependent regulator (IdeR) protein is a member of the diphtheria toxin repressor (DtxR) family of metal responsive proteins that regulates genes involved in iron homeostasis, oxidative stress response, and virulence in Mycobacterium tuberculosis. The roles of metals involved in activating IdeR are not completely understood. A better understanding of the metal binding process could possibly provide insight to a metal's role in virulence and other cell functions. The biological activating metal for IdeR is Fe(II), but several divalent metals including Ni(II), Co(II), Zn(II), Mn(II), and Cd(II) have been shown to activate the protein in vitro. The current model for the activation process involves IdeR selectively binding Fe(II) from cytosolic pools of free iron and becoming competent to bind DNA by structural organization and formation of a homodimer. The metal selection and dimerization processes are not well understood. Two drawbacks from previous work on this system are the use of in vivo assays which lack the details of metal binding and the absence of in vitro studies using Fe(II) as the metal ligand. This work provides a better understanding of the selection process of Fe(II) versus other metals as well as the dimerization effects of different metals. Thermodynamics of metal selection by Fe(II) and other metals was investigated using equilibrium dialysis. The results showed that Zn(II) and Fe(II) bound two equivalents of metal per monomer with each equivalent's affinity differing by 65-fold and 24-fold, respectively. Site mutant studies clearly showed Fe(II) bound to the ancillary site with high affinity while the Zn(II) results were less clear, but suggested that Zn(II) bound the primary site with high affinity. The equilibrium assays was modified to determine binding of more than one metal species. The mixed metal binding studies clearly showed that IdeR bound 1 equivalent each of Zn(II) and Fe(II). The fxbA promoter binding assay with 1 equivalent of Zn(II) and Fe(II) bound exhibited 30-fold tighter DNA binding than with Fe(II) alone. While the Fe(II) alone affinity is fairly weak, the enhanced affinity is in the range of most DNA binding regulators from other systems. Dimerization is important in the function of most DNA binding regulators. The enhancement of the dimerization of IdeR due to metal binding was studied using Analytical Ultracentrifugation. The sedimentation velocity analysis revealed a larger fraction of dimer present in IdeR samples when Zn(II) was bound versus Fe(II). The metal binding results of both Fe(II) and Zn(II) is novel to this family of repressor proteins which lead to questions about the role of each metal in IdeR. The binding processes of Fe(II) and Zn(II) were studied using Molecular Dynamic simulations. The free energies of Fe(II) and Zn(II) were computed using the CHARMM software and compared for relative differences in affinity at both sites between monomer and dimer configurations. The analysis of the trajectory data concluded that there are small subtle structural changes that are initiated by Zn(II) binding leading to dimerization. The information obtained in the studies shed new light on the metal binding selectivity of IdeR which was previously thought to only bind Fe(II). Zn(II) plays an important role in IdeR activation by enhancing DNA binding by stabilizing the dimer form.
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Date Issued: 2013
Identifier: FSU_migr_etd-8032
Format: Thesis

Title: Characterization of Polyelectrolyte Multilayers and Complexes: Towards Bio-Implant Applications.
Creator: Rmaile, Amir H., Schlenoff, Joseph B., Marshall, Alan, Striegel, André, Safron, Sanford, Alamo, Rufina, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: A self assembled multilayer technique based on alternating deposition of oppositely charged polyelectrolytes onto charged solid substrates is described in this thesis. The main principles and methodologies of this technique are explained in details in an effort to develop new technologies that would be beneficial for making new products or enhancing the quality of existing ones. In this thesis, fundamental studies to characterize the water content, swelling by different salts, annealing...
Show moreA self assembled multilayer technique based on alternating deposition of oppositely charged polyelectrolytes onto charged solid substrates is described in this thesis. The main principles and methodologies of this technique are explained in details in an effort to develop new technologies that would be beneficial for making new products or enhancing the quality of existing ones. In this thesis, fundamental studies to characterize the water content, swelling by different salts, annealing effect, viscoelastic and mechanical properties of polyelectrolyte multilayers and complexes are illustrated and conducted. Using the attenuated total reflectance Fourier Transform Infrared, ATR-FTIR, spectroscopy it is possible to probe the bulk of the PDADMA/PSS and P4VMP/PSS multilayers thus gaining valuable information about their water and counter-ion content. The multilayer resembles a polymeric network comprising regions of varying intensities of polyion-polyion interactions the extent and distribution of which are sensitive to the type of the polyelectrolyte used and to the type and concentration of the salt in the surrounding medium. A distinction is afforded in terms of the swelling and doping abilities of 14 different salts based on their hydration state. For both PDADMA/PSS and P4VMP/PSS multilayers, one can cautiously generalize that hydrophobic ions are more effective dopers than hydrophilic ions. Water content of those two multilayers was studied extensively using a range of different experiments conducted by ATR-FTIR technique. Osmotic pressure experiment is a good approach to characterize the water content inside a PEMU. Polyethylene glycol, PEG, a highly water-soluble polymer, was used as an osmotic stressing agent. Unlike salt solutions, PEG does not diffuse inside the PEMU from solution. It rather creates enough osmotic pressure to pull water out from the PEMU. A good estimate of the number of water molecules inside a multilayer was achieved using PEG. Ion-pairing and water content are key factors influencing permeability of molecules into polyelectrolyte multilayers. They are also important in controlling the multilayer mechanical properties which are especially important when considering the use of PEMUs in biomaterials applications. Layer by layer buildup of polyelectrolyte multilayers on a germanium crystal and ATR-FTIR measurements of ratios of water to sulfonate and azide to sulfonate peaks allowed us to monitor the annealing of PDADMA-PSS multilayers of different thicknesses in different ionic strength solutions. The film loses extrinsic sites as it anneals. PDADMA/PSS complexes were produced and their mechanical and thermal properties were characterized using different analytical methods. Dynamic mechanical analysis (DMA), thermogravimetric analysis (TGA), and mechanical stretching machines were used to conduct different experiments on those complexes. Swelling of the PDADMA/PSS complexes was investigated. We were able to get preliminary data about both the elastic modulus and the shear modulus of those complexes. Their water content, mechanical properties, thermal properties, and swelling behavior all encourage us to apply them in the biomedical field, in particular as bio-implants to replace intervertebral disc in the spinal cord. We are still investigating the different properties of those complexes and working on enhancing their structure and biocompatibility. We are synthesizing PAA-co-PAEDAPS and PDADMA-co-PAEDAPS for that purpose. The synthesis of those two polyzwetterions PZs is one of many steps taken towards producing biocompatible complexes that can be used as bio-implants.
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Date Issued: 2007
Identifier: FSU_migr_etd-1877
Format: Thesis

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

Title: Characterization of Soil Organic Phosphorus and Carbon by 31P and 13C Nuclear Magnetic Resonance Spectroscopy and Liquid Chromatography-Mass Spectrometry.
Creator: Imvittaya, Aopeau, Cooper, William T., Landing, William M., Dorsey, John G., Goldsby, Kenneth A., Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Phosphorus is an essential nutrient in ecosystems and exists in both inorganic and organic forms. The important role of organic phosphorus in the phosphorus cycle cannot be fully understood unless the quantitative abundances of individual organic phosphorus compounds in various ecosystems are better known. This study investigated the relationship between soil depth, soil carbon quality and types and amounts of soil phosphorus in three locations, including a subtropical treatment wetland in...
Show morePhosphorus is an essential nutrient in ecosystems and exists in both inorganic and organic forms. The important role of organic phosphorus in the phosphorus cycle cannot be fully understood unless the quantitative abundances of individual organic phosphorus compounds in various ecosystems are better known. This study investigated the relationship between soil depth, soil carbon quality and types and amounts of soil phosphorus in three locations, including a subtropical treatment wetland in the Florida Everglades, a boreal peatland in northern Minnesota, and an ombrotropic tropical wetland in northwest Panama. The three wetlands differ in vegetation, climate, and land use. Soil samples in the three wetlands were collected at three depths. Phosphorus compounds in these soil samples were extracted using the two-step extraction with ethylenediamine tetraacetic acid (EDTA) and sodium hydroxide solution. Solution 31P NMR spectroscopy was applied to quantitate phosphorus compounds in the extracts. Variations in amounts of inorganic phosphorus (ortho-, pyro-, and polyphosphates) and organic phosphorus (phosphonates, phosphate monoesters and phosphate diesters) at different soil depths in the three wetlands are presented. Hydrofluoric acid (HF) pretreatment was used to differentiate phosphorus chelated to metals or sorbed to anionic sorption sites from phosphorus incorporated into soil organic matter. In addition, solid-state 13C NMR spectroscopy was applied to distinguish soils sharing similar physical properties but differing in the quality of soil carbon. A humification index (HI) based on the ratio of O-alkyl to alkyl carbon was used to define soil organic matter diagenetic state (i.e. extent of decomposition). The combination of phosphorus speciation and carbon humification in these soils illustrates how microbial processing of organic matter and phosphorus turnover are linked in the wetlands. In addition, special attention was given to one particularly important form of organic phosphorus, inositol hexakisphosphate (IP6). Synthesized by plants and strongly complexed by metals, IP6 is the dominant form of identifiable organic phosphorus in most organic-enriched soils. However, most 31P NMR spectrometers cannot distinguish IP6 from other organic phosphate monoesters. Therefore, the method using liquid chromatography coupled with time-of-flight mass spectrometry (LC-MS) was developed to separate and quantify IP6 from other phosphorus compounds which appear in the same chemical shift region in a 31P NMR spectrum. Quantitative estimates of IP6 were used to better understand the role of IP6 in the phosphorus cycle in the Florida Everglades.
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Date Issued: 2014
Identifier: FSU_migr_etd-8812
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: Characterization of the Iron Dependent Regulator and Its Hyperactive Mutant from Mycobacterium Tuberculosis.
Creator: Walker, Lawrence, Logan, Timothy M., Blaber, Michael, Miller, Brian, Zhu, Lei, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: The Iron-dependent Regulator (IdeR) is a 230-amino acid transcriptional repressor that regulates iron homeostasis, oxidative stress response and virulence in Mycobacterium tuberculosis. IdeR binds to DNA as a dimer of dimers to repress gene expression and as four dimers to upregulate gene expression (upregulation is only for iron storage genes). IdeR contains two metal binding sites both of which have always been assumed to bind Fe(II) in vivo and it is the binding of metal by IdeR which...
Show moreThe Iron-dependent Regulator (IdeR) is a 230-amino acid transcriptional repressor that regulates iron homeostasis, oxidative stress response and virulence in Mycobacterium tuberculosis. IdeR binds to DNA as a dimer of dimers to repress gene expression and as four dimers to upregulate gene expression (upregulation is only for iron storage genes). IdeR contains two metal binding sites both of which have always been assumed to bind Fe(II) in vivo and it is the binding of metal by IdeR which induces the dimerization. IdeR is able to be activated for DNA binding in vitro by various divalent transition metals including: Mn(II), Fe(II), Co(II), Ni(II), Zn(II) and Cd(II). The model for activation of IdeR has always been investigated using divalent metals other than Fe(II) because of the rapid oxidation of Fe(II) to Fe(III). The objective of our work was to use Fe(II) to investigate the metal activation and DNA binding properties of IdeR. We also wanted to characterize a hyperactive mutant of IdeR (IdeR(D177K)) to better understand the cause of the hyperactive phenotype. To investigate the Fe(II) binding of IdeR we directly measured the Fe(II) bound using equilibrium dialysis and we investigated DNA binding using a fluorescence anisotropy assay. We determined that IdeR has the ability to bind Fe(II) into both metal binding sites, but that both sites bind the Fe(II) with affinities that differ by a factor of 25. We also determined that IdeR with only one Fe(II) bound per monomer was inactive and not able to bind to DNA. Through the use of IdeR metal binding site mutants we were able to determine that the first equivalent of Fe(II) bound to the ancillary metal site. This result was in contrast to all other papers published on IdeR, or its closest homologue (DtxR), that have shown that mutations to the primary metal binding site completely knock out repressor activity and mutations to the ancillary metal binding site only abrogate repressor activity. We determined that IdeR activation for promoter binding is enhanced when either Mn(II) or Zn(II) is present in solution before titrating in Fe(II), with Zn(II) having a much greater enhancement compared to Mn(II). We also determined that Zn(II) is able to bind at both metal binding sites in IdeR, but with KD values that differ by a factor of 70 and that the first equivalent of Fe(II) and the first equivalent of Zn(II) bind to different metal binding sites and the first equivalent of Zn(II) binding is ~36 picomolar. We determined that having Zn(II) present in solution before titrating in Fe(II) reduced the amount of Fe(II) required to activate promoter binding by 30 fold. These results suggest that IdeR is a mixed-metal repressor, where Zn(II) acts as a structural metal and Fe(II) acts to trigger the physiologically relevant promoter binding. We also set out to characterize the hyperactive repressor (IdeR(D177K)). We determined that this hyperactive mutant binds Fe(II) with a lower affinity that wild-type. The Fe(II) dependent promoter binding affinity is lower for IdeR(D177K) than wild-type IdeR. When Zn(II) is present we see that IdeR(D177K) also requires 30 fold less Fe(II) to activate promoter binding, but that IdeR(D177K) still requires a higher concentration of Fe(II) than wild-type IdeR. We also looked at the promoter binding affinity while keeping the metal concentrations constant and we saw that regardless of the metal species present (Fe(II), Zn(II), or Fe(II)-Zn(II)) IdeR(D177K) and wild-type IdeR had similar affinities for the promoter. IdeR(D177K) does not have a decrease sensitivity to changes in the free metal concentration once bound to the promoter as evidenced by our EDTA titration assay. The hyperactivity of IdeR(D177K) is due to it being bound to the promoter for a longer period of time compared to wild-type IdeR when both Zn(II) and Fe(II) are present.
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Date Issued: 2013
Identifier: FSU_migr_etd-8061
Format: Thesis

Title: Characterization of the Pro-Apoptotic Protein, Bad, and Its Protein Interactions.
Creator: Grimley, Alix, Miller, Brian, Lee, Choogon, Logan, Timothy, Zhu, Lei, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: BAD is a pro-apoptotic member of the Bcl-2 family of cell death regulators. The Bcl-2 family controls the permeability of the outer mitochondrial membrane via interactions between pro- and anti-apoptotic members of the family. Once the mitochondrion has been permeabilized, apoptotic factors are released into the cytoplasm. The cellular concentrations, locations and modifications of the family members determine which protein complexes are formed and, ultimately, whether a cell lives or dies....
Show moreBAD is a pro-apoptotic member of the Bcl-2 family of cell death regulators. The Bcl-2 family controls the permeability of the outer mitochondrial membrane via interactions between pro- and anti-apoptotic members of the family. Once the mitochondrion has been permeabilized, apoptotic factors are released into the cytoplasm. The cellular concentrations, locations and modifications of the family members determine which protein complexes are formed and, ultimately, whether a cell lives or dies. Human BAD is a 21 kDa BH3-only protein that functions to initiate the apoptotic cascade when the cell is stressed. In healthy cells, however, kinases phosphorylate BAD and allow for regulatory proteins to bind and sequester the death promoter. Under these conditions, BAD has long been thought to play little role in cellular regulation and metabolism. Recently, BAD was identified as part of a five-membered complex at the mitochondrial membrane of hepatic and pancreatic β-cells. Unlike previously detected BAD complexes, this contained no other Bcl-2 family members but instead included the body's glucose sensor, glucokinase. Glucokinase is a 50 kDa enzyme that phosphorylates glucose in the liver and pancreas. Unlike other hexokinases, glucokinase has a low affinity for glucose and a K0.5 value that lies in the physiological range of blood glucose concentration (4 - 10 mM), allowing the enzyme to be ultra sensitive to small changes in glucose levels. Glucokinase also shows kinetic cooperativity with its principal substrate, which is especially unusual given that glucokinase functions as a monomer. This cooperativity is thought to arise from slow conformational changes upon glucose binding. As a member of the mitochondrial complex, BAD has been shown to be crucial to full glucokinase activity in the cell. Although the complex has been detected at the mitochondria regardless of phosphorylation status, studies suggest that phosphorylation of one or more serine residues on BAD is required to confer the highest level of glucokinase activity. We hypothesize that BAD functions to stabilize glucokinase in a conformation(s) that increases its affinity for glucose at low glucose concentrations. BAD is a small protein that degrades quickly upon purification. Containing only a single notable domain, the Bcl-2 homology 3 (BH3) domain, BAD is predicted to be largely unstructured outside of this short helix. Purification of the protein has generally resulted in an impure and unstable protein that aggregates, and for this reason, most studies with the protein have focused on peptides containing the BH3 domain. Extensive rounds of purification and protocol optimization have yielded the first known samples of full length BAD in milligram quantities. This has allowed for extensive kinetic and thermodynamic studies on BAD and its protein partners, including glucokinase and the Bcl-2 family member Bcl-xL. Furthermore, phosphorylation of BAD at serine residues 75, 99 and 118 has resulted in a two-fold higher yield of monomeric protein, suggesting that phosphorylation stabilizes the protein. Secondary structure analysis from circular dichroism experiments confirms that the phosphorylated protein contains a higher proportion of secondary structure. Kinetic characterization of the BAD- glucokinase complex demonstrates that BAD increases glucokinase activity by ten percent at glucose concentrations below the K0.5 value. Phosphorylation, however, does not significantly increase this activation. This data provides the first biochemical evidence that BAD interacts with and affects glucokinase in vitro and that the native interaction could have important practical implications for diabetes drug targets.
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Date Issued: 2013
Identifier: FSU_migr_etd-8703
Format: Thesis

Title: Characterization of the Solvation Properties of Subcritical Water and Its Application as a Mobile Phase in Reversed-Phase Liquid Chromatography.
Creator: Alghoul, Zahra M., Dorsey, John G., Landing, William M., Roper, Michael G., Stiegman, Albert E., Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Reversed-phase liquid chromatography, RPLC, continues to be one of the most widely used techniques in the fields of separation and quantitative analysis of complex samples. With the advancement in column technology, faster and more efficient analysis have been attained. This led to an expanded use of the technique in fields such as proteomics, environmental studies, and pharmaceutical analysis. Along with the expanded use of RPLC, another field which is green chemistry has been developed and...
Show moreReversed-phase liquid chromatography, RPLC, continues to be one of the most widely used techniques in the fields of separation and quantitative analysis of complex samples. With the advancement in column technology, faster and more efficient analysis have been attained. This led to an expanded use of the technique in fields such as proteomics, environmental studies, and pharmaceutical analysis. Along with the expanded use of RPLC, another field which is green chemistry has been developed and which is focused on finding more environmentally friendly solvents or techniques than the one used in laboratories. Water is one of the most likable green solvents, due its non-toxic nature. Because of its high polarity, water is classified as a weak mobile phase in RPLC and is usually mixed with varying percentages of organic solvents such as acetonitrile and methanol to obtain a less polar mobile phase. In order to keep the percentage of organic modifier minimum, temperature has been explored as a means for decreasing the polarity of the mobile phase. Temperature has been explored as an experimental variable in RPLC since it has a direct effect on the mechanism and thermodynamic properties of a retention mechanism. Until few years ago, the temperature range explored was limited to a narrow range below boiling point of water. Experimental conditions such as temperature and pressure can be used to tune the properties of subcritical water, making it less polar. The effect of temperature and pressure on the solvation properties of subcritical water are explored using solvatochromic methods. The dipolarity/polarizability of water is found to be relatively constant over the temperature range studied. The hydrogen-bond donating ability of water decreases with temperature whereas its hydrogen-bond accepting ability shows a slight increase under the same conditions. The effect of temperature on the solvation properties of subcritical water are more significant than pressure, in contrast to what is usually observed in supercritical water. This effect is mainly due to an alteration in the hydrogen-bond network that makes up the structure of water. Compared to some of the organic solvents and hydro-organic solvent mixtures, subcritical water is found to have its own set of properties and does not resemble a particular solvent or mixture. Temperature is used as a variable for controlling the properties of subcritical water as a mobile phase. A study of the retention process through van Deemter analysis is performed. The effect of temperature is compared to the effect of percentage of organic modifier on the column efficiency of a separation. Higher flow rates can be used in subcritical water chromatography without the loss of efficiency, commonly encountered with the more viscous hydro-organic solvents used in conventional mobile phases.
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Date Issued: 2012
Identifier: FSU_migr_etd-6879
Format: Thesis

Title: Chromatographic Silanol Activity Tests: The Development of a Comprehensive Test Procedure.
Creator: Rogers, Sydana D., Dorsey, John G., McDuffie, Ernest L., Vickers, Thomas J., Goldsby, Kenneth A., Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: There is great interest in the analysis of stationary phases used in eversed-phase liquid chromatography, with a strong emphasis on the evaluation of residual silanols. A number of techniques have been developed in order to assess the level of silanol activity in reversed-phased columns; however, to date there is no universally accepted test procedure. The overall goal of this work was to develop such a test. The invention of a universally accepted test would allow for a classification system...
Show moreThere is great interest in the analysis of stationary phases used in eversed-phase liquid chromatography, with a strong emphasis on the evaluation of residual silanols. A number of techniques have been developed in order to assess the level of silanol activity in reversed-phased columns; however, to date there is no universally accepted test procedure. The overall goal of this work was to develop such a test. The invention of a universally accepted test would allow for a classification system for reversed-phase columns that would enable researchers to select columns more appropriately. The project consisted of two phases. In phase I of this study, several different silanol activity tests were compared. Test procedures were performed on a large collection of columns, including some of the most popular C18 phases used for the analysis of basic solutes. Column rankings, ranging from a low silanol activity to a high silanol activity, were determined according to the specifications of each test. Column rankings varied significantly among tests, suggesting that the current chromatographic silanol activity tests are not all testing for the same property. A number of unresolved issues from the literature were also addressed in this study. It was revealed that the older test procedures were inept at distinguishing between newer generation phases. Newer test procedures were shown to be more in agreement with each other than older test procedures. Test procedures were examined as candidates for a comprehensive procedure; however, none of the current tests were found to include all of the necessary components. Results indicated the need for a new approach in the development of a universal test procedure. In Phase II of this study, an attempt has been made to develop a universally accepted chromatographic silanol activity test procedure. This procedure analyzed all of the major interactions which comprise the term 'silanol activity', namely hydrogen bonding and ionic interactions. Aromatic compounds with nitrogen groups were selected as test solutes. These compounds had pKa values between 4 and 11. The new procedure took into account the pKa shifts that occur upon the addition of organic modifier. This test analyzed the basic compounds at mobile phase pH values ranging from 2.5 to 7.5 by utilizing a single mobile phase system which can be used in a pH gradient. This approach allowed for the analysis of the columns performance at all possible pH ranges for nondestructive use of the column. Results from the isocratic portion of this procedure illustrated the ability of the procedure to predict silanol activity at different pH levels when compared to results from everyday analyses. This work further demonstrated the need for testing columns over their entire useful pH range. An attempt was made to shorten the procedure by using the mobile phase system to create a pH gradient. Although analysis times were decreased greatly, the ability of the test to predict a column's performance according to silanol activity was also diminished. Overall, gradient experiments provided solid foundational investigations into a radically different approach to silanol activity assessment.
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Date Issued: 2003
Identifier: FSU_migr_etd-1844
Format: Thesis

Title: Cloning and Expression of Notch ELRs 10-13 and Delta EBD.
Creator: Kitchen, Jacqueline R., Logan, Timothy, Greenbaum, Nancy, Dorsey, John, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Notch signaling plays an important role in tissue differentiation and remodeling and has been extensively studied. Much has been learned about the Notch signaling pathway and consequences of defects within the path, yet little is understood about the structure and biochemistry of the Notch receptor/Notch ligand interaction responsible for initiating the signaling events. Our goal is to investigate the biophysical interactions of Notch with its ligands Delta and Serrate. Here we describe the...
Show moreNotch signaling plays an important role in tissue differentiation and remodeling and has been extensively studied. Much has been learned about the Notch signaling pathway and consequences of defects within the path, yet little is understood about the structure and biochemistry of the Notch receptor/Notch ligand interaction responsible for initiating the signaling events. Our goal is to investigate the biophysical interactions of Notch with its ligands Delta and Serrate. Here we describe the cloning and expression of Drosophila Notch ELRs 10-13 and Delta EBD, as well as initial isolation schemes for a Delta EBD-His construct.
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Date Issued: 2006
Identifier: FSU_migr_etd-2825
Format: Thesis

Title: Coatings from Polyelectrolytes: Fundamentals of Buildup and Control over Mechanical Properties for Bioapplications.
Creator: Lehaf, Ali, Schlenoff, Joseph B., Ma, Teng, Alabugin, Igor, Roper, Mike, Marshall, Alan, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Controlling cell fate via mechanical properties of the culture substrates provides an important tool for biomedical applications. Thin films of polyelectrolyte multilayers (PEMUs) have been used as substrates for cell culture due to their biocompatibility. Photocrosslinkable polyelectrolyte multilayers were prepared from poly(acrylic acid) grafted with photosensitive benzophenone (PAABp) as the polyanion and poly(allylamine hydrochloride) (PAH) as the polycation. Young's modulus measured by...
Show moreControlling cell fate via mechanical properties of the culture substrates provides an important tool for biomedical applications. Thin films of polyelectrolyte multilayers (PEMUs) have been used as substrates for cell culture due to their biocompatibility. Photocrosslinkable polyelectrolyte multilayers were prepared from poly(acrylic acid) grafted with photosensitive benzophenone (PAABp) as the polyanion and poly(allylamine hydrochloride) (PAH) as the polycation. Young's modulus measured by force spectroscopy using nano-indentation showed smooth controlled increase after irradiation with UV light. The permeability of PEMUs for iodide ions, measured with a rotating disc electrode, decreased significantly. The surface wettability and charge density were not affected by irradiation, suggesting that surface chemistry and charge remained essentially unaltered. This provides substrate material for cell culture applications where the only variable is the mechanical stiffness. Since photocrosslinking provided control over the region of crosslinking, photomasks were used to prepare substrates with gradient elasticity. The behavior of rat aortic smooth muscle cells (A7r5) and osteosarcoma (U2OS) was followed on uniform substrates of increasing stiffness and on substrates with gradient elasticity. A7r5 cells detected the elasticity gradient and those that were on the soft side could polarize and orient towards the stiff side, where they showed better adhesion. U2OS cells also showed preference to the stiff side; however U2OS cells that were on the soft side did not adhere and underwent apoptosis. Nano-indentation was used to study some important fundamental properties of polyelectrolyte multilayer from poly(diallyldimethylammounium chloride) PDADMAC as the polycation and sodium poly(styrene sulfonate) (NaPSS) as the polyanion, which allowed proposing mechanism for PEMU build-up. The degree of swelling varied depending on the type of polyelectrolyte on the surface, with PDADMA-ending films more swollen than PSS ending films. This observation correlated well with the change in modulus. This suggested that PDADMA-ending films are extrinsically compensated while PSS-ending films are more intrinsically compensated. Also using nano-indentation, we demonstrated that the surface features and roughness of polyelectrolyte multilayers were not due to phase separation. Using nano-indentation, we demonstrated the role of water in controlling the mechanical properties of PEMUs. Previously, it was shown that salt plasticizes PEMUs and they were termed "saloplastics". A complimentary study was done to investigate the plasticizing effect of water. We used osmotic stress to control the amount of water in the films without affecting the degree of ionic crosslinks. Poly(ethylene glycol) (PEG) was used as the osmotic stressor. We showed that water increases free volume between the polyelectrolyte chains and acted as lubricant and the elastic modulus increases with decreasing water content. Water was also found to contribute to the viscoelastic properties of the polyelectrolyte multilayer, suggesting that water acts in plasticizing polyelectrolyte multilayers.
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Date Issued: 2012
Identifier: FSU_migr_etd-6954
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: A Completed KLK Activome Profile: Investigation of Activation Profiles of KLK9, 10, and 15..
Creator: Yoon, Hyesook, Blaber, Sachiko, Debela, Mekdes, Goettig, Peter, Scarisbrick, Isobel, Blaber, Michael
Abstract/Description: We previously reported the activation profiles of the human kallikrein-related peptidases (KLKs) as determined from a KLK pro-peptide fusion-protein system. That report described the activity profiles of 12 of the 15 mature KLKs versus the 15 different pro-KLK sequences. The missing profiles in the prior report, involving KLK9, 10, and 15, are now described. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, mass spectrometry, and N-terminal sequence analyses show that KLK9 and 10...
Show moreWe previously reported the activation profiles of the human kallikrein-related peptidases (KLKs) as determined from a KLK pro-peptide fusion-protein system. That report described the activity profiles of 12 of the 15 mature KLKs versus the 15 different pro-KLK sequences. The missing profiles in the prior report, involving KLK9, 10, and 15, are now described. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, mass spectrometry, and N-terminal sequence analyses show that KLK9 and 10 exhibit low hydrolytic activities towards all of the 15 pro-KLK sequences, while KLK15 exhibits significant activity towards both Arg- and Lys-containing KLK pro-sequences. The ability of KLK15 to activate pro-KLK8, 12, and 14 is confirmed using recombinant pro-KLK proteins, and shown to be significant for activation of pro-KLK8 and 14, but not 12. These additional data for KLK9, 10, and 15 now permit a completed KLK activome profile, using a KLK pro-peptide fusion-protein system, to be described. The results suggest that KLK15, once activated, can potentially feed back into additional pro-KLK activation pathways. Conversely, KLK9 and 10, once activated, are unlikely to participate in further pro-KLK activation pathways, although similar to KLK1 they may activate other bioactive peptides.
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Date Issued: 2009
Identifier: FSU_migr_biomed_faculty_publications-0012
Format: Citation

Title: Complex Mixture Analysis by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: Applications for the Fuel Industry.
Creator: Jarvis, Jacqueline Marie, Marshall, Alan G., Chanton, Jeffery P., Dorsey, John G., Cooper, William T., Dalal, Naresh S., Rodgers, Ryan P., Department of Chemistry and...
Show moreJarvis, Jacqueline Marie, Marshall, Alan G., Chanton, Jeffery P., Dorsey, John G., Cooper, William T., Dalal, Naresh S., Rodgers, Ryan P., Department of Chemistry and Biochemistry, Florida State University
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Abstract/Description: As the world's reserves of light crude oil are depleted, the fuel industry will have to find other sources to generate transportation fuels. There are known supplies of unconventional crude oil (bitumen) in Canada, but it is more difficult and costly to generate fuels from bitumen. In addition, bitumen is still a fossil fuel, which means that the supply is finite. Ultimately, development of an alternative fuel from renewable resources, such as biofuel, would be the best option. Development of...
Show moreAs the world's reserves of light crude oil are depleted, the fuel industry will have to find other sources to generate transportation fuels. There are known supplies of unconventional crude oil (bitumen) in Canada, but it is more difficult and costly to generate fuels from bitumen. In addition, bitumen is still a fossil fuel, which means that the supply is finite. Ultimately, development of an alternative fuel from renewable resources, such as biofuel, would be the best option. Development of a cost efficient biofuel would lessen the demand for fossil fuels and benefit the environment at the same time. However, at this time, it is still more cost-effective to produce fuels from unconventional crude oils than biofuels. Petroleomics has utilized Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to successfully link the chemical composition of conventional petroleum crude oil to the behavior of that feed during production and processing. However, the chemical composition of unconventional crude oils and biofuels is still relatively unknown due to their complex nature and more recent usage compared to light crude oils. The ultrahigh resolving power and mass accuracy of FT-ICR MS can be used to determine the chemical composition of extremely complex unconventional crude oil and relatively unknown biofuels. From the insight gained by FT-ICR MS, predictions on the best sources for future fuels can be made. Chapter 1 presents the basics about petroleum needed to understand petroleomics, including classification, terminology, and composition. Some of the problems associated with the use of bitumen are also described. This information is presented before the basics of biofuels (Chapter 2), especially bio-oil, to gain an understanding of how and why bio-oils were analyzed as they are. The techniques (FT-ICR MS and ionization methods) used to analyze complex mixtures, specifically bio-oil and petroleum interfacial material, are described in Chapter 3. The first analysis of bio-oil by FT-ICR mass spectrometry is presented in Chapter 4. Here, the chemical composition of the oily and aqueous phases of the bio-oil generated from the slow pyrolysis of pine pellet and peanut hulls is determined. Chapter 5 presents an analysis of bio-oils generated from different source material under different pyrolysis conditions than the study in Chapter 3. The chemical composition of the final product (bio-oil) depends of the source material and pyrolysis conditions. Boron-containing compounds were also discovered for the first time in bio-oils by FT-ICR MS. Bio-oils are too polar in their raw form to be used as a co-feed alongside petroleum in refineries without reducing their oxygen content. Chapter 6 studies the changes that occur to a raw bio-oil as it is upgraded over zeolite catalysts to promote deoxygenation. To gain more understanding of the oxygenated species present within bio-oils, a fractionation technique was applied to bio-oil samples to generate fractions of increasing polarity (Chapter 7). Some of the most dominate peaks present in FT-ICR mass spectra were correlated to possible structures from compounds that had previously been identified by GC-MS. Chapter 8 takes the information gained from analysis of highly oxygenated species (bio-oils) and applies this knowledge to the analysis of petroleum emulsions. The species thought to exist at the oil/water interface have higher oxygen content than the species typically identified in whole petroleum crude oils. A new method for isolating interfacial material from petroleum crude oil is described and validated in this chapter.
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Date Issued: 2013
Identifier: FSU_migr_etd-7853
Format: Thesis

Title: Composition Control and Localization of S²⁻ in CdSSe Quantum Dots Grown from Li₄[Cd₁₀Se₄(SPh)₁₆].
Creator: Oyler, Ryan Edward, Strouse, Geoffrey F., Safron, Sanford A., Steinbock, Oliver, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Reproduced with permission from Journal of the American Chemical Society, submitted for publication. Unpublished work copyright 2008 American Chemical Society. The development of ternary nanoscale materials with controlled cross-sectional doping is an important step in the use of chemically prepared quantum dots for nanoscale engineering applications. We report cross-sectional, elemental doping for the formation of an alloyed CdSSe nanocrystal. The nanocrystal is prepared from the thermal...
Show moreReproduced with permission from Journal of the American Chemical Society, submitted for publication. Unpublished work copyright 2008 American Chemical Society. The development of ternary nanoscale materials with controlled cross-sectional doping is an important step in the use of chemically prepared quantum dots for nanoscale engineering applications. We report cross-sectional, elemental doping for the formation of an alloyed CdSSe nanocrystal. The nanocrystal is prepared from the thermal decomposition of Li4[Cd10Se4(SPh)16]. The sulfur incorporation arises from a surface mediated degradation of a [Cd(SPh)4]2- tetrahedral passivant tightly bound to the growing quantum dot surface. In the alloy, we identify a pure CdSe nucleus of ~ 1.5 nm consistent with the predictions of nucleation theory. As the particle grows, S2- incorporation increases until ~3.5 nm, at which point an equilibrium of the S2-/Se2- incorporation rate is attained. The use of molecular clusters to allow controlled defect ion incorporation can open new pathways to more complex nanomaterials.
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Date Issued: 2008
Identifier: FSU_migr_etd-2437
Format: Thesis

Title: A Comprehensive Assessment of the Effects of Ozone and Uv Radiation on the Molecular Weights and Optical Properties of Natural Water and Wastewater Effluent Dissolved Organic Matter.
Creator: Paul, Allison L. (Allison Lee), Cooper, William T., Huettel, Markus, Watts, Michael, Dorsey, John G., Dudley, Gregory B., Department of Chemistry and Biochemistry, Florida State...
Show morePaul, Allison L. (Allison Lee), Cooper, William T., Huettel, Markus, Watts, Michael, Dorsey, John G., Dudley, Gregory B., Department of Chemistry and Biochemistry, Florida State University
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Abstract/Description: The work presented concentrations on better understanding the physical and chemical properties of dissolved organic matter (DOM) in two distinctly different aquatic environments. More specifically, examining the degradation of DOM before and after oxidation treatments with ozone, ultra violet radiation (UVR), and combination of the two. DOM consists of low and high molecular weight species such as humic substances, hydrophilic acids, carboxylic acids, amino acids, carbohydrates, and...
Show moreThe work presented concentrations on better understanding the physical and chemical properties of dissolved organic matter (DOM) in two distinctly different aquatic environments. More specifically, examining the degradation of DOM before and after oxidation treatments with ozone, ultra violet radiation (UVR), and combination of the two. DOM consists of low and high molecular weight species such as humic substances, hydrophilic acids, carboxylic acids, amino acids, carbohydrates, and hydrocarbons [1]. DOM is a component of the carbon cycle, serves as a nutrient source, impacts treatment processes, and plays an important role in the transportation of aquatic contaminates. However, the exact structure of DOM is still not fully understood because of its complex nature and origin. Chapter 1 is a brief introduction to DOM, including its role in natural and wastewater systems. This chapter also discusses the different types of oxidation processes and the interactions of oxidation with DOM. Chapter 2 is a brief introduction to the four analytical techniques that will be used in the analysis of natural water DOM (NDOM) and wastewater effluent DOM (efDOM). The four analytical techniques include: size exclusion chromatography (SEC), time-of-flight mass spectrometry (TOF MS), ultra-violet visible (UV-Vis) absorption spectroscopy, and excitation-emission matrix fluorescence spectroscopy (EEMS). Chapter 3 is understanding DOM in a natural water system. DOM is a key component in freshwater ecosystems, and strongly influences the optical, chemical, and biological environment. Therefore, it becomes important to understand the nature of DOM within this system. Many natural water systems are treated for drinking water purposes at water treatment facilities with the use of chlorine. The formation of hazardous disinfection by-products (DBPs) from the interaction of chlorine with DOM has lead to finding alternative methods for disinfection. The use of ozone, UVR, and the combination of these two will be examined on the degradation and removed of DOM in a natural water system. Chapter 4 is understanding the degradation DOM in a wastewater system after oxidation treatment. Natural water systems are known to be more of terrestrial origin, whereas wastewater is of microbally-derived origin. Therefore, it is important to understand the effects of oxidants in terrestrially-derived and microbally-derived systems. Chapter 5 aims at comparing NDOM and efDOM before and after advanced oxidation treatment (AOP). AOP is the combination of ozone and UVR. More specifically, this research examines two types of AOP treatments: (1) ozone and UVR from an artificial radiation source and (2) ozone and UVR from a natural radiation source. The will be determined which type of AOP is best suited for the degradation and removal of DOM in both systems and to determine if AOP has the same or different effect of DOM of two distinctly different systems. Four analytical techniques will be used in combination in order to better understand DOM characteristics for the studies performed in chapters 3, 4, and 5. Multimethod analysis will be used to develop a broad view of the DOM characteristics and will aid in revealing similarities and differences in NDOM and efDOM before and after advanced oxidation treatments. These four techniques include: (1) size exclusion chromatography (SEC) to qualitatively and quantitatively understand the molecular weight distribution of DOM, (2) time-of-flight mass spectrometry (TOF MS) to separate DOM based on the mass-to-charge ratio, (3) ultraviolet-visable (UV-Vis) absorption spectroscopy to understand the chromophoric character of DOM, and (4) exitation-emission matrix fluorescence spectroscopy to understand the fluorophoric origin of DOM. Chapter 6 is understanding the effects of microalgae bioremediation on wastewater DOM. Microalgae serve a dual role: they are environmentally-friendly alternatives to disinfection/oxidation of wastewater and produce biomass that can be used as biofuels and feeds. The most suitable conditions for maximum microalgae growth, and therefore maximum biomass and feed production is still largely unknown because microalgae growth depends on factors such as pH and temperature, concentration of essential nutrients, including nitrogen, phosphorus, and organic carbon, availability of light, oxygen, and carbon dioxide. Therefore, it is important to determine where in the treatment process would be suitable for maximum algae growth and greatest degradation of DOM. Two analytical techniques will be used collectively to better understand the interactions of microalgae and DOM: size exclusion chromatography and excitation emission matrix fluorescence spectroscopy.
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Date Issued: 2012
Identifier: FSU_migr_etd-5099
Format: Thesis

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

Title: Computational Simulations of Vortex Waves Interacting with Heterogeneities: from Chemical Media to Cardiac Tissues.
Creator: Zhang, Zhihui, Steinbock, Oliver, Rikvold, Per Arne, Bertram, R. (Richard), Yang, Wei, Knappenberger, Kenneth L., Florida State University, College of Arts and Sciences, Program...
Show moreZhang, Zhihui, Steinbock, Oliver, Rikvold, Per Arne, Bertram, R. (Richard), Yang, Wei, Knappenberger, Kenneth L., Florida State University, College of Arts and Sciences, Program in Molecular Biophysics
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Abstract/Description: Vortex waves are striking spatial-temporal patterns and observed in diverse systems, such as active galaxies, catalytic reactions, and even biological systems. In the heart, vortex patterns of electrical waves are linked to ventricular arrhythmia. The fast rotating vortices, once created, repetitively excite the cardiac tissues and act as a pacemaker with a high frequency. The resulting heartbeat is much faster than the normal rhythm and termed ventricular tachycardia (VT). If the single...
Show moreVortex waves are striking spatial-temporal patterns and observed in diverse systems, such as active galaxies, catalytic reactions, and even biological systems. In the heart, vortex patterns of electrical waves are linked to ventricular arrhythmia. The fast rotating vortices, once created, repetitively excite the cardiac tissues and act as a pacemaker with a high frequency. The resulting heartbeat is much faster than the normal rhythm and termed ventricular tachycardia (VT). If the single vortex is unstable and fragmented, it leads to an often fatal ventricular fibrillation (VF). In this case, the heart does not pump but quivers asynchronously and causes sudden cardiac death, which accounts for 325,000 deaths in the United States each year. Most excitable media, particularly biological systems, are not homogeneous. For example, in the heart, the heterogeneity can refer to dead tissues, collagen fibers, or blood vessels. Indeed, the inhomogeneity of the media plays a critical role in the behavior of the vortex wave. Many experimental results have shown that the presence of the uncoupling substrates changes the activation pattern of excitation waves. In both chemical media and cardiac tissues, these changes result in abnormal conductance locally and a rise in the susceptibility to vortices. On the contrary, an unexcitable or impermeable heterogeneity can anchor the vortex wave. The anchored nonlinear waves are stabilized and even suppress the turbulence in their surrounding space. To date, the complex relationship between the uncoupling/insulating heterogeneity and the vortex wave has not been elucidated and clearly requires more in-depth studies. A major driving force is computer simulations that have greatly contributed to a deeper understanding of observations and continue to guide the design of new experiments. With even greater technological advances in the future, computer simulations will be indispensable tools to interpret dynamics of excitable media. In this thesis, I described computer simulations of both chemical and cardiac models in two- as well as three-dimensional spaces to study the dynamics of the vortex waves in presence of heterogeneities. For chemical media, my studies focus on the interaction between the vortex wave and a moving inert heterogeneity. For two-dimensional systems, I designed simulations to investigate the impact of a moving disk with varying velocities on a rigidly rotating wave. Many other factors that may contribute to the dynamics were also discussed, such as the phase of the spiral wave and the radius of the disk. Additionally, I integrated local fluid dynamics into the model to accord with the realistic chemical solution. The results suggested that the Stokes flow generated by the moving disk can be a very weak perturbation to the wave pattern. In three-dimensional simulations, I further explored the dynamics of a scroll wave that was partially pinned to the cylindrical heterogeneity. The scroll wave was thus stretched and deformed. Moreover, for systems with step-like heterogeneities, the simulations predicted that over short distances scroll waves are attracted towards the step and then rapidly commence a steady drift along the step line. For my studies of cardiac systems, I primarily modeled a static heterogeneity in a slab of cardiac tissue with a single reentrant wave or turbulence. Important properties of the heterogeneity as well as biological features of the cardiac fibers were considered in my research. For example, I locally changed the conductions within the cylindrical heterogeneity, which represents the tissues damaged by gap junctional uncoupling in the real heart. In addition, the role of an insulating heterogeneity in systems with scroll wave turbulence was also investigated. By anchoring to a thin cylinder, the stabilized wave rotates sufficiently fast to repel the free segments of the turbulent filament tangle, which then annihilate at the system boundaries. Furthermore, the results show that even thicker cylinders can suppress analogous forms of tachycardia by forming pinned multi-armed vortices. In this process, the observed number of wave arms depends on the cylinder radius.
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Date Issued: 2016
Identifier: FSU_FA2016_Zhang_fsu_0071E_13604
Format: Thesis

Title: Controlling Surface Energy in Polyelectrolyte Multilayers.
Creator: Jisr, Rana M., Schlenoff, Joseph B., Xiong, Peng, Dorsey, John G., Cooper, William T., Logan, Timothy M., Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: The performance of a material is often dictated by surface and interfacial properties such as wettability, friction and adhesion. The ability to modify the wetting properties of surfaces by using self assembled polyelectrolyte multilayers, PEMUs, provides a straightforward mechanism for controlling the surface chemistry and therefore the properties of the modified surface for a variety of applications such as self cleaning surfaces, biosensing and separation. Wettability of surfaces is...
Show moreThe performance of a material is often dictated by surface and interfacial properties such as wettability, friction and adhesion. The ability to modify the wetting properties of surfaces by using self assembled polyelectrolyte multilayers, PEMUs, provides a straightforward mechanism for controlling the surface chemistry and therefore the properties of the modified surface for a variety of applications such as self cleaning surfaces, biosensing and separation. Wettability of surfaces is directly related to the surface energy. Among the various methods used for surface modification, PEMUs assembled by the layer-by-layer technique are gaining considerable attention due to the ease with which these multilayer coatings can be constructed. PEMUs are produced by sequential adsorption of oppositely charged polyelectrolytes where control over the film thickness at the molecular level is achieved. Surface properties ranging from hydrophobic to hydrophilic and smooth to rough can be controlled by varying the molecular weight of the polyelectrolyte and the pH, temperature and ionic strength used for multilayer fabrication. The overall objective of this dissertation is to control the wetting behavior of surfaces through using polyelectrolyte multilayer thin films assembled with novel fluorinated and fluorinated/zwitterionic copolyelectrolytes. The synthesis of these polyelectrolytes was discussed and wettability of the multilayer surfaces was studied using contact angle measurements. Also the flux of charged probe molecules, ferricyanide and potassium iodide, through the fluorinated multilayers was assessed with rotating disk electrode voltammetry. Moreover, the hydrophobic properties of the fluorinated surfaces were enhanced by increasing the surface roughness through the use of attapulgite clay nanorods. This study allowed us to mimic the "Lotus leaf" and produce ultrahydrophobic surfaces with very high contact angle and low contact angle hysteresis. The thermal stability of these surfaces was also tested. In another study, a series of fluorinated/zwitterionic polyelectrolytes with different charge densities were synthesized and used to generate arrays of patterns with different wetting properties on polyelectrolyte surfaces using microcontact printing technique. Friction measurements for these surfaces were assessed with lateral force microscopy. A simple method to modify the surface of polydimethylsiloxane, PDMS, to make it more wettable to trifuoroethanol solvent, TFE, is also discussed. The effect of hydrophobicity on cell adhesion, morphology and motility was examined in this work. Moreover, Functionalization of the surface of single wall carbon nanotube buckypaper, SWNT-buckypaper, by introducing carboxyl and amine functional groups through the use of PEMUs to enhance the binding between the SWNT and the resin matrix is demonstrated. The efficiency of the interfacial bonding between the multilayer and the resin was monitored by FTIR, TGA and SEM.
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Date Issued: 2007
Identifier: FSU_migr_etd-4653
Format: Thesis

Title: Controlling the Nanoscopic and Macroscopic Properties of Inorganic Tubes: Towards Advanced Applications in Materials Science.
Creator: Makki, Rabih Mohammad, Steinbock, Oliver, Trombley, Paul, Dalal, Naresh, Latturner, Susan, Department of Chemistry and Biochemistry, Florida State University
Abstract/Description: Materials synthesis far from thermodynamic equilibrium can yield hierarchical order that spans from molecular to macroscopic length scales. Here we study the formation of microscopic and macroscopic inorganic tubes during spatially controlled reaction processes. The underlying chemistry involves the precipitation of amorphous silica and metal hydroxides during the mixing of silicate solutions and metal ions with insoluble hydroxides. The conventional method involves the addition of a metal...
Show moreMaterials synthesis far from thermodynamic equilibrium can yield hierarchical order that spans from molecular to macroscopic length scales. Here we study the formation of microscopic and macroscopic inorganic tubes during spatially controlled reaction processes. The underlying chemistry involves the precipitation of amorphous silica and metal hydroxides during the mixing of silicate solutions and metal ions with insoluble hydroxides. The conventional method involves the addition of a metal salt crystal into an aqueous silicate solution. However the random shape and size as well as the continuous, and hence transient, dissolution of the crystal hamper the quantitative and systematic analysis of the system, and limit the potential applications of the obtained structures. To overcome these problems, we employ small (micro-scale) polymer beads. These beads are produced by emulsification techniques and then loaded with metal salt by simple equilibration in aqueous solution. Once exposed to the sodium silicate solution, a thin colloidal membrane starts to grow around the entire bead from which microtubes emerge. For the copper sulfate-silicate system, tube formation is only observed above a critical bead radius that is inversely proportional to the initial loading concentration. The formed tubes have inner radii down to 3 ïm, reach lengths of 0.5 mm, and grow at speeds of up to 50 ïm sï1. Moreover, tubes pinned to air bubbles can induce directional bead motion. We also describe a new approach that establishes control over the growth velocities of macroscopic silica-metal oxide/hydroxide tubes. Our approach is demonstrated for the injection of acidic cupric sulfate solution into a large volume of basic sodium silicate solution. The forming tube is pinned to a gas bubble that is held at the end of a hollow glass rod. The tube's linear growth follows the speed of the glass rod while its radius is self-selected according to volume conservation of the injected solution. Depending on the experimental conditions, tube growth occurs at either the moving gas bubble or the stationary glass capillary. Oscillatory modulations of the growth velocity provoke the formation of hollow nodules on the outer tube surface. These nodules form after each rapid velocity decrease at exponentially decaying rates and seem to be energetically favored over a sudden isotropic increase in tube radius. We also demonstrate a simple and facile method for the production of straight copper(oxide)-silica tubes. The tubes are produced for different oxidation states of copper (i.e., Cu, CuO, and Cu2O). For these experiments, copper hydroxide-silica tubes are first prepared by injecting cupric sulfate solution into sodium silicate solution and pinning a growing tube to a "free" rising air bubble. The obtained tubes are then heated to form copper(II), or copper(I), oxide-silica tubes. Finally, copper-silica tubes are produced by adding a diluted sulfuric acid solution over the copper(I) oxide-silica tubes. Thermogravimetric analyses confirm the heat-induced decomposition of copper hydroxide within the original tubes while X-ray diffraction certifies the composition of the modified tubes. Lastly, the velocity-controlled approach is applied to the synthesis of iron oxide-silica tubes. Transmission electron microscopy and X-ray diffraction, as well as Raman and MÃ¶ssbauer spectroscopy reveal magnetite nanoparticles in the range of 5−15 nm. Optical data suggest that the magnetite particles follow first-order nucleation−growth kinetics. The hollow tubes exhibit superparamagnetic behavior at room temperature, with a transition to a blocked state at TB = 95 K for an applied field of 200 Oe. Heat capacity measurements yield evidence for the Verwey transition at 20 K. Finally, we show a remarkable dependence of the tubes' magnetic properties on the speed of the pinning rod and the injection rate employed during synthesis
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Date Issued: 2012
Identifier: FSU_migr_etd-6963
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

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