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- Title
- DEVELOPMENT OF A FLUORESCENT DRUG SCREENING PLATFORM FOR INHIBITORS OF MYCOBACTERIUM TUBERCULOSIS PROTEIN-PROTEIN INTERACTIONS.
- Creator
-
Versfeld, Zina, Rohde, Kyle, University of Central Florida
- Abstract / Description
-
Tuberculosis (TB) is a respiratory disease caused by Mycobacterium tuberculosis (Mtb) that kills around 1.3 million people annually. Multi-drug resistant TB (MDR-TB) strains are increasingly encountered, in part resulting from shortcomings of current TB drug regimens that last between six to nine months. Patients may stop taking the antibiotics during their allotted regimen, leading to drug resistant TB strains. Novel drug screening platforms are therefore necessary to find drugs effective...
Show moreTuberculosis (TB) is a respiratory disease caused by Mycobacterium tuberculosis (Mtb) that kills around 1.3 million people annually. Multi-drug resistant TB (MDR-TB) strains are increasingly encountered, in part resulting from shortcomings of current TB drug regimens that last between six to nine months. Patients may stop taking the antibiotics during their allotted regimen, leading to drug resistant TB strains. Novel drug screening platforms are therefore necessary to find drugs effective against MDR-TB. In order to discover compounds that target under-exploited pathways that may be essential only in vivo, the proposed screening platform will use a novel approach to drug discovery by blocking essential protein-protein interactions (PPI). In Mtb, PPI can be monitored by mycobacterial protein fragment complementation (M-PFC). This project will re-engineer the M-PFC assay to include the red fluorescent mCherry reporter for increased efficiency and sensitivity in high-throughput screening applications. To optimize the mCherry assay, we have developed fluorescent M-PFC reporter strains to monitor distinct PPI required for Mtb virulence: homodimerization of the dormancy regulator DosR. A drug screen will then identify novel compounds that inhibit this essential PPI. The screen will involve positional-scanning combinatorial synthetic libraries, which are made up of chemical compounds with varying side chains. This work will develop novel tools for TB drug discovery that could identify new treatments for the emerging world threat of MDR-TB.
Show less - Date Issued
- 2015
- Identifier
- CFH0004785, ucf:45369
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004785
- Title
- VALIDATING DRUG TARGETS THROUGH INHIBITION OF PROTEIN-PROTEIN INTERACTIONS IN MYCOBACTERIUM TUBERCULOSIS.
- Creator
-
Driscoll, Erin C, Rohde, Kyle, University of Central Florida
- Abstract / Description
-
Tuberculosis is the leading cause of death by single infectious disease worldwide; novel antibiotics are needed to continue to treat this disease. To goal of this project is to provide proof-of-principle support for the idea that targeting protein-protein interactions (PPI) is an appropriate course for the discovery of new drugs. This study optimized the M-PFC assay, which allows detection of PPI in Mycobacteria, through the use of stronger promoters and inducible expression of a peptide...
Show moreTuberculosis is the leading cause of death by single infectious disease worldwide; novel antibiotics are needed to continue to treat this disease. To goal of this project is to provide proof-of-principle support for the idea that targeting protein-protein interactions (PPI) is an appropriate course for the discovery of new drugs. This study optimized the M-PFC assay, which allows detection of PPI in Mycobacteria, through the use of stronger promoters and inducible expression of a peptide blocker by riboswitch. To accomplish this, promoter induction studies were used to find stronger promoters for the M-PFC, optimization of the riboswitch as a method for inducible protein expression within this system, and the addition of both elements to the existing version of the M-PFC. This M-PFC targets DosR homodimerization; this process is known to be essential for survival within the host. This study optimizes a system that may be used to screen for drugs that are capable of interrupting this interaction.
Show less - Date Issued
- 2017
- Identifier
- CFH2000190, ucf:46030
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH2000190
- Title
- DEVELOPMENT OF LUMINESCENT TOOLS FOR USE IN THE STUDY OF MYCOBACTERIUM TUBERCULOSIS.
- Creator
-
Moore, Krista A, Rohde, Kyle, University of Central Florida
- Abstract / Description
-
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a growing problem worldwide due to the emergence of multi-drug resistant and extensively-drug resistant strains of the bacteria. A key to combatting the spread of these strains lies in the understanding of gene expression occurring in Mtb. This study focuses on the development and optimization of a luciferase-based bioluminescent transcriptional reporter that can be used to monitor gene expression in Mtb. The...
Show moreMycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a growing problem worldwide due to the emergence of multi-drug resistant and extensively-drug resistant strains of the bacteria. A key to combatting the spread of these strains lies in the understanding of gene expression occurring in Mtb. This study focuses on the development and optimization of a luciferase-based bioluminescent transcriptional reporter that can be used to monitor gene expression in Mtb. The luminescent signal emitted from the reporter can be measured and correlated with the level of transcription of certain genes. This study focuses specifically on a gene called whiB7 which encodes a transcription factor known to contribute to the drug resistance of Mtb. The drug-inducible whiB7 promoter was cloned into various locations in the luciferase plasmid in order to determine the ideal configuration of the reporter for maximum luminescence. The optimized luciferase reporter was then compared with a fluorescent transcriptional reporter, mCherry, also under control of the whiB7 promoter. Fluorescent reporters present some disadvantages including delayed kinetics and inability to accurately reflect gene downregulation due to long half-life of reporter proteins. It was hypothesized that the luciferase reporter would solve these problems by offering a more sensitive and dynamic tool to monitor gene expression. Quantitative real-time PCR was used to measure whiB7 mRNA present in cultures containing either the luciferase or mCherry reporters. The luminescent and fluorescent signal given from these reporters was then compared to actual mRNA expression. It was observed that the signal from the luciferase reporter more closely matched mRNA expression at each timepoint, indicating that the luciferase reporter is a better gauge of actual gene expression levels than the mCherry reporter.
Show less - Date Issued
- 2019
- Identifier
- CFH2000478, ucf:45912
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH2000478
- Title
- DETECTION AND CHARACTERIZATION OF PATHOGENIC MYCOBACTERIA USING BINARY DEOXYRIBOZYMES.
- Creator
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Rosenkrantz, Bradley, Rohde, Kyle, University of Central Florida
- Abstract / Description
-
The genus Mycobacterium contains many pathogenic bacteria that are known to cause serious diseases in humans. One of the most well-known of these bacteria is Mycobacterium tuberculosis, or Mtb, which is the causative agent of tuberculosis. It infects nearly one-third of the world's population and kills 1.4 million people annually. Another important mycobacterial pathogen is Mycobacterium abscessus, or Mabs, which causes respiratory infections in cystic fibrosis patients. One of the biggest...
Show moreThe genus Mycobacterium contains many pathogenic bacteria that are known to cause serious diseases in humans. One of the most well-known of these bacteria is Mycobacterium tuberculosis, or Mtb, which is the causative agent of tuberculosis. It infects nearly one-third of the world's population and kills 1.4 million people annually. Another important mycobacterial pathogen is Mycobacterium abscessus, or Mabs, which causes respiratory infections in cystic fibrosis patients. One of the biggest difficulties in combating these pathogens is the lack of effective diagnostics, as current strategies hold many pitfalls and can be unreliable. One common method used is sputum smear microscopy which involves acid fast staining of the bacteria present in a patient's sputum. This method of detection fails to detect more than 50% of infections and is unable to differentiate between species of mycobacterium. This project introduces a novel method of mycobacterial diagnostics using binary deoxyribozymes (DNAzymes). Binary DNAzymes recognize bacteria-specific nucleic acid sequences and bind to them, forming a catalytic core which cleaves a substrate molecule. This cleavage separates a quencher molecule from a fluorophore, which results in a fluorescent output. This flexible assay platform has great potential for the detection of Mtb or Mabs. Our data shows the specificity of the DNAzymes allowing for a differential diagnosis of various species of Mycobacteria. It also shows the limit of detection of this technology and its additional utility in molecular typing of Mtb clinical isolates as well as drug resistance characterization. This multipurpose tool can contribute to disease management in multiple ways.
Show less - Date Issued
- 2015
- Identifier
- CFH0004758, ucf:45343
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004758
- Title
- DETECTION OF DRUG-RESISTANCE CONFERRING SINGLE NUCLEOTIDE POLYMORPHISMS IN MYCOBACTERIUM TUBERCULOSIS USING BINARY DNAZYMES.
- Creator
-
Addario, Marina, Rohde, Kyle, University of Central Florida
- Abstract / Description
-
Mycobacterium tuberculosis (Mtb) is the pathogen that causes Tuberculosis (TB) and is responsible for an average of 1.5 million deaths annually. Although a treatment regimen does exist, Multi-Drug Resistant (MDR-TB) and eXtremely Drug Resistant (XDR-TB) TB strains are becoming a more prevalent concern partly due to failure of patient compliance with the current six to nine month drug treatment regimen. The current diagnostic methods are not able to identify these MDR and XDR-TB strains...
Show moreMycobacterium tuberculosis (Mtb) is the pathogen that causes Tuberculosis (TB) and is responsible for an average of 1.5 million deaths annually. Although a treatment regimen does exist, Multi-Drug Resistant (MDR-TB) and eXtremely Drug Resistant (XDR-TB) TB strains are becoming a more prevalent concern partly due to failure of patient compliance with the current six to nine month drug treatment regimen. The current diagnostic methods are not able to identify these MDR and XDR-TB strains efficiently therefore more effective point-of-care (POC) diagnostics and drug susceptibility testing (DST) are urgently needed to detect drug resistance and facilitate prompt, appropriate treatment plans. In order to detect TB and efficiently identify drug resistance, this project seeks to develop a novel diagnostic technology based on deoxyribozyme (DNAzyme) sensors. The overall goal of this project is to create an assay which combines Polymerase Chain Reaction (PCR) and DNAzymes to identify drug resistance conferring Single Nucleotide Polymorphisms (SNPs). To safely test the ability of DNAzyme sensors to detect SNPs indicative of multi-drug resistant TB, we have constructed a panel of drug resistant (drugR) nonpathogenic M. bovis BCG. We have designed a multiplex PCR that amplifies 6 chromosomal regions of the genome necessary for the species specific detection of TB and determination of a drug susceptibility profile based on the presence of SNPs. To improve the sensitivity and selectivity of the detection and DST of Mtb, we have designed and optimized DNAzyme sensor assays combined with multiplex PCR analytes that will enable the rapid, POC detection of drug resistance. This work aims to develop novel tools for the prompt and specific diagnosis of TB allowing for the implementation of an iv effective treatment regimen that will ultimately lessen transmission and control the emerging global threat of MDR and XDR-TB.
Show less - Date Issued
- 2015
- Identifier
- CFH0004844, ucf:45435
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004844
- Title
- DEVELOPMENT OF NOVEL FLUORESCENT TOOLS FOR INVESTIGATING VIRULENCE FACTORS AND DRUG SUSCEPTIBILITY IN MYCOBACTERIUM TUBERCULOSIS.
- Creator
-
Wilburn, Kaley, Rohde, Kyle, University of Central Florida
- Abstract / Description
-
Mycobacterium tuberculosis (Mtb) is the causative agent of Tuberculosis (TB), a life-threatening disease primarily affecting the lungs that infects about one third of the world's population and causes 1.3 million deaths annually. It is estimated that TB has been infecting humans for around 70,000 years and has killed more people than any other infectious disease. The highly effective, persistent, and multifaceted virulence strategies that have allowed Mtb to continue to spread and thrive for...
Show moreMycobacterium tuberculosis (Mtb) is the causative agent of Tuberculosis (TB), a life-threatening disease primarily affecting the lungs that infects about one third of the world's population and causes 1.3 million deaths annually. It is estimated that TB has been infecting humans for around 70,000 years and has killed more people than any other infectious disease. The highly effective, persistent, and multifaceted virulence strategies that have allowed Mtb to continue to spread and thrive for so long are still poorly understood at the molecular level. This lack of knowledge contributes to ongoing challenges to curing TB. Although drugs capable of killing Mtb exist, even strains that are susceptible to these drugs remain so difficult to treat that stringent six- to nine-month courses of four-drug cocktails are required. Practical difficulties in administering full treatments and patient noncompliance have contributed to a rise in drug-resistant TB cases globally. To combat this increasing world health problem, new antibiotic treatments that kill Mtb and drug-resistant Mtb more effectively via new mechanisms of action are necessary. Discovering these antibiotics expediently requires that innovative Mtb-specific drug-screening assays are developed. An ideal and innovative TB drug screening method would target validated protein-protein interactions (PPI) essential to Mtb's pathogenesis and would be performed on whole Mtb cells under relevant in vivo-like conditions. This project focused on engineering several tools relevant to creating an ideal TB drug screen. A protein fragment complementation assay capable of studying PPI of the TB gyrase complex was created, and this assay was assessed for future HTS applications. To streamline the readout, this assay was re-engineered to include green fluorescent protein. Modifications to the red fluorescent protein mCherry, including the creation of a large Stokes shift mutant mCherry and an mCherry bimolecular fluorescence complementation assay, were also engineered and investigated.
Show less - Date Issued
- 2015
- Identifier
- CFH0004843, ucf:45473
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004843
- Title
- MYCOBACTERIUM TUBERCULOSIS REGULATION OF EFFLUX PUMP TAP BY TRANSCRIPTIONAL ACTIVATOR WHIB7.
- Creator
-
Pollock, Aaron, Rohde, Kyle, University of Central Florida
- Abstract / Description
-
Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), remains a debilitating disease that affects the health of millions annually. Understanding its ability to persist within host and resist eradication by antibiotics is of utmost importance in the effort to develop new interventions. This study will focus on the transcriptional activator WhiB7 and its regulation of the multidrug Tap efflux pump encoded by Rv1258c. WhiB7 is thought to respond to redox stress induced by antibiotics and a...
Show moreTuberculosis, caused by Mycobacterium tuberculosis (Mtb), remains a debilitating disease that affects the health of millions annually. Understanding its ability to persist within host and resist eradication by antibiotics is of utmost importance in the effort to develop new interventions. This study will focus on the transcriptional activator WhiB7 and its regulation of the multidrug Tap efflux pump encoded by Rv1258c. WhiB7 is thought to respond to redox stress induced by antibiotics and a variety of in vivo stresses by activating multiple genes including Rv1258c. Much remains to be determined regarding the role of WhiB7 and downstream genes in Mtb virulence and drug resistance. We will create a tool for studying WhiB7-mediated gene regulation by engineering a strain of the nonpathogenic bacterium Msm expressing the mCherry fluorescent protein controlled by the Rv1258c promoter. Knocking out the native WhiB7 gene in Msm via homologous recombination will allow clear introduction of wild type and mutant versions of Mtb WhiB7. Changes in the fluorescent activity of Rv1258c promoter fusion to mCherry will indicate the effects of WhiB7 mutagenesis. Secondly, we can also use this system to confirm additional genes identified by microarray analysis that are potentially regulated by WhiB7. This will be done by cloning other promoters in front of mCherry in the Msm strain containing wild-type Mtb WhiB7. Understanding WhiB7's role in Mycobacterium tuberculosis macrophage survival and antibiotic resistance may provide new strategies for developing drugs that can lead to a cure.
Show less - Date Issued
- 2014
- Identifier
- CFH0004654, ucf:45260
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004654
- Title
- UNDERSTANDING THE ROLE OF A HEMERYTHRIN-LIKE PROTEIN IN MYCOBACTERIUM TUBERCULOSIS.
- Creator
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Herndon, Caitlyn, Rohde, Kyle, University of Central Florida
- Abstract / Description
-
According to the Centers for Disease Control and Prevention (CDC), 8 million people each year are infected with Mycobacterium tuberculosis (Mtb) leading to 1.5 million deaths annually. This staggering number calls for advancements in understanding this bacterium so progress can be made in treating and preventing the disease. It is particularly important to understand mechanisms by which TB survives inside hostile host immune cells known as macrophages and within hypoxic granuloma lesions of...
Show moreAccording to the Centers for Disease Control and Prevention (CDC), 8 million people each year are infected with Mycobacterium tuberculosis (Mtb) leading to 1.5 million deaths annually. This staggering number calls for advancements in understanding this bacterium so progress can be made in treating and preventing the disease. It is particularly important to understand mechanisms by which TB survives inside hostile host immune cells known as macrophages and within hypoxic granuloma lesions of the lung. Preliminary microarray data has shown that a TB gene known as Rv2633c is induced upon macrophage invasion. Bioinformatic analysis of Rv2633c coding sequence shows the product of Rv2633c has homology with hemerythrin-like proteins. Hemerythrins are a class of proteins commonly used to bind oxygen and sense nitric oxide and iron, leading us to hypothesize a role for Rv2633c in surviving hypoxic or nitrosative stress encountered within macrophages and granulomas. My first aim will be to generate a reporter strain of Mycobacterium smegmatis (Msm) expressing the mCherry fluorescent protein driven by the Rv2633c promoter. This tool will allow us to determine the stress conditions (i.e. hypoxia, nitric oxide treatment, acid pH) that activate expression of this gene by measuring the change in fluorescence. Linking the regulation of Rv2633c to specific environmental cues relevant to infections in vivo will provide insight into the role of this unique protein. Secondly, a knockout mutant of Rv2633c in the attenuated M. bovis BCG will be constructed and characterized to determine the importance and function of this protein during TB infections.
Show less - Date Issued
- 2014
- Identifier
- CFH0004647, ucf:45291
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004647
- Title
- Biochemical Characterization of Rv2633c from Mycobacterium tuberculosis and the Effects of Mutagenesis on Iron Binding.
- Creator
-
Strickland, Kyle, Self, William, Rohde, Kyle, Davidson, Victor, University of Central Florida
- Abstract / Description
-
Mycobacterium tuberculosis (Mtb) is a pathogenic bacterium that is the causative agent of the disease Tuberculosis (TB). TB kills an estimated 1.8 million people annually and roughly one third of the world's population carries Mtb in a dormant state. Drug resistant Mtb strains are on the rise, thus a new method of combating this disease is paramount. Mtb survival inside of macrophages requires overcoming various stressors such as; iron restriction, reactive oxygen species, and hypoxic...
Show moreMycobacterium tuberculosis (Mtb) is a pathogenic bacterium that is the causative agent of the disease Tuberculosis (TB). TB kills an estimated 1.8 million people annually and roughly one third of the world's population carries Mtb in a dormant state. Drug resistant Mtb strains are on the rise, thus a new method of combating this disease is paramount. Mtb survival inside of macrophages requires overcoming various stressors such as; iron restriction, reactive oxygen species, and hypoxic conditions. Mtb employs the use of catalases, nitric oxide reductase, superoxide dismutase, and siderophores to aid in survival. These functions have also been found in a novel group of non-heme diiron binding proteins called hemerythrin-like proteins.The gene Rv2633c encodes a protein with the hemerythrin-like domain and has been shown to be upregulated under acidic or nutrient deficient conditions which coincides with Mtb infection of a macrophage. It has also been shown to be regulated by PhoP, Whib3, and DosR. In this work we expressed the wild type protein and several mutants heterologously in E. coli. The purified proteins were studied via UV-visible spectroscopic analysis, native polyacrylamide gel electrophoresis (native-PAGE) and analyzed for iron content.Our refined expression and purification protocol led to a significant increase in soluble protein with a di-iron cofactor. We found that mutagenesis of 11th amino acid, a histidine, led to the absence of the diiron co-factor. Reduction and autoxidation of protein was also achieved and characterized through UV-visible absorption. Native-PAGE gel analysis indicated only the dimeric form contained iron. This research is the first to produce large quantities of soluble iron laden protein, demonstrate that Rv2663c is capable of both reduction and autoxidation, and show it does not bind oxygen in a functional capacity. This information will enable future studies in protein crystallization, ligand interaction and in vivo studies.
Show less - Date Issued
- 2019
- Identifier
- CFE0007729, ucf:52456
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007729
- Title
- Political, Economic, and Health Determinants of Tuberculosis Incidence.
- Creator
-
Rutherford, Ashley, Unruh, Lynn, Rohde, Kyle, Wan, Thomas, Nobles, Matt, University of Central Florida
- Abstract / Description
-
The epidemiologic transition has shifted major causes of mortality from infectious disease to chronic disease; however, infectious diseases are again re-emerging as a major global concern (Diamond, 1997; Karlen, 1995; McNeil, 1976). This research aimed to identify potential areas of infectious disease influence that are not health-related in order to help governments and policymakers establish new policies, correct current policies, or further address these issues in order to effectively...
Show moreThe epidemiologic transition has shifted major causes of mortality from infectious disease to chronic disease; however, infectious diseases are again re-emerging as a major global concern (Diamond, 1997; Karlen, 1995; McNeil, 1976). This research aimed to identify potential areas of infectious disease influence that are not health-related in order to help governments and policymakers establish new policies, correct current policies, or further address these issues in order to effectively prevent and combat infectious disease. This study employed a retrospective, cross-sectional, non-experimental design via structural equation modeling (SEM) and examined tuberculosis incidence rates at the country-level. Secondary data from open-source, international databases like World Bank's World Development Indicators, World Governance Indicators, and World Health Organization for the year 2014 was utilized. Results revealed that the latent constructs of political stability, health system indicators, and detection policies directly affected tuberculosis incidence rates; they also exhibited an indirect effect due to covariation. Economic stability did not direct affect tuberculosis incidence, but it indirectly influenced incidence through the covariation of political stability, health system indicators, and detection policies. As a country's political stability increased, tuberculosis incidence decreased. As positive health system indicators increased, tuberculosis incidence decreased. Countries with more Xpert detection policies in place experienced an apparent increase in tuberculosis incidence.
Show less - Date Issued
- 2016
- Identifier
- CFE0006842, ucf:51798
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006842
- Title
- The mauC gene encodes a versatile signal sequence and redox protein that can be utilized in native and non-native protein expression and electron trnasfer systems.
- Creator
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Dow, Brian, Davidson, Victor, Self, William, Rohde, Kyle, Tatulian, Suren, University of Central Florida
- Abstract / Description
-
The redox-active type 1 copper site of amicyanin is composed of a single copper ion that is coordinated by two histidines, a methionine, and a cysteine residue. This redox site has a potential of +265 mV at pH7.5. Over ten angstroms away from the copper site resides a tryptophan residue whose fluorescence is quenched by the copper. The effects of the tryptophan on the electron transfer (ET) properties were investigated by site-directed mutagenesis. Lessons learned about the hydrogen bonding...
Show moreThe redox-active type 1 copper site of amicyanin is composed of a single copper ion that is coordinated by two histidines, a methionine, and a cysteine residue. This redox site has a potential of +265 mV at pH7.5. Over ten angstroms away from the copper site resides a tryptophan residue whose fluorescence is quenched by the copper. The effects of the tryptophan on the electron transfer (ET) properties were investigated by site-directed mutagenesis. Lessons learned about the hydrogen bonding network of amicyanin from the aforementioned study were attempted to be used as a model to increase the stability of another beta barrel protein, the immunoglobulin light chain variable domain (VL). In addition, amicyanin was used as an alternative redox partner with MauG. MauG is a diheme protein from the mau gene cluster that catalyzes the biogenesis of the tryptophan tryptophylquinone cofactor of methylamine dehydrogenase (MADH). The amicyanin-MauG complex was used to study the free energy dependence and impact of reorganization energy in biological electron transfer reactions.The sole tryptophan of amicyanin was converted to a tyrosine via site-directed mutagenesis. This mutation had no effect on the electron transfer parameters with its redox partners, methylamine dehydrogenase and cytochrome c-551i. However, the pKa of the pH-dependence of the redox potential of the copper site was shifted +0.5 pH units. This was a result of an additional hydrogen bond between Met51 and the copper coordinating residue His95 in the reduced form of amicyanin. This additional hydrogen bond stabilizes the reduced form. Also, the stability of the copper site and the protein overall was significantly decreased, as seen by the temperature dependence of the visible spectrum of the copper site and the circular dichroism spectrum of the protein. This destabilization is attributed to the loss of an interior, cross-barrel hydrogen bond.The VL is structurally similar to amicyanin, but it does not contain any cross-barrel hydrogen bonds. The importance of the cross-barrel hydrogen bond in stabilizing amicyanin is evident. A homologous bond in VL was attempted to be engineered by using site-directed mutagenesis to insert neutral residues with protonatable groups into the core of the protein. Wild-type (WT) VL was purified from the periplasm and found to be properly folded as determined by circular dichroism and size exclusion chromatography. Mutants were expressed in E. coli using the amicyanin signal sequence for periplasmic expression. Folded mutant protein could not be purified from the periplasm.When amicyanin is used in complex with MauG, it retains the pH-dependence of the redox potential of its copper site due to the looseness of the interprotein interface. The free energy of the reaction was manipulated by variation in pH from pH 5.8 to 8.0. The ET parameters are reorganization energy of 2.34 eV and an electronic coupling constant of 0.6 cm-1. P94A amicyanin has a potential that is 120 mV higher than WT amicyanin and was used to extend the range of the free energy dependence studied. The ET parameters of the reaction of WT and P94A amicyanin with MauG were within error of each other. This is significant because the ET reaction of P94A amicyanin with its natural electron acceptor was not able to be studied due to a kinetic coupling of the ET step with a non-ET step. This kinetic coupling obscured the parameters of the ET step because it is not kinetically distinguishable from the ET step.A Y294H MauG mutant was also studied. This mutation replaced the axial tyrosine ligand of the six-coordinate heme of MauG with a histidine. No reaction is observed with Y294H MauG in its native reaction. However, the high valent oxidation state of the five-coordinate heme of Y294H MauG reacts with reduced amicyanin. The ET rate was analyzed by ET theory to study the high valent heme in Y294H MauG. The reorganization energy of Y294H MauG was calculated to be nearly 20% lower as compared to the same reaction with WT MauG. These results provide insight into the obscured nature of reorganization energy of large redox cofactors in proteins, particularly heme cofactors, as well as to how the active sites of enzymes are optimized to perform long range ET vs catalysis with regard to balancing redox potential and reorganization energy.
Show less - Date Issued
- 2016
- Identifier
- CFE0006100, ucf:51192
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006100
- Title
- Purification and Characterization of a Novel Selenocysteine Lyase from Enterococcus faecalis.
- Creator
-
Nelson, Samantha, Self, William, Moore, Sean, Rohde, Kyle, University of Central Florida
- Abstract / Description
-
A previous study identified Enterococcus faecalis as one of two bacteria known to have the selD gene and other selenium related genes without having the genes necessary to make selenocysteine or selenouridine. EF2570, a gene in the cluster, was later shown to be upregulated during biofilm formation and also responsible for a selenite- and molybdate-dependent increase in biofilm formation in vitro. The protein encoded was identified as a selenium dependent molybdenum hydroxylase (SDMH),...
Show moreA previous study identified Enterococcus faecalis as one of two bacteria known to have the selD gene and other selenium related genes without having the genes necessary to make selenocysteine or selenouridine. EF2570, a gene in the cluster, was later shown to be upregulated during biofilm formation and also responsible for a selenite- and molybdate-dependent increase in biofilm formation in vitro. The protein encoded was identified as a selenium dependent molybdenum hydroxylase (SDMH), enzymes that contain a labile selenium atom required for activity. While the process of inserting selenocysteine into a protein is well known, the process by which a SDMH acquires a labile selenium atom has not yet been described. To begin unraveling this pathway, the nifS-like EF2568 from the gene cluster will be characterized. Some NifS-like proteins have been shown to have selenocysteine lyase activity, providing a source of selenium for selenophosphate synthetase, the selD gene product. Study of EF2568 has shown that it specifically reacts with L-selenocysteine to form selenide and alanine with L-cysteine inhibiting the reaction. Guided by homology to the well-characterized human and E. coli NifS-like proteins, mutants of the active site and substrate discerning residues were also characterized for activity with L-selenocysteine and L-cysteine. While mutation of the residue at position 112 thought to be responsible for substrate specificity did not affect reactivity of the enzyme with L-cysteine, it did affect reactivity with L-selenocysteine. Studying the characteristics of this novel group II selenocysteine lyase will provide a foundation for studying the remaining pathway.
Show less - Date Issued
- 2014
- Identifier
- CFE0005388, ucf:50455
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005388
- Title
- Validation of a novel hypothesis of generating foam cells by its use to study reverse cholesterol transport.
- Creator
-
Sengupta, Bhaswati, Parthasarathy, Sampath, Singla, Dinender, Jewett, Mollie, Rohde, Kyle, University of Central Florida
- Abstract / Description
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Generation of foam cells, an essential step for reverse cholesterol transport (RCT) studies, uses the technique of receptor dependent macrophage loading with radiolabeled acetylated Low Density Lipoprotein (Ac-LDL). In this study, we used the ability of a biologically relevant detergent molecule, Lysophosphatidylcholine (Lyso PtdCho), to form mixed micelles with cholesterol or cholesteryl ester (CE) to generate macrophage foam cells. Fluorescent or radiolabelled cholesterol / Lyso PtdCho...
Show moreGeneration of foam cells, an essential step for reverse cholesterol transport (RCT) studies, uses the technique of receptor dependent macrophage loading with radiolabeled acetylated Low Density Lipoprotein (Ac-LDL). In this study, we used the ability of a biologically relevant detergent molecule, Lysophosphatidylcholine (Lyso PtdCho), to form mixed micelles with cholesterol or cholesteryl ester (CE) to generate macrophage foam cells. Fluorescent or radiolabelled cholesterol / Lyso PtdCho mixed micelles were prepared and incubated with RAW 264.7 or mouse peritoneal macrophages. Results showed that such micelles were quite stable at 4(&)deg;C and retained the solubilized cholesterol during one month storage. Macrophages incubated with cholesterol or CE (unlabeled, fluorescently labeled or radiolabeled) / Lyso PtdCho mixed micelles accumulated CE as documented by microscopy, lipid staining, labeled oleate incorporation, and by thin layer chromatography (TLC). Such foam cells unloaded cholesterol when incubated with high density lipoprotein (HDL) and not with oxidized HDL (Ox-HDL). We propose that stable cholesterol or CE / Lyso PtdCho micelles would offer advantages over existing methods.Oxidative stress is associated with heart failure (HF). Previously our research group observed that the patients with low left-ventricular ejection fraction showed accumulation of high level of oxidized LDL (Ox-LDL) when compared with the heart failure patients with normal range of ejection fraction (EF). HDL is known to be atheroprotective and one of its important antioxidative functions is to protect LDL from oxidative modifications. However, HDL itself undergoes oxidation and Ox-HDL becomes functionally poor. It is expected to have a diminished ability to promote reverse cholesterol transport. Therefore, it was hypothesized that the quality of HDL present in the patients with EF would more compromised than those present in the patients with normal EF. Functionality of HDL was evaluated by measuring its cholesterol efflux capacity from foam cells generated in vitro. Functionality of HDL, which is strongly related to the oxidative modifications of HDL was further estimated by measuring paraoxonase 1 (PON1) enzyme activity associated with HDL. Higher the PON1 activity and RCT ability, better is the functionality of HDL.
Show less - Date Issued
- 2014
- Identifier
- CFE0005250, ucf:50596
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005250
- Title
- Characterization of Novel Borrelia burgdorferi Transcripts Expressed during Tick and Mammalian Infection.
- Creator
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Adams, Philip, Jewett, Mollie, Rohde, Kyle, Moore, Sean, Fernandez-Valle, Cristina, University of Central Florida
- Abstract / Description
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The purpose of this dissertation is to characterize the transcriptome of Borrelia (Borreliella) burgdorferi to discover novel transcripts, important for pathogenesis. As a spirochete and the etiological agent of Lyme disease, the foremost vector-borne bacterial infection in the world, B. burgdorferi fulfills a distinctive niche among bacterial pathogens. Persisting in the disparate environments of a tick vector and mammalian reservoirs, it is absolutely dependent on its hosts for transmission...
Show moreThe purpose of this dissertation is to characterize the transcriptome of Borrelia (Borreliella) burgdorferi to discover novel transcripts, important for pathogenesis. As a spirochete and the etiological agent of Lyme disease, the foremost vector-borne bacterial infection in the world, B. burgdorferi fulfills a distinctive niche among bacterial pathogens. Persisting in the disparate environments of a tick vector and mammalian reservoirs, it is absolutely dependent on its hosts for transmission and nutrient acquisition. B. burgdorferi harbors a complex fragmented genome which is largely linear, unlike that of most prokaryotes, lacks an array of classically described metabolic genes, and contains an unusually large percentage of unique genomic sequences specific to Borrelia (Borreliella) species. To date, few regulatory mechanisms have been identified which contribute to the ability of the spirochete to sense and respond to its environment. Efforts to use global transcript analysis to elucidate the molecular mechanisms of B. burgdorferi host adaptation have proven challenging due to the low numbers of the pathogen present during infection. Previously, our laboratory successfully developed an in vivo expression technology based approach for B. burgdorferi (BbIVET) to identify spirochete promoter sequences that are active during a murine infection. This screen identified 233 unique putative promoters which mapped to locations across the entire genome. These putative infection-active B. burgdorferi promoters were not only located at the 5' end of annotated open reading frames (ORFs), but also mapped to unannotated locations antisense, intergenic, and intragenic to ORFs. Given the limited characterization of the B. burgdorferi transcriptome, this dissertation applies an RNA sequencing approach (5'RNA-seq) to globally annotate the transcriptional start sites (TSSs) and 5' processed ends of the spirochete's RNA during in vitro cultivation. This resulted in the discovery of numerous novel internal, intergenic, and antisense transcripts. Synergistic analysis combining Northern blotting techniques, alignments of these transcripts to BbIVET proposed promoters, and interrogation of promoter activity via in vivo live imaging of mice, confirmed the expression of a variety of RNAs during laboratory culture and mammalian infection. Further, as a means to improve quantitation of the expression of these transcripts, a new methodology was developed and applied to measure B. burgdorferi promoter activity during tick-pathogen interactions, in a strand specific manner. Finally, because the Lyme disease spirochete harbors many unclassified and unique genomic sequences, the mammalian infection-expressed gene bb0562, identified through BbIVET and 5'RNA-seq, was selected for targeted deletion and evaluation throughout B. burgdorferi's infectious cycle. This demonstrated that gene bb0562 encodes a membrane associated protein, whose presence is critical for establishing murine infection through the bite of an infected tick. In sum, this work contributes significant insight into the transcriptome of B. burgdorferi, provides an innovative approach for the analysis of RNA transcripts at the tick-pathogen interface, and identifies a novel gene critical for Lyme disease pathogenesis.
Show less - Date Issued
- 2017
- Identifier
- CFE0006707, ucf:51915
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006707
- Title
- Discovery and characterization of novel antimicrobials against Mycobacterium tuberculosis.
- Creator
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Rodrigues Felix, Carolina, Rohde, Kyle, Jewett, Mollie, Self, William, Phanstiel, Otto, University of Central Florida
- Abstract / Description
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Tuberculosis disease is currently a global health emergency, causing the most deaths worldwide due a single infectious agent. Eradication of TB is hampered by lack of an effective vaccine and poor treatment options. During infection, host-derived cues such as hypoxia and starvation induce Mycobacterium tuberculosis to halt replication and become dormant, which leads to tolerance to front-line antibiotics used in the TB treatment. This dormant phenotype causes delayed clearance of M....
Show moreTuberculosis disease is currently a global health emergency, causing the most deaths worldwide due a single infectious agent. Eradication of TB is hampered by lack of an effective vaccine and poor treatment options. During infection, host-derived cues such as hypoxia and starvation induce Mycobacterium tuberculosis to halt replication and become dormant, which leads to tolerance to front-line antibiotics used in the TB treatment. This dormant phenotype causes delayed clearance of M. tuberculosis, therefore a long treatment time is required for stable cure without relapse. Poor patient compliance increases the emergence of drug resistant strains, posing yet another challenge for the eradication of TB. There is dire need for novel compounds targeting not only drug-resistant, but also dormant bacteria so as to effectively eliminate drug-resistant strains and also shorten treatment time. This requires compounds with novel modes of action and novel drug screening approaches which focus on dormant M. tuberculosis. In the current work a method was optimized which induces the dormant phenotype of M. tuberculosis in vitro allowing large scale screening of compounds against these tolerant bacteria. The high chemical diversity of marine natural products was explored to increase the chances of finding novel compounds with novel mechanisms of action. Additionally, gold-complexed scaffolds were examined for their putative ability to inhibit topoisomerase 1, which is a highly conserved and essential protein of mycobacteria, not currently targeted in classical treatment regimens. Several marine natural products were identified with selective bactericidal activity against dormant bacteria, emphasizing the powerful tool that was developed for drug screening. Moreover, the gold-complexes were also bactericidal against not only replicating and dormant bacilli, but also mycobacteria resistant to front-line TB drugs. Compounds characterized in this study represent a promising starting point for the development of novel TB therapeutics and discovery of new conditionally essential pathways of dormant bacteria.
Show less - Date Issued
- 2017
- Identifier
- CFE0007294, ucf:52172
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007294
- Title
- Development of Molecular Diagnostic Tools for Mycobacterium Species.
- Creator
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Bengtson, Hillary, Kolpashchikov, Dmitry, Rohde, Kyle, Self, William, Jewett, Travis, Masternak, Michal, University of Central Florida
- Abstract / Description
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This dissertation focuses on the development of diagnostic tools for mycobacteria using hybridization based technologies including binary deoxyribozyme (BiDz) sensors and microarrays. The genus Mycobacterium, is a diverse group of bacteria containing 150+ species including M. tuberculosis (M.tb) and non-tuberculous mycobacteria (NTM) which exhibit a range of pathogenicity, drug susceptibility and growth characteristics. M. tuberculosis (M.tb) is the causative agent of tuberculosis (TB) and...
Show moreThis dissertation focuses on the development of diagnostic tools for mycobacteria using hybridization based technologies including binary deoxyribozyme (BiDz) sensors and microarrays. The genus Mycobacterium, is a diverse group of bacteria containing 150+ species including M. tuberculosis (M.tb) and non-tuberculous mycobacteria (NTM) which exhibit a range of pathogenicity, drug susceptibility and growth characteristics. M. tuberculosis (M.tb) is the causative agent of tuberculosis (TB) and the leading cause of infectious disease related deaths worldwide. The control of TB is limited by the lack of sensitive and specific diagnostic tools available at the point of care (POC). The studies presented here illustrate the advances in our technology for the detection and differentiation of M.tb and NTM. The use of BiDz sensors enables the selective recognition of DNA/RNA analytes containing single nucleotide polymorphisms associated with species-specific identification, drug susceptibility testing (DST) and strain typing. First, we developed a platform for the detection of M.tb and drug susceptibility using multiplex PCR and BiDz sensors. However, this method relies on the use of expensive instrumentation which is often not available in high TB burden countries. Therefore, additional studies focused on the development of tools for the detection of isothermal amplification products and the direct detection of rRNA. Based on these findings, we also developed an NTM species typing tool using BiDz sensors for species identification in ~1 hour. Despite the advantages of BiDz sensor technology, their use is limited to the detection of a few selected mutations. To address this limitation, we developed a 15-loci multiplex PCR followed by analysis with a custom microarray for high-throughput identification of SNPs. The work presented in this dissertation has the potential to enable the rapid, specific and sensitive identification of mycobacterial species necessary to reduce the diagnostic delay, ensure initiation of effective therapy, and prevent further transmission.
Show less - Date Issued
- 2017
- Identifier
- CFE0006856, ucf:51735
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006856