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Title: CHARACTERIZATION OF HEMERYTHRIN-LIKE PROTEIN RV2633C.
Creator: Cherne, Michelle D, Self, William, University of Central Florida
Abstract / Description: Hemerythrin-like protein Rv2633c is a small 18 kDa protein that is expressed in Mycobacterium tuberculosis (Mtb). Sequence analysis of Rv2633c predicts the presence of a hemerythrin-like domain, which binds dioxygen using a �-oxo-bridge (Fe-O-Fe), rather than a heme group. Though it is noticeably upregulated during macrophage infection and during in vitro acidification, the role of Rv2633c in Mtb survival has yet to be elucidated. This project aims to characterize the function of Rv2633c by...
Show moreHemerythrin-like protein Rv2633c is a small 18 kDa protein that is expressed in Mycobacterium tuberculosis (Mtb). Sequence analysis of Rv2633c predicts the presence of a hemerythrin-like domain, which binds dioxygen using a �-oxo-bridge (Fe-O-Fe), rather than a heme group. Though it is noticeably upregulated during macrophage infection and during in vitro acidification, the role of Rv2633c in Mtb survival has yet to be elucidated. This project aims to characterize the function of Rv2633c by studying the in vitro response of the recombinant protein to conditions present in the macrophage lysosome, such as reduced oxygen levels or the presence of reactive oxygen species. UV-visible spectroscopy is used to observe these changes, as the spectrum shows a characteristic peak at 330 nm that likely corresponds to the diiron cofactor in its native state. Our results show this spectrum shifts in response to hydrogen peroxide addition, showing the proposed environmental conditions can affect the active site. Bioinformatics techniques, such as the 3D modeling program SWISS-MODEL, have been used to hypothesize possible structure and function. Determining the function of Rv2633c may help explain how Mtb so readily evades the human immune system to reside in the macrophage.
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Date Issued: 2016
Identifier: CFH2000011, ucf:45581
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH2000011

Title: MODELING TRANSMISSION DYNAMICS OF TUBERCULOSIS INCLUDING VARIOUS LATENT PERIODS.
Creator: Atkins, Tracy, Mohapatra, Ram, University of Central Florida
Abstract / Description: The systems of equations created by Blower et al. (1995) and Jia et al. (2007) designed to model the dynamics of Tuberculosis are solved using the computer software SIMULINK. The results are first employed to examine the intrinsic transmission dynamics of the disease through two models developed by Blower et al. (1995). The "simple transmission model" was used primarily to give insight to the behavior of the susceptible, latent, and infectious groups of individuals. Then, we consider a more...
Show moreThe systems of equations created by Blower et al. (1995) and Jia et al. (2007) designed to model the dynamics of Tuberculosis are solved using the computer software SIMULINK. The results are first employed to examine the intrinsic transmission dynamics of the disease through two models developed by Blower et al. (1995). The "simple transmission model" was used primarily to give insight to the behavior of the susceptible, latent, and infectious groups of individuals. Then, we consider a more detailed transmission model which includes several additional factors. This model captures the dynamics of not only the susceptible, latent and infectious groups but also the non-infectious cases and the recovered cases. Using the SIMULINK results, it can be shown that the intrinsic dynamics of the disease contribute to the rise and decline of the disease seen in historical accounts. Next, the simulation results are used to study the equilibrium points of the disease which can be obtained by varying the parameters and therefore changing the value for the basic reproduction ratio (R0 ). Our model uses the system of equations developed by Jia et al. (2007). The SIMULINK results are used to visually confirm the hypothesis proposed by Jia et al. (2007) that the equilibrium behavior of the system when R0 > 1 is globally asymptotically stable.
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Date Issued: 2008
Identifier: CFE0002030, ucf:47606
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0002030

Title: Discovery and characterization of novel antimicrobials against Mycobacterium tuberculosis.
Creator: Rodrigues Felix, Carolina, Rohde, Kyle, Jewett, Mollie, Self, William, Phanstiel, Otto, University of Central Florida
Abstract / Description: 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.
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Date Issued: 2017
Identifier: CFE0007294, ucf:52172
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007294

Title: UNDERSTANDING THE ROLE OF A HEMERYTHRIN-LIKE PROTEIN IN MYCOBACTERIUM TUBERCULOSIS.
Creator: 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.
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Date Issued: 2014
Identifier: CFH0004647, ucf:45291
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH0004647

Title: Expression of Lipase from Mycobacterium tuberculosis in Nicotiana tobacum and Lactuca sativa Chloroplasts.
Creator: Lloyd, Bethany, Daniell, Henry, Kolattukudy, Pappachan, Self, William, University of Central Florida
Abstract / Description: Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (M. tuberculosis), is a global threat and the leading cause of death among individuals infected with HIV. TB treatment requires multi-drug cocktails, due to the increasing rates of drug resistance of the bacterium. With multi-drug cocktails, strains have been documented to be resistant to all major drugs in the fight against TB. Since the strains are drug resistant, it calls for an increasing need for vaccine and treatment...
Show moreTuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (M. tuberculosis), is a global threat and the leading cause of death among individuals infected with HIV. TB treatment requires multi-drug cocktails, due to the increasing rates of drug resistance of the bacterium. With multi-drug cocktails, strains have been documented to be resistant to all major drugs in the fight against TB. Since the strains are drug resistant, it calls for an increasing need for vaccine and treatment development for the purpose of preventing and managing the disease. The most widely distributed vaccine against TB is Bacillus Calmette-Gue(&)#180;rin (BCG). Apart from being ineffective in certain individuals, BCG offers only a limited timeframe of protection, is unable to serve as a booster for extending this timeframe and due to the intradermal route of administration requires costly refrigeration and syringes.LipY protein, a M. tuberculosis cell wall lipase, may play a potential role as not only a drug target but a potential vaccine antigen. LipY is known to be up-regulated during both active infection and dormancy. In a previous study, sera from TB patients had shown an IgG and IgM response against it. In this study transplastomic Lactuca sativa and Nicotiana tabacum plants were generated by transforming the chloroplasts through the particle delivery system with pLsDv-LipY and pLD-LipY vectors respectively. The vectors were flanked by the native trnI and trnA gene sequence to facilitate homologous recombination into the chloroplast genome. The vector also contained the 16S rRNA promoter, the selectable marker gene, aadA for specitinomycin resistance, the rbcL untranslated region, the LsPpsbA (PpsbA in N. tabacum) promoter, and LsTpsbA (tpsbA in N. tabacum) untranslated region. Site specific integration of the LipY gene into the chloroplast genome was confirmed by PCR. Homoplasmy of transplastomic plants was confirmed by Southern blot analysis. These plants showed normal growth and were fertile, producing seeds. Once germinated, these seeds did not show Mendelian segregation of the transgene. Immunoblot analysis was performed to analyze the expression of the LipY protein. A 40kDa protein was produced in E.coli, and a 25kDa protein was produced in chloroplasts; a cleaved product in chloroplasts is still valuable as an antigen for vaccine production. Future studies will include testing this chloroplast derived antigen in animal models for vaccine development. ?
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Date Issued: 2012
Identifier: CFE0004502, ucf:49289
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0004502

Title: THE EFFECTS OF SITE-DIRECTED MUTAGENESIS ON HEMERYTHRIN-LIKE PROTEIN RV2633C.
Creator: Rosch, Kelly M, Self, William, University of Central Florida
Abstract / Description: Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the top ten causes of death worldwide. One of the genes upregulated in Mtb during macrophage infection is rv2633c, but the structure and function of its gene product remain unknown. Preliminary research has indicated that Rv2633c is a hemerythrin-like protein that exhibits catalase activity and binds two iron atoms using an HHE domain. Additionally, Rv2633c appears to exist as a dimer. The purpose of this project...
Show moreMycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the top ten causes of death worldwide. One of the genes upregulated in Mtb during macrophage infection is rv2633c, but the structure and function of its gene product remain unknown. Preliminary research has indicated that Rv2633c is a hemerythrin-like protein that exhibits catalase activity and binds two iron atoms using an HHE domain. Additionally, Rv2633c appears to exist as a dimer. The purpose of this project is to identify specific residues outside of the HHE domain that contribute to the protein's iron-binding ability and/or catalase activity, and to determine whether residues on the C terminus are required for dimerization. Conserved residues D37, E42, and E95 were selected due to their proximity in the amino acid sequence to the HHE domain. Each residue was mutated to alanine using site-directed mutagenesis and the mutations were confirmed using Sanger sequencing. The E95A mutant and the C-terminal truncation mutant were expressed in Escherichia coli using the T7 expression system and purified using affinity chromatography. While wild-type Rv2633c eluted as a soluble protein, the C-terminal truncation mutant was not soluble, indicating that the C terminus may be required for Rv2633c folding. The E95A mutant eluted as a soluble protein, but may have lower iron content than wild-type Rv2633c, indicating that this glutamic acid residue could contribute to iron-binding, despite being outside the HHE domain.
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Date Issued: 2018
Identifier: CFH2000438, ucf:45794
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH2000438

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.
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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.
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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: 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.
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Date Issued: 2015
Identifier: CFH0004758, ucf:45343
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH0004758

Title: Stable expression of tuberculosis vaccine antigen in lettuce chloroplasts.
Creator: Saikumar Lakshmi, Priya, Daniell, Henry, Khaled, Annette, Naser, Saleh, University of Central Florida
Abstract / Description: Tuberculosis (TB) is caused by Mycobacterium tuberculosis and is one of the leading reasons of death by an infectious bacterial pathogen. The development of TB vaccines has been recognized as a major public health priority by the World Health Organization. In this study, a potential candidate antigen, ESAT-6 (6 kDa early secretory antigenic target) was fused with cholera toxin B subunit (CTB). Transplastomic lettuce plants were generated expressing these fusion proteins. Site-specific...
Show moreTuberculosis (TB) is caused by Mycobacterium tuberculosis and is one of the leading reasons of death by an infectious bacterial pathogen. The development of TB vaccines has been recognized as a major public health priority by the World Health Organization. In this study, a potential candidate antigen, ESAT-6 (6 kDa early secretory antigenic target) was fused with cholera toxin B subunit (CTB). Transplastomic lettuce plants were generated expressing these fusion proteins. Site-specific transgene integration into the chloroplast genome was confirmed by polymerase chain reaction and Southern blot analysis. In transplastomic leaves, expression levels of fusion protein (CTB-ESAT6) varied depending upon the developmental stage and time of leaf harvest with highest-level of accumulation in mature leaves harvested at 6PM. Transplastomic CTB-ESAT6 lettuce plants accumulated up to 0.75% of total leaf protein. Lyophilization increased CTB-ESAT6 protein content per gram of leaf material by 22 fold. Western blot analysis of lyophilized lettuce leaves showed that the CTB-ESAT6 fusion protein was stable and can be stored for prolonged period at RT. Hemolysis assay with purified CTB-ESAT6 protein showed partial hemolysis of red blood cells and confirmed functionality of ESAT-6 antigen. GM-1 binding assay demonstrated that the CTB-ESAT6 fusion protein formed pentamers to interact with GM1 ganglioside receptor. The expression of functional Mycobacterium tuberculosis antigens fused to CTB in transplastomic plants should facilitate development of a cost-effective and orally deliverable TB vaccine with potential for long term storage at room temperature.
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Date Issued: 2011
Identifier: CFE0004487, ucf:49303
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0004487

Title: Development of Molecular Diagnostic Tools for Mycobacterium Species.
Creator: Bengtson, Hillary, Kolpashchikov, Dmitry, Rohde, Kyle, Self, William, Jewett, Travis, Masternak, Michal, University of Central Florida
Abstract / Description: 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.
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Date Issued: 2017
Identifier: CFE0006856, ucf:51735
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006856

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.
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Date Issued: 2019
Identifier: CFE0007729, ucf:52456
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007729

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.
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Date Issued: 2015
Identifier: CFH0004785, ucf:45369
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH0004785

Title: SYSTEMATIC REVIEW AND META-ANALYSIS: TUBERCULOSIS, TNFΑ INHIBITORS, AND CROHN'S DISEASE.
Creator: Cao, Brent L, Naser, Saleh A., University of Central Florida
Abstract / Description: Inflammation is often a protective reaction against harmful foreign agents. However, in many disease conditions, the mechanisms behind the inflammatory response are poorly understood. Often times, the inflammation causes adverse effects, such as joint pain, abdominal pain, fever, fatigue, and loss of appetite. Thus, many treatments aim to inhibit the inflammatory response in order to control adverse symptoms. Such treatments include TNFα inhibitors. However, a major risk associated with drugs...
Show moreInflammation is often a protective reaction against harmful foreign agents. However, in many disease conditions, the mechanisms behind the inflammatory response are poorly understood. Often times, the inflammation causes adverse effects, such as joint pain, abdominal pain, fever, fatigue, and loss of appetite. Thus, many treatments aim to inhibit the inflammatory response in order to control adverse symptoms. Such treatments include TNFα inhibitors. However, a major risk associated with drugs inhibiting tumor necrosis factor alpha (TNFα) is serious infection, including tuberculosis (TB). Anti-TNFα therapy is used to treat patients with Crohn's disease, for which the risk of tuberculosis may be even more concerning. Recent literature suggests Crohn's might involve Mycobacterium avium subspecies paratuberculosis (MAP), an intracellular TB-like bacterium. This study seeks to investigate the risk of developing TB in patients with Crohn's disease treated with TNFα inhibitors. A meta-analysis synthesized existing evidence. Evidence came from published randomized, double-masked, placebo-controlled trials of TNFα inhibitors for treatment of adult Crohn's disease. Twenty-three trials were identified, including 5,669 patients. The risk of tuberculosis was significantly increased in anti-TNFα treated patients, with a risk difference of 0.028 (95% confidence interval [CI], 0.0011-0.055). The odds ratio was 4.85 (95% CI, 1.02-22.99) when all studies were included and 5.85 (95% CI, 1.13-30.38) when studies reporting zero tuberculosis cases were excluded. The risk of tuberculosis is increased in patients with Crohn's disease treated with TNFα inhibitors. The medical community should be alerted about this risk and the potential for TNFα inhibitor usage favoring granulomatous infections and worsening the patient condition.
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Date Issued: 2018
Identifier: CFH2000360, ucf:45909
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH2000360

Title: USING ANTENNA TILE-ASSISTED SUBSTRATE DELIVERY TO IMPROVE THE DETECTION LIMITS OF DEOXYRIBOZYME BIOSENSORS.
Creator: Cox, Amanda, Kolpashchikov, Dmitry, University of Central Florida
Abstract / Description: One common limitation of enzymatic reactions is the diffusion of a substrate to the enzyme active site and/or the release of the reaction products. These reactions are known as diffusion-controlled. Overcoming this limitation may enable faster catalytic rates, which in the case of catalytic biosensors can potentially lower limits of detection of specific analyte. Here we created an artificial system to enable deoxyribozyme (Dz) 10-23 based biosensor to overcome its diffusion limit. The sensor...
Show moreOne common limitation of enzymatic reactions is the diffusion of a substrate to the enzyme active site and/or the release of the reaction products. These reactions are known as diffusion-controlled. Overcoming this limitation may enable faster catalytic rates, which in the case of catalytic biosensors can potentially lower limits of detection of specific analyte. Here we created an artificial system to enable deoxyribozyme (Dz) 10-23 based biosensor to overcome its diffusion limit. The sensor consists of the two probe strands, which bind to the analyzed nucleic acid by Watson-Crick base pairs and, upon binding re-form the catalytic core of Dz 10-23. The activated Dz 10-23 cleaves the fluorophore and quencher-labeled DNA-RNA substrate which separates the fluorophore from the quencher thus producing high fluorescent signal. This system uses a Dz 10-23 biosensor strand associated to a DNA antenna tile, which captures the fluorogenic substrate and channels it to the reaction center where the Dz 10-23 cleaves the substrate. DNA antenna tile captures fluorogenic substrate and delivers it to the activated Dz 10-23 core. This allows for lower levels of analyte to be detected without compromising the specificity of the biosensor. The results of this experiment demonstrated that using DNA antenna, we can create a synthetic environment around the Dz 10-23 biosensor to increase its efficiency and allow for lower levels of analyte to be detected without using amplification techniques like PCR.
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Date Issued: 2015
Identifier: CFH0004887, ucf:45432
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH0004887

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.
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Date Issued: 2014
Identifier: CFH0004654, ucf:45260
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH0004654

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.
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Date Issued: 2016
Identifier: CFE0006842, ucf:51798
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006842

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.
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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.
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Date Issued: 2015
Identifier: CFH0004843, ucf:45473
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH0004843

tuberculosis (10)	+ -
Mycobacterium tuberculosis (8)	+ -
M-PFC (3)	+ -
mycobacterium (3)	+ -
Hemerythrin (2)	+ -

Hemerythrin-like (2)	+ -
Rv2633c (2)	+ -
SNP (2)	+ -
TB (2)	+ -
drug discovery (2)	+ -
mycobacteria (2)	+ -
mycobacterium tuberculosis (2)	+ -
Bacterial infection (1)	+ -
BiFC (1)	+ -
Biosensors (1)	+ -
Crohn's disease (1)	+ -
DNA nanotechnology (1)	+ -
DNAzyme (1)	+ -
Deoxyribozyme (1)	+ -
Diffusion Limitation (1)	+ -
Dz 10-23 (1)	+ -
Edible vaccine (1)	+ -
Lactuca sativa (1)	+ -
Large Stokes Shift (1)	+ -
Limit of Detection (1)	+ -
Lyophilized leaves (1)	+ -
Nicotiana tabacum (1)	+ -
Non-heme diiron carboxylate (1)	+ -
Oral delivery (1)	+ -
SEIR (1)	+ -

Honors Collection (12)	+ -
ETD Collection (7)	+ -

University of Central Florida (19)	+ -
Rohde, Kyle (12)	+ -
Self, William (6)	+ -
Daniell, Henry (2)	+ -
Kolpashchikov, Dmitry (2)	+ -

Addario, Marina (1)	+ -
Atkins, Tracy (1)	+ -
Bengtson, Hillary (1)	+ -
Cao, Brent L (1)	+ -
Cherne, Michelle D (1)	+ -
Cox, Amanda (1)	+ -
Davidson, Victor (1)	+ -
Driscoll, Erin C (1)	+ -
Herndon, Caitlyn (1)	+ -
Jewett, Mollie (1)	+ -
Jewett, Travis (1)	+ -
Khaled, Annette (1)	+ -
Kolattukudy, Pappachan (1)	+ -
Lloyd, Bethany (1)	+ -
Masternak, Michal (1)	+ -
Mohapatra, Ram (1)	+ -
Moore, Krista A (1)	+ -
Naser, Saleh (1)	+ -
Naser, Saleh A. (1)	+ -
Nobles, Matt (1)	+ -
Phanstiel, Otto (1)	+ -
Pollock, Aaron (1)	+ -
Rodrigues Felix, Carolina (1)	+ -
Rosch, Kelly M (1)	+ -
Rosenkrantz, Bradley (1)	+ -

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