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DEVELOPMENT OF NOVEL FLUORESCENT TOOLS FOR INVESTIGATING VIRULENCE FACTORS AND DRUG SUSCEPTIBILITY IN MYCOBACTERIUM TUBERCULOSIS

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Date Issued:
2015
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 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.
Title: DEVELOPMENT OF NOVEL FLUORESCENT TOOLS FOR INVESTIGATING VIRULENCE FACTORS AND DRUG SUSCEPTIBILITY IN MYCOBACTERIUM TUBERCULOSIS.
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Name(s): Wilburn, Kaley, Author
Rohde, Kyle, Committee Chair
University of Central Florida, Degree Grantor
Type of Resource: text
Date Issued: 2015
Publisher: University of Central Florida
Language(s): English
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 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.
Identifier: CFH0004843 (IID), ucf:45473 (fedora)
Note(s): 2015-08-01
B.S.
Medicine, Dept. of Molecular Biology and Microbiology
Bachelors
This record was generated from author submitted information.
Subject(s): mycobacterium tuberculosis
M-PFC
BiFC
fluorescent tools
TB
mCherry
Large Stokes Shift
Persistent Link to This Record: http://purl.flvc.org/ucf/fd/CFH0004843
Restrictions on Access: campus 2020-07-01
Host Institution: UCF

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