Browsing by Subject "Tuberculosis"
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Item A comparison of the rate of recovery and personality factors in tuberculosis patients(Texas Tech University, 1967-05) Coffman, DavidNot availableItem Crystal Structures of Binary and Ternary Complexes of Thymidylate Synthase (ThyA) from Mycobacterium tuberculosis: Insights into Selectivity and Inhibition(2012-10-19) Harshbarger, WayneThymidylate synthase (TS), encoded by the ThyA gene, is essential for the growth and survival of Mycobacterium tuberculosis and therefore is a potential drug target. Thymidylate synthase binds both a substrate, 2'-deoxyuridine-5'monophosphate (dUMP) as well as a cofactor, (6R,S)-5,10-methylenetetrahydrofolate (mTHF), providing the ability to inhibit a single target by two separate classes of molecules. 5'-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) is a very tight binding mechanism based inhibitor, shown to have a Ki of 2nM for Mtb TS. Pemetrexed and Raltitrexed are both anti-folates, targeting the cofactor binding site of thymidylate synthase. The x-ray crystal structures of Mycobacterium tuberculosis thymidylate synthase were solved in the binary complexes ThyA-dUMP and ThyA-FdUMP at 2.5 A and 2.4 A resolutions, respectively. The ternary complex, ThyA-dUMP-Pemetrexed was solved to a resolution of 1.7 A. The enzyme is comprised of 8 alpha-helices as well as 23% of the protein formed by beta-sheets, including the dimer interface which is a beta-sandwich. Examination of the dUMP binding site allowed the identification of key conserved residues that play a role in ligand binding and catalysis. Comparison of the dUMP-Pemetrexed ternary complex with that of the human crystal structure shows two fewer interactions in the Mtb enzyme. One is due to the replacement of a Met with a Val which doesn't allow hydrophobic interactions with the ring system of Pemetrexed, and the other is the replacement of an Asn with a Trp, depriving the Mtb protein of a hydrogen bond at the N7 of the pyrrolo ring. A spectrophotometric assay that monitored DHF formation was used to determine the inhibition of Pemetrexed and Raltitrexed on Mtb TS. Both were verified as noncompetitive inhibitors, and Pemetrexed was found to have an IC50 of 17muM and a Ki of 16.8muM, while Raltitrexed had an IC50 of 3.5muM and a Ki of 3.2muM.Item DNA-based molecular circuits for diagnostics and therapeutics(2013-08) Codrea, Vlad Alexandru; Ellington, Andrew D.Nucleic acids are a uniquely flexible and multi-faceted class of molecules that fulfill fundamental and defining tasks such as replication and determination of heritable characteristics in every living organism. From the microscopic to the gigantic, from the most primitive to the most complex, life has been both molded and served by nucleic acids. Nucleic acid circuits straddle the realm of nature and technology. The elegance of interaction between nucleic acid molecules invites us to gain a deeper understanding of the naturally occurring systems they compose and to apply our ingenuity and foresight toward developing ever more complex synthetic systems. Nature has provided these basic building blocks, which we can now arrange – and augment – for the purpose of creating molecular-level machinery. Here we describe some ways in which we have rationally harnessed nucleic acids. In preparation for outbreaks of novel and deadly avian influenza viruses, we used quantitative polymerase chain reaction (qPCR) to track the number of flu virus particles surviving in the presence of potential antiviral drugs. We engineered tunable on/off switches that can be used to evaluate a series of conditions for diagnostic applications or to enable ‘smart’ drugs that sense, analyze, and respond to their microenvironment. We optimized the conditions for, and used, a unique set of guanine-rich DNA sequences called G-quadruplexes, whose enzymatic and structural properties make them prime effector candidates in diagnostic platforms. G-quadruplex folding powers isothermal DNA amplification, and the small organic molecules they bind endow G-quadruplexes with expanded catalytic abilities. We genotyped drug resistance mutations in tuberculosis via visually detectable color changes in the reaction buffer. We developed a paper fluidics assay that employs soluble and bead-immobilized nucleic acids to scan for genes in tuberculosis, and upon detection, to generate a readily observable discoloration on the paper strip. Finally, we probed the boundary of nucleic acid circuitry by attempting to expand its language via the incorporation of unnatural nucleobases into oligonucleotide components of a catalytic hairpin assembly (CHA) circuit. We subsequently evaluated the resilience of the unnatural CHA circuit to contamination by random DNA species, such as may be encountered in clinical samples.Item Fiber Optic Micro-endoscopy for Detection of Bacteria in Early Stages of Infection(2012-02-14) Mufti, Nooman SadatMycobacterium tuberculosis, the bacterium that causes tuberculosis, has an incubation period ranging from a few months to several years following infection via inhalation into the lungs. Whole body fluorescence scanners are used to image and monitor the growth of fluorescent protein expressing strains of M. tuberculosis in the lungs of animal models. Accurate quantitative analysis of bacterial growth during the early stages of infection inside lungs remains elusive, due to tissue absorption and scattering of photons emitted by the low numbers of bacteria deep in tissue. Fiber optic micro-endoscopy is uniquely suited to provide a novel solution to this problem by delivering light excitation directly to and collecting fluorescence from the infection site located in the lungs of an animal model, thereby enabling detection of fluorescent bacteria during the early stages of infection. In this thesis, I present a contact probe fiber bundle fluorescence micro-endoscope with a range of LED based excitation wavelengths, 4 ?m resolution, a 750 ?m field of view, and a 1 mm outer diameter. This system has detected tdTomato and GFP expressing Bacillus Calmette-Gu?rin (BCG) bacteria in vitro. Additionally, images of bacterial regions of infection obtained in mice subcutaneously infected with tdTomato expressing bacteria at concentrations ranging from 106 to 101 Colony Forming Units (CFU) and intra-tracheally infected mice at 106 CFU demonstrate the micro-endoscope?s capability to detect and resolve regions of bacterial infection in vivo. By relaying the bacterial fluorescence image from the infection site to an external detector, we are able to increase the sensitivity to early stages of infection.Item The lived experience of tuberculosis treatment for Mexican Americans living on the US-Mexico border(2013-05) Zuniga, Julie Ann; García, Alexandra Anne, 1964-This study produced a rich description of the lived experiences of tuberculosis (TB) treatment among Mexican Americans with TB living in the Lower Rio Grande Valley (LRGV) of Texas. This phenomenological study was guided by Merleau-Ponty's philosophical framework, particularly his theories on mind-body influence, fabric of relationships, importance of culture, and equilibrium. A purposeful sample was recruited through TB clinics in four south Texas border counties: Hidalgo, Cameron, Starr, and Willacy, which make up the LRGV. Interviews from 18 participants were conducted in the participants' preferred language and analyzed. There were five women and 13 men. The majority of interviews (n=16) were conducted in Spanish. Five themes were discovered: a) being observed taking pills everyday b) signs and symptoms, c) importance of family, d) stigma; and e) border living. Stigma has four subconcepts: masks, interactions with others, internalization of stigma, and actions to limit exposure to stigma. The overarching theme was a struggle to find a balance during treatment between being exposed to stigma and isolation from social support. Recommendations have been made in regard to education, practice, and research, and health policy.Item Multi-Scale Imaging of Respiratory Bacterial Infection Using Fiber Microendoscopy and Whole-Animal Imaging(2014-08-27) Bixler, Joel NathanWe have integrated a fluorescence microendoscope into a whole-animal optical imaging system, allowing for simultaneous microscopic and macroscopic imaging of tdTomato expressing BCG in vivo. A 535 nm LED was collimated and launched into a 10,000 element fiber bundle can be inserted through an intra-tracheal catheter into the lung of a mouse. Fluorescence emission can either be (1) collected by the bundle and imaged onto the surface of a CCD camera for localized detection or (2) the fluorescence can be imaged by the whole animal imaging stystem providing macroscopic information. Results from internal localized excitation and external whole body detection indicate the potential for imaging bacterial infections down to 100 colony forming units. This novel imaging technique has the potential to allow for functional studies, enhancing the ability to assess new therapeutic agents.Item Structure, function, and inhibition of enoyl reductases(2009-05-15) Kuo, Mack RyanMalaria and tuberculosis constitute two of the world?s deadliest infectious diseases. Together, they afflict over one third of the world?s population. Once thought of as one of a group of nearly vanquished diseases only 50 years ago, malaria and tuberculosis have experienced renewed prominence due to issues such as multi-drug resistance and a lack of responsiveness by the global community. Fatty acid biosynthesis has been shown to be an essential pathway to the causative organisms of malaria and tuberculosis. One integral component of the fatty acid biosynthesis pathway, enoyl acyl-carrier-protein (ACP) reductase, has repeatedly been validated as an appropriate drug target in other organisms. The 2.4 ? crystal structure of the enoyl-ACP reductase from the human parasite Plasmodium falciparum (PfENR) reveals a nucleotide-binding Rossmann fold, as well as the identity of several active site residues important for catalysis. The 2.43 ? crystal structure of PfENR bound with triclosan, a widely utilized anti-bacterial compound, provides new information concerning key elements of inhibitor binding. Applying knowledge attained from these initial crystal structures, several triclosan derivatives were synthesized, and subsequently PfENR:inhibitor co-crystal structures were determined to extend our knowledge of protein:inhibitor interactions within the active site. Additionally, the crystal structures of the enoyl-ACP reductase from the mouse parasite Plasmodium berghei (PbENR), in apo-form and in complex with triclosan, were refined to 2.9 ? and 2.5 ? resolution, respectively. These structures confirm the structural and active site conservation between the human and mouse parasite enoyl-ACP reductases, suggesting that utilizing a murine model for in vivo testing of promising inhibitors is viable. The 2.6 ? crystal structure of the enoyl-ACP reductase from Mycobacterium tuberculosis (InhA) in complex with triclosan reveals a novel configuration of triclosan binding, where two molecules of triclosan are accommodated within the InhA active site. Finally, high-throughput screening approaches using enoyl acyl-carrier-protein reductases as the targets were utilized to identify new lead compounds for future generations of drugs. The 2.7 ? crystal structure of InhA bound with Genz-10850 confirms the value of this technique.Item The Biochemical Investigation and Isolation of Small Molecule Inhibitors for Two Essential Proteins of Mycobacterium tuberculosis H37Rv: IspD and Wag31(2014-06-12) Joseph, SoniaTuberculosis is one of the leading causes of death due to infectious disease. The causative agent, Mycobacterium tuberculosis, is a facultative intracellular parasite with a slow regeneration rate. Though there is a decline in the overall TB incidence since 2005, the emergence of resistant strains that are impervious to existing treatment regimens make the discovery and development of new drug leads crucial. To this end, exploiting key differences between the biology of the host and the pathogen can generate novel lead molecules with minimal side effects. This thesis details the study of two proteins that are essential for the survival of M. tuberculosis (M.tb) but are not present in the host, making them potential drug targets. The first protein, IspD (2-C-methyl-D-erythritol-4-phosphate (MEP) cytidyl transferase), is a part of the non-mevalonate pathway for the synthesis of isoprenoids and catalyzes the condensation of MEP and cytidine triphosphate (CTP) to form 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) and pyrophosphate (PPi). A medium-throughput enzyme assay was developed to identify inhibitors for this protein. It was screened against 3550 compounds drawn from five different M. tb whole-cell active small molecule libraries generating a total of five hits. These molecules were then assessed for their potency against IspD as measured by their IC50, their activity against M. tb whole cells and their cytotoxicity. Of the five hits, two compounds inhibited M. tb whole cell growth at a concentration below 50 ?M while exhibiting no general cytotoxicity to human dermal fibroblasts (HDF). They each had an IC50 of 26.1 ?M and 37.8?M and preliminary SAR studies were performed on the latter. These molecules could prove to be a viable starting point for the rational design of IspD inhibitors. The second protein, Wag31 is a cell division associated protein that regulates mycobacterial cell size and septum formation. Wag31 exhibited a propensity for gel formation both alone and in association with other cellular proteins. A purification strategy was developed to circumvent this tendency and generate soluble protein. It was found that mutations within the wag31 protein coding sequence conferred resistance to a whole-cell active small molecule (MIC99=6.25 ?g/ml) in both M. tuberculosis and M. smegmatis. Moreover, all of the discovered mutations were clustered within the C-terminal coiled-coil domain of the protein. It was established that this compound binds to Wag31 and seems to shift the equilibrium of the protein solution towards gel formation. The mutated protein does not form gel and seems to bind to the compound at a significantly reduced rate. To further confirm that Wag31 was indeed the target of this small molecule, whole cell viability assays were performed to establish whether the over-expression of Wag31 in M. smegmatis would shift the EC50 and MIC99 values. Wag31 over-expression reduced both the EC50 and MIC99 values providing further proof that Wag31 is the target for this compound. The compound appears to act by shifting the equilibrium of the protein towards a gelatinous state which proves inhibitory for cell growth.Item Using High Throughput Screening to Acquire Promising Drug Candidates Against Mycobacterium tuberculosis(2011-07-27) LaiHing, Steven 1983-Mycobacterium tuberculosis currently affects 1/3 of the world's population. Over the past 20 years tuberculosis has become more resistant to all front line drugs used against it. Because of this, the threat of Multi Drug Resistant (MDR-TB) and Extensive Drug Resistant (XDR-TB) strains has grown greater and emerges as a world health issue. Modern travel has greatly facilitated the spread of these resistant strains. For this reason, more front line drugs are urgently needed in the fight against TB infection. High Throughput Screening can be used to both find and analyze promising drug candidates. Using automation, thousands of compounds can be tested against an attenuated strain of Tuberculosis and separate the promising compounds from the ineffective ones. We have found a select subset of candidates from our custom built ~52,000 compound diversity library which show potent inhibitory effects against our mc^2-7000 attenuated TB strain. These compounds have IC50s ranging from 1.98 muM to 11.3 muM and should be considered for future development as drugs against TB. Among the active compounds, we have found enrichment for hydrazines, as well as representation of several chemi-classes including quinolones. To determine possible toxicity issues, we have also vetted these compounds against a strain of human lymphoma; all of our promising compounds meet the threshold for non-cytoxicity.