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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
201

Regulation and trafficking of the iron export protein, ferroportin1, in Mycobacterium tuberculosis-infected macrophages

Van Zandt, Kristopher Edward, January 2007 (has links)
Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 93-119).
202

Structural Studies On Mycobacterial RecA And RuvA

Rajan Prabhu, J 01 1900 (has links)
Homologous recombination is a fundamental cellular process evolved to maintain genomic integrity and to generate genetic diversity. It plays a crucial role in DNA repair, correct segregation of meiotic chromosomes and resumption of the stalled replication forks. In vitro, the homologous recombination pathway is kinetically separable into a four step process involving initiation, homologous pairing, branch migration and junction resolution. The process of pairing and strand exchange between two homologous double-stranded DNA molecules leads to the formation of an intermediate structure called the Holliday junction (HJ). The crucial enzyme involved in this step in bacteria is RecA. In eubacteria, the junction is processed by three proteins, collectively referred to as the RuvABC protein complex. RuvA binds to the HJ, while RuvB, a helicase, binds to the RuvA-HJ complex and pumps the duplex DNA thus facilitating branch migration. The work reported here is concerned with structural studies on mycobacterial RecA and RuvA. X-ray crystallography was used to solve the protein crystal structures. The hanging drop vapour diffusion method was used for crystallization in all cases. X-ray intensity data were collected on a MAR Research imaging plate mounted on a Rigaku RU200 X-ray generator except for two data sets collected using synchrotron radiation. The data were processed mostly using Mosflm and Scala and few data sets were processed using the HKL program suite. The molecular replacement method using programs Phaser and AMoRe was used for structure solution. Structure refinements were carried out using programs CNS and PHENIX. Model building was performed using COOT and O. PROCHECK, MOLPROBITY, ALIGN and NACCESS were used for structure validation and analysis of the refined structures. Mycobacterium smegmatis RecA (MsRecA) and its nucleotide complexes crystallize in three different, but closely related, forms characterized by specific ranges of unit cell dimensions. The six crystals discussed in the earlier part of the thesis and the five reported earlier, all grown under the same or very similar conditions, belong to these three forms, all in space group P61. They include one obtained by reducing the relative humidity around the crystal. In all crystals, RecA monomers form filaments around a 61 screw axis. Thus, the c-dimension of the crystal corresponds to the pitch of the RecA filament. As reported in the case of E.coli RecA, the variation in the pitch among the three forms correlate well with the motion of the C-terminal domain of the RecA monomers with respect to the main domain. The domain motion is compatible with formation of inactive as well as active RecA filaments involving monomers with a fully ordered C-domain. It does not appear to influence the movement upon nucleotide-binding of the switch residue Gln 196, which is believed to provide the trigger for transmitting the effect of nucleotide-binding to the DNA-binding region. Interestingly, partial dehydration of the crystal results in the movement of the residue, in a way similar to that caused by nucleotide-binding. The ordering of the DNA-binding loops L1 and L2, which present an ensemble of conformations, is also unaffected by domain motion. The conformation of loop L2 appears to depend upon nucleotide-binding presumably on account of the movement of the switch residue which forms part of the loop. The conformations of loops L1 and L2 are correlated and have implications to intermolecular communications within the RecA filament. The structures resulting from different orientations of the C-domain and different conformations of the DNA-binding loops appear to represent snapshots of the RecA molecule at different phases of activity and provide insights into the mechanism of action of RecA. Crystal structures of mutants of MsRecA involving changes of Gln 196 from glutamine to alanine, asparagine and glutamic acid, wild type MsRecA and several of their nucleotide complexes were subsequently determined using mostly low temperature and partly room temperature X-ray data. At both the temperatures, nucleotide binding results in a movement of Gln 196 towards the bound nucleotide in the wild type protein. This movement is abolished in the mutants, thus establishing the structural basis for the triggering action of the residue in terms of the size, shape and the chemical nature of the side chain. The 25 crystal structures reported in this thesis, along with the 5 MsRecA structures reported earlier, provide further elaboration of the relation among the pitch of the `inactive´ RecA filament, the orientation of the C-terminal domain with respect to the main domain and the location of the switch residue. The low temperature structures define one extreme of the range of positions the C-domain can occupy. The movement of the C-domain is correlated to those of the LexA binding loop and the loop that connects the main and the N-terminal domains. These elements of molecular plasticity are made use of in the transition to the `active´ filament, as evidenced by the recently reported structures of RecA-DNA complexes. The available structures of RecA resulting from X-ray and electron microscopic studies appear to represent different stages in the trajectory of the allosteric transformations of the RecA filament. This work contributes to the description of the early stages of this trajectory and provides insights into structures relevant to the later stages. The interesting results observed in the case of MsRecA prompted similar studies on the RecA from Mycobacterium tuberculosis (MtRecA). In this study, the crystals were grown at slightly different conditions and examined at different relative humidities and temperatures. Surprisingly, in spite of the 92% sequence identity between the two proteins, the structures indicated MtRecA to be substantially less plastic than MsRecA. The crystal structures do not provide an obvious explanation for this difference. Further studies are warranted to explain the molecular basis of the difference. RuvA, along with RuvB, is involved in branch migration of heteroduplex DNA in homologous recombination. The structures of four crystal forms of RuvA from Mycobacterium tuberculosis (MtRuvA) have been determined. The RuvB-binding domain is cleaved off in one of them. Detailed models of the complexes of octameric RuvA from different species with the Holliday junction have also been constructed. A thorough examination of the structures determined as part of the doctoral programme and those reported earlier bring to light the hitherto unappreciated role of the RuvB-binding domain in determining inter-domain orientation and oligomerization. These structures also permit an exploration of the interspecies variability of structural features such as oligomerization and the conformation of the loop that carries the acidic pin, in terms of amino acid substitutions. These models emphasize the additional role of the RuvB-binding domain in HJ binding. This role along with its role in oligomerization could have important biological implications. In addition to the work on RecA and RuvA, which forms the body of the thesis, the author was also involved in a structural bioinformatics study in which several carbohydrate binding proteins were probed to identify common minimum principles required for binding mannose, glucose and galactose. The study, presented in an Appendix, identified interactions that were specific to particular sugars, leading to individual fingerprints. These fingerprints were then used for exploring lead compounds, using a fragment based approach. This investigation helped the author to familiarize himself with the analysis of protein structures and ligand design based on them.
203

Structural and Functional Studies of Mycothiol Biosynthesis Precursor Enzyme in Mycobacterium tuberculosis

Zhu, Wan Wen 2011 August 1900 (has links)
MshA is a glycosyltransferase that synthesizes the precursor of mycothiol, a low-molecular-weight thiol found exclusively in Actinomycetes, including the virulent pathogen Mycobacterium tuberculosis (Mtb). The structure of MshA from Mtb (herein coined as TbMshA) and its complex with uridine diphosphate N-acetyl-glucosamine (UDP-GlcNAc) have been solved to resolutions of 2.32 A and 2.89 A respectively. Both structures form two monomers in the asymmetric unit cell and exhibit typical beta/alpha/beta Rossmann folds. Upon binding of UDP-GlcNAc, the C-terminal domain of TbMshA undergoes conformational changes in order to interact with UDP-GlcNAc at the binding site. In addition, ligand-bound TbMshA structure enables the identification of critical residues for enzymatic interactions, especially the residue Glu-353 (E353) at the active site that is believed to serve as a nucleophile in the sugar transfer of TbMshA. In order to verify this, a mutant of TbMshA with a single amino acid mutation from glutamate to glutamine at residue 353 is generated. The mutant (E353Q) has shown reduced enzyme activity by more than four-fold compared to the wild-type TbMshA (Vmax for wild-type is 0.17 plus/minus 0.02 microM sec^-1, whereas Vmax for E353Q is 0.04 plus/minus 0.01 microM sec-1). The kcat/Km for wild-type TbMshA (3.5 plus/minus 1.1 * 10^3 M^-1 sec^-1) is an order of magnitude higher than that of the mutant (0.3 plus/minus 0.1 * 10^3 M^-1 sec^-1), indicating the catalytic efficiency is greatly suppressed by the mutation. Mass spectrometry data also reveals that E353Q is unable to form the product of the reaction catalyzed by the wild-type TbMshA. These findings suggest the important role of Glu-353 in the structure and activity of TbMshA.
204

Relaciones filogenéticas entre algunos Telmatobinidos (Anura, Leptodactylidae, Telmatobiinae) de Perú basado en la morfología de los estados larval y adulto

Pandia Fajardo, Elma Alcira January 2006 (has links)
La finalidad del presente estudio fue evaluar el medio de cultivo Löwenstein - Jensen modificado con nitrato de potasio (KNO3) para el diagnóstico de Mycobacterium tuberculosis, compararando la sensibilidad y especificidad de este medio modificado con el medio Löwenstein - Jensen convencional y evaluando los factores importantes que influyen en el proceso como por ejemplo el tiempo. En el presente estudio, se evaluó un total de 120 muestras provenientes de pacientes diagnosticados con tuberculosis pulmonar por baciloscopía, las muestras (esputo, lavado bronquial, aspirado bronquial, aspirado gástrico) se trataron por el método Petroff modificado para su descontaminación y homogenización y se sembraron en los medios Löwenstein - Jensen convencional y modificado con nitrato de potasio al 35%. Luego estos cultivos fueron incubados a 37°C durante 7,10, 14 días, después de los cuales se determinó la reducción de nitratos a nitritos. El nitrito producido por Mycobacterium tuberculosis permitió identificar la presencia de la bacteria empleando el reactivo de Peter Griess, detectado por medio de un viraje de color del medio de cultivo. En el medio Löwenstein - Jensen modificado, se determinó que la sensibilidad era del 41% y la especificidad del 100%, con un valor predictivo positivo de 100%, y valor predictivo negativo de 34%. Al comparar estadísticamente la sensibilidad y especificidad de la prueba en el medio modificado y el cultivo convencional o Estándar de oro, se observó que los factores predominantes en el proceso de evaluación son: tipo (esputo, lavado bronquial, aspirado bronquial, aspirado gástrico) y calidad de la muestra (Carga Bacilar), obtenidas adecuadamente. Los resultados fueron obtenidos en su mayoría a los 10 días, con un promedio ponderado de 12 días para el diagnóstico de Mycobacterium tuberculosis. El estudio realizado justifica el empleo del medio Löwenstein - Jensen modificado conteniendo nitrato de potasio (KNO3), presentándose como una alternativa para el diagnóstico presuntivo de Mycobacterium tuberculosis. / The tuberculosis in anyone of his manifestations, one becomes of without a doubt the public- health problems more important at the present time and it has been a diagnostic shoot's quest and it is, one join of the objective main things in many parts worldly, most of all in countries as the our that he presents high incidence rates and whose more population once was affected does not count on the economic means to accede to anyone of the diagnostic- shoot methods existents. The present study's purpose was to evaluate Löwensein-Jensen's midway modified with nitrate reductasa in order to the pulmonary tuberculosis's diagnosis, Mycobacterium tuberculosis's identification, comparing sensibility and modified specificity of this midway with the cultivation conventional Löwenstein-Jensen and evaluating the mains factors that have influence in the process for example theTime. In the present study evaluated 120 samples coming from patients diagnosed with pulmonary tuberculosis for baciloscopy; the samples them tried to him for the method Petroff once was modified in order to his decontamination and homogenization before being sown in the Löwensein-Jensen's midway conventional and modified , with potassium nitrate to 37 per cent.. The cultures were incubates went to 37 ºC for 7, 10, 14 days; then the nitrate reduction was suggesting growth to nitrite. To positive reaction was detected by means of a color turn. In the Löwensein-Jensen's midway conventional obtained a sensibility of 41 per cent and specificity of 100 per cent in the Löwenstein-Jensen midway modificated, with a positive value once of 100 per cent and negative value once of 34 per cent. To the comparing statistically sensibility and specificity of the essay test with nitrate reductasa and the conventional cultivation or Gold Standard heeded that the prevailing factors in the evaluation process are: the type and quality of the sample quality (charges Bacilar), fitting to obtain. The results were obteined aftermaths in the main to the 10 days, with a 12 days average prudent in order to TB'S diagnosis – Pulmonar The study once was accomplished justifies test's job Löwenstein – Jensen modified with potassium of Nitrate (KNO3), encountering as an alternative in order to the Mycobacterium tuberculosis's diagnosis.
205

Étude de la tuberculose chez l'éléphant importance en parc zoologique /

Delnatte, Pauline Ducos de Lahitte, Jacques January 2008 (has links) (PDF)
Reproduction de : Thèse d'exercice : Médecine vétérinaire : Toulouse 3 : 2008. / Titre provenant de l'écran titre. Bibliogr. p. 207-217.
206

Molecular characterization of isoniazid-resistant mycobacterium tuberculosis in Hong Kong

Woo, Wai-lan., 胡慧蘭. January 2005 (has links)
published_or_final_version / Medical Sciences / Master / Master of Medical Sciences
207

Direct detection of rifampin-resistant mycobacterium tuberculosis in clinical specimens by DNA sequencing

梁秀敏, Leung, Sau-man. January 2001 (has links)
published_or_final_version / Medical Sciences / Master / Master of Medical Sciences
208

A metabolomics approach for characterising tuberculosis / Ilse Olivier

Olivier, Ilse January 2012 (has links)
In 2001, the WHO declared tuberculosis (TB) a global emergency, as one third of the world‟s population suffered from latent M. tuberculosis infection. Today, a decade later, millions of people still die worldwide as a result of this disease. This growing TB incidence may be ascribed to a variety of reasons, including, amongst others, the inadequacies associated with the currently available diagnostic methods and TB treatment regimes, especially when considering the growing MDR-TB and HIV epidemics. This study investigated the potential of metabolomics as a tool for characterising TB and various TB-causing bacteria, allowing for a better understanding of TB disease mechanisms, which may ultimately lead to improved diagnostic and treatment regimens. Firstly, we investigated the potential of a fatty acid, metabolomics approach to characterise various cultured Mycobacterium species. For this exploration, three fatty acid extraction procedures, prior to GC-MS analyses, were compared based on their respective repeatability and extraction capacities. Using the data obtained from the analyses done with the most optimal extraction approach (the modified Bligh-Dyer method), multivariate statistical analyses were able to differentiate between the various Mycobacterium species at a detection limit of 1 x 103 bacterial mL-1, in 16 hours. Subsequently, the compounds best describing the variation between the sample groups were identified as potential metabolite markers and were discussed in the light of previous studies. The most optimal GC-MS, fatty acid metabolomics approach, mentioned above, was then applied to analyse and characterise a wild-type M. tuberculosis parent strain and two rifampicinresistant conferring rpoB mutants (S522L and S531L). Due to the variation in their fatty acid profiles, a clear differentiation was achieved between these M. tuberculosis sample groups, and those metabolites contributing most to this variation were identified as metabolite markers characteristic for rifampicin-resistance. The altered metabolite markers detected in the rpoB mutants propose a decreased synthesis of various 10-methyl branched-chain fatty acids and cell wall lipids, and an increased use of the shorter-chain fatty acids and alkanes as alternative carbon sources. Furthermore, the rpoB S531L mutant, previously reported to occur in well over 50% of all clinical rifampicin-resistant M. tuberculosis strains, showed a better capacity for using these alternative energy sources, in comparison to the less frequently detected rpoB S522L mutant. The developed fatty acid GC-MS metabolomics approach was then successfully adapted in order to improve its speed, cost and complexity. This improved fatty acid extraction method was furthermore compared to another, similar approach (total metabolome extraction method), developed for the extraction of a much wider variety of compounds, prior to GC-MS and statistical data analyses. Although both these methods show promise for bacterial characterisation using matabolomics, the total metabolome extraction method proved the better of the two methods because it is comparatively simpler, faster (taking less than 4 hours), more repeatable, better differentiates between sample groups due to less within group variation, has a lower detection limit, and isolates a wider variety of biologically relevant metabolites (as opposed to fatty acids alone). We, furthermore, identified and described the occurrence of those compounds, extracted by both methods, which contribute most to the variation between the bacterial groups, in order to validate these methods for metabolomic applications and the isolation of compounds with biological relevance. In order to evaluate the potential of this developed metabolomics approach for application to biological samples other than bacteriological cultures, it was adapted for the direct analyses of complex sputum samples. For this application, four sputum pre-extraction preparation methods, including three standard Mycobacterium cell isolation procedures (Sputolysin, NALC-NaOH, and NaOH) and a fourth, applying only a simple ethanol homogenisation step, prior to direct sputum extraction, were compared. Of these methods, the ethanol homogenisation method proved to have the best comparative extraction efficiency, repeatability and differentiation capacity, when used in combination with the previously developed metabolomics methods. Subsequently, when applying this approach to patient collected sputum samples, a set of metabolite markers, differentiating the TB-positive from the TB-negative samples, were identified. These markers could directly be linked to: 1) the physical presence of the M. tuberculosis in these samples; 2) changes in the bacterial metabolome due to in vivo growth conditions and; 3) changes in the human metabolome due to pulmonary M. tuberculosis infection. In addition to the proposal of a number of new hypotheses, explaining various mechanisms of TB and drug-resistant TB, the mapping of the newly identified metabolite markers to known metabolic pathways led to the confirmation of various previously suggested metabolic pathways and alterations thereof due to an assortment of perturbations. Therefore, this study significantly contributes to the characterisation of various TB causing bacteria, rifampicin-resistant M. tuberculosis strains and the TB disease state, which may in future lead to the development of innovative TB vaccination, diagnostic and treatment protocols. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2012
209

A metabolomics approach for characterising tuberculosis / Ilse Olivier

Olivier, Ilse January 2012 (has links)
In 2001, the WHO declared tuberculosis (TB) a global emergency, as one third of the world‟s population suffered from latent M. tuberculosis infection. Today, a decade later, millions of people still die worldwide as a result of this disease. This growing TB incidence may be ascribed to a variety of reasons, including, amongst others, the inadequacies associated with the currently available diagnostic methods and TB treatment regimes, especially when considering the growing MDR-TB and HIV epidemics. This study investigated the potential of metabolomics as a tool for characterising TB and various TB-causing bacteria, allowing for a better understanding of TB disease mechanisms, which may ultimately lead to improved diagnostic and treatment regimens. Firstly, we investigated the potential of a fatty acid, metabolomics approach to characterise various cultured Mycobacterium species. For this exploration, three fatty acid extraction procedures, prior to GC-MS analyses, were compared based on their respective repeatability and extraction capacities. Using the data obtained from the analyses done with the most optimal extraction approach (the modified Bligh-Dyer method), multivariate statistical analyses were able to differentiate between the various Mycobacterium species at a detection limit of 1 x 103 bacterial mL-1, in 16 hours. Subsequently, the compounds best describing the variation between the sample groups were identified as potential metabolite markers and were discussed in the light of previous studies. The most optimal GC-MS, fatty acid metabolomics approach, mentioned above, was then applied to analyse and characterise a wild-type M. tuberculosis parent strain and two rifampicinresistant conferring rpoB mutants (S522L and S531L). Due to the variation in their fatty acid profiles, a clear differentiation was achieved between these M. tuberculosis sample groups, and those metabolites contributing most to this variation were identified as metabolite markers characteristic for rifampicin-resistance. The altered metabolite markers detected in the rpoB mutants propose a decreased synthesis of various 10-methyl branched-chain fatty acids and cell wall lipids, and an increased use of the shorter-chain fatty acids and alkanes as alternative carbon sources. Furthermore, the rpoB S531L mutant, previously reported to occur in well over 50% of all clinical rifampicin-resistant M. tuberculosis strains, showed a better capacity for using these alternative energy sources, in comparison to the less frequently detected rpoB S522L mutant. The developed fatty acid GC-MS metabolomics approach was then successfully adapted in order to improve its speed, cost and complexity. This improved fatty acid extraction method was furthermore compared to another, similar approach (total metabolome extraction method), developed for the extraction of a much wider variety of compounds, prior to GC-MS and statistical data analyses. Although both these methods show promise for bacterial characterisation using matabolomics, the total metabolome extraction method proved the better of the two methods because it is comparatively simpler, faster (taking less than 4 hours), more repeatable, better differentiates between sample groups due to less within group variation, has a lower detection limit, and isolates a wider variety of biologically relevant metabolites (as opposed to fatty acids alone). We, furthermore, identified and described the occurrence of those compounds, extracted by both methods, which contribute most to the variation between the bacterial groups, in order to validate these methods for metabolomic applications and the isolation of compounds with biological relevance. In order to evaluate the potential of this developed metabolomics approach for application to biological samples other than bacteriological cultures, it was adapted for the direct analyses of complex sputum samples. For this application, four sputum pre-extraction preparation methods, including three standard Mycobacterium cell isolation procedures (Sputolysin, NALC-NaOH, and NaOH) and a fourth, applying only a simple ethanol homogenisation step, prior to direct sputum extraction, were compared. Of these methods, the ethanol homogenisation method proved to have the best comparative extraction efficiency, repeatability and differentiation capacity, when used in combination with the previously developed metabolomics methods. Subsequently, when applying this approach to patient collected sputum samples, a set of metabolite markers, differentiating the TB-positive from the TB-negative samples, were identified. These markers could directly be linked to: 1) the physical presence of the M. tuberculosis in these samples; 2) changes in the bacterial metabolome due to in vivo growth conditions and; 3) changes in the human metabolome due to pulmonary M. tuberculosis infection. In addition to the proposal of a number of new hypotheses, explaining various mechanisms of TB and drug-resistant TB, the mapping of the newly identified metabolite markers to known metabolic pathways led to the confirmation of various previously suggested metabolic pathways and alterations thereof due to an assortment of perturbations. Therefore, this study significantly contributes to the characterisation of various TB causing bacteria, rifampicin-resistant M. tuberculosis strains and the TB disease state, which may in future lead to the development of innovative TB vaccination, diagnostic and treatment protocols. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2012
210

Protein-ligand interactions of arylamine N-acetyltransferase from Mycobacterium smegmatis

Brooke, Edward W. January 2003 (has links)
Tuberculosis is the world's largest cause of death from an infectious agent. Treatment is by an extended period of combination chemotherapy. Drug resistance is an increasing problem in tuberculosis therapy, particularly to the frontline anti-tubercular drug isoniazid (INH). Recombinant arylamine N-acetyltransferase (NAT) of Mycobacterium tuberculosis N-acetylates INH using the cofactor Acetyl Coenzyme A. NAT from M. tuberculosis is a polymorphic enzyme and also acetylates INH in vivo. Acetylated INH is inactive therapeutically against M. tuberculosis both in vivo and in vitro. The acetylation of isoniazid in the mycobacterial cell may compete with the activation of INH by the catalase-peroxidase, katG, and hence contribute to INH resistance in clinical isolates. Inhibition of NAT in M. tuberculosis may thus increase the efficacy of INH therapy. A novel assay based around the detection of free Coenzyme A released during the acetylation reaction was used to determine the substrate specificity of recombinant NAT from the related Mycobacterium M. smegmatis (MSNAT). A relationship was observed between the lipophilicity of simple arylamine substrates and the rate of acetylation by MSNAT. Several MSNAT substrates possess antibacterial activity. The assay could also be used to screen compound libraries for MSNAT inhibitors. Synthesis of seventeen thiazolidinedione sultams in collaboration with Dr.Vickers (Dyson Perrins), identified as weak inhibitors of MSNAT, gave a minimum competitive inhibitory constant of 14μM. Screening a library of 5,074 drug-like compounds for inhibition of MSNAT identified thirteen compounds with semi-maximal inhibition constants (IC<sub>50</sub>) of below 10μM. Based on this, fifteen maleimides were synthesised and were irreversible inhibitors of MSNAT with submicromolar potency. Similarly, ninety-six aminothiazoles were synthesised by Dr. Vickers and were uncompetitive inhibitors of MSNAT with a minimum IC<sub>50</sub> of 1.5μM. The most potent aminothiazole showed no effect on the growth of M. smegmatis or M. bovis BCG or the sensitivity of the bacteria to isoniazid. However the aminothiazoles were shown not to penetrate the cells.

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