Orientador: Marcelo de Carvalho Ramos / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-09T07:44:31Z (GMT). No. of bitstreams: 1 Panunto_AlessandraCosta_D.pdf: 4929023 bytes, checksum: e09c1fb4f0b9ff64c05e8133763291d7 (MD5) Previous issue date: 2007 / Resumo: O M. Avium é um microrganismo oportunista e sua infecção é feeqüentemente encontrada em pacientes com aids no Brasil, apesar do largo uso da quimioterapia antiretroviral altamente efetiva. Este estudo documenta a relevância desse problema. Dentro de uni número significante de pacientes (n=39) infectados com o M. avium, os isolados puderam ser recuperados de uma variedade de espécimes clínicos. Todos os isolados (n=45) foram tipados pela técnica de RFLP usando a seqüência 1S1245. A maioria dos pacientes (n=35) eram infectados pelo HIV. Somente duas cepas não puderam ser tipadas por causa da ausência da seqüência detectável pela 1S1245. Nas 43 cepas restantes os "blots" apresentaram de 6 a 23 bandas. Uma média de 17 seqüências foram observadas para cada cepa. Para alguns pacientes, mais de um isolado pode ser recuperado. Em dois pacientes deste grupo com doença disseminada, o M. avium pode ser recuperado mais de uma vez. De cada paciente, pelo menos duas amostras com diferentes genótipos foram recuperadas de locais estéreis, indicando que eles tinham infecções policlonais. Esses achados têm sido relatados por outros autores. Em um estudo recente, SAAD et aI., 2000, demonstrou que isolados de infecções policlonais e diferentes "fmgerprints" podem apresentar diferentes suscetibilidade antimicrobiano. Quatro "clusters" de pacientes puderam ser identificados. O maior "cluster" foi composto de oito pacientes. Estes resultados indicam que um mecanismo de transmissão recente ocorreu. A fonte de contaminação desses microrganismos não pode ser determinada. Assim, a transmissão pessoa a pessoa não apresentou uma importância significativa na transmissão desse microrganismo. Nós supomos que esses pacientes adquiriram o microrganismo de fontes hospitalares como água, alimento ou mesmo do ambiente / Abstract: not informed. / Doutorado / Ciencias Basicas / Doutor em Clínica Médica

A Mycobacterium abscessus subspécie abscessus é um pesadelo quando envolvida em infecção pulmonar que são incuráveis, a despeito do uso de antimicrobianos com atividade in vitro, caso o tratamento não inclua a ressecção cirúrgica da área afetada. É a micobactéria patogência de crescimento rápido mais frequentemente isolada de culturas de sítios pulmonares. Há um número reduzido de opções terapêuticas para o tratamento dessas infecções, e é ainda mais reduzido o número de antimicrobianos que atingem concentrações terapêuticas no compartimento intracelular, em particular no fagossomo. O número limitado de antimicrobianos disponíveis para tratamento apontam a necessidade de determinação do perfil de susceptibilidade frente a antimicrobianos isolados e em combinação, nos compartimentos intra e extracelular. Os objetivos deste estudo foram avaliar: a sensibilidade de M. abscessus estruturadas em biofilmes e presentes no interior dos macrófagos; a ocorrência de sinergismo quando da associação entre fármacos, inibidores de betalactamase e o anti-inflamatório. As combinações entre os antimicrobianos foram apenas indiferente quanto ao FIC e a atividade dos fármacos em biofilme e em macrófagos é bacteriostático. / Mycobacterium abscessus subspecies abscessus is a nightmare when involved in lung infection that is incurable, despite the use of antibiotics with in vitro activity, if the treatment does not include surgical resection of the affected area. It is a MCR - rapidly growing mycobacteria pathogenic most frequently isolated from cultures of lung sites. There are a small number of therapeutic options for the treatment of such infections is further reduced and the number of drugs that reach therapeutic concentrations in the intracellular compartment, particularly in the phagosome. The limited number of antimicrobials available for treatment indicate the need for determining the susceptibility profile against antimicrobials alone and in combination, in the intra and extracellular compartments. The objectives of this study were sensitivity of MCR structured biofilms and present in macrophages, the occurrence of synergism when the association between drugs, beta-lactamase inhibitors and anti-inflammatory. Combinations of antimicrobials were just indifferent and the activity of drugs on biofilms and macrophages was bacteriostatic.

A tuberculose continua sendo uma importante doença infecciosa, tanto nos humanos quanto nos animais, com índices de morbidade e mortalidade significativos e perdas econômicas em todo o mundo. A tuberculose bovina é uma doença infecciosa causada pela bactéria Mycobacterium bovis, que gera perdas na produção nos rebanhos infectados, sendo também considerada uma importante zoonose. Os métodos de diagnóstico direto têm fundamental importância para um sistema de vigilância para a tuberculose bovina e a agregação de métodos moleculares, notadamente aqueles que têm aplicação em epidemiologia, traz maior precisão diagnóstica para esses sistemas. Dentre as técnicas moleculares, destacam-se o TB Multiplex PCR, o RD Multiplex PCR e o Multispacer Sequence Typing, para a identificação dos isolados e o Variable Number Tandem Repeat (VNTR) e o Spoligotyping, como técnicas de fingerpint de M. bovis. Assim, o presente estudo teve como objetivo a identificação molecular de amostras oriundas de várias regiões do Brasil utilizando estes padrões de técnicas moleculares. Os espoligotipos identificados em maior abundância foram o SB0121, o qual apresentou-se amplamente distribuído entre as amostras, seguido pelo SB0295, SB1380, SB0140 e SB1050. Além disso, foram detectados quatro perfis nunca antes descritos na literatura, sendo que um deles foi o terceiro mais frequente entra as amostras pesquisadas. Os resultados observados neste trabalho demonstraram ainda que a tipagem pelo MIRU-VNTR revelou-se superior ao Spoligotyping para discriminar os isolados. Nesta perspectiva, acredita-se que as pesquisas moleculares voltadas a identificação de micobactérias, aliadas as técnicas epidemiológicas tradicionais, possam melhorar sensivelmente a performance dos sistemas de vigilância para tuberculose bovina no Brasil. / Tuberculosis remains a major infectious disease in both humans and animals, with morbidity and mortality and significant economic losses. Bovine tuberculosis is an infectious disease caused by Mycobacterium bovis, with yield losses in infected herds and is also considered an important zoonosis. The methods of direct diagnosis are important for a surveillance system for bovine tuberculosis and aggregation of molecular methods brings greater diagnostic accuracy for these systems, especially those that have application in epidemiology. Among them, TB Multiplex PCR, RD Multiplex PCR, and Multispacer Sequence Typing for the identification of strains, and Variable Number Tandem Repeat (VNTR) and Spoligotyping, for fingerprint of M. bovis. Thus, the present study aimed to identify molecular samples from different regions of Brazil using these molecular techniques. The most abundant were the spoligotype SB0121, which has become widely distributed among the samples, followed by SB0295, SB1380, SB0140, and SB1050. In addition, four profiles never before described in the literature were detected, one of which was the third most frequent. The results of this study also showed that the MIRU-VNTR typing has proved superior to Spoligotyping to discriminate the isolates. In this perspective, it is believed that the research focused on molecular identification of mycobacteria, combined traditional epidemiological techniques, can significantly improve the performance of surveillance systems for bovine tuberculosis in Brazil.

O Mycobacterium bovis é o agente causador da tuberculose em bovinos, zoonose que produz prejuízos para a produção de carne e leite em muitos países. Para apoiar estudos epidemiológicos no âmbito dos programas de controle, recentemente surgiram vários métodos de discriminação molecular de isolados de M. bovis. Os mais utilizados são o Spoligotyping, Mycobacterial Interspersed Repetitive Units (MIRU) e Exact Tandem Repeat (ETR), que apresentam diferentes poderes de discriminação. No presente estudo calculou-se a diversidade alélica para cada locus de MIRU e de ETR e o índice discriminatório de Hunter-Gaston (HGI) para o Spoligotyping, 10 MIRU e 3 ETR em 116 amostras de M. bovis isoladas de bovinos. Além disso, verificou-se se a capacidade de detectar agrupamentos espaciais de focos aumenta na medida em que é aumentado o poder discriminatório das análises moleculares. Comparou-se a utilização dessas três técnicas moleculares em análises espaciais para verificação de agrupamentos de focos da doença. A análise da diversidade alélica indicou que os MIRU 16, 26 e 27 e os ETR A, B e C foram os que apresentaram maior diversidade dentre os ensaiados. O HGI para cada uma das técnicas foi de: Spoligotyping = 0,738381; MIRU = 0,829835 e ETR = 0,825337. A associação desses métodos aumentou o poder discriminatório: Spoligotyping + MIRU = 0,930585; Spoligotyping + ETR = 0,931034; MIRU + ETR = 0,953373. O maior poder discriminatório foi alcançado quando as três técnicas foram associadas (HGI = 0,98051). Considerando as análises realizadas no presente estudo, o método inicial deveria ser o Spoligotyping, por diferenciar o M. bovis dos outros integrantes do complexo Mycobacterium tuberculosis. Como as associações do MIRU e do ETR com o Spoligotyping resultaram em HGI praticamente idênticos, depois do Spoligotyping, o método ETR parece ser a melhor escolha, pois é mais rápido e econômico do que o MIRU. Finalmente, o MIRU deve ser o último método a ser realizado. Assim, a escolha do método depende do poder discriminatório necessário para o objetivo em questão. Embora a capacidade de detectar agrupamentos espaciais de focos não tenha sido aumentada na medida em que cresceu o poder discriminatório das análises moleculares, a premissa continua válida. / Mycobacterium bovis is the causative organism of bovine tuberculosis, has zoonotic character and leads economic losses in livestock production in a lot of countries. To support the epidemiological researches on the disease control programs several molecular methods has been used to detect M. bovis strains. Among them, Spoligotyping, Mycobacterial Interspersed Repetitive Units (MIRU) and Exact Tandem Repeat (ETR) are the molecular analyses that present different powers of Mycobacterium bovis discrimination. In this study, allelic diversity of MIRU and ETR locus and Hunter-Gaston discriminatory index (HGI) were calculated for Spoligotyping, 10 MIRU and 3 ETR of 116 isolates of M. bovis from Brazilian bovines. The use of those three molecular techniques was compared in space analyses for verification of groupings of focuses of the disease. Besides, it was verified if the capacity to cluster of focuses increases in the measure in that the discriminatory power of the molecular analyses is increased. The analysis of the allelic diversity indicated that MIRU 16, 26, 27 and ETR A, B, C presented larger diversity among the rehearsed. The HGI for each individual technique was: Spoligotyping = 0.738381; ETR = 0.825337 and MIRU = 0.829835. The associations of these methods increased the discriminatory power: Spoligotyping + MIRU = 0.930585; Spoligotyping + ETR = 0.931034; MIRU + ETR = 0.953373. The greater discriminatory power was verified when the three techniques were associated (HGI = 0,98051). Considering the analyses performed, the initial method for differentiating M. bovis from the other species of the M. tuberculosis complex should be Spoligotyping. The association of MIRU and ETR with Spoligotyping resulted in HGI almost identical, after Spoligotyping, the ETR technique showed to be the best choice, due the shorter time to process and economic benefits when compared to MIRU. Finally, MIRU is the last method to be performed. Thus, the choice among the techniques depends on the discriminatory power necessary for the task. Although the capacity to detect clusters of focuses has not been increased in the measure in that it increased the discriminatory power of the molecular analyses, the premise continues valid.

Mycobacter avium complex serovars 4 and 20 were cultured in the presence of [3H] fucose, [3H]-methionine, and [3H]-mannose to specifically radiolabel the oligosaccharide of the glycopeptidolipid (GPL) antigens. Distribution of radioactivity in lipid was determined by thin-layer chromatographic methods. Examination of acid hydrolysates from radiolabeled antigens revealed that [3H]-methionine incorporated into methylated sugars in polar and apolar GPL components, whereas [3H]-mannose incorporated exclusively into the oligosaccharide of polar GPL antigens. Least incorporation of radiolabel into antigens was observed with [3H]-fucose. Use of radiolabeled serovar 4 antigens in macrophage uptake studies revealed maximum uptake to be slightly above 250 gg/ 3.2 x 105 cells. Timed experiments demonstrated that GPL antigens were relatively inert to degradation by resident peritoneal macrophages.

La tuberculose est une maladie contagieuse qui s’attaque habituellement aux poumons, mais parfois aussi à d’autres parties du corps, comme les reins, les ganglions et les os. La tuberculose tue près 1,8 millions de personnes chaque année dans le monde. Il y a par conséquent un besoin urgent de mettre des moyens analytiques pour détecter l’ADN de la bactérie Mycobacterium tuberculosis, responsable de la propagation de la tuberculose. La recherche développée dans le cadre de cette thèse consiste en l'élaboration d'un outil de diagnostic pour la détection d’ADN génomique de bactérie M.Tuberculosis après la lyse et amplification par PCR. Dans cette perspective, nous nous sommes intéressés au développement des biocapteurs basés sur des différentes morphologies de polypyrrole comme transducteur pour la détection électrochimique d'ADN du gène rpob de Mycobacterium tuberculosis. Une étude de l’impact des différentes morphologies sur les propriétés électriques des matériaux et également sur les performances des biocapteurs électrochimiques d’ADN a été effectuée. Nous avons aussi étudié l’effet de la fonctionnalisation par différentes chaine linéaire et ramifiée sur la structure des nanomatériaux de polypyrroles et leurs effets sur la quantité de biomolécules immobilisées. La stratégie adoptée pour le développement de ces matériaux est une construction du biocapteur réalisée étape par étape. Les différentes couches le constituant ont été caractérisées par différentes techniques de surface telles que les techniques électrochimiques et optiques, la microscopie électronique à balayage et la microscopie électronique à force atomique. Les propriétés des biocapteurs ont été suivies à travers les propriétés redox des groupes ferrocényles et naphtoquinone. La détection électrochimique de l’ADN cible évaluée avec ces biocapteurs à base de polypyrrole nanostructure montre une sensibilité de détection plus élevée que celle du biocapteur à base de polypyrrole couche compacte. Cela démontre le potentiel d'utilisation de la surface élevée des nanostructures polypyrrole comme transducteur et l’utilisation des approches de modification douce. Les biocapteurs ont été appliqués à la détection de l'ADN dans des échantillons réels d'ADN génomique de Mycobacterium tuberculosis et de l'ADN muté présentant la résistance de la rifampicine. Les biocapteurs basés sur des nanostructures de polypyrrole démontre une application potentielle dans la détection d'ADN et la capacité à discriminer le gène rpoB du mutant et pourrait être utilisé comme plate-forme dans la technologie des biocapteurs. / Tuberculosis is a contagious disease that usually attacks the lungs but can sometimes reach other parts of the body such as the kidneys, ganglia and bones. This disease is responsible for killing nearly 1.8 million people each year worldwide. Therefore, we have an urgent need to put analytical means to detect the DNA of the bacterium Mycobacterium tuberculosis, responsible for the spread of tuberculosis. The research developed in this thesis consists in the development of a diagnostic tool for the detection of genomic DNA of M.Tuberculosis bacteria after lysis and amplification by PCR. In this perspective, we focused on the development of biosensors based on different polypyrrole morphologies as transducers for the electrochemical detection of DNA of the rpob gene of Mycobacterium tuberculosis. A study of the impact of different morphologies on the electrical properties of materials as well as the performance of electrochemical DNA biosensors was carried out. We also studied the effect of the functionalization by different linear and branched chains on the structure of polypyrrole nanomaterials and their effects on the number of immobilized biomolecules. The strategy adopted for the development of these materials is a biosensor construction carried out step by step. The various layers constituting it have been characterized by different surface techniques such as electrochemical and optical techniques, scanning electron microscopy and atomic force electron microscopy. The properties of the biosensors were monitored through the redox properties of the ferrocenyl groups. The electrochemical detection of the target DNA evaluated with these biosensors based on polypyrrole nanostrcutrue compared to the results obtained with a biosensor based on polypyrrole compact layer shows a higher detection sensitivity.This demonstrates a potential for using the high surface area of polypyrrole nanostructures as a transducer and the use of soft modification approaches. The biosensors were applied to the detection of DNA in real samples of genomic DNA of Mycobacterium tuberculosis and mutated DNA with resistance to rifampicin. The biosensors based on polypyrrole nanostructures demonstrate potential application in DNA detection and the ability to discriminate the mutant rpoB gene and could be used as a platform in biosensor technology.

Thesis (M.Sc. (Chemistry)) -- University of Limpopo, 2020 / In an attempt to synthesise quinoxaline-ferrocene compounds with antimycobacterial activity; a series of quinoxaline alkynyl derivatives were successfully synthesised from 3- (quinoxalin-3-yl)prop-2-yn-1-ol 86A and 3-(6-chloroquinoxalin-2-yl)prop-2-yn-1-ol 86B. In this series compounds 87A – B, 90A – B, and 93A – C were intermediates obtained in an effort to synthesise quinoxaline-ferrocene compounds. Treatment of either 86A or 86B with various acid chlorides afforded quinoxaline alkynyl ester derivatives 97A - 97B. Within this series, two quinoxaline-ferrocene compounds 3-(quinoxalin-3-yl)prop-2-ynyl ferrocetate 97A-iv and 3-(6-chloroquinoxalin-2-yl)prop-2-ynyl ferrocetate 97B-iv were successfully incorporated with ferrocenoyl chloride and obtained in 42 - 43% yield. The reactions of 3-chloroquinoxaline-2-carbonyl chloride 99 with ferrocenyl alcohol and ferrocenyl amine were unsuccessful. However, 3-chloroquinoxaline-2-carbonyl ester 100A - C and amide 101A - D derivatives with various alcohols and amines were obtained. The structures of all the compounds were confirmed by spectroscopic analysis (NMR, FT-IR and HRMS). The synthesised compounds were all evaluated for preliminary in-vitro antimycobacterial activity. The results obtained exhibited compound 90B with the highest activity against Mtb H37RV strain at MIC90 of 1.13 µM, followed by 90A and 87A exhibiting MIC90 of 4.55 and 6.47 µM, respectively. The quinoxaline alkynyl ester derivatives were found to exhibit poor to good activity. Within this series, three compounds were found to exhibit antimycobacterial activity at MIC90 ˂ 20 µM with compound 97A-ii showing the highest activity at MIC90 of 16.18 µM, followed by 97A-i and 97B-iii showing MIC90 of 18.05 and 19.36 µM, respectively. From the two quixonaline-ferrocene compounds, compound 97A iv was found to exhibit antimycobacterial activity at MIC90 of 39.90 µM. However, compound 97B-iv was found to be inactive. The 3-chloroquinoxaline-2-carbonyl ester 100A - C and amide 101A - D derivatives were found to be inactive. However, compound 99-C was found to exhibit antimycobacterial activity at MIC90 of 40.66 µM. Compounds 86A, 86C, 87A and 90A were evaluated for in-vitro antiproliferative activity against cancer cell lines. The results of antiproliferative activity showed that compounds 86A and 87A exhibited excellent activity against A549 lung cancer cell lines. Compound 87A was found to be the most active against A549 cell line showing 50% viability-inhibition at 25 µM / National Research Foundation (NRF)

Please read the abstract in the 00 front of this document. / Thesis (PhD)--University of Pretoria, 2009. / Veterinary Tropical Diseases / unrestricted

Yes / Mycobacterium africanum lineage (L) 6 is an important pathogen in West Africa, causing up to 40% of pulmonary tuberculosis (TB). The biology underlying the clinical differences between M. africanum and M. tuberculosis sensu stricto remains poorly understood. We performed ex vivo expression of 2179 genes of the most geographically dispersed cause of human TB, M. tuberculosis L4 and the geographically restricted, M. africanum L6 directly from sputa of 11 HIV-negative TB patients from The Gambia who had not started treatment. The DosR regulon was the most significantly decreased category in L6 relative to L4. Further, we identified nonsynonymous mutations in major DosR regulon genes of 44 L6 genomes of TB patients from The Gambia and Ghana. Using Lebek's test, we assessed differences in oxygen requirements for growth. L4 grew only at the aerobic surface while L6 grew throughout the medium. In the host, the DosR regulon is critical for M. tuberculosis in adaptation to oxygen limitation. However, M. africanum L6 appears to have adapted to growth under hypoxic conditions or to different biological niches. The observed under expression of DosR in L6 fits with the genomic changes in DosR genes, microaerobic growth and the association with extrapulmonary disease. / European Research Council-INTERRUPTB starting grant nr.311725 (to BdJ, BO, FG, MA, CM).

Mycobacterium tuberculosis atteint le tiers de la population mondiale et cause plus de 1.5 millions de décès chaque année. L’augmentation des infections chez les patients immuno-compromis et l’émergence d’infections à de nouvelles souches multirésistantes aux antibiotiques somme la communauté scientifique à la découverte de nouvelles cibles thérapeutiques ainsi qu’au développement de nouveaux antibiotiques, vaccins et thérapies. Parmi les ~ 4000 gènes du génome de Mycobacterium tuberculosis, un d’entre eux, glbN, code pour l’hémoglobine tronquée N. Les hémoglobines sont de petites métalloprotéines qui fixent réversiblement l’oxygène et qui effectuent plusieurs activités catalytiques importantes. Ainsi, nos recherches s’inscrivent dans le cadre d’une approche biochimique qui vise à définir une fonction pour l’hémoglobine tronquée N de Mycobacterium tuberculosis à l’aide de techniques biochimiques modernes et de la spectroscopie à flux-arrêté. Nos recherches nous amènent également à résoudre la structure tridimensionnelle du complexe oxygéné de trHbN, à caractériser les interactions protéines-ligands au site actif de l’hémoglobine et à définir leurs rôles dans l’établissement du potentiel fonctionnel de l’enzyme en utilisant les spectroscopies d’absorption, de résonance Raman et de diffraction des rayons X. Nous avons découvert que l’activité rapide et efficace de détoxication aérobie du •NO de l’hémoglobine tronquée N mesurée chez Mycobacterium bovis BCG pourrait remplir un rôle similaire chez Mycobacterium tuberculosis et permettre la persistance de l’infection tuberculeuse dans l’hôte par la prévention des effets cytotoxiques associés au •NO et à ses dérivés réactifs azotés. De plus, l’architecture et la polarité du site actif de l’hémoglobine tronquée N définissent le potentiel fonctionnel de la protéine et contrôlent la diffusion, la fixation, la stabilisation, l’activation des ligands coordonnées au fer de l’hème et assurent le maintien d’une activité catalytique rapide et efficace. Finalement, nos découvertes suggèrent que l’hémoglobine tronquée N pourrait constituer une nouvelle cible thérapeutique pour le développement éventuel de drogues inhibitrices qui inactiveraient la première ligne de défense du parasite et perturberaient son adaptation métabolique face aux stress oxydatifs. / Mycobacterium tuberculosis infects over one-third of the human population, causing 1.5 millions deaths each year. The increase incidence of infections among immunocompromised patients and the emergence of strains with resistance to multiple antibiotics urge the scientific community to discover new therapeutic targets as well as develop new antibiotics, vaccines and therapies. Among the ~ 4000 genes that compose the genome of Mycobacterium tuberculosis, one of them, glbN, encodes the truncated hemoglobin N. Hemoglobins are small metalloproteins that reversibly bind oxygen and perform a wide array of important catalytic activities. Thus, our research aims at defining a function for Mycobacterium tuberculosis truncated hemoglobin N using modern biochemical techniques and stopped-flow spectroscopy. Our research also leads us to solve the three-dimensional structure of the oxygenated complex of trHbN, characterize the proteins-ligands interactions within the active site of the hemoglobin and define their roles in establishing the functional potential of the enzyme using absorption, resonance Raman and x-rays diffraction spectroscopies. We discovered that the fast and efficient •NO detoxification activity of truncated hemoglobin N measured in Mycobacterium bovis BCG could fulfill a similar role in Mycobacterium tuberculosis and allow the persistence of the tuberculous infection in the host by preventing the cytotoxic effects associated with •NO and its reactive nitrogen derivatives. Moreover, the architecture and polarity of truncated hemoglobin N active site define the functional potential of the protein and control the diffusion, binding, stabilization, and activation of the heme-iron coordinated ligands and ensure the maintenance of a fast and efficient catalytic activity. Finally, our discoveries suggest that truncated hemoglobin N could constitute a new therapeutic target for the development of inhibitors that would inactivate the first line of defence of the parasite and disturb its metabolic adaptation to nitrosative stresses.