Made available in DSpace on 2015-04-22T22:00:46Z (GMT). No. of bitstreams: 1 Pamela Campos Levy.pdf: 4863540 bytes, checksum: 820e34768b005399acf73dec3e491ae5 (MD5) Previous issue date: 2012-08-24 / FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas / Tuberculosis (TB) is an infectious disease transmitted by Koch's bacillus, or Mycobacterium tuberculosis. An estimated 1.4 million people died of tuberculosis in 2010. About 95% of these deaths occurred in developing countries, or development. In Brazil, each year are registered more than 68,000 new cases. Currently, Amazon is the Brazilian state with the highest incidence rate of the disease. a of TB diagnostic methods, adopted by the Ministry of Health is examining smear of bright field. The smear is the count of bacilli in slides containing sputum samples of the patient, prepared and stained according to the methodology standard. Over the past five years, research related to the recognition of bacilli tuberculosis, using images obtained by microscopy bright field, has been carried out with a view to automating this diagnostic method, given the fact that the number high smear tests performed by professional induce eyestrain and due to diagnostic errors. This paper presents a new method of recognition and targeting of tubercle bacilli in slides fields of images, containing pulmonary secretions of the patient, stained by Kinyoun method. From these bacilli images of pixels and background samples were extracted for training classifier. Images were automatically broken down into two groups, according with substantial content. The developed method selects an optimal set of color characteristics of the bacillus and of the background, using the method of selection climbing characteristics. These features were used in a pixel classifier, a multilayer perceptron, trained by backpropagation algorithm. The optimal set of features selected, {GI, Y-Cr, La, RG, a}, from the RGB color spaces, HSI, YCbCr and Lab, combined with the network perceptron with eighteen (18) neurons in first layer three (3) and the second one (1) in the third (18-3-1), resulted in an accuracy of 92.47% in the segmentation of bacilli. The image discrimination method in relation to automated background content contributed to affirm that the method described in this paper it is more appropriate to target bacilli images with low content density background (more uniform background). For future work, new techniques to remove noise present in images with high density of background content (containing background many artifacts) should be developed. / A tuberculose (TB) é uma doença infectocontagiosa, transmitida pelo bacilo de Koch, ou Mycobacterium tuberculosis. Estima-se que 1,4 milhões de pessoas morreram de tuberculose em 2010. Cerca de 95% dessas mortes ocorreram em países subdesenvolvidos ou em desenvolvimento. No Brasil, a cada ano são registrados mais de 68 mil novos casos. Atualmente, o Amazonas é o estado brasileiro com a maior taxa de incidência da doença. Um dos métodos de diagnóstico da TB, adotado pelo Ministério da Saúde, é o exame de baciloscopia de campo claro. A baciloscopia consiste na contagem dos bacilos em lâminas contendo amostras de escarro do paciente, preparadas e coradas de acordo com metodologia padronizada. Nos últimos cinco anos, pesquisas relacionadas ao reconhecimento de bacilos da tuberculose, utilizando imagens obtidas por microscopia de campo claro, tem sido realizadas com vistas a automatização desse método diagnóstico, em face do fato de que o número elevado de exames de baciloscopia realizado pelos profissionais induzirem a fadiga visual e em consequência a erros diagnósticos. Esse trabalho apresenta um novo método de reconhecimento e segmentação de bacilos da tuberculose em imagens de campos de lâminas, contendo secreção pulmonar do paciente, coradas pelo método de Kinyoun. A partir dessas imagens foram extraídas amostras de pixels de bacilos e de fundo para treinamento do classificador. As imagens foram automaticamente discriminadas em dois grupos, de acordo com o conteúdo de fundo. O método desenvolvido seleciona um conjunto ótimo de características de cor do bacilo e do fundo da imagem, empregando o método de seleção escalar de características. Essas características foram utilizadas em um classificador de pixels, um perceptron multicamada, treinado pelo algoritmo backpropagation. O conjunto ótimo de características selecionadas, {G-I, Y-Cr, L-a, R-G, a}, proveniente dos espaços de cores RGB, HSI, YCbCr e Lab, combinado com a rede perceptron com 18 (dezoito) neurônios na primeira camada, 3 (três) na segunda e 1 (um) na terceira (18-3-1), resultou em uma acurácia de 92,47% na segmentação dos bacilos. O método de discriminação de imagens em relação ao conteúdo de fundo automatizado contribuiu para afirmar que o método descrito neste trabalho é mais adequado para segmentar bacilos em imagens com baixa densidade de conteúdo de fundo (fundo mais uniforme). Para os trabalhos futuros, novas técnicas para remover os ruídos presentes em imagens com alta densidade de conteúdo de fundo (fundo contendo muitos artefatos) devem ser desenvolvidas.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Com o objetivo de enfatizar a importÃncia do diagnÃstico laboratorial de micobactÃrias em sÃtios extrapulmonares, exceto renal, utilizou-se neste trabalho a reaÃÃo em cadeia da polimerase (PCR). Na PCR foram empregados iniciadores especÃficos para a detecÃÃo de micobactÃrias do complexo Mycobacterium tuberculosis (MTB) e nÃo pertencentes ao complexo Mycobacterium tuberculosis (MNTB). Foram comparados os resultados obtidos na PCR com os mÃtodos microbiolÃgicos convencionais. Foram analisadas 114 amostras clÃnicas (lÃquidos corporais) provenientes de pacientes internados ou em atendimento ambulatorial, com suspeita clÃnica de tuberculose extrapulmonar, exceto renal. As amostras foram coletadas no perÃodo de 2001 a 2003 e cedidas pelo LaboratÃrio de Microbiologia do LaboratÃrio Central do Hospital UniversitÃrio Walter CantÃdio (HUWC-UFC) e pelo LaboratÃrio Central de SaÃde PÃblica da Secretaria de SaÃde do Estado do Cearà (LACEN-CE). A baciloscopia e a cultura foram negativas em todas as amostras. A pesquisa molecular para o gÃnero Mycobacterium foi positiva em dez amostras e, para o complexo Mycobacterium tuberculosis, foi negativa em todas as amostras. / In order to focus on the importance of laboratorial diagnosis of extrapulmonary mycobacteria, except renal, we employed the polymerase chain reaction (PCR). Clinical samples (body fluids) were tested by PCR for the presence of Mycobacterium tuberculosis complex (MTB) and nontuberculous mycobacteria (MNTB). We compared the results obtained through the PCR with the ones obtained through conventional microbiological methods. The 114 clinical samples were obtained from internal patients or from or in ambulatorial attendance with clinical suspicion of extrapulmonary tuberculosis, except renal. The samples were collected from 2001 to 2003, and ceded to this research by the LaboratÃrio de Microbiologia do LaboratÃrio Central do Hospital UniversitÃrio Walter CantÃdio (HUWC-UFC), and by the LaboratÃrio Central de SaÃde PÃblica da Secretaria de SaÃde do Estado do Cearà (LACEN-CE). The direct microscopy and culture were negative in all the samples. The molecular research, for the Mycobacterium genus was positive in 10 samples, and for the Mycobacterium tuberculosis complex was negative in all the samples.

Made available in DSpace on 2015-04-11T13:54:24Z (GMT). No. of bitstreams: 1 Marcia Alves de Souza.pdf: 1923965 bytes, checksum: 3ad1484efad384495579203b0e85259c (MD5) Previous issue date: 2013-05-30 / FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas / A Tuberculose (TB) é uma doença infecciosa causada pelo complexo Mycobacterium tuberculosis, sendo considerada um grave problema de saúde pública mundial. Atualmente, isolados de M. tuberculosis resistentes a pelo menos um medicamento utilizado no tratamento da TB tem sido documentados em todos os países. De acordo com a Organização Mundial de Saúde (OMS) a TB multirresistente (TBMR) é definida quando, isolados de M. tuberculosis de pacientes apresentam resistência a pelo menos Isoniazida e Rifampicina, os dois fármacos fundamentais no tratamento da TB. A resistência à Rifampicina tem sido associada às mutações gênicas no bacilo, no gene rpoB (referentes aos códons 531, 526 e 516). Para a Isoniazida, as mutações associadas à resistência têm sido relatadas nos genes katG, inhA, ahpC e kasA, sendo que a mutação no gene katG, referente ao códon 315, tem sido a mais citada para resistência a este fármaco. Neste contexto, métodos moleculares têm sido propostos pra detecção de mutações gênicas, em isolados de M. tuberculosis, que possam estar associadas à resistência aos fármacos. O presente estudo avaliou o desempenho da PCR multiplex alelo específico (PCR-MAS), diretamente em 86 amostras de escarro de pacientes com TB pulmonar multibacilares (n=42) e paucibacilares (n=44) da Policlínica Cardoso Fonte. A PCR-MAS teve como alvos: os genes katG ,inhA e rpoB. A concordância entre a PCR-MAS e o Método de Redução de Nitrato foi avaliada utilizando o teste kappa e a associação entre as mutações gênicas e a resistência fenotípica aos fármacos foi realizada pelo teste exato de Fisher. A análise de concordância, pelo teste kappa, foi realizada entre as PCR-MAS a partir de amostra de escarro e de isolados de M. tuberculosis. A PCR-MAS apresentou fraca concordância com o Método de Redução de Nitrato, pois de 18 amostras resistentes à Isoniazida, apenas em 4 (22,2%) foram detectadas as mutações para o gene katG ou inhA. No entanto, a avaliação da sensibilidade fenotípica à Rifampicina, apresentou boa concordância com a PCR-MAS (kappa = 0,7237), quando as amostras foram de pacientes de TB pulmonar multibacilar. Além disso, houve associação da presença de mutações no gene rpoB com resistência fenotípica à Rifampicina (p = 0.0014). Em relação a concordância entre as PCR-MAS, de amostras de escarro e seus respectivos isolados de M. tuberculosis, o desempenho foi excelente quando testados em amostras de pacientes com TB pulmonar multibacilar, para detecção de mutações no gene rpoB (kappa = 0,7742). Portanto, os resultados obtidos com a PCR-MAS, a partir de amostras de escarros, foram satisfatórios e poderão ser utilizados para monitorar e pesquisar as mutações associada à resistência à Rifampicina em pacientes de TB multibacilar na rede básica de saúde, pois é um teste rápido, reprodutível e de menor custo.

Made available in DSpace on 2014-07-29T15:30:40Z (GMT). No. of bitstreams: 1 Dissertacao Alyne Melo Pereira.pdf: 2782543 bytes, checksum: 9841bfc0f181c8d2c6af086d835eadc3 (MD5) Previous issue date: 2012-03-07 / Tuberculosis (TB) is a chronic infectious bacterial disease, very contagious, that, despite almost 130 years of biomedical research since the discovery of the tubercle bacillus, it continues to be a major threat to global health and is one of the leading causes of death by a bacterial organism, particularly in the developing world. The etiologic agent, Mycobacterium tuberculosis, is a bacterium that is easily transmitted by air. The disease control depends on several factors, being the correct diagnosis; efficient treatment; and active disease patient management to avoid transmission, essential ones. In order to improve the success in diagnosis, the knowledge of genotypic profiles by molecular techniques has provided positive results. Additionally, the correlation between the geographical locations of TB cases is a useful tool to aid epidemiological control strategies, especially when it is performed together with the knowledge of genetic variability of the studied TB strains. In this study we used the 15 loci MIRU-VNTR technique to identify polymorphisms among Mycobacterium tuberculosis isolates. A total of 119 M. tuberculosis samples, isolated between 2006 and 2007 from patients attending two reference hospitals of Goiânia city, were genotyped and the results compared with the gold standard RFLP-IS6110 technique. The 15 loci MIRU-VNTR analysis of 119 TB isolates provided 110 distinct genotypes, 105 of which contained only one isolate while 14 isolates were grouped in five genetic groups (clusters). This technique showed a good discriminatory power (0.9986). The 15 loci MIRU-VNTR technique was more discriminatory than RFLP-IS6110 (0.9942). We also performed the georeferencing of 241 TB cases, corresponding to all clinical forms reported in Goiânia during the year 2007 by the City´s Secretary of Health Department. The cases were randomly distributed throughout the city. The distribution of cases showed that, visually, there was no relationship between disease and socio-economic situation of the population. Among the georeferenced cases, we were able to genotype 50 isolates by 15 loci MIRU-VNTR. A great genetic variability of genotypes among the 50 isolates was observed, and the few ones that were clustered did not show epidemiological links. Based on the observed data, no transmission source the TB was identified in the city of Goiânia, and consequently we can infer that TB in the City of Goiânia could be resulted from a previously acquired infection due to the high degree of heterogeneous genetic profiles observed. / A tuberculose (TB) é uma doença bacteriana crônica infecto-contagiosa que, apesar de quase 130 anos de pesquisas desde a descoberta do bacilo, continua a ser um importante agravo à saúde global e uma das principais causas de morte, particularmente nos países em desenvolvimento. A doença tem como agente etiológico o Mycobacterium tuberculosis, uma bactéria de fácil transmissão uma vez que os bacilos se propagam pelo ar. O controle da doença depende de vários fatores, dentre os quais, o correto diagnóstico, o tratamento completo e o manejo adequado dos pacientes com a doença ativa para evitar a transmissão são fundamentais. Neste cenário, o conhecimento dos perfis genotípicos dos microorganismos circulantes em uma região, através de técnicas moleculares apropriadas, tem contribuído com bons resultados. Além disso, a correlação entre o espaço geográfico de casos da doença é uma ferramenta útil para a definição de estratégias epidemiológicas, sobretudo quando se conhece a variabilidade genética das cepas presentes em uma determinada população associada a sua localização geográfica. Neste estudo, foi empregada a análise de MIRU-VNTR para identificar o polimorfismo de 15 loci em amostras de Mycobacterium tuberculosis. Foram analisadas 119 amostras, coletadas entre 2006 e 2007, de pacientes com TB pulmonar em dois hospitais de referência no município de Goiânia. Os resultados obtidos pela técnica de 15 loci MIRU-VNTR foram posteriormente, comparados com resultados gerados pela técnica padrão ouro RFLP-IS6110. Pela análise dos padrões moleculares dos 119 isolados, foram encontrados 110 genótipos distintos. Destes, 105 continham um único isolado enquanto que 14 amostras se agruparam em cinco grupos (cluster) genéticos. Esta técnica apresentou um bom poder discriminatório (0,9986). Nossos resultados mostraram que a técnica de tipagem por 15 loci MIRU-VNTR se mostrou mais discriminatória que a técnica de RFLP-IS6110 (0,9942). Adicionalmente, foram georreferenciadas 241 amostras de pacientes diagnosticados com qualquer forma clínica de TB em 2007 de acordo com a Secretaria Municipal de Saúde e destas, 50 amostras foram genotipadas por 15 loci MIRU-VNTR. No georreferenciamento de 241 casos da doença, observou-se uma distribuição aleatória destes pelo município sem aglomerados significativos. A distribuição dos casos mostrou que, visualmente, não houve relação entre a doença e a situação sócio-ecônomica da população. O estudo genotípico de 50 isolados georreferenciados demonstrou que existe grande variabilidade genética de cepas circulantes na cidade e essas, quando agrupadas, não apresentam associação geo-espacial que possibilite estabelecer uma ligação epidemiológica entre os casos. Podemos concluir que não existem focos recentes de transmissão da doença, podendo esta ser proveniente de reativação endógena de infecção latente adquirida anteriormente, já que a maioria dos isolados apresentaram perfis únicos.

Como entre 25-50% dos isolados de Mycobacterium tuberculosis resistentes à INH não apresentam mutações nos genes katG, inhA, ahpC e kasA que possam justificar sua resistência, foi proposta a influência de mutações específicas no gene nat nos mecanismos de resistência e atividade da NAT. Todos os isolados obtidos (n=125) foram identificados e caracterizados através da amplificação pela PCR da IS6110 e por MIRU-VNTR, respectivamente. A determinação da concentração inibitória mínima (CIM) foi realizada pelo método REMA. Após triagem de mutações nos genes caracteristicamente envolvidos com resistência pela PCR-SSCP, seguida de seqüenciamento de DNA, foram selecionados 45 isolados para o estudo de mutações específicas (pela PCR e sequenciamento) e expressão gênica do mRNA do gene nat através da RT-PCR em tempo real. Confirmou-se que mutação no gene katG é a mais correlacionada com a resistência à INH, pois 68,4% das cepas resistentes apresentaram mutação neste gene. Mutações na região promotora do gene inhA, na região intergênica oxyR-ahpC e no gene kasA foram encontradas em 8,8%, 5,6% e 21,6% dos isolados, respectivamente. Mutações no gene nat, das quais 4 não haviam sido descritas previamente, foram encontradas em 40,0% dos isolados. Apesar dessas mutações causarem alterações na proteína, não foi observada uma relação direta com aumento na CIM. Também não foi observada relação entre a variação da expressão do mRNA do gene nat com os valores de CIM e com o número de mutações, tanto específicas do gene nat como em outros genes caracteristicamente relacionados com resistência à INH. / Since around 25-50% of the Mycobacterium tuberculosis strains resistant to INH do not present any mutation in katG, inhA, ahpC and kasA genes that could explain their resistance, we proposed to evaluate the influence of specific mutations in the nat gene in the mechanisms of resistance and in the activity of NAT. All strains were identified and characterized molecularly by the amplification by PCR of the IS6110 region and by MIRU-VNTR, respectively. The minimal inhibitory concentration (MIC) was performed using the REMA method. After screening of mutations in the resistant-related genes by PCR-SSCP followed by DNA sequencing, 45 strains were selected to be evaluated for specific mutations (by PCR and sequencing) and mRNA expression of nat by real time RT-PCR. It was showed that mutations in the katG gene were the most frequent and related to INH resistance since 68.4% of all resistant strains presented mutation in this gene. Mutations in the promoter region of inhA gene, oxyR-ahpC intergenic region and kasA gene were found in 8.8%, 5.6% and 21.6% of the strains, respectively. Mutations in the nat gene, four of them not previously described, were found in 40.0% of the strains. Although those mutations influence in the protein produced it was not observed a direct relation in an increase in CIM. It was also noted no relation between the expression of mRNA of nat gene neither with the MIC values nor with the number of mutations, both specific of nat gene as well in the other genes characteristically related to INH resistance.

All organisms must sense and respond to their environment in order to survive. The processes that allow a living cell to sense changes in its environment, and respond appropriately are collectively referred to as ‘signal transduction’. Cyclic AMP is a ubiquitously used second messenger molecule that plays diverse roles from hormone signalling in mammalian cells to catabolite repression in enteric bacteria. In several bacterial pathogens such as Pseudomonas aeruginosa, cAMP has also been found to mediate pathogenesis, usually by regulating the production of several virulence factors aiding in colonisation of the host. Cyclic AMP signalling has been suggested to regulate the virulence of the obligate intracellular Mycobacterium tuberculosis. Mycobacteria, including M. tuberculosis, code for a large number of adenylyl cyclases, enzymes that synthesise cAMP. Of the 16 putative adenylyl cyclases encoded by M. tuberculosis H37Rv, 10 have received extensive biochemical attention. A knockout of one of these cyclases, Rv0386, resulted in compromised virulence of M. tuberculosis. Ten proteins predicted to bind cAMP and mediate its cellular roles have also been identified in M. tuberculosis. Among these are the cAMP-regulated transcription factor, CRPMt, and cAMP-regulated protein acetyl transferase, KATmt. Comparatively little information is available, however, regarding the roles of cAMP-degrading machinery in mycobacteria. Two phosphodiesterases, with modest activity against cAMP in vitro, have been identified from M. tuberculosis, and are encoded by the Rv0805 and Rv2795c loci. Of these, Rv2795c has orthologs in all sequenced mycobacterial genomes. However, Rv0805-like proteins are coded only by slow growing mycobacteria such as the M. tuberculosis-complex, M. marinum and M. leprae, several of which are human or animal pathogens. Rv0805 belongs to the metallophosphoesterase superfamily of proteins, consisting of metal-dependent phosphoesterases with substrates ranging from large polymers like nucleic acids to small molecules like cAMP and glycerophospholipids. Like other metallophosphoesterases, Rv0805 displays promiscuous substrate utilisation and efficient hydrolysis of 2’3’-cAMP in vitro. Rv0805 also hydrolyses 3’5’-cAMP in vitro. Overexpression of Rv0805 is reported to lead to reduction in intracellular cAMP levels in M. smegmatis and M. tuberculosis, suggesting that it is capable of hydrolysing cAMP in the bacterial cell as well. The structure of Rv0805 revealed a sandwich-like α/β fold, typical of metallophosphoesterases, along with a relatively flexible C-terminal domain of unknown function. Despite extensive biochemical and structural information on Rv0805 however, its roles in mycobacteria remain unknown. In this study, the cellular roles of Rv0805 are explored and using information from biochemical and structural analyses, novel activities and interactions of Rv0805 have been identified. Rv0805, when expressed in M. smegmatis, led to a reduction in intracellular cAMP, as previously reported. However, the extent of reduction was modest (~30 %) and limited to the exponential phase of growth when both Rv0805 and intracellular cAMP are at their highest levels. Overexpression of Rv0805 also resulted in hypersensitivity to cell wall perturbants like crystal violet and sodium dodecyl sulphate (SDS) indicative of a change in the properties of the cell envelope of M. smegmatis. Importantly, these effects were independent of cAMP-hydrolysis by Rv0805, as overexpression of catalytically inactive Rv0805N97A also elicited similar changes. Unexpectedly, Rv0805 was localised to the cell envelope, both in M. tuberculosis as well as in M. smegmatis. The ability of Rv0805 to localise to the cell envelope was dependent on it C-terminus, as truncation of Rv0805 in this region (Rv0805Δ10, Rv0805Δ20 and Rv0805Δ40) resulted in progressively greater enrichment in the cytosol of M. smegmatis. Overexpression of Rv0805Δ40, which was localised almost completely to the cytosol, did not result in hypersensitivity to SDS, suggesting that cell envelope localisation, rather than cAMP-hydrolysis, was crucial for the cell envelope modifying roles of Rv0805. A possible mechanism behind the cell envelope-related effects of Rv0805 overexpression was the ability of the protein to interact with the cell wall of mycobacteria in a C-terminus-dependent manner. Purified Rv0805, but not Rv0805Δ40, could associate with crude mycobacterial cell wall as well as purified cell wall core polymer (mycolyl-arabinogalactan-peptidoglycan complex) in vitro. In addition to the C-terminus, the architecture of the active site was also crucial for this interaction as mutations in the active site that compromised metal-binding also resulted in poor interaction with the cell wall. Most significant among these residues was His207, which when mutated to Ala almost completely abrogated interaction with the cell wall in vitro. Further, Rv0805H207A was unable to localise to the cell envelope when expressed in M. smegmatis, even in the presence of the C-terminus, highlighting the importance of this residue in maintaining the structural integrity of Rv0805, and demonstrating that the structure of the C-terminus, rather than its sequence alone, played a role in cell envelope localisation and interaction. In order to verify that the observed sensitivity of Rv0805-overexpressing M. smegmatis to cell wall perturbants was due to a change in cell envelope properties atomic force microscopy was employed. Two distinct modes of operation were used to analyse surface and bulk properties of the mycobacterial cell envelope. These were tapping mode phase imaging, and contact mode force spectroscopy. Using tapping mode phase imaging, it was found that the cell surface of M. smegmatis was inherently heterogeneous in its mechanical properties. Further, contact mode force-spectroscopy revealed that the cell envelope of M. smegmatis in cross-section had at least three layers of varying stiffness. Typically, a middle layer of high stiffness was observed, sandwiched between two lower stiffness layers. This organisation is reminiscent of the current model of the mycobacterial cell envelope, possessing a central polysaccharide rich layer and outer and inner lipid rich layers. Treatment of wild type M. smegmatis with cell wall-perturbing antibiotics isoniazid and ethambutol resulted in markedly altered phase images, as well as significantly lower stiffness of the bacterial cell envelopes, validating that the methodology employed could indeed be used to assess cell wall perturbation in mycobacteria. Further, M. smegmatis harbouring deletions in cell envelope biosynthesis related genes, MSMEG_4722 and aftC, showed significantly lower cell wall stiffness than wild type M. smegmatis, providing evidence that genetic perturbation of the cell wall of mycobacteria could also be studied using atomic force microscopy. While phase imaging revealed similar surface properties of Rv0805-overexpressing and control M. smegmatis, force spectroscopy revealed significantly lower cell envelope stiffness, particularly of the middle layer, of the former. Cell envelope stiffness was, however, unaffected by expression of Rv0805Δ40 in M. smegmatis, providing direct evidence for C-terminus-dependent cell envelope perturbation upon Rv0805 overexpression. Additionally, overexpression of Rv0805N97A, but not Rv0805H207A led to reduced stiffness of the cell envelope of M. smegmatis, demonstrating that the cell wall remodelling activity of Rv0805 was independent of cAMP-hydrolysis, but dependent on cellular localisation and cell wall interaction. Like in M. smegmatis, overexpression of Rv0805 also led to lower cAMP levels in M. tuberculosis. Using a microarray-based transcriptomics approach, pathways affected by Rv0805 overexpression were identified. Rv0805 overexpression elicited a transcriptional response, leading to the down-regulation of a number of virulence associated genes such as whiB7, eis, prpC and prpD. Importantly, Rv0805-overexpression associated gene expression changes did not include genes regulated by CRPMt, the primary cAMP-regulated transcription factor in M. tuberculosis. Further, Rv0805N97A overexpression in M. tuberculosis led to similar changes in gene expression as overexpression of the wild type protein. These observations reiterated that, at least upon overexpression, the effects of Rv0805 were largely independent of cAMP-hydrolysis. Using overexpression in M. smegmatis and M. tuberculosis, cAMP-hydrolysis independent roles of Rv0805 in mycobacteria were identified. To further validate these observations, a knockout strain of the Rv0805 gene was generated in M. bovis BCG, a well-established model to study M. tuberculosis. Curiously, deletion of Rv0805 did not lead to a change in intracellular cAMP levels, demonstrating that cAMP-hydrolysis by Rv0805 may not contribute to the modulation of mycobacterial cAMP levels under standard laboratory growth conditions. Rv0805 deletion led to altered colony morphology and possible reduction in cell wall thickness, reaffirming the roles of this phosphodiesterase in regulating cell envelope physiology of mycobacteria. Additionally, Rv0805 deletion also resulted in compromised growth of M. bovis BCG in fatty acid-deficient media, implicating Rv0805 as a possible regulator of carbon metabolism. In summary, this thesis explores novel links between Rv0805 and the mycobacterial cell wall and elucidates the critical importance of the C-terminus domain of this metallophosphodiesterase in modulating its cellular localisation to, and interaction with, the mycobacterial cell envelope. En route to understanding the effects of Rv0805 overexpression on the cell wall of M. smegmatis, an atomic force microscopy-based methodology to assess perturbation of the cell envelope of mycobacteria was also developed. Finally, using a combination of biochemical and genetic analyses, cellular roles of Rv0805, independent of cAMP-hydrolysis, were identified in slow-growing mycobacteria. This study therefore provides direct evidence against the sole role of this mycobacterial phosphodiesterase as a regulator of intracellular cAMP levels, and opens up new avenues to understanding the cellular functions of Rv0805 and indeed other members of the metallophosphoesterase superfamily.

La tuberculose est encore aujourd’hui un problème de santé majeur et l’augmentation des cas de tuberculoses résistantes au niveau mondial, associé au dépistage et diagnostic insuffisants, suggère une éradication totale encore lointaine. La compréhension des relations hôte-pathogène est essentielle pour permettre d’établir de nouvelles stratégies thérapeutiques. Ces travaux de thèse ont mis en exergue la contribution limitée de la voie de signalisation IL-36, cytokine de la famille de l’IL-1, au cours de l’infection mycobactérienne. Ces données permettent d’envisager sérieusement l’IL-36 en tant que cible thérapeutique pour le psoriasis sans risquer la réactivation de tuberculose latente chez les patients. De plus, nous avons également démontré le rôle différentiel de la voie de signalisation TNF au sein de populations cellulaires spécifiques lors de l’infection par M. tuberculosis, et nos résultats apportent des connaissances solides pour envisager des stratégies immuno-modulatoires qui pourraient constituer l’avenir des traitements antituberculeux. D’autre part, nous avons caractérisé un modèle murin humanisé pour une étude facilitée des anticorps anti-TNF humains actuels. L’utilisation de ce modèle pourrait permettre, à termes, d’identifier et de valider de nouveaux candidats d’anticorps anti-TNF de deuxième génération. Enfin, nous avons montré que des anticorps neutralisants l’IL-17 ne perturbent pas la réponse immunitaire à la tuberculose, contrairement aux anticorps anti-TNF, et que l’absence de l’IL-17 n’est pas compensée par l’IL-22 puisque des animaux déficients pour ces deux voies de signalisation sont capables de contrôler l’infection. / Tuberculosis remains a major health problem in the world nowadays. The increasing incidence of resistant tuberculosis is associated with a poor diagnosis, reflecting important difficulties for total eradication. Understand host-pathogen interactions is crucial to establish new therapeutic strategies. This work first shows the limited contribution of IL-36 pathway during mycobacterial infection. These results suggest that IL-36 could be targeted for the treatment of psoriasis without a high risk of tuberculosis reactivation. We then demonstrate the differential role of TNF pathway among myeloid or lymphoid cells during M. tuberculosis infection, and our data support the development of immunomodulatory strategies to boost host immune response, thus helping to clear the infection. Moreover, we characterize a humanized murine model allowing the study of new anti-TNF candidates in the context of M. tuberculosis infection. Finally, we show that antibodies targeting IL-17 does not dampen host control of M. tuberculosis infection, unlike anti-TNF, and that IL-22 does not compensate absence of IL-17 for this control.

A ce jour, l’Organisation Mondiale de la Santé estime qu’un tiers de la population mondiale est infectée par Mycobacterium tuberculosis (Mtb), l’agent étiologique de la tuberculose. De par un taux de mortalité et de morbidité élevé, cette maladie infectieuse constitue un véritable fléau sanitaire au niveau mondial. <p>Mtb est un pathogène intracellulaire qui infecte son hôte par voie aérienne. In vivo, il doit faire face à une série d’environnements (phagosome, granulome) stressants de par leurs activités antimicrobiennes et par leur composition en nutriments relativement pauvre. Pour pouvoir survivre et se multiplier dans ces conditions, Mtb possède un panel de transporteurs spécifiques ainsi que toute une série de mécanismes pour contrer, voire détourner les défenses immunitaires de l’hôte. Malgré cette oppostion, les mycobactéries finissent séquestrées à l’intérieur de granulomes, structure caractéristique de la tuberculose, dans un état de dormance. La mise au point de nouveaux traitements prophylactiques et thérapeutiques nécessite la compréhension des mécanismes mis en œuvre par Mtb pour survivre et se multiplier au sein de son hôte.<p><p>Le présent travail a consisté en l’étude de mécanismes favorisant la dispersion et la survie intracellulaire de Mtb chez son hôte via: <p>1)\ / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Malgré la disponibilité d’un traitement curatif et un vaccin largement utilisé, l’OMS estime qu’approximativement un tiers de la population mondiale est infectée par Mycobacterium tuberculosis, l’agent étiologique de la tuberculose, et qu’environ 2 millions de personnes en meurent chaque année. La compréhension de l’épidémiologie de la tuberculose et les actions de contrôle de la maladie ont été, récemment, compliquées par l’émergence de bacilles tuberculeux résistants aux antibiotiques et par la synergie fournie par la co-infection avec le VIH. Une tendance alarmante pour la santé publique est l’émergence de souches résistantes à plusieurs antibiotiques (multi-résistantes, MDR), définies comme des isolats résistants au moins à l’isoniazide (INH) et la rifampicine (RIF), les deux agents anti-tuberculeux les plus puissants.<p><p>La sélection de mutants résistants se produit chez le patient lorsque les taux d’antibiotiques présents dans le corps sont sub-thérapeutiques ou lorsque la thérapie est inappropriée. Un des facteurs favorisant est l’exceptionnelle durée de la chémothérapie. Le besoin de maintenir des taux élevés d’antibiotiques pendant des mois, combiné avec la toxicité inhérente des agents, résultent en une observance incomplète du traitement par le patient et le risque d’acquérir des résistances. La résistance aux antibiotiques chez M. tuberculosis résulte d’altérations dans des gènes chromosomiques spécifiques. Les causes génétiques de la résistance ont été définies pour certains antibiotiques bien que plusieurs inconnues persistent. <p><p>Le présent travail a consisté en l’étude du problème de la résistance aux antibiotiques anti-tuberculeux par différentes approches :l’analyse génétique des mécanismes de résistance, l’évaluation de l’activité thérapeutique de nouvelles molécules et la caractérisation du profil de résistance de souches cliniques. <p><p>L’acide p-aminosalicylique (PAS) est un antibiotique bactériostatique de deuxième ligne dont le mécanisme d'action sur le bacille tuberculeux est incompris. Récemment, en utilisant la mutagenèse par transposon, la résistance au PAS fut associée à des mutations de la thymidylate synthase encodée par le gène thyA. Suite à cette découverte, nous avons entrepris une étude moléculaire de souches cliniques et de mutants spontanés résistants au PAS. Des mutations du gène thyA furent identifiées chez seulement 37% des souches. En tout, vingt-quatre mutations différentes furent identifiées dans le gène thyA. Les séquences nucléotidiques de cinq autres gènes de la voie de synthèse du folate et de la thymine (dfrA, folC, folP1, folP2, et thyX) ainsi que de 3 gènes encodant des N-acétyltransférases (nhoA, aac1 et aac2) furent également analysées mais aucune mutation associée à la résistance au PAS n’a pu être mise en évidence. L’utilisation de techniques bioinformatiques de prédiction structurelle révèle que les mutations identifiées affectent soit la structure soit le site fonctionnel de ThyA. L’étude des profils de croissance des organismes résistants au PAS nous permit de constater que les organismes porteurs d’une mutation de la protéine ThyA présentent un profil de croissance constant en présence de concentrations croissantes de PAS. Les organismes résistants au PAS possédant une protéine ThyA sauvage répondent, quant à eux, aux concentrations croissantes de PAS de façon dose-dépendante, indiquant que le(s) mécanisme(s) alternatif(s) de résistance au PAS est (sont) dose-dépendant(s).<p><p>La thymidylate synthase est également une des cibles du 5-fluorouracil (5-FU), l’agent chimiothérapeutique le plus largement utilisé pour le traitement du cancer colorectal avancé. Etant donné l’augmentation du nombre de souches résistantes de M. tuberculosis, de nouveaux composés anti-tuberculeux sont nécessaires de façon urgente. Ici, nous avons évalué l’efficacité in vitro et in vivo du 5-FU sur M. tuberculosis. La concentration minimale inhibitrice du 5-FU fut déterminée sur une collection de souches cliniques sensibles et multi-résistantes ainsi que sur des mutants spontanés résistants au PAS. Tous les isolats montrèrent une sensibilité au 5-FU à des concentrations allant de 0.4 à 1.8 µg/ml, et ce indépendamment de leur profil de sensibilité/résistance aux agents anti-tuberculeux actuels. Les études in vivo du 5-FU (sur un modèle murin de tuberculose active) montrèrent une efficacité de celui-ci durant les deux premières semaines de traitement puis une perte d’activité à la troisième semaine, vraisemblablement engendrée par les effets secondaires du 5-FU.<p><p>L’éthionamide (ETH) est un autre antibiotique de deuxième ligne dont l’utilisation est limitée aux tuberculoses multi-résistantes étant donné les effets secondaires qu’il engendre. Ces dernières années, les études ont montré que l’ETH est un pro-médicament, transformé en forme active par l’enzyme monooxygénase EthA dont l’expression est contrôlée par le répresseur transcriptionnel EthR. Notre étude décrit l’élaboration d’inhibiteur d’EthR capable d’augmenter la sensibilité de M. tuberculosis à l’ETH suite à l’amélioration de son activation. Les composés synthétisés et sélectionnés pour leur capacité à inhiber l’interaction EthR-ADN furent co-cristallisés avec EthR. Les structures tridimensionnelles des complexes furent utilisées pour la synthèse d’analogues capables d’améliorer la puissance de l’ETH en culture. Les molécules les plus prometteuses furent testées sur un modèle murin de tuberculose. Pour un des inhibiteurs d’EthR testés, nous avons montré que sa co-administration avec l’ETH permet une réduction de la dose d’ETH utilisée de 3 fois, pour l’obtention d’une même réduction de charge mycobactérienne pulmonaire. Ce travail démontre la possibilité d’augmenter l’index thérapeutique de l’éthionamide en agissant pharmacologiquement sur le mécanisme régulateur de son activation.<p><p>Dans certaines régions du monde, le problème de la multi-résistance devient très présent. Nous avons étudié l’état de la situation à Mourmansk (Fédération russe), une région à haute incidence de tuberculose. La résistance aux antibiotiques et l’épidémiologie moléculaire de la tuberculose furent étudiées sur des isolats collectés en 2003 et 2004 dans cette région. Une extrêmement haute prévalence de tuberculose multi-résistante (MDR-TB) fut constatée à la fois pour les nouveaux cas (primaires) (26%) et les cas précédemment traités (72.9%). Le typage des souches MDR primaires révèle une appartenance au génotype Beijing pour la plupart des isolats (79.8%) et l’homogénéité génétique des souches suggère une transmission active au sein de la population. L’analyse moléculaire des gènes impliqués dans la résistance à l’INH et à la RIF montre la présence des mutations katG codon 315 et rpoB codon 531 chez, respectivement, 98,2% et 76,3% des isolats MDR-TB primaires. La haute fréquence de ces mutations « communes » suggère la possible utilisation de tests moléculaires ciblant spécifiquement ces mutations pour détecter rapidement la plupart des cas de MDR-TB.<p><p>Nos travaux illustrent les différentes voies à suivre pour maitriser le problème de la résistance aux antibiotiques :l’élucidation des mécanismes de résistance, le développement de nouveaux médicaments et la détection rapide des cas de résistance.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished