Submitted by Pós Imunologia (ppgimicsufba@gmail.com) on 2017-03-13T17:29:54Z No. of bitstreams: 1 Dissertação_Flávia.pdf: 1128860 bytes, checksum: 42e7e0ff6c678d2edae8e8d9c7311c94 (MD5) / Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2017-03-23T13:26:19Z (GMT) No. of bitstreams: 1 Dissertação_Flávia.pdf: 1128860 bytes, checksum: 42e7e0ff6c678d2edae8e8d9c7311c94 (MD5) / Made available in DSpace on 2017-03-23T13:26:19Z (GMT). No. of bitstreams: 1 Dissertação_Flávia.pdf: 1128860 bytes, checksum: 42e7e0ff6c678d2edae8e8d9c7311c94 (MD5) / FAPESB / Introdução: Têm-se debatido o papel de microrganismos na imunomodulação de doenças alérgicas atópicas cuja prevalência tem aumentado. A Micobactéria emerge, então, como um potencial agente imunomodulador. Objetivo: Identificar os perfis de citocinas induzidos pelo extrato de Mycobacterium bovis e pelo PPD e como elas são induzidas em adolescentes atópicos e não atópicos de Salvador, Bahia. Metodologia: O estudo utilizou amostra de conveniência de 55 indivíduos cujo sangue periférico coletado fora utilizado no cultivo de sangue total. As células foram estimuladas com 5μg do extrato de M. bovis ou 5μg do PPD. Utilizou-se o Multiplex para verificar o perfil de citocinas (IFNγ, IL-10, TNFα, IL-13, IL-5 e IL-17) induzido em 24h. A atopia foi determinada através da presença de IgE acima de 0,7 kU/mL para pelo menos um alérgeno (Blomia tropicalis, Dermatophagoides pteronissinus, Blatella germanica e Periplameta americana). A amostra foi dicotomizada em respondedor e não respondedor com base no limite mínimo de detecção do kit para cada citocina e o teste Exato de Fisher foi aplicado. Resultados: O estímulo com PPD e com extrato induziu mudanças significativas nas frequências de respondedores à IL-5, IL-13 e IL- 17, e à IL-5, IL-13, IL-17 e IFNγ, respectivamente. 97,7% dos indivíduos foram produtores de IL-10 em resposta ao extrato. Os compostos não induziram alterações significativas nas frequências de respondedores entre os grupos de atopia. Conclusão: As dosagens de citocinas serão finalizadas, incluindo-se a do sobrenadante de 120h, permitindo a aplicação de teste estatístico com maior poder para verificação dos efeitos dos compostos utilizados / Introduction: The role of microrganisms in immunomodulation of atopic allergic diseases whose prevalence has increased has been discussed. Mycobacteria emerges as a potential immunomodulatory agent. Objective: Identify the cytokine profile inducede by Mycobacterium bovis extract and PPD and how they are induced in adolescentes atopic and non-atopic from Salvador, Bahia. Methodology: This study used a convenience sample of 55 individuals. Pefipheral blood samples were used in the whole blood culture. The cells where stimulated with 5μg of M. bovis extract or 5μg of PPD. Multiplex was used to verify cytokine profile (IFNγ, IL-10, TNFα, IL-13, IL-5 e IL-17) induced in 24h. Atopy was determined by the presence of IgE above 0.7 kU / mL for at least one allergen (Blomia tropicalis, Dermatophagoides pteronissinus, Blatella germanica e Periplameta americana). The sample was dichotomized into responder and non-responder based on the minimum limit of kit detection for each cytokine and Fisher's Exact Test was applied. Results: The stimulation with PPD and with the extract induced significant changes in the frequency of responders to IL-5, IL- 13 and IL-17, and the frequency of responders to IL-5, IL-13, IL-17 and IFNγ, respectively. 97,7% of subjects were producers of IL-10 in response to the extract. The compounds did not induce significant changes in the frequency of responders between atopy groups. Conclusion: Cytokines dosages will be finalized including the 120h supernatant, allowing the application of statistical analysis with the largest verisimilitude as the power and verify the effects of these compounds used.

Made available in DSpace on 2014-07-28T18:09:56Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 107.pdf: 2520157 bytes, checksum: 93334c0f181c08b7be000b7a1441e401 (MD5) Previous issue date: 2008 / Made available in DSpace on 2014-12-22T16:56:36Z (GMT). No. of bitstreams: 2 107.pdf: 2520157 bytes, checksum: 93334c0f181c08b7be000b7a1441e401 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / A quantidade de bacilos viáveis presentes na vacina BCG é um importante requisito de qualidade deste produto, pois permite avaliar a consistência de produção. A contagem de colônias em meio sólido é o método oficial recomendado pela Organização Mundial da Saúde e pela Farmacopéia Brasileira para a determinação da viabilidade do BCG. A difícil dispersão do BCG e seu crescimento lento conferem limitações ao método oficial, como a variabilidade dos resultados, a baixa reprodutibilidade e o prolongado tempo de análise. Este trabalho avaliou a aplicabilidade da técnica de redução do sal de tetrazol XTT como método alternativo para a determinação do número de viáveis em vacinas produzidas com o BCG Moreau-RJ. Foi demonstrada a linearidade das técnicas de redução do XTT realizadas com base em Kairo et al. (1999) e em Argüelles et al (2005) em 3, 24 e 48 horas de incubação. Os ajustes lineares foram obtidos pela técnica de redução do XTT a 1 mg/mL baseada em Kairo et al. (1999), com coeficiente de determinação (RP2P=0,99), em conformidade com a Resolução N. 899/2003 da ANVISA referente à validação de métodos bioanalíticos. O melhor ajuste abrangeu as concentrações de vacina de 2,0 a 16,0 x 10P6 PUFC/mL após 24 e 48 horas de incubação a 37C. Nestas condições de ensaio os números de viáveis obtidos preliminarmente para cinco lotes de vacina BCG intradérmica empregados na rotina de imunização do país e para três amostras da vacina de referência BRABCG003 foram fortemente correlacionados (r=0,96) àqueles fornecidos pelo método tradicional de cultivo em meio de Lowenstein-Jensen. Com base nos valores de referência preconizados pela Farmacopéia Brasileira, observou-se 100 por cento de concordância na interpretação final dos resultados fornecidos pelos dois métodos para os cinco lotes de vacina avaliados. / The number of culturable particles in the BCG vaccine is an important requirement of quality control, therefore it allows to evaluate the production consistency. The colony counting on solid medium is the official method recommended by the World Health Organization and by the Brazilian Pharmacopeia to determine the viability of the BCG on final lots. The nature of the organism (difficult dispersion and slow growth) confers inter-laboratory variation of results and time consuming characteristics to the current test. The present work evaluated the applicability of the reduction of the salt of tetrazol XTT as an alternative method to determine the number of viable units in vaccines produced with the BCG Moreau-RJ. The method was linear by the techniques of reduction of the XTT based on Kairo et. (1999) and on Argüelles (2005) after 3, 24 and 48 hours of incubation. The linear adjustment was obtained by assays of reduction of XTT at 1 mg/mL based on Kairo et al. (1999), with coefficient of determination (RP2P=0,99) required by the Resolution N° 899/2003 / ANVISA concernem to the validation of bioanalitical assays. Optimal calibration curves enclosed vaccine concentrations from 2,0 x 10P6 Pto 16,0 x 10P6 PCFU/mL after 24 and 48 hours of incubation at 37 °C. With these conditions the numbers of viable units obtained from five lots of intradermal vaccines used in the brazilian routine of immunization and from three samples of the reference vaccine BRABCG003 had been strongly correlated (r=0,96) to those provided by the current method of culture in Lowenstein-Jensen. Thus, we observed 100% of agreement between the final interpretations of the two methods of analysis considering the range of values referred by the Brazilian Pharmacopeia to BCG vaccine. Therefore, beyond the strong correlation with the official method, the reduction length of assay from 4 weeks to 48 hours, the relative low cost and the easiness of execution represent strong incentives to the validation of the reduction of XTT as an alternative method to replace the counting of colonies in Lowenstein-Jensen.

Made available in DSpace on 2016-05-11T13:01:05Z (GMT). No. of bitstreams: 2 natalia_roque_ioc_dout_2015.pdf: 13428594 bytes, checksum: 3b9534ef5ab8b753d140d8d49bb0c9a0 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Os corpúsculos lipídicos (CL), também chamados de gotas lipídicas, são organelas ricas em lipídios que tem sido frequentemente associadas às condições inflamatórias e infecciosas. O aumento no número e tamanho dessas organelas é um fenômeno bem regulado que tem sido associado com a persistência bacteriana. Nesse trabalho, nós investigamos os mecanismos pelos quais os CL induzidos pela micobactéria podem atuar em favor da infecção. Por microscopia eletrônica de transmissão (MET), nós mostramos que os CLs marcados com ADRP interagem com fagossomas durante a infecção por M. bovis bacillus Calmette-Guérin, (BCG) in vivo. Por microscopia de fluorescência, nós observamos que a interação entre os CL e os fagossomas não é dependente da viabilidade bacteriana. Nós mostramos uma maior frequência de CLs em proximidade com os fagossomas contendo partículas de látex revestidas com Lipoarabinomanana (LAM) ou Fosfatidilinositol manosídeo (PIM) de M. tuberculosis (M.tb), mas não com as partículas de látex não revestidas, sugerindo um mecanismo de recrutamento de CL para próximo dos fagossomas. Partículas de látex revestidas com LAM interagem com os CL, 102,60% mais do que as partículas de látex não revestidas em macrófagos carregados com ácido oleico. Além disso, nós observamos que a Rab7, uma importante GTPase endocítica e, marcador de endossoma tardio, assim como seu efetor RILP, mas não a Rab5 co-localizou com os CL marcados por Bodipy® induzidos pela infecção com BCG em 24 h. Por MET, nós observamos a imunolocalização da Rab7 co-localizada com os CL no local de interação com um fagossoma infectado durante a infecção experimental por BCG in vivo. De maneira interessante, nós observamos uma diminuição na aproximação das partículas revestidas com LAM e os CL após os macrófagos terem sidos tratados com CID1067700, um inibidor competitivo da Rab7 Nós observamos que o BCG e o LAM, mas não, a micobactéria não patogênica M. smegmatis, ou partículas de látex não revestidas, foram capazes de induzir a formação de CL em macrófagos in vitro. O papel dos CL na modulação da infecção micobacterianas foi avaliado pelo tratamento com C75 (inibidor de ácido graxo sintase) que modulou negativamente a formação dos CL e a síntese de PGE2 induzidos por BCG. Além disso, esse tratamento foi capaz de aumentar o TNF-\03B1 enquanto inibiu a produção de IL-10 e a sobrevivência micobacteriana, determinado por contagens de UFC. Esses resultados sugerem que a interação dos CLfagossomas é um fenômeno bem regulado e modulado pelo componente da parede micobacterina, LAM e dependente de Rab7. O sequestro da Rab7 ativa para os CL pode permitir a interação com o fagossomas e favorecer a troca de lipídios atuando como nutrientes para sobrevivência micobacteriana. Além disso, nossos resultados indicam que os CL modulam mediadores pro- e anti- inflamatórios em favor da replicação e sobrevivência do BCG. Assim, sugerindo que a inibição da formação e/ou função dos corpúsculos lipídicos como um alvo promissor para intervenções terapêuticas na infecção micobacteriana / Lipid bodies (LB) also named lipid droplets, are lipid rich organelles that have been often associated with inflammatory and infectious conditions. The increase in number and size of these organelles is a well-regulated phenomenon that have been involved with bacterial persistence. In this work, we investigated the mechanisms by which mycobacteria induced -lipid bodies may act in favor of infection. By Transmission electron microscopy (TEM), we showed ADRP-marked LB interacting with phagosomes during M. bovis bacillus Calmette- Guérin, (BCG) infection in vivo. By fluorescence microscopy we observed that the interaction between LB and phagosomes is not dependent of bacterial viability. We showed an increase in the frequency of LB proximity with phagosomes containing beads coated with Lipoarabinomannan (LAM) or Phosphatidylinositol mannoside (PIM) from M. tb, but not with non-coated beads suggesting a recruitment mechanism for LB-phagosome proximity. LAM coated beads interacts with LB 102,60% more than non- coated beads in macrophages loaded with oleic acid. Moreover, we observed that Rab7, an important endocytic GTPase and late endosome marker, as well as its effector RILP but not Rab5 was co-localized with LB stained with Bodipy® induced by BCG infection at 24 h. By MET, we observed an immunolocalization of Rab7 co-localized with LB in the site of interaction with an infected phagosome during the experimental BCG infection in vivo. Interestingly, we observed a decrease of LAM coated bead and LB apposition after macrophages were treated with CID1067700, a competitive inhibitor of Rab7. We observed that BCG and LAM, but not nonpathogenic mycobacteria M. smegmatis or non-coated latex beads, was able to induce LB formation in macrophages in vitro. Next, we evaluated the role of LB modulation on mycobacterial infection Treatment with C75 (fatty acid synthase inhibitor) down-regulated LB formation and PGE2 synthesis induced by BCG. Also, these treatment were able to enhance TNF-\03B1 while inhibit the IL-10 production and down-modulation of mycobacterial survival and growth assessed by CFU count. These results suggest that LBphagosome interaction are well-regulated phenomena modulated by mycobacteria cell wall component, LAM and dependent on Rab7. The hijack of active Rab7 to LB may enable the interaction with phagosome, and may favour the exchange of lipids acting as nutrients to mycobacterial survival. In addition, our results indicate that LB modulate proand anti- inflammatory mediators in favor of BCG survival and replication. Thus suggesting that inhibition of LB formation and/or function as a promising target for therapeutic interventions in mycobacterial infection

Made available in DSpace on 2014-06-11T19:26:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-06-28Bitstream added on 2014-06-13T20:55:07Z : No. of bitstreams: 1 tiveron_tg_me_botfmvz.pdf: 943287 bytes, checksum: 0700299366e235cf95f5bad0fd1b22ff (MD5) / Universidade Estadual Paulista (UNESP) / A tuberculose bovina é causada principalmente pelo Mycobacterium bovis, uma enfermidade mundialmente reconhecida que além de ser um problema de saúde pública proporciona grandes perdas econômicas e impactos negativos ao mercado internacional da carne. O objetivo deste trabalho foi observar a prevalência de lesões sugestivas de tuberculose em um frigorífico localizado na região sudeste do estado de São Paulo, Brasil. Os dados foram coletados a partir das papeletas do serviço de Inspeção Federal, nas quais observou-se a proporção de positividade de tuberculose bovina quanto ao sexo do animal, ao tipo de lesão e a classificação dos linfonodos. Os resultados avaliados demonstraram que não houve uma diferença relevante nos índices de positividade quanto ao sexo masculino e feminino nos animais abatidos nem quanto às lesões calcificadas e caseosas. Porém, os linfonodos mais acometidos foram os retrofaringeanos e mediastínicos; que entre os anos de 2.007 e 2.011, respectivamente, os retrofaríngeos apresentaram prevalência de 55,42%, 53,62%, 53,20%, 47,01%, 44,16% e os mediastínicos, revelou uma prevalência de 36,06%; 39,05%, 38,43%, 36,53% e 35,93%. No entanto, os demais linfonodos como traqueo-brônquico, parotidiano, esofagiano, hepático, apical e mesentéricos não obtiveram grandes índices. O presente estudo demonstra a importância do exame post-mortem e dos mapas nosográficos nos estabelecimentos matadouro-frigorífico para identificar os animais infectados, controlar a doença e promover a Saúde Pública / Bovine tuberculosis is caused mainly by Mycobacterium bovis, a globally recognized disease that besides being a public health problem is of great economic loss and negative impact on the international meat market. This study aimed to observe the prevalence of lesions that suggest tuberculosis in a refrigerator located in the southeastern state of Sao Paulo, Brazil. Data were collected from the ballots of Federal Inspection and a proportion of positive bovine tuberculosis on the sex of the animal, the type of injury and classification of lymph nodes were observed. The evaluated results showed that there was no significant difference in rates of positivity about the male and female animals slaughtered and the calcified and caseous lesions. But the lymph nodes were the most affected retropharyngeal and mediastinal that between the years 2007 and 2011, respectively, the retropharyngeal presented the prevalence of 55.42%, 53.62%, 53.20%, 47.01%, 44, 16% and mediastinal revealed a prevalence of 36.06%, 39.05%, 38.43%, 36.53% and 35.93%. Further as tracheo-bronchial lymph nodes, parotid, esophageal, liver, and mesenteric apical was also evaluated but did not get great rates. The present study demonstrates the importance of post-mortem examination and maps nosographic slaughter and fridgeestablishments to identify infected animals to control disease and to promote public health

Tesis para optar al Grado de Magíster en Ciencias Animales y Veterinarias mención Medicina Preventiva Animal. / Con el objetivo de implementar métodos que disminuyan los tiempos de respuesta diagnóstica y por ende proporcionen rapidez en la emisión del resultado de tuberculosis bovina, se evaluaron y compararon los resultados obtenidos por los métodos BACTEC MGIT 960, cultivo bacteriológico en medio Stonebrink (ST), reacción en cadena de la polimerasa en tiempo real (RT-PCR), e Histopatología. Todo el trabajo se realizó en el Laboratorio de Micobacterias del Subdepartamento Laboratorios y Estación Cuarentenaria Pecuaria del Servicio Agrícola y Ganadero (SAG), siguiendo los protocolos establecidos por el laboratorio. Se utilizaron 100 muestras de tejido obtenidas de nódulos linfáticos y/u órganos con lesiones del tipo granulomatosas (LTG), provenientes de bovinos de plantas faenadoras o por inspección de seguimiento. Los resultados mostraron que el cultivo en el sistema BACTEC MGIT 960 puede disminuir el tiempo de detección de Mycobacterium bovis y favorecer la rapidez en la emisión del resultado en el diagnóstico de la Tuberculosis Bovina (TBB). En combinación las cuatro pruebas detectaron 89 muestras positivas y todas tuvieron concordancia positiva entre sí, que varió entre débil a moderada, según los índices de Kappa, con significancia estadística (p < 0,05). En cuanto a la eficiencia, cada método sobresalió por un parámetro diferente, siendo RT-PCR menos compleja y más rápida, los cultivos los más económicos comercialmente y de mayor certidumbre, aunque la Histopatología resultó con el mejor nivel de eficiencia combinado. Se propone implementar un esquema que incluya los cuatro métodos (histopatológico, molecular, un cultivo en medio sólido y un cultivo en medio líquido), que permita detectar el número máximo de resultados positivos, en el menor tiempo posible y con alta certidumbre. / In order to implement methods to shorten the time of growth and thus provide speed in issuing the results in the diagnosis of bovine tuberculosis were evaluated and compared the results obtained by the methods BACTEC MGIT 960, bacteriological culture amid Stonebrink (ST ), polymerase chain reaction in real time (RT-PCR), and histopathology. All work was performed in the Laboratory of mycobacteria at Subdepartment of Laboratories and Quarantine Station of the Agricultural and Livestock Service (SAG), following the protocols established by the Laboratory. 100 tissue samples obtained from bovine lymph nodes and / or organs with granulomatous lesions (LTG) were used. The results showed that cultivation in the MGIT 960 system can shorten the growth of Mycobacterium bovis and the time required for diagnosis of bovine tuberculosis. In combination, the four tests detected 89 positive samples and all had positive agreement among themselves, with significant (p<0,05) Kappa indexes which ranged from weak to middle values. Regarding efficiency, each method excelled some parameter, with RT-PCR being less complex and faster, bacteriological culture were the cheapest, although histopathology had the highest combined efficiency. These results support a diagnostic scheme that includes the four methods (histopathological, molecular, solid medium and a liquid medium) for achievement the maximum number of isolates, in the shortest time and with highest certainty.

Made available in DSpace on 2014-06-11T19:30:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2003Bitstream added on 2014-06-13T19:18:36Z : No. of bitstreams: 1 cavalheiro_js_me_botfmvz.pdf: 256330 bytes, checksum: f23556a464c6107559d352bd6a674722 (MD5) / Universidade Estadual Paulista (UNESP) / A tuberculose bovina é uma doença transmissível de natureza granulomatosa crônica e caráter progressivo, apresentando tubérculos característicos, tendo como agente etiológico o Mycobacterium bovis - um patógeno intracelular. A imunidade celular é o mecanismo mais efetivo para o controle da infecção por Mycobacterium spp. Esta resposta resulta no acúmulo de fagócitos e na formação de lesão macroscópica (tubérculo ou granuloma tuberculoso), na tentativa de impedir a proliferação e disseminação bacteriana. Neste trabalho foram utilizados modelos experimentais murinos, obtidos por seleção genética bidirecional de camundongos bons (High=H) e maus (Low=L) produtores de anticorpos (Seleção IVA) contra antígenos naturais complexos. Trabalhos anteriores demonstraram alterações funcionais de células imunocompetentes, particularmente de macrófagos, sugerindo que os animais HIV-A seriam mais susceptíveis à infecção causada por patógenos intracelulares, enquanto que ocorreria maior susceptibilidade dos LIV-A à infecção por patógenos extracelulares, quando a imunidade humoral seria o mecanismo mais importante... . / The bovine tuberculosis is a transmissible disease, showing a chronic and progressive character, with typic tubercles. Its etiological agent, Mycobacterium bovis, is an intracellular pathogen. Cell-mediated immunity is the most effective mechanism for this infection control, with phagocytes accumulum and formation of a macroscopic lesion, aiming to avoid the bacteria proliferation and dissemination. In this work, a murine experimental model was used, with mice genetically selected for high (H) and low (L) antibody production (Selection IV-A). Previous works related functional alterations in immunecompetent cells, manily macrophages, suggesting that HIV-A mice were more susceptible to intracellular pathogens, whereas LIV-A were more susceptible to extracellular ones, when humoral-mediated immunity would be the most important mechanism. The goal of this work was to evaluate the macrophagic activity and to characterize the immune response of Mycobacterium bovis-AN5-infected mice. The response prolife previously observed in these strains was not similar in the Mycobacterium bovis infection; however, it was in agreement with works carried out with selection I, which is very similar the selection IV-A with regards to infection with Mycobacterium tuberculosis and Bacillus Calmette-Guérin. As to the bacterial recovery, LIV-A selection showed a higher containnment of the infectious process in the lung, but not in the spleen, where HIV-A selection was more resistant. With respect the macrophagic activity, H2O2 seemed to be not involved in the containment of the infection in both strains, since there was an inibition of this metabolite generation. NO and TNF- production was also analysed, and seemed to be important in the control of bacterial replication, varying according to the strain, time period and compartiment. Antibody production was also evaluated... (Complete abstract, click electronic address below).

Submitted by Mario BC (mario@bc.ufrpe.br) on 2018-03-07T12:29:19Z No. of bitstreams: 1 Renata Gomes Revoredo.pdf: 1561355 bytes, checksum: 4aee3bbcbec49d64292b65d441e7c880 (MD5) / Made available in DSpace on 2018-03-07T12:29:19Z (GMT). No. of bitstreams: 1 Renata Gomes Revoredo.pdf: 1561355 bytes, checksum: 4aee3bbcbec49d64292b65d441e7c880 (MD5) Previous issue date: 2016-02-26 / The Mycobacterium bovis belongs to the Mycobacterium tuberculosis Complex and is responsible for tuberculosis in bovines and buffaloes, as well as in humans. This study aimed to carry out biomolecular assays using PCR for investigate M. bovis in serum samples of bovine subjected to the tuberculin test. A hundred samples, from seemingly healthy bovines and previously submitted to the tuberculin test, raised in six properties from different cities of the State of Pernambuco, were examined for Genomic identification of M. bovis. The samples were chosen at random by non-probability convenience and distributed in six groups. From the samples subjected to PCR, 5% (100/05) resulted positive for M. Bovis. The tuberculin test showed a positive reaction on 19% (19/100) of the animals. Was possible to conclude that the real time PCR, when applied directly in blood serum, showed low sensitivity to be used as a tool in routine diagnosis of tuberculosis in bovine. / O Mycobacterium bovis integra o Complexo Mycobacterium tuberculosis e é responsável pela tuberculose nos bovinos e bubalinos (TB), entre outras espécies, inclusive a humana. Objetivou-se com este estudo realizar ensaios biomoleculares investigativos usando PCR para pesquisa de Mycobacteruim bovis em amostras séricas de bovinos previamente submetidos ao teste da tuberculina. Foram examinadas 100 amostras séricas para identificação genômica do M. bovis, escolhidas ao acaso por conveniência não probabilística e distribuídas em seis grupos experimentais, provenientes de bovinos aparentemente hígidos e previamente submetidos ao Teste da Tuberculina, criados em seis propriedades de diferentes municípios do estado de Pernambuco. Das 100 amostras de soro sanguíneo submetidas à PCR, 5% (05/100) apresentaram positividade ao M. Bovis. Quanto ao Teste da Tuberculina, 19% (19/100) dos animais apresentaram reação positiva. Concluiu-se que a PCR em tempo real, quando aplicada diretamente em amostras de soro sanguineo, demonstrou baixa sensibilidade para ser empregada como ferramenta na rotina diagnóstica da tuberculose em bovinos.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-02-15T13:08:10Z No. of bitstreams: 1 gabrielsantoscruzrodrigues.pdf: 1333283 bytes, checksum: 46d4dabb8192501d9b5b879ecdd9a3e4 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-02-26T12:23:19Z (GMT) No. of bitstreams: 1 gabrielsantoscruzrodrigues.pdf: 1333283 bytes, checksum: 46d4dabb8192501d9b5b879ecdd9a3e4 (MD5) / Made available in DSpace on 2016-02-26T12:23:19Z (GMT). No. of bitstreams: 1 gabrielsantoscruzrodrigues.pdf: 1333283 bytes, checksum: 46d4dabb8192501d9b5b879ecdd9a3e4 (MD5) Previous issue date: 2014-04-03 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A obesidade é um grande problema de saúde mundial e com os hábitos e estilos de vidas atuais a sua importância social só tende a crescer. Hoje associa-se a obesidade com o mau prognóstico de diversas doenças, como câncer, diabetes e doenças auto-imune brandas, além disso, ela também está associada a uma resposta imune deficitária frente a infecções por diversos patógenos, como alguns vírus e bactérias. A tuberculose é uma doença infecto-contagiosa, mais comumente pulmonar, causada pelo Mycobacterium tuberculosis (ou bacilo de Koch) e outros membros do complexo Mycobacterium tuberculosis. Esta doença causa cerca de 1,4 milhões de morte ao ano, sendo constatados cerca de 8 milhões de novos casos ao ano , estando apenas atrás da AIDS na taxa de mortalidade por doenças infecciosas e seu sucesso se deve em parte à capacidade de viver de meses à décadas no hospedeiro, em estado assintomático. Neste trabalho, avaliamos a relação entre a obesidade e o desenvolvimento da tuberculose, através do modelo de infecção experimental por Mycobacterium bovis BCG em camundongos C57/BL6 controles e tratados com dieta hiperglicídica. Os resultados demonstraram que os animais obesos apresentaram um menor recrutamento leucocitário, dentre estes, menor influxo de neutrófilos e eosinófilos. Além disso, animais obesos apresentaram menor formação de corpúsculos lipídicos, menor produção de PGE2, maior produção de mediadores anti-inflamatórios, como IL-10 e adiponectina, e menor produção de mediadores pró-inflamatórios, como leptina, em comparação aos animais controles. De maneira geral, nossos resultados apontam que a obesidade induz um perfil mais anti-inflamatório, inibindo a expressão de fatores de favorecimento do patógeno, como a formação de corpúsculos lipídicos, PGE2 e eosinofilia, sugerindo que a obesidade, nos estágios iniciais de infecção, pode modular negativamente a resposta inflamatória induzida pela infecção micobacteriana. / Obesity is a huge world health problem and with the habits and lifestyles of these days, it’s important only tend to grown. Today obesity is associated with a bad prognosis of a large number of diseases , like cancer, diabetes and light self-immune diseases, and besides that, its also associated with an uncompleted response to several pathogens, like some virus and bacteria. The tuberculosis is an infect-contagious disease, commonly pulmonary, caused by Mycobacterium tuberculosis (or, Koch bacillus), and other members of the Mycobacterium tuberculosis complex. This illness cause about 1,4 million deaths per year, being registered 8 million new cases per year, being only behind AIDS on the mortality ratio by infectious diseases in the world and ur success can be related to your capacity to live from months to decades on the host , in a asymptomatic state. In this work, we evaluated the relationship between the obesity and the development of tuberculosis, by using the experimental infection model by Mycobacterium bovis BCG. C57/BL6 mice, male, with about 4 to 6 weeks of life, had being fed with standard ration (control group) or hiperglicidic ration (obese group), for 0, 2 or 3 months. The body weight was monitored during this period, and the fat gain and glucose tolerance was measured at the end, so the state of obesity could be characterized. At the end of these periods, this animals received a intraperitoneal inoculation of 100 mL of saline solution (infection control) or 100 mL of BCG solution (infected group) and characteristic processes of this infection were observed, like leukocitary recruitment , lipid bodies formation on these cells, the production of pro and anti-inflammatory cytokines, eicosanoids (PGE2) and adipokines. Was observed then, that the obese animals, presented a lower leukocitary recruitment , among then, less influx of neutrophils and eosinophils, lower formation of lipid bodies and production of PGE2 , higher production of anti-inflammatory mediators (IL-10 and adipokine) and lower production of pro-inflammatory mediators (leptin) . Overall , our data points to a more anti-inflammatory profile on the obese animals , occurring in a minor expression of factors that are important to the pathogen survival , like the formation of lipid bodies and eosinophilia , indicating that, the obesity, on the early stages of the infection, may be representing a disadvantaging factor to the disease.

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

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.