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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Avaliação da expressão de um suposto gene responsável pela síntese de sideróforo em mycobacterium massiliense, em diferentes condições de disponibilidade de ferro / Evaluation of the expression of a putative gene responsible for the synthesis of siderophore in Mycobacterium massiliense under different conditions of iron availability

Rocha, V. L. 21 May 2014 (has links)
Submitted by Luanna Matias (lua_matias@yahoo.com.br) on 2015-02-04T16:18:28Z No. of bitstreams: 2 Dissertação - Viviane Lopes Rocha - 2014.pdf: 3025545 bytes, checksum: 8144fc57a9ecf1ebc51b1286fa161d44 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-02-05T10:07:09Z (GMT) No. of bitstreams: 2 Dissertação - Viviane Lopes Rocha - 2014.pdf: 3025545 bytes, checksum: 8144fc57a9ecf1ebc51b1286fa161d44 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-02-05T10:07:09Z (GMT). No. of bitstreams: 2 Dissertação - Viviane Lopes Rocha - 2014.pdf: 3025545 bytes, checksum: 8144fc57a9ecf1ebc51b1286fa161d44 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-05-21 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Mycobacterium massiliense (MM) has been associated as the causative agent of many nosocomial outbreaks related to laparoscopy, arthroscopic and wound infections. Several outbreaks have been reported in Brazil. The cities of Goiânia, Rio de Janeiro and Belem reported a high number of cases in 2006 and 2007. The iron ion (Fe) is extremely important for many biochemical processes in all organisms and, in the case of microorganisms, the success of the infection. Microorganisms synthesize molecules called siderophores (SD) to aid Fe uptake. Micobactin and carboximicobactin are the SDs that has been described in mycobacteria. One of the genes responsible for the assembly of these SD in M. tuberculosis is the mbtb. A MM strain, which belongs to the outbreak that happened in Goiânia (MM GO06) had its genome sequenced and the analysis revealed that the species has a putative gene with high similarity to M. tuberculosismbtb, which could be an indication that MM also synthesizes a siderophore molecule and that this can be helping this mycobacteria to install the infection in the host. It is known that in the absence of mbtb gene M. tuberculosis do not synthesize their SD. To estimate whether a gene similar to mbtb and with the same function is present in MM (smbtb) will assist in the understanding of the infection mechanisms of MM and discover new drug targets for treating infections with this microorganism. Total RNA was obtained from cultures grown at different concentrations of Fe. Real time PCR was performed targeting the smbtb to evaluate the expression of this gene during bacterial growth in each condition. The expression of smbtb was higher with the increase of the availability of iron. In vivo studies with mice supplemented with or chelated fromiron showed expression profile of smbtb different from those obtained in in vitro studies. In mice, M. massiliense smbtb expressed at higher levels when the animal were treated for iron depletion. Thus, we have evidence that smbtb is involved in iron uptake both for subsequent storage, when this ion is available, and for prompt use in the metabolism of the bacteria when it is not in an environment where there is availability of this ion. / Mycobacterium massiliense (MM) tem sido associado como agente causador de vários surtos nosocomiais relacionados à laparoscopia, artroscopia e infecções de feridas. Inúmeros surtos têm sido reportados no Brasil. As cidades de Goiânia, Rio de Janeiro e Belém apresentaram um alto número de casos em 2006 e 2007. O íon ferro é extremamente importante para vários processos bioquímicos em todos os organismos e, no caso dos microrganismos, para o sucesso da infecção. Para auxiliar a captação de Fe durante este processo, os microrganismos sintetizam moléculas chamadas sideróforos, que desempenham esta função. Micobactina e carboximicobactina são os sideróforos que já foram descritos em micobactérias. Um dos genes responsáveis pela síntese dos sideróforos em M. tuberculosis é o mbtb. Um isolado de MM, com origem no surto que aconteceu em Goiânia (MM GO06) teve seu genoma sequenciado e sua análise revelou que esta espécie possui um gene putativo com alta similaridade com o mbtb de M. tuberculosis, o que poderia indicar que MM também sintetiza sideróforos e que está molécula poderia estar auxiliando esta micobactéria a instalar a infecção no hospedeiro. Sabemos que a ausência do gene mbtb em M. uberculosis torna esta micobactéria incapaz de sintetizar sideróforos. Avaliar se um gene similar ao mbtb e com a mesma função está presente em MM (smbtb) irá auxiliar o entendimento dos mecanismos de infecção de MM e a descoberta de novos alvos para drogas para o tratamento de infecções causadas por MM. RNA total foi obtido de culturas onde MM foi crescido em diferentes concentrações de ferro. Realizou-se Real Time PCR para o gene smbtb a fim de avaliar a expressão deste gene durante o crescimento bacteriano em cada condição. A expressão do sbmtb aumentou com o aumento da disponibilidade de ferro. Estudos in vivo com camundongos suplementados ou privados do íon ferro apresentaram um perfil de expressão diferente daquele obtido nos estudos in vitro. Em camundongos, MM expressou smbtb em altos níveis nos animais que foram tratados com quelante para o íon ferro. Evidenciamos então, que smbtb pode estar envolvido na captação de ferro tanto para armazenamento deste íon, quando o mesmo está isponível, quanto para a utilização imediata no metabolismo da bactéria, quando há uma baixa disponibilidade de ferro no ambiente que MM se encontra.
2

Chimie innovante en série dioxyde de quinoxaline : vers de nouveaux antituberculeux, inhibiteurs de la biosynthèse des mycobactines / Innovative quinoxaline dioxide chemistry : toward new antitubercular drugs, inhibitors of mycobactin biosynthesis

Dahbi, Samir 21 November 2012 (has links)
Afin d'internaliser le fer, un micronutriment essentiel pour sa survie, Mycobacterium tuberculosis, la mycobactérie responsable de la tuberculose, biosynthétise des composés ayant une très grande affinité pour Fe3+ appelés les mycobactines. Ces sidérophores sont biosynthétisés par voie non­ ribosomale, la synthèse débutant par l'activation d'une molécule d'acide salicylique par l'enzyme d'adénylation mbtA sous la forme d'un dérivé ester d'adénosylmonophosphate (salicyl-AMP). Notre laboratoire a déjà préparé des analogues stables de salicyl-AMP, inhibiteurs potentiels de la biosynthèse des mycobactines. Durant la thèse, nous nous sommes attachés à la préparation de phosphonamidates et de sulfonamides porteurs d'un motif dioxyde de quinoxaline en tant qu'analogues de salicyl-AMP, qui devraient présenter une excellente affinité avec le site actif de l'enzyme mbtA. Nous avons développé une extension à la réaction de Beirut, permettant la préparation, pour la première fois, de phosphonates de dioxyde de quinoxaline, précurseurs de nos analogues phosphonamidates. Lorsque le dioxyde de quinoxaline était substitué par un groupement aryle en position 3, un réarrangement du phosphonate en phosphate de monoxyde de quinoxaline a été observé. Des études RMN ont permis de mettre en évidence le caractère intramoléculaire de ce réarrangement, inédit en série N-oxyde d'aryle. La suite de la synthèse pour accéder à nos analogues phosphonamidate s'est en revanche avérée difficile. D'autre part, afin d'accéder à nos analogues sulfonamides, nous avons développé une voie de synthèse utilisant des conditions douces, et qui nous a permis de préparer les premiers exemples de sulfonamide en série dioxyde de quinoxaline. Ces derniers devraient être testés afm d'évaluer l'influence du motif dioxyde de quinoxaline sur l'activité antituberculeuse, et ainsi confirmer le potentiel de nos analogues-cibles, dont la synthèse est à terminer. Enfin, en parallèle de la synthèse des analogues sulfonamides, nous avons développé avec succès une nouvelle préparation de dioxydes de quinoxaline 2,3-disubstituée via un couplage de type Liebeskind-Srogl, qui représente le premier exemple de couplage organométallique en série dioxyde de quinoxaline. / In order to intemalize iron, a vital micronutriment, Mycobacterium tuberculosis, the causative agent of tuberculosis, biosynthesizes compounds with extremely Fe (III) affinity, called mycobactins. The biosynthesis of these compounds is a non ribosomal process initiated by the adenylation enzyme mbtA, which activates a molecule of salicylic acid to the corresponding adenosylmonophosphate ester (salicyl-AMP). Our laboratory has already prepared various hydrolytically-stable analogues of salicyl-AMP as potential inhibitors of mycobactin biosynthesis. Lately, we have been working on the preparation of phosphonamidate and sulfonamide analogues with a quinoxaline 1,4-dioxide moiety, which should display a very good affinity with the active site of the enzyme mbtA. We successfully developed an extension to the Beirut reaction to access the first phosphonates of quinoxaline 1,4-dioxides, precursors of our phosphonamidates analogues. When the phopshonylated quinoxaline 1,4-dioxide was substituted with an aryl group on position 3, a rearrangement of the phosphonate into a phosphate of quinoxaline 1-monoxide was observed. NMR studies of this rearrangement, new in the N-aryl oxide series, suggested that it was intramolecular. The end of the synthesis to get our phosphonamidate analogues, however, proved difficult. Also, in order to prepare our sulfonamide analogues, we developed a synthesis that uses mild conditions and allowed us to access the first examples of sulfonamide in the quinoxaline 1,4-dioxide series, which should be tested to evaluate the influence of the quinoxaline 1,4-dioxide moiety on the antitubercular activity and confirm the potency of our targeted sulfonamide analogues, which have yet to be synthesized. Finally, while working on the synthesis of our sulfonamide analogues, we successfully developed a new preparation of 2,3-disubstituted quinoxaline 1,4-dioxide via a Liebeskind-Srogl-like cross-coupling reaction, which represents the first example of organometallic cross-coupling reaction in the quinoxaline 1,4-dioxide series.

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