Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O cÃncer à caracterizado por ser um conjunto de doenÃas que envolve crescimento descontrolado, surgimento e espalhamento de cÃlulas anormais, e à considerada uma das principais causa de morte por doenÃa no mundo. AscÃdias e microorganismos marinhos sÃo profÃcuos produtores de substÃncias com atividade citotÃxica e antitumoral. Estudos preliminares com a ascÃdia Eudistoma vannamei, endÃmica do nordeste brasileiro, identificaram uma potente atividade anticÃncer de seu extrato e, a partir dele, isolaram-se esteuroporinas inÃditas. Curiosamente, tais molÃculas sÃo comumente produzidas por bactÃrias. No presente trabalho, os extratos dos microorganismos isolados da ascÃdia foram testados quanto a sua citotoxicidade e o mais potente deles foi selecionado e indentificado como actinomiceto pertencente ao gÃnero Streptomyces sp. A partir da purificaÃÃo desse extrato, foi isolada uma ditiolpirrolona citotÃxica que apresentou valores de concentraÃÃo inibitÃria mÃdia variando de 1,05 a 6,39 μM, em diferentes linhagens tumorais. Estudos realizados em cÃlulas de carcinoma prostÃtico humano metastÃtico indicaram que a ditiolpirrolona causou despolarizaÃÃo mitocondrial e induziu o aparecimento de figuras mitÃticas, apÃs 24 e 48 horas de tratamento. AlÃm disso, detectou-se alteraÃÃo no ciclo celular, como atraso nas fases final do ciclo, S e G2/M. A ditiolpirrolona alterou a expressÃo de algumas proteinas relacionadas ao ciclo celular e principalmente à citocinese, como Prc1, Plk-1 e RhoA, tais proteÃnas estÃo diretamente envolvidas com a formaÃÃo do anel contrÃtil, imprescindÃvel para a conclusÃo da divisÃo celular / Ascidians and marine microorganisms are prolific producers of cytotoxic and antitumor compounds. As part of a study to examine Brazilian species, we examined microbiota associated with ascidian, Eudistoma vannamei as potential sources for active natural product leads Earlier revisions with this specie showed a potent anticanceractivity of its extract; and two unpublished staurosporines were isolated. Curiously, these classes of compounds are generally produced by bacteria. So, in order to evaluate the biomedical potential of these compounds the microbiota associated to the ascidian Eudistoma vannamei was investigated. From this effort we isolated a number of bacterial strains and after screening for cytotoxicity using a panel of tumor cell lines, we identified a Streptomyces sp.,that presented significant bioactivity. Using bioactivity fractionation, we identified the active compound as, dithiolopyrrolone N-(4,5-dihydro-5-oxo-1,2- dithiolo[4,3-b]pyrrol-6-yl)-N-methyl-formamide also know as VD846. The compound presented IC50 values ranging from 1.1 to 6.4 μM across a panel of cell lines. Further biological studies, indicated that this compound induced cell cycle arrest during mitosis, an observation that was confirmed by evaluating the effects on a series of cell lines using mitotic index analyses, flow cytometry, and confocal microscope. Western blot analyses indicated that cells treated with VD846 resulted in reduced expression of Plk1 and RhoA, proteins that are necessary for cleavage furrow assembly and exit from cytokinesis.

Du fait de l’apparition de microorganismes pathogènes ayant une résistance aux antibiotiques actuels, la recherche de nouvelles molécules bioactives possédant une application médicale est devenue une préoccupation mondiale. Saccharothrix algeriensis, une bactérie filamenteuse de l’ordre des actinomycètes a montré une étonnante capacité à produire des molécules bioactives qui appartiennent aux dithiolopyrrolones, ayant de remarquables propriétés à la fois antibiotiques et anticancéreuses. Lors de ce projet de thèse, l’identification du cluster de gènes de la voie de biosynthèse des dithiolopyrrolones chez Sa. algeriensis est envisagée. Suite au séquençage du génome de Sa. algeriensis, une approche génomique ou « genome mining » est suivie, cette approche a révélé un cluster thi potentiellement responsable de la voie de biosynthèse des dithiolopyrrolones chez Sa. algeriensis. Ce cluster contient 12 gènes, dont 8 gènes de biosynthèse, 3 gènes régulateurs et un gène transporteur. Les analyses in silico des gènes ont montré que la cystéine est le substrat d’une NRPS. Les analyses transcriptionelles ont montré que les trois gènes clés codent pour une NRPS, une thiorédoxine et une thioestérase qui pourraient être impliquées dans la biosynthèse des dithiolopyrrolones. Deux gènes actA et actB codant pour des acyltransférases putatives ont été identifiés. Les analyses transcriptionelles suggèrent qu’actA et actB pourraient être responsables de l’acylation de la pyrrothine. Finalement, la caractérisation de deux activités enzymatiques, acétyltransférase et benzoyltransférase, présentes dans l’extrait brut de Sa. algeriensis, ont permis de déterminer les paramètres optimaux (pH et T °C) de la réaction enzymatique. Enfin, les paramètres cinétiques de ces activités ont des valeurs complètements différentes, ce qui confirme la présence d’au moins deux activités différentes chez Sa. algeriensis. / Due to the emergence in the last decades of new and old infectious diseases to existing antibiotics, the research for new bioactive molecules which possess medical applications become a global occupation. Saccharothrix algeriensis, filamentous bacteria of actinomycetes order showed a surprising ability to produce bioactive molecules belongs to dithiolopyrrolones with remarkable properties of both antibiotics and anticancer. In this thesis, the identification of dithiolopyrrolones biosynthetic gene cluster in Sa. algeriensis was investigated. Through S. algeriensis genome sequencing, a genomics approach "genome mining" was followed, this approach has revealed a potentially thi cluster responsible for dithiolopyrrolones biosynthesis pathway in Sa algeriensis. This cluster contains 12 genes, including 8 biosynthesis genes, three regulatory genes and one transporter gene. The in silico analysis of this cluster showed that the cysteine is the substrate of the NRPS. The transcriptional analyzes showed that the three key genes which encode for NRPS, thioredoxin and thioesterase could be involved in dithiolopyrrolone biosyntheses. Two genes, actA and actB, encode for two putative acyltransferases were identified, the transcriptional analyzes suggests that these genes may be responsible for the acylation of pyrrothine core. The characterizations of two activities, acetyltransferase and benzoyltransferase, in the crude extract of Sa. algeriensis led the determination of the optimal parameters (pH and T °C) to detect these activities. Moreover, the effect of temperature and pH on these activities was determined. Finally, the kinetic parameters of these activities showed different values, which confirm the presence of, at least, two activities in Sa. algeriensis.