Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-09-04T20:22:35Z No. of bitstreams: 1 LucasLuizVieiraDissertacao2016.pdf: 2717272 bytes, checksum: b547db9dacaad5a2da23826b5d215ccb (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-09-04T20:23:00Z (GMT) No. of bitstreams: 1 LucasLuizVieiraDissertacao2016.pdf: 2717272 bytes, checksum: b547db9dacaad5a2da23826b5d215ccb (MD5) / Made available in DSpace on 2017-09-04T20:23:00Z (GMT). No. of bitstreams: 1 LucasLuizVieiraDissertacao2016.pdf: 2717272 bytes, checksum: b547db9dacaad5a2da23826b5d215ccb (MD5) Previous issue date: 2016-04-14 / Invasive fungal infections are a major public health problem in the world, since they increase morbidity and patients??? hospitalization time. In Brazil, the highest mortality rate among the systemic mycoses is caused by an endemic disease named paracoccidioimycosis, which the etiologic agent is the dimorphic fungus Paracoccidioides spp. The worldwide increased resistance to the commercially available antifungal agents, their limited spectrum of activity against some fungal pathogens and concerns with their toxic side effects are reasonable evidence of the necessity of novel therapeutic strategies, especially the development of new antifungal agents. Thus, the essential gene rim8 was identified by comparative genomics as an orthologous sequence in the genome of human pathogenic fungi absent in the human genome. In filamentous fungi and yeasts, gene expression regulation by the ambient pH involves components of a signaling pathway that mediate proteolytic activation of the transcription factor PacC/Rim101 in response to alkaline environmental pH. The rim8 gene in yeasts, also called palF in filamentous fungi, performs an essential step in this signaling pathway. It is important for host-pathogen interaction, leading to increased virulence and pathogen survival. Thus, Rim8 is a very interesting and promising drug target. The aim of this work is to optimize heterologous expression and purification of Rim8 protein from P. luzii to further perform its structural and functional characterization and also, to use it as a molecular target for drugs development. The rim8 gene was chemically synthesized with a histidine tag and cloned into a pET-21a vector. Heterologous expression of the gene was made using two strains of E. coli, obtaining the best conditions using LB medium with 0.5% glucose at 30 ??C for 2 hours and 0.25 mM IPTG and adding protease inhibitor cocktail. The Rim8 heterologous protein was purified using a nickel affinity chromatography column. Expression and purification results were analyzed by SDS-PAGE and in some cases, confirmed by western blot. Immunization of BALB/c mice was performed with the purified protein to obtain anti- Rim8 antibodies. The antibody production was confirmed by ELISA test. The protein immunocitolocalization in P. lutzii cells showed a difuse protein-plasma membrane association at acidic pH. At neutral pH the fluorescence pattern is showed as localized foci in plasma membrane and later, with extracellular alcalinization, it migrates into the cytosol. Thus, it can be inferred that this work gives some contribution to the development of new antifungal drugs, but still must undergo further production steps, proteolysis reduction and protein purification to allow structural and functional characterization of Rim8. / Infec????es f??ngicas invasivas s??o um problema de sa??de p??blica no mundo, j?? que aumentam a morbidade e o tempo de interna????o de pacientes. A micose sist??mica com o maior ??ndice de mortalidade no Brasil, onde ?? considerada end??mica, ?? a paracoccidioidomicose, causada pelo fungo dim??rfico Paracoccidioides spp. A tend??ncia global de aumento da resist??ncia aos agentes antif??ngicos dispon??veis comercialmente, o espectro limitado de atividade contra alguns fungos patog??nicos e a preocupa????o com a toxicidade s??o evid??ncias da necessidade de novas estrat??gias terap??uticas, sobretudo do desenvolvimento de novas drogas antif??ngicas. Nesse sentido, o gene essencial rim8 foi identificado por gen??mica comparativa como uma sequ??ncia ort??loga no genoma de fungos patog??nicos humanos e ausente em humanos. Em fungos filamentosos e leveduras, a regula????o da express??o g??nica pelo pH envolve componentes de uma via de sinaliza????o que levam ?? ativa????o proteol??tica do fator de transcri????o PacC/Rim101 em resposta ?? alcaliniza????o do pH externo. O gene rim8 em leveduras ou palF em fungos filamentosos possui um papel essencial nessa cascata de sinaliza????o, sendo importante para a intera????o pat??geno-hospedeiro, aumento de virul??ncia e sobreviv??ncia do pat??geno. Por isso, Rim8 ?? um alvo molecular promissor e bastante interessante para o desenvolvimento de drogas. O objetivo deste trabalho ?? otimizar a express??o heter??loga e purifica????o da prote??na Rim8 de P. lutzii, visando realizar a caracteriza????o estrutural e funcional da prote??na para futuramente utiliz??-la como alvo molecular para o desenvolvimento de drogas. O gene rim8 foi sintetizado quimicamente com cauda de histidinas e foi clonado em vetor pET-21a. A express??o heter??loga do gene foi padronizada usando duas estirpes de E. coli, obtendo as melhores condi????es para purifica????o com o uso de meio LB contendo 0,5% de glicose a 30??C por 2 h e 0,25 mM de IPTG e coquetel de inibidores de proteases. Os resultados da express??o e purifica????o foram analisados por SDS-PAGE e/ou confirmados por western blot. Realizou-se a imuniza????o de camundongos BALB/c com a prote??na purificada para obten????o de anticorpos anti-Rim8. A produ????o dos anticorpos foi confirmada por ELISA. A imunocitolocaliza????o em c??lulas de P. lutzii mostrou que a prote??na se encontra associada ?? membrana plasm??tica de forma difusa em pH ??cido, em seguida apresenta focos localizados em resposta a um pH pr??ximo da neutralidade e, por fim, migra para o interior da c??lula em pH alcalino. Sendo assim, pode-se inferir que o trabalho apresenta contribui????es para o desenvolvimento de novas drogas antif??ngicas, al??m de auxiliar o entendimento do processo biol??gico no qual a prote??na RIM8 est?? envolvida.