Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-08-01T13:42:41Z No. of bitstreams: 1 RenataCarolineCostaDeFreitas_DISSERT.pdf: 1600372 bytes, checksum: 4d58eee589d9f02fdfaf2ffcdc800d96 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-08-07T12:11:18Z (GMT) No. of bitstreams: 1 RenataCarolineCostaDeFreitas_DISSERT.pdf: 1600372 bytes, checksum: 4d58eee589d9f02fdfaf2ffcdc800d96 (MD5) / Made available in DSpace on 2017-08-07T12:11:18Z (GMT). No. of bitstreams: 1 RenataCarolineCostaDeFreitas_DISSERT.pdf: 1600372 bytes, checksum: 4d58eee589d9f02fdfaf2ffcdc800d96 (MD5) Previous issue date: 2017-06-16 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / O clopidogrel ? um f?rmaco utilizado na terapia para preven??o de trombose e aterosclerose. Contudo, a hepatotoxicidade induzida por clopidogrel ? um potencial efeito adverso relacionado com les?es hep?ticas e resposta antiplaquet?ria. Considerando a falta de diagn?stico precoce para esse efeito adverso, miRNAs derivados de exossomos podem ser ?teis para contribuir para o monitoramento da resposta antiplaquet?ria e na predi??o do risco de hepatotoxicidade. Portanto, primeiramente objetivamos investigar as intera??es miRNA-mRNA com a toxicidade do f?rmaco in silico utilizando dados de microarrays dispon?veis e software Ingenuity Pathways Analysis 6 (IPA). Em seguida, o perfil de express?o exossomal dos miR-26a-5p, miR-145-5p, miR-15b-5p e miR-4701-3p, bem como os mRNAs alvos (PLOD2, SENP5, EIF4G2, HMGA2, STRADB e TLK1) derivados de c?lulas foram avaliadas in vitro, uma vez que estes foram os alvos moleculares principalmente associados ao efeito adverso. Assim, c?lulas HepG2 foram incubadas com clopidogrel a 6,25, 12,5, 25, 50 e 100 ?M durante 24 e 48 h. A citotoxicidade foi avaliada por citometria de fluxo pela an?lise da fragmenta??o do DNA e do ciclo celular. A express?o de miRNA derivados de exossomos e de mRNA derivados de c?lulas foi avaliado pela RT-qPCR. As c?lulas tratadas com concentra??es mais elevadas de 50 e 100 ?M causaram maior fragmenta??o do DNA ap?s 24 e 48 h sugerindo ser uma concentra??o t?xica. A express?o do miR-26a-5p foi elevada e a express?o do miR-15b-5p foi diminu?da na concentra??o t?xica 100 ?M em rela??o ao controle em 24 e 48 h. HMGA2, EIF4G2, STRADB e SENP5 alvos do miR-26a-5p foram pouco expressos nas concentra??es t?xicas em 24 h e o HMGA2 se manteve pouco expresso depois de 48 h de tratamento, em rela??o ao controle. TLK1, alvo do miR-15b-5p, teve express?o diminu?da em 24 h na concentra??o t?xica. Os resultados sugerem que concentra??es t?xicas de clopidogrel podem modular a express?o de miR-26a-5p e miR-15b-5p e seus alvos de mRNA. Al?m disso, o miR-26a pode representar um marcador importante para prever a hepatotoxicidade induzida por clopidogrel / Clopidogrel is an essential therapy for prevention of thrombosis and atherosclerosis. However, clopidogrel-induced hepatotoxicity is a potential adverse effect related to liver damage and antiplatelet response. Considering the lack of diagnostic for this adverse effect, new exosomes-derived miRNAs may be useful for improve the monitoring of response and hepatotoxicity risk. Therefore, we first aimed to investigate the miRNA-mRNA interactions with drug toxicity by in silico using available microarray data and Ingenuity Pathways Analysis 6 (IPA) software. After, the exosomal-expression profile of miR-26a-5p, miR-145-5p, miR-15b-5p and miR-4701-3p, as well as the cell-derived mRNAs target PLOD2, SENP5, EIF4G2, HMGA2, STRADB and TLK1 were evaluated in vitro, once they were the molecular targets mainly associated with the adverse effect. Thus, HepG2 cells were incubated with clopidogrel at 6.25, 12.5, 25, 50, and 100 ?M for 24 and 48 h. The cytotoxicity was evaluated by flow cytometry to analyze DNA fragmentation and cell cycle. Profile expression of exosomes-derived miRNAs obtained by column methods and cell-derived mRNAs was evaluated by RT-qPCR. Cells treated with higher concentrations of 50 and 100 ?M caused an increased DNA fragmentation after 24 and 48 h suggesting a toxic concentration for the cells. The miR-26a-5p upregulation in toxic concentration of 100 ?M of clopidogrel and a downregulation of miR-15b-5p in comparison to control were observed in both period of 24 and 48 h. HMGA2, EIF4G2, STRADB and SNP5 targets of miR-26a-5p were downregulated in toxic concentrations at 24 h and HMGA2 remains downregulated after 48 h of clopidogrel treatment. TLK1, a target of miR-15b-5p, was downregulated at 24 h in toxic concentration. The results are suggestive that toxic concentrations of clopidogrel may modulate the miR-26a-5p and miR-15b-5p expression and their mRNA targets. Moreover, miR-26a may represent an important marker to predict clopidogrel-induced hepatotoxicity.