Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-09-06T17:52:33Z No. of bitstreams: 1 DiannyElizabethJimenezJojoaTese2017.pdf: 3946754 bytes, checksum: 81cb9190de587ca935463b13f7a89e30 (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-09-06T17:52:38Z (GMT) No. of bitstreams: 1 DiannyElizabethJimenezJojoaTese2017.pdf: 3946754 bytes, checksum: 81cb9190de587ca935463b13f7a89e30 (MD5) / Made available in DSpace on 2017-09-06T17:52:38Z (GMT). No. of bitstreams: 1 DiannyElizabethJimenezJojoaTese2017.pdf: 3946754 bytes, checksum: 81cb9190de587ca935463b13f7a89e30 (MD5) Previous issue date: 2017-03-23 / The physiological and pathological processes are leaded by infinity of mechanisms and metabolic pathways. In the last decades, researches have focused on to small vesicles, the exosomes, from endosomal origin that could be involved in these processes. These vesicles can be released by different cell types carrying important components that could become biomarkers for the diagnosis of diseases or as moldable tools for controlled release of bioactive substances for clinical and therapeutic application. However, one of the challenges in the study of these interesting and versatile microvesicles is the purification process. Currently, significant difficulties are found as the lack of purity, yield and reproducibility revealed in the unreliable data at both the protein and transcriptional levels. Therefore, our aim in the present study was to compare in human serum samples the currently used purification techniques: ExoQuick??? (Systems Biosciences), Total Exosome Isolation (Life Technologies), Ultrafiltration and Ultracentrifugation and Size Exclusion Chromatography as regards to yield, size and purity evaluating, in addition, the reproducibility. The results obtained revealed the existence of differences between the techniques in the amount of particle recovery with sizes corresponding to the exosomes. Ultrafiltration was the most efficient technique followed by polymer precipitation methods and Ultracentrifugation. Protein content diversified considerably, with the Ultrafiltration almost ten times more than Ultracentrifugation. Different profiles of proteins in acrylamide gel were found, with 50 kDa to 70 kDa bands suggesting the co-precipitation of abundant contaminating proteins as albumin. When the chromatography was used to evaluate the recovery efficiency of the other techniques, the result showed no interference in the separation of populations by size, but could represent an additional tool for techniques as Ultrafiltration in the isolation of albumin as the main interfering in the serum samples. Important data were found when particle number normalization was performed in the analysis by the TRPS method, where a reduction of the aggregation and a change in the particle distribution were visible. In our results it was possible to confirm the need for the new reproducible and reliable protocols that can allow the isolation of exosomes for clinical application. On the other hand, our data provide guidance on the use of current methods for obtaining exosomes from serum and other biological fluids. / Os processos fisiol??gicos e patol??gicos s??o regidos por uma infinidade de mecanismos e vias metab??licas. Nas ??ltimas d??cadas, as pesquisas dirigiram-se a pequenas ves??culas, os exossomas, de origem endossomal que poderiam estar involucradas nestes processos. Estas ves??culas podem ser liberadas por diferentes tipos de c??lulas carregando no seu interior componentes importantes que poderiam transformar-se em biomarcadores para o diagn??stico de doen??as ou podem servir como ferramentas mold??veis para libera????o controlada de subst??ncias bioativas de aplica????o cl??nica e terap??utica. No entanto, um dos grandes desafios no estudo destas interessantes e vers??teis microves??culas constitui o processo de purifica????o. Atualmente, dificuldades significantes s??o encontradas no que se refere ?? falta de pureza, rendimento e reprodutibilidade refletidos nos dados pouco confi??veis tanto a n??vel proteico como transcricional. Por conseguinte, o nosso objetivo no presente estudo foi comparar em amostras de soro humano as t??cnicas de purifica????o atualmente utilizadas: ExoQuick??? (Systems Biosciences), Total Exosome Isolation (Life Technologies), Ultrafiltra????o e Ultracentrifuga????o e Cromatografia de exclus??o pelo tamanho em termos de rendimento, tamanho e pureza avaliando, al??m disso, a sua reprodutibilidade. Os resultados obtidos demonstraram a exist??ncia de diferen??as marcantes entre as t??cnicas na quantidade de recupera????o de part??culas com tamanhos correspondentes aos exossomas. A Ultrafiltra????o foi a t??cnica mais eficiente seguida pelos m??todos de precipita????o com pol??mero e a Ultracentrifuga????o. O conte??do proteico tamb??m variou consideravelmente sendo na Ultrafiltra????o quase dez vezes maior que a Ultracentrifuga????o. Perfis diferentes de prote??nas em gel de acrilamida foram encontrados, ressaltando a presen??a de bandas de 50 kDa a 70 kDa sugerindo a coprecipita????o de prote??nas contaminantes abundantes como a albumina. Quando a cromatografia foi usada para avaliar a efici??ncia de recupera????o das outras t??cnicas, o resultado obtido revelou que a mesma n??o auxilia na separa????o de popula????es pelo tamanho, mas que poderia representar uma ferramenta adicional para t??cnicas como a Ultrafiltra????o no isolamento de albumina como principal interferente nas amostras de soro. Dados relevantes foram encontrados quando foi realizada a normaliza????o do n??mero de part??culas na an??lise pelo m??todo de TRPS onde foi vis??vel uma redu????o da agrega????o e uma mudan??a da distribui????o das part??culas. Nos nossos resultados foi poss??vel evidenciar a necessidade da cria????o de novos protocolos reprodut??veis e confi??veis que podem permitir o isolamento de exossomas para a sua aplica????o cl??nica. Por outro lado, nossos dados fornecem uma orienta????o no uso dos m??todos atuais para a obten????o de exossomas derivados de soro e outros fluidos biol??gicos.