Submitted by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2014-12-18T17:01:07Z No. of bitstreams: 2 carlos_felipe_reis_costa_guimaraes.pdf: 3938293 bytes, checksum: e0cdf597f1d9513ef2531c8a0cd6278f (MD5) license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2014-12-18T17:56:25Z (GMT) No. of bitstreams: 2 carlos_felipe_reis_costa_guimaraes.pdf: 3938293 bytes, checksum: e0cdf597f1d9513ef2531c8a0cd6278f (MD5) license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2014-12-18T18:01:44Z (GMT) No. of bitstreams: 2 carlos_felipe_reis_costa_guimaraes.pdf: 3938293 bytes, checksum: e0cdf597f1d9513ef2531c8a0cd6278f (MD5) license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) / Made available in DSpace on 2014-12-18T18:01:44Z (GMT). No. of bitstreams: 2 carlos_felipe_reis_costa_guimaraes.pdf: 3938293 bytes, checksum: e0cdf597f1d9513ef2531c8a0cd6278f (MD5) license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) Previous issue date: 2013 / Neste trabalho foi estudada a influ?ncia dos efeitos estruturais na rea??o de dimeriza??o associada ? obten??o do pept?deo antimicrobiano homotarsinina (Htr), bem como foram realizados ensaios hemol?ticos e de toxicidade desse pept?deo perante c?lulas humanas. Primeiramente foi realizada a s?ntese da cadeia monom?rica da homotarsinina (Htr-M), empregando-se a estrat?gia Fmoc de s?ntese em fase s?lida. Em seguida, o produto foi purificado por cromatografia l?quida de alta efici?ncia em fase reversa (CLAE-FR), tendo sido o homod?mero obtido sequenciado e caracterizado por espectrometria de massas (MALDI-ToF). Foram realizados estudos das prefer?ncias conformacionais da Htr-M por dicro?smo circular (CD) em diferentes meios e condi??es, como na presen?a de tamp?o Tris-HCl aquoso, em diferentes misturas de TFE-H2O e na presen?a de micelas de SDS. Realizou-se ent?o a rea??o de dimeriza??o do mon?mero em tr?s meios: (i) solu??o tamp?o Tris-HCl 100 mmol?L-1 pH 8,5, (ii) solu??o micelar de SDS 350 mmol?L-1 em tamp?o Tris-HCl 100 mmol?L-1 pH 8,5 e (iii) solu??o TFE:H2O 40:60 (v/v) em Tris-HCl 100 mmol?L-1 pH 8,5. O acompanhamento cin?tico das rea??es de dimeriza??o foi realizado por CLAE-FR. Como resultado, foi observado que em solu??o micelar de SDS a rea??o se completa em aproximadamente 6h (rendimento de 62%), enquanto que em solu??o tamp?o e em solu??o de TFE:H2O s?o necess?rias 24h e 48h (rendimentos de 66% e 85%), respectivamente. Os resultados de CD obtidos para a Htr-M em condi??es similares ?s empregadas nas rea??es de dimeriza??o mostraram que a Htr-M apresenta predominantemente conforma??o rand?mica em tamp?o Tris-HCl aquoso, enquanto adota predominantemente estrutura de h?lice ? em solu??o de TFE:H2O (40:60) ou na presen?a de micelas de SDS. Assim, os resultados do acompanhamento cin?tico da rea??o de dimeriza??o indicam que a conforma??o predominantemente rand?mica da Htr-M em tamp?o Tris-HCl aquoso, bem como a diminui??o de sua mobilidade e maior intera??o com a superf?cie das micelas de SDS e os efeitos de agrega??o que ocorrem em solu??o de TFE:H2O influenciam diretamente na forma de aproxima??o das cadeias monom?ricas, atuando de maneiras diferentes na forma??o da liga??o dissulfeto. Por fim, foram realizados ensaios de toxicidade em c?lulas humanas, os quais mostraram que Htr-M e Htr n?o apresentam toxicidade a leuc?citos mononucleares, al?m do que tamb?m n?o apresentam atividade hemol?tica. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Qu?mica, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2013. / ABSTRACT This dissertation is mainly related to studies concerning the role of the structural effects on the dimerization reaction that leads to the antimicrobial peptide homotarsinin (Htr). Besides, hemolytic assays as well as the toxicity of this peptide against human cells have also been investigated. Firstly, the monomeric chain (Htr-M) was obtained by Fmoc solid-phase synthesis. The product was purified by reverse phase high performance liquid chromatography (RP-HPLC) and then sequenced and characterized by mass spectrometry (MALDI-ToF). Circular dichroism (CD) studies regarding the Htr-M conformational preferences were performed for the peptide in different media and conditions, such as Tris-HCl aqueous buffer, TFE:H2O mixtures, as well as in the presence of SDS micelles. The dimerization reaction was then performed in three distinct media, namely: (i) Tris-HCl aqueous buffer 100 mmol?L-1 pH 8.5, (ii) SDS micellar solution of 350 mmol?L-1 in Tris-HCl 100 mmol?L-1 pH 8.5 and (iii) TFE:H2O 40:60 in Tris-HCl 100 mmol?L-1 pH 8.5. CD studies performed in similar conditions indicated that Htr-M presents predominantly random conformations in aqueous Tris-HCl buffer, whereas it adopts ?-helical arrangements in either TFE:H2O (40:60) solution or in the presence of SDS micelles. The kinetic monitoring of the dimerization reactions was carried out by RP-HPLC. The results indicated that the reaction is completed in about 6 h (yield 62%) in the presence of SDS micelles, whereas 24 h and 48 h (yields 66% and 85%) are necessary for the dimerizations performed in the aqueous Tris-HCl and in the TFE:H2O solutions, respectively. Therefore, the kinetic studies indicated that the predominantly random conformation of Htr-M in Tris-HCl aqueous buffer, as well as the mobility decrease alongside with the peptide-micelle interactions in the presence of SDS, as well as the aggregation effects in the TFE:H2O solution directly affect the inter-chain approach process, which leads to different pathways of the disulfide bond formation. Finally, toxicity assays were performed in human cells and the results showed that both Htr-M and Htr are not toxic against mononuclear leukocytes and also do not show hemolytic activity.