Submitted by Divis??o de Documenta????o/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-03-16T14:11:57Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Reprodu????o N??o Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) Approved for entry into archive by Divis??o de Documenta????o/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-03-16T14:12:14Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Reprodu????o N??o Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) Approved for entry into archive by Divis??o de Documenta????o/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-03-16T14:15:32Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Reprodu????o N??o Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) Made available in DSpace on 2017-03-16T14:15:32Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Reprodu????o N??o Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) Previous issue date: 2016-06-24 CAPES - Coordena????o de Aperfei??oamento de Pessoal de N??vel Superior The Bothrops asper is one of the main species of Central America because of its medical importance in countries such as Panam?? and Costa Rica, where they cause a high number of accidents. A great number of basic phopholipase A2 (PLA2s, EC 3.1.1.4) this species have been characterized, but a few number of studies have been carried out with acidic isoforms. Moreover, as the snake venom is a rich source of bioactive, the investigation of the biotechnological potential is required. Therefore, from the B. asper venom, this study shows three main lines of research: the caracterization of novel PLA2s isoforms; the study of PLA2s in poisoning pathophysiology and the study of potential antiparasitic of PLA2s. The snake venom was fractionated on three chromatographic methods that have proved effective in purification: ion exchange, hydrophobic interaction and reversed phase, giving rise to nine PLA2, four acidic (BaspAc-I, BaspAc-II, BaspAc-III e BaspAc-IV) and five basic (BaspB-I, BaspB-II, BaspB-III, BapsB-IV e BaspB-V). The molecular weight was determined by mass spectrometry (MALDI-TOF) showed values ranging between 13.8 and 14.3 kDa. The isoelectric point of the acidic PLA2 were determined by 2D SDS-PAGE, showing pI values between 4.6 and 5.0. The N-terminal sequence of nine PLA2 was determined, and 05 are new [four Asp49 (BaspAc-I, BaspAc-II, BaspAc-III and BaspAc-IV) and one Lys49 (BaspB-II)]. The enzymatic activity and the evaluation of the preferred substrates by PLA2 Asp49 was performed using fluorescent phospholipids which was observed that, among the tested phospholipids, acid PLA2 catalyzed all substrates, however, the basic PLA2 showed the highest specificity for phosphatidyl glycerol and phosphatidic acid. A discussion about the role of acidic PLA2 in case of poisoning by snake bite is performed based on myotoxicity assay. Moreover, it is demonstrated the antiparasitic action of these molecules, since the acidic PLA2 (BaspAc-I, BaspAc-II, BaspAc-III and BaspAc-IV) showed effects against epimastigote and pomastigotes forms of Trypanossoma cruzi and Leishmania infantum respectively. On the other hand, the basic PLA2, BaspB-II and IV-BaspB showed activity against Plasmodium falciparum. Furthermore, for the first time it demonstrated the synergistic action between Lys49 and Asp49 PLA2s against the malaria parasite, demonstrating that these molecules might be important targets in the development of new therapeutics. Keywords: Snake Venoms, Bothrops Asper, Phospholipase A2, Venomics, Synergism in Parasiticide Action A serpente Bothrops asper ?? uma das esp??cies mais importantes da Am??rica Central, principalmente, por sua import??ncia m??dica em pa??ses como Panam?? e Costa Rica, onde causam um n??mero elevado de acidentes. Diversas fosfolipases A2 (PLA2s, EC 3.1.1.4) b??sicas desta esp??cie j?? foram caracterizadas, por??m poucos estudos foram realizados com as isoformas ??cidas. Al??m disso, sendo o veneno de serpente uma rica fonte de bioativos, faz-se necess??ria a investiga????o de seu potencial biotecnol??gico. Desta forma, a partir do veneno de B. asper, o presente trabalho teve objetivos principais: a caracteriza????o de novas isoformas de PLA2s, o estudo das PLA2s na fisiopatologia do envenenamento e o estudo do potencial antiparasit??rio das PLA2s. Para tanto, o veneno foi fracionado em tr??s m??todos cromatogr??ficos que se mostraram eficientes na purifica????o: troca i??nica, intera????o hidrof??bica e fase reversa, dando origem a nove PLA2 isoladas, quatro ??cidas (BaspAc-I, BaspAc-II, BaspAc-III e BaspAc-IV) e cinco b??sicas (BaspB-I, BaspB-II, BaspB-III, BapsB-IV e BaspB-V). As massas moleculares foram determinadas por espectrometria (MALDI TOF) com valores variando entre 13,8 e 14,3 kDa. Os pontos isoel??tricos das PLA2s ??cidas foram determinados por 2D-SDS-PAGE com valores de pI variando entre 4,6 e 5,0. A sequ??ncia N-terminal das nove PLA2s foram determinadas, sendo observado que dentre as nove, 05 s??o in??ditas [quatro Asp49 (BaspAc-I, BaspAc-II, BaspAc-III e BaspAc-IV) e uma Lys49 (BaspB-II)]. A atividade enzim??tica e a avalia????o dos substratos preferenciais das PLA2s Asp49 foram realizadas utilizando-se fosfolip??deos fluorescentes e observou-se que, dentre os fosfolip??deos testados, as PLA2s ??cidas catalisaram a degrada????o de todos os substratos, entretanto, para a PLA2 b??sica houve prefer??ncia para o fosfatidilglicerol e ??cido fosfat??dico. Uma discuss??o a respeito do papel das PLA2s ??cidas no envenenamento of??dico ?? conduzida com base no ensaio de miotoxicidade. Ademais, ?? demostrado a a????o antiparasit??ria dessas mol??culas, j?? que as PLA2s ??cidas (BaspAc-I, BaspAc-II, BaspAc-III e BaspAc- IV) tiveram a????o contra as formas epimastigotas de T. cruzi e formas promastigotas de L. infantum e as PLA2s b??sicas BaspB-II e BaspB-IV apresentaram atividade contra Plasmodium falciparum. Posto isto, pela primeira vez, foi demonstrado a a????o sin??rgica entre uma PLA2 Lys49 e uma PLA2 Asp49 contra o parasita da mal??ria, demonstrando que estas mol??culas podem ser importantes alvos no desenvolvimento de novos terap??uticos.