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Previous issue date: 2015-07-10 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / As esponjas marinhas se apresentam como uma rica fonte de novos compostos bioativos de grande interesse biotecnol?gico, vimos, neste trabalho, relatar a purifica??o e caracteriza??o de uma lectina da esp?cie Aplysina fulva, denominada AFL com atividade imunomoduladora, bem como avaliar se a mesma possue efeito citot?xico sobre c?lulas em cultura (normais, tumorais e leishmanias). As prote?nas totais foram extra?das da esponja com tamp?o Tris-HCl 20 mM, pH 7,5, e fracionadas por precipita??o com acetona. A fra??o obtida com 1 volume de acetona possu?a maior atividade hemaglutinante e foi submetida a uma cromatografia em matriz de goma guar com tamp?o tetraborato de s?dio 20 mM, pH 7,5. Em seguida foi aplicada em uma coluna de Superdex 75 (FPLC-AKTA) contendo tamp?o Tris- HCl 20 mM, pH 7,5. A pureza da lectina foi atestada em uma coluna HILIC (HPLC). A sequ?ncia de 15 res?duos de amino?cidos do N-terminal da lectina foi determinada por degrada??o de Edman e n?o apresentou similaridade com nenhuma outra prote?na dos bancos de dados mais conhecidos. A massa foi estabelecida em 62 kDa, por gel filtra??o em Superdex 75. An?lise ap?s tratamento com agentes redutores permitiu deduzir que AFL ? uma prote?na homotetram?rica. Sua atividade hemaglutinante foi reduzida quando a lectina foi exposta em meio ?cido (pH < 4) ou quando submetida a temperaturas acima de 60 ?C. AFL apresentou especificidade para lactose e parcial para D-glicose e D-galactose. A lectina purificada AFL n?o apresentou efeito citot?xico para as linhagens de c?lulas cancer?genas (PANC, HeLa, HT-29, Bf16F10, A549) e de fibroblastos (MRC5). AFL foi capaz de aglutinar formas promastigotas vivas de Leishmania amazonensis e Leishmania. braziliensis,e teve sua atividade revertida pela adi??o de lactose ao ensaio. AFL n?o diminuiu de forma significante a viabilidade de Leishmania amazonensis. A lectina AFL apresentou atividade mitog?nica em concentra??es a partir de 40 ?g/mL para c?lula mononucleares do sangue perif?rico (mon?citos e linf?citos). A lectina AFL foi capaz de induzir na linhagem de macr?fagos (RAW264.7) de murinos a libera??o de TNF- ?, mas n?o de IL-6, na concentra??o de 10,0 ?g/mL. Quando AFL foi incubada com c?lulas espl?nicas de camundongos BALB/c, foi capaz de estimular a produ??o de IFN-? e promover a diferencia??o de c?lulas T para uma resposta imune celular do tipo Th1. A capacidade de modular a resposta imune do tipo Th1 jamais foi relatada em lectinas de esponjas marinhas. Todos os resultados corroboram com o alto potencial que a lectina AFL apresenta como uma nova mol?cula capaz de modular o sistema imune, podendo ser utilizada como adjuvante de vacinas contra microrganismos, dentre eles, os do g?nero leishmania. / Marine sponges are presented as a rich source of new bioactive compounds of great biotechnological interest. Here in this research we report the purification and characterization of a lectin from Aplysina fulva species, called AFL, that presents immunomodulatory activity, and it was also evaluated whether it possesses cytotoxic effects on cultured cells (normal, tumor and Leishmania). Total proteins were extracted from the sponge with Tris-HCl buffer 20 mM, pH 7.5, and fractionated by precipitation with acetone. The fraction obtained with 1 volume of acetone had greater hemagglutinating activity and was subjected to a column chromatography on guar gum matrix with 20 mM sodium tetraborate buffer, pH 7.5. It was then applied to a Superdex 75 column (AKTA-FPLC) containing 20 mM Tris-HCl buffer, pH 7.5. The purity of the lectin was attested in a HILIC (HPLC) column. The sequence of 15 residues of N-terminal amino acid of the lectin was determined by Edman degradation and showed no similarity to any other protein of the known databases. The molecular mass was set at 62 kDa by gel filtration on Superdex 75. Analysis after treatment with reducing agents enabled to deduce that the AFL is a homotetrameryc protein. Their hemagglutinating activity was reduced when the lectin was exposed in an acidic medium (pH <4) or when subjected to temperatures above 60 ? C. AFL showed specificity for partial lactose and D-glucose and D-galactose. The purified lectin LFA showed no cytotoxic effect on cancer cell lines (PANC, HeLa, HT-29, Bf16F10, A549) and fibroblast (MRC5). AFL was able to agglutinate living promastigotes of Leishmania amazonensis and Leishmania braziliensis, and had their activity reversed by the addition of lactose to the test. AFL did not decrease significantly the viability of Leishmania amazonensis. The AFL lectin showed mitogenic activity at concentrations from 40 ug / ml for mononuclear peripheral blood cells (lymphocytes and monocytes). The AFL lectin was able of induce the macrophage murine lineage (RAW264.7) release of TNF-?, but not IL-6 at a concentration of 10.0 mg / mL. When LPA was incubated with spleen cells from BALB / c mice was able to stimulate the production of IFN-? and to promote the differentiation of T cells to a cellular Th1 immune response. The ability to modulate the immune response of the Th1-type lectins has never been reported in marine sponges. All results confirm the high potential that the lectin AFL, that appears as a new molecule capable of modulating the immune system and can be used as an adjuvant for vaccines against microorganisms, including the gender Leishmania.
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.ufrn.br:123456789/22224 |
| Date | 10 July 2015 |
| Creators | Santos, Paula Ivani Medeiros dos |
| Contributors | 41305655400, http://lattes.cnpq.br/6762251930590306, Queiroz, Alexandre Fl?vio Silva de, 67218709400, http://lattes.cnpq.br/4447420857221826, Souto, Janeusa Trindade de, 61689858400, http://lattes.cnpq.br/6501426772186111, Souza, Djair dos Santos de Lima e, 02395757411, http://lattes.cnpq.br/4456114894660929, Amorim, Ticiana Maria L?cio de, 05002240488, http://lattes.cnpq.br/6216030147805627, Santos, Elizeu Antunes dos |
| Publisher | PROGRAMA DE P?S-GRADUA??O EM BIOQU?MICA, UFRN, Brasil |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Source | reponame:Repositório Institucional da UFRN, instname:Universidade Federal do Rio Grande do Norte, instacron:UFRN |
| Rights | info:eu-repo/semantics/openAccess |
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