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Purificação e caracterização de proteínas Gla nos modelos peixe e mamífero
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Authors
Advisor(s)
Abstract(s)
O presente trabalho teve três objectivos distintos, mas todos eles relacionados
com proteínas contendo resíduos Gla.
O primeiro objectivo consistiu na purificação e caracterização de duas isoformas
de osteocalcina (OC1 e OC2), aparentemente apenas existentes na classe Osteichthyes à
qual pertencem os peixes teleósteos. As extracções foram realizadas a partir do osso de
cinco peixes teleósteos. Em Solea senegalensis (linguado) obteve-se a sequência de
aminoácidos completa da OC2 e uma sequência parcial correspondente à OC1. Para
Diplodus sargus (sargo) obteve-se a sequência completa da OC1 e uma sequência quase
completa correspondente à OC2. Para Oncorhynchus mykiss (truta) obtiveram-se duas
sequências completas correspondentes à OC2. Estes resultados demonstraram pela
primeira que nesta classe além de existirem duas isoformas de osteocalcina a nível
genético, também ocorre a produção e acumulação das mesmas.
O segundo objectivo deste trabalho foi a identificação e caracterização da
proteína GRP, recentemente descoberta e isolada a partir da cartilagem calcificada do
condrósteo Acipenser nacarii (esturjão), em mamíferos. Os mamíferos analisados foram
o Sus domesticus (porco), o Mus musculus (ratinho) e o Oryctolagus cuniculus (coelho)
e os resultados obtidos demonstraram a presença de GRP em todas as espécies
examinadas e validaram os anticorpos específicos disponíveis (CTerm-GRP e M3B).
O terceiro objectivo consistiu no estudo da osteocalcina produzida por um
sistema celular que se pretendia viabilizar como modelo in vitro. Neste sistema a
expressão constitutiva de osteocalcina havia sido induzida. O sistema foi caracterização
na presença e ausência de tratamento mineralizante. Utilizaram-se os clones derivados
de Sparus aurata VSa16B (linha osteoblástica) e VSa13B (linha condrocítica), nos
quais foi inserido cDNA de osteocalcina da mesma espécie (SaOC1). Ambos os clones
expressaram osteocalcina transgénica, tanto com ou sem o tratamento mineralizante,
tendo sido obtidos níveis de proteína mais elevados no clone VSa16B. Verificou-se
ainda que a proteína produzida pelos clones VSa16B era γ-carboxilada, mas a produzida
pelos clones VSa13B não o era.
This experimental work had three different goals but all of them related with proteins containing Gla residues. The first goal consisted in the purification and characterization of two osteocalcin isoforms (OC1 and OC2), apparently only existing in Osteichthyes class to which teleost fish belong. The extractions were performed from bones of five teleost fish. For Solea senegalensis (sole) OC2 full amino acid sequence was obtained and also a partial sequence that matched OC1. For Diplodus sargus (white bream) OC1 full sequence was obtained and also an almost complete sequence that matched OC2. In Oncorhynchus mykiss (rainbow trout) two OC2 full sequences were obtained. These results demonstrated for the first time that in this class not only there exists to osteocalcin isoforms at a genetic level but also they are produced and accumulated. The second goal was the identification and characterization in mammals of GRP, which had been recently identified and isolated from the calcified cartilage of the condrostean Acipenser nacarii (sturgeon). The analyzed mammals were Sus domesticus (pig), Mus musculus (mouse) and Oryctolagus cuniculus (rabbit) and the obtained results demonstrated the presence of GRP in all the examined species and validated the used available antibodies (CTerm-GRP and M3B). The third aim of this work was to study the osteocalcin produced by a cellular system which we pretended to validate as an in vitro model. The constitutive expression of osteocalcin had been induced in this system. The system was characterized either with or without a mineralizing treatment. There were used the Sparus aurata derived clones VSa16B (osteoblastic line) and VSa13B (condrocytic line) in which cDNA from the same specie (SaOC1) was inserted. Both clones expressed transgenic osteocalcin, either with or without the mineralizing treatment, expressing VSa16B the highest levels of protein. It was also verified that the protein produced by VSa16B was γ-carboxylated but the protein produced by VSa13B clones wasn´t.
This experimental work had three different goals but all of them related with proteins containing Gla residues. The first goal consisted in the purification and characterization of two osteocalcin isoforms (OC1 and OC2), apparently only existing in Osteichthyes class to which teleost fish belong. The extractions were performed from bones of five teleost fish. For Solea senegalensis (sole) OC2 full amino acid sequence was obtained and also a partial sequence that matched OC1. For Diplodus sargus (white bream) OC1 full sequence was obtained and also an almost complete sequence that matched OC2. In Oncorhynchus mykiss (rainbow trout) two OC2 full sequences were obtained. These results demonstrated for the first time that in this class not only there exists to osteocalcin isoforms at a genetic level but also they are produced and accumulated. The second goal was the identification and characterization in mammals of GRP, which had been recently identified and isolated from the calcified cartilage of the condrostean Acipenser nacarii (sturgeon). The analyzed mammals were Sus domesticus (pig), Mus musculus (mouse) and Oryctolagus cuniculus (rabbit) and the obtained results demonstrated the presence of GRP in all the examined species and validated the used available antibodies (CTerm-GRP and M3B). The third aim of this work was to study the osteocalcin produced by a cellular system which we pretended to validate as an in vitro model. The constitutive expression of osteocalcin had been induced in this system. The system was characterized either with or without a mineralizing treatment. There were used the Sparus aurata derived clones VSa16B (osteoblastic line) and VSa13B (condrocytic line) in which cDNA from the same specie (SaOC1) was inserted. Both clones expressed transgenic osteocalcin, either with or without the mineralizing treatment, expressing VSa16B the highest levels of protein. It was also verified that the protein produced by VSa16B was γ-carboxylated but the protein produced by VSa13B clones wasn´t.
Description
Dissertação de Mestrado, Biologia Molecular e Microbiana, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2010
Keywords
Proteínas Gla Osteocalcina Peixes Mamíferos GRP