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Authors
Abstract(s)
A Doença de Parkinson é a segunda doença neurodegenerativa mais comum no
mundo e é caracterizada pela perda seletiva dos neurónios dopaminérgicos na substantia
nigra. A sua principal característica patológica é a formação de inclusões neuronais como
os corpos de Lewy, constituídas por espécies proteicas misfolded agregadas. A alfasinucleína é uma proteína pequena envolvida no tráfico vesicular no cérebro, que
compreende o modelo de polimerização-conversão-nucleação durante o seu processo de
agregação. A agregação anormal promove a conversão de monómeros em fibrilas
preformadas, que se acumulam nos neurónios. Deste modo, foram criados modelos
animais que apresentam a forma patológica da alfa-sinucleína, cujo intuito é mimetizar
a patologia, para melhor a compreender, e desenvolver novos tratamentos. A produção
de fibrilas tem como objetivo a criação de fibrilas a partir de monómeros in vitro e após
vários testes de controlo de qualidade das fibrilas preformadas, poder usá-las em
modelos celulares ou modelos animais.
Neste trabalho foi proposta a otimização de um método de preparação e formação
das fibrilas preformadas com base em monómeros da alfa-sinucleína para posterior
desenvolvimento de um modelo celular e animal da doença. Para avaliar a qualidade das
fibrilas preformadas realizou-se o ensaio da Tioflavina T e estudou-se a estrutura
secundária da proteína com base na técnica de dicroísmo circular.
Os resultados deste estudo mostraram que para haver agregação proteica é
necessário que a proteína se encontre na conformação correta, desordenada, desde a sua
comercialização. Neste caso não foi possível obter fibrilas preformadas devido à
qualidade dos monómeros comercializados, uma vez que a conformação apresentada era
de a-hélice. De forma a garantir que o processo de fibrilização seja executado com
sucesso é necessário refazer todo o processo de formação e avaliação de novos
monómeros e fibrilas preformadas. Por outro lado, para otimizar e proporcionar a
indução patológica com recurso às fibrilas preformadas da alfa-sinucleína num modelo
animal é necessário fazer mais estudos que avaliem a sua caraterização e formação.
Parkinson’s Disease is the second most common neurodegenerative disease in the world and it is characterized by the selective loss of dopaminergic neurons in the substantia nigra. Its main pathological feature is the formation of neuronal inclusions such as Lewy bodies, which are constituted of misfolded protein aggregates. Alphasynuclein is a small protein involved in vesicular trafficking in the brain, which follows the polymerization-conversion-nucleation model during its aggregation process. The abnormal aggregation promotes the conversion of monomers into preformed fibrils, that accumulate in the neurons. Therefore, the animal models that exhibit the pathological form of alpha-synuclein were created, in order to mimic the pathology, for a better understanding and to develop new treatments. The aim of the synthesis of preformed fibrils is to create fibrils from monomers in vitro and after preforming control tests on them, be able to use them on cellular and animal models. In this dissertation it was proposed an optimization of a method of preparation and synthesis of preformed fibrils from monomers to develop a posterior cell and animal model of the disease. In an effort to validate its quality, it was conducted the Thioflavin T assay and a protein secondary structure study through circular dichroism technique. The results showed that for the propose of protein aggregation is necessary that the protein has the correct conformation- random coil- since its commercialization. In this case it was not possible to obtain preformed fibrils, due to the quality of the commercialized monomers, that presented an a-helix conformation. For the purpose of securing the success of the fibrillization process, it is necessary to redo the all-synthesis process and the evaluation of new monomers and preformed fibrils. Besides, as a means to optimize and provide pathologic induction with the use of alpha-synuclein preformed fibrils in animal models, it is necessary to make more studies regarding its characterization and synthesis.
Parkinson’s Disease is the second most common neurodegenerative disease in the world and it is characterized by the selective loss of dopaminergic neurons in the substantia nigra. Its main pathological feature is the formation of neuronal inclusions such as Lewy bodies, which are constituted of misfolded protein aggregates. Alphasynuclein is a small protein involved in vesicular trafficking in the brain, which follows the polymerization-conversion-nucleation model during its aggregation process. The abnormal aggregation promotes the conversion of monomers into preformed fibrils, that accumulate in the neurons. Therefore, the animal models that exhibit the pathological form of alpha-synuclein were created, in order to mimic the pathology, for a better understanding and to develop new treatments. The aim of the synthesis of preformed fibrils is to create fibrils from monomers in vitro and after preforming control tests on them, be able to use them on cellular and animal models. In this dissertation it was proposed an optimization of a method of preparation and synthesis of preformed fibrils from monomers to develop a posterior cell and animal model of the disease. In an effort to validate its quality, it was conducted the Thioflavin T assay and a protein secondary structure study through circular dichroism technique. The results showed that for the propose of protein aggregation is necessary that the protein has the correct conformation- random coil- since its commercialization. In this case it was not possible to obtain preformed fibrils, due to the quality of the commercialized monomers, that presented an a-helix conformation. For the purpose of securing the success of the fibrillization process, it is necessary to redo the all-synthesis process and the evaluation of new monomers and preformed fibrils. Besides, as a means to optimize and provide pathologic induction with the use of alpha-synuclein preformed fibrils in animal models, it is necessary to make more studies regarding its characterization and synthesis.
Description
Keywords
Alfa-Sinucleína Doença de Parkinson Modelo das Fibrilas Preformadas Modelo de Polimerização-Conversão-Nucleação Monómeros