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Thèse de Doctorat
DOI
10.11606/T.10.2016.tde-16092015-171546
Document
Auteur
Nom complet
Isabella Rodrigues Fernandes
Adresse Mail
Unité de l'USP
Domain de Connaissance
Date de Soutenance
Editeur
São Paulo, 2015
Directeur
Jury
Braga, Patricia Cristina Baleeiro Beltrao (Président)
Castelucci, Patricia
Han, Sang Won
Pignatari, Graciela Conceição
Zanoteli, Edmar
Titre en portugais
Modelagem neuronal de pacientes com distrofia muscular de Duchenne utilizando células pluripotentes induzidas
Mots-clés en portugais
Distrofia muscular de Duchenne
Distrofina
iPSC
Modelagem de doenças
Neurônios
Reprogramação celular
SHED
Resumé en portugais
A Distrofia Muscular de Duchenne (DMD) é uma patologia neuromuscular causada pela mutação ou deleção do gene da distrofina, localizado no cromossomo X, levando a degeneração muscular ao longo da vida do paciente. A doença também tem sido associada a déficit cognitivo e falta de habilidade comportamental. Pesquisas com células neurais de pacientes com DMD poderiam ajudar a elucidar os sintomas neurológicos associados. Neste trabalho, através de células-tronco pluripotentes induzidas (iPSC) derivadas da polpa de dente decíduo esfoliado (SHED) de pacientes com DMD modelamos a DMD produzindo células neurais vivas in vitro. A expressão da distrofina foi verificada durante e após a diferenciação neuronal e nos ensaios de imunofluorescência, mostrando que essa proteína está presente em células do SNC. Na análise gênica através do qPCR, a Dp71 e a Dp140, isoformas da distrofina, apresentavam uma expressão menor do que os controles. Além disso, as análises das sinapses baseada na colocalização de marcadores pré e pós-sinápticos (Sinapsina1 e Homer 1) revelaram que os neurônios dos pacientes com DMD tinham menor quantidade de sinapses que os controles, reforçando o papel da distrofina no SNC. Logo, a expressão de genes relacionados a plasticidade sináptica revelou 10 genes alterados nos neurônios dos pacientes DMD, sugerindo que a mutação no gene da distrofina possivelmente altera a plasticidade sináptica e pode estar envolvida na habilidade cognitiva destes pacientes. Desta forma, com base nos nossos achados, a modelagem neuronal de DMD é factível e pode auxiliar a elucidar os mecanismos da fisiopatologia da doença
Titre en anglais
Neuronal modelling with Duchenne muscular dystrophy patients using pluripotent stem cells
Mots-clés en anglais
Cell reprogramming
Duchenne muscular dystrophy
Dystrophin
iPSC
Modeling diseases
Neurons
SHED
Resumé en anglais
The Duchenne muscular dystrophy (DMD) is a neuromuscular disorder caused by a mutation or deletion of the dystrophin gene located on the X chromosome, leading to muscle degeneration throughout the patient's life. The disease has also been associated with cognitive impairment and lack of behavioral skill. Research on neural cells from patients with DMD could help to elucidate the neurological symptoms associated. In this work, through induced pluripotent stem cells (iPSC) derived from dental pulp exfoliated (SHED) of patients with DMD model the DMD producing living neural cells in vitro. The dystrophin expression was observed during and after neuronal differentiation and immunofluorescence assays, showing that this protein is present in CNS cells. In gene analysis by qPCR, the Dp71 and Dp140, isoforms of dystrophin, had a lower expression than controls. Furthermore, based on analysis of synapses colocalization pre and postsynaptic markers (Synapsin1 and Homer 1) showed that neurons of DMD patients had lower number of synapses controls, supporting a role for dystrophin in the CNS. Finally, the expression of synaptic plasticity related genes wasfound in 10 genes altered in neurons of DMD patients, suggesting that the mutation of the dystrophin gene possibly alters synaptic plasticity and may be involved in cognitive ability of these patients. Finally, based on our findings, neuronal modeling DMD is feasible and may help elucidate the mechanisms of pathophysiology of the disease
 
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Date de Publication
2016-04-04
 
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