Detalhes bibliográficos
| Ano de defesa: |
2016 |
| Autor(a) principal: |
Marinowic, Daniel Rodrigo
 |
| Orientador(a): |
Costa, Jaderson Costa da
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| Banca de defesa: |
Não Informado pela instituição |
| Tipo de documento: |
Tese
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| Tipo de acesso: |
Acesso aberto |
| Idioma: |
por |
| Instituição de defesa: |
Pontifícia Universidade Católica do Rio Grande do Sul
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| Programa de Pós-Graduação: |
Programa de Pós-Graduação em Medicina e Ciências da Saúde
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| Departamento: |
Faculdade de Medicina
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| País: |
Brasil
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| Palavras-chave em Português: |
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| Área do conhecimento CNPq: |
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| Link de acesso: |
http://tede2.pucrs.br/tede2/handle/tede/6778
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Resumo: |
Changes in the cerebral cortex development are presented as a group of distinct defects with a not well-defined pathogenesis. The focal cortical dysplasia (FCD) is one of the most frequent form of cortical development malformation, that encompasses multiple types of changes both in the cortical architecture and cytological abnormalities. It is an underlying pathology of a significant proportion of partial epilepsy refractory to drug treatment. Especially the limited number of cases and the lack of suitable experimental models rarely document the mechanisms involved with the genesis of FCD. The scarse predictive preclinical models that can be used to study the pathophysiology and to translate the therapeutic discovery from animal models to human use, strengthens the need to study the brain development from cells originated from patients that harbor these central nervous systems diseases. The generation of iPSC cells and the differentiation into specific cells and tissues will provide important testing and an unique ability to study the development and progress of CNS diseases. The objective of this study was to establish a cellular model of focal cortical dysplasia through the generation of induced pluripotent stem cells (iPSC) from fibroblasts derived from affected patients. Human fibroblasts were obtained from skin biopsies from two patients and cultured up to the fifth passage. Immunofluorescence analysis of AKT/mTOR signaling pathways was performed in the dysplastic brain tissue. iPSC cells were generated from fibroblasts through transfection with a viral vector containing the genes OCT4, KLF4, SOX2, and C-MYC and characterized by immunohistochemistry. Cell migration co-cultured with fibroblast and iPSC was investigated. Morphological and molecular characterization of neurodifferentiated iPSC were performed by immunohistochemistry and PI3K/ATK/mTOR signaling pathway analysis. Both patients were diagnosed with FCD type IIb. The AKT and mTOR phosphorylated and non-phosphorylated was higher in brain sections from patient 01. iPSC clones from patients and controls were generated from skin fibroblasts. Both iPSC from patients and controls showed polarization and morphology similar to nerve cells. In the cell migration assay, fibroblasts derived from FCD migrate with greater intensity within 24 hours (p<0,0001) and 48 hours (p<0,001), and iPSC cells did not present difference in cell migration. During neurodifferentiation, iPSC cells from patients with FCD showed lower values of 4EBP-1, β-catenin, CIAP-1, CIAP-2 and PI3K, and higher values of MCL 1 gene expression. Changes in cell migration in adult tissue, uncontrolled cell proliferation, cell adhesion protein deficiency, and alterations in the expression of genes responsible for apoptosis and PI3K pathway that are implicated with an complete and well successful CNS formation. Alterations in some of these were detected in cells and iPSC from patients with FCD and may be related to the ethiopathology of the disease. |
