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Previous issue date: 2016-03-03 / 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, ?-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. / As altera??es do desenvolvimento do c?rtex cerebral apresentam-se como um grupo de malforma??es com uma distin??o e patog?nse ainda n?o bem definidas. A displasia cortical focal (DCF) ? uma das formas mais frequentes de malforma??es do desenvolvimento cortical, sendo a patologia subjacente a uma parcela significativa de epilepsias parciais refrat?rias ao tratamento medicamentoso. A displasia cortical focal engloba m?ltiplos tipos de altera??es tanto na arquitetura cortical quanto em anormalidades citol?gicas. Palmini e colaboradores classificaram as displasias corticais focais de acordo com observa??es na subst?ncia branca e na arquitetura da camada cortical. Os mecanismos envolvidos na g?nese da DCF s?o pouco investigados, principalmente pelo n?mero limitado de casos e a falta de modelos experimentais adequados e suas causas provavelmente est?o relacionadas a muta??es som?ticas. A falta de modelos pr?-cl?nicos preditivos que possam ser utilizados no estudo da fisiopatologia e o hist?rico enfraquecido quando se trata em traduzir a descoberta terap?utica de modelos animais para o uso humano, fortalece a necessidade de estudar o desenvolvimento cerebral a partir de c?lulas originadas do pr?prio paciente. A gera??o de c?lulas iPSC e diferencia??o tecidual espec?fica de c?lulas de pacientes acometidos por doen?as neurol?gicas relevantes possui um valor inestim?vel para a realiza??o de testes al?m de fornecerem uma capacidade adicional e ?nica para estudar o desenvolvimento inicial e a progress?o das patologias associadas ao SNC. O objetivo do presente trabalho ? estabelecer um modelo celular de Displasia Cortical Focal atrav?s da gera??o de c?lulas-tronco pluripotentes induzidas (iPSC) a partir de fibroblastos de pacientes afetados. Fibroblastos humanos foram obtidos de bi?psias de pele de dois pacientes com DCF e foram cultivados at? a quinta passagem. Foi realizada an?lise imunopatol?gica e das vias de sinaliza??o AKT/mTOR no tecido cerebral displ?sico. C?lulas iPSC foram geradas a partir dos fibroblastos atrav?s da exposi??o a vetores virais contendo os genes OCT4, KLF4, SOX2, e C-MYC e caracterizadas por imunohistoqu?mica. Foi realizado ensaio de migra??o celular dos fibroblastos (24h, 48h e 72h) e das iPSC (3 dias e 7 dias). As c?lulas iPSC foram neurodiferenciadas e analisadas nos per?odos de 14 dias, 22 dias e 35 dias. Nesses per?odos, foram realizadas an?lises morfol?gicas, imunohistoqu?mica (polariza??o) e moleculares (genes relacionados a via PI3K/ATK/mTOR). Ambos os pacientes foram diagnosticados com DCF tipo IIb. O paciente 01 apresentou valores maiores que o paciente 02 em rela??o ? ?rea marcada das vias AKT e mTOR, tanto na via fosforilada como n?o fosforilada no tecido cerebral, com diferen?as estatisticamente significativas. Clones iPSC dos pacientes e controles foram gerados e caracterizados a partir dos fibroblastos de pele. Tanto as iPSC dos pacientes quanto do controle apresentaram morfologia e polariza??o semelhante a c?lulas nervosas ap?s protocolo de neurodiferencia??o. No ensaio de migra??o celular, fibroblastos com DCF migraram com mais intensidade em 24 horas (p<0,0001) e 48 horas (p<0,001). As c?lulas iPSC n?o apresentaram diferen?a no potencial de migra??o celular nos per?odos analisados. Durante o protocolo de neurodiferencia??o, as c?lulas iPSC dos pacientes com DCF apresentaram valores menores de express?o dos genes 4EBP-1, ?-Catenina, CIAP-1, CIAP-2 e PI3K, e valores mais elevados da express?o de MCL 1. Altera??es na migra??o celular no tecido adulto e em processos como de prolifera??o celular acentuada, defici?ncia de prote?na de ades?o celular, altera??o na express?o de genes respons?veis pelo controle de apoptose e altera??o na via PI3K respons?vel no sistema nervoso central pela sobreviv?ncia celular, controle de apoptose, migra??o neuronal, desenvolvimento morfol?gico dos neur?nios e forma??o das neurotransmiss?es, puderam ser evidenciadas nas c?lulas dos pacientes com DCF em rela??o aos pacientes controle e podem estar relacionadas com a forma??o do c?rebro com displasia.
| Identifer | oai:union.ndltd.org:IBICT/oai:tede2.pucrs.br:tede/6778 |
| Date | 03 March 2016 |
| Creators | Marinowic, Daniel Rodrigo |
| Contributors | Costa, Jaderson Costa da |
| Publisher | Pontif?cia Universidade Cat?lica do Rio Grande do Sul, Programa de P?s-Gradua??o em Medicina e Ci?ncias da Sa?de, PUCRS, Brasil, Faculdade de Medicina |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | Portuguese |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da PUC_RS, instname:Pontifícia Universidade Católica do Rio Grande do Sul, instacron:PUC_RS |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | 7620745074616285884, 600, 600, 600, -8624664729441623247, -969369452308786627 |
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