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Master's Dissertation
DOI
10.11606/D.42.2012.tde-25052012-083840
Document
Author
Full name
Ana Maria Marques Orellana
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2012
Supervisor
Committee
Scavone, Cristoforo (President)
Camarini, Rosana
Quintas, Luis Eduardo Menezes
Title in Portuguese
Administração intrahipocampal de Ouabaína ativa o NF - kB e a sinalização da proteína WNTem ratos.
Keywords in Portuguese
Ativação enzimática
Neurofarmacologia
Ouabaína
Ratos
Sistema nervoso central
Transdução de sinal celular
Abstract in Portuguese
A enzima Na+, K+-ATPase é uma proteína de membrana altamente conservada em eucariotos, capaz de gerar um gradiente eletroquímico, fundamental para o balanço osmótico das células, o potencial de repouso das membranas e a propriedade excitatória das células musculares e nervosas. Além de seu papel regulatório na homeostasia iônica, desempenha um papel na transdução de sinal e na ativação de transcrição gênica, modulando na presença de ouabaína o crescimento celular, migração e morte celular programada. A Ouabaína (OUA) é um esteróide cardiotônico, produzido no córtex da adrenal e no hipotálamo. Em linhas gerais, a sinalização da Na+, K+-ATPase promovida pela OUA parece ativar vias associadas à modulação de fatores de transcrição como a via da Src, MAPK, Ca2+ e NF-kB. Evidências indicam que o NF-kB exerça algum tipo de modulação na via canônica do WNT, no entanto os mecanismos moleculares ainda são desconhecidos. A via de sinalização WNT desempenha função importante na embriogênese e na homeostase de tecidos adultos. Assim, o objetivo do presente projeto é verificar se a administração intrahipocampal de OUA é capaz de modular a atividade das vias canônicas do NF-kB e da WNT. Estas vias foram estudadas em um decurso temporal imediato (1 -2 horas) e tardio (10, 24 e 48 horas) utilizando técnicas como Western Blotting, RT-PCR e EMSA. Os resultados encontrados mostram que a OUA (10 nM) foi capaz de ativar a via de sinalização NF-kB, após 1 hora, 10, 24 e 48 horas. A OUA também foi capaz de ativar a via canônica do WNT, sendo que após 10 horas ocorreu aumento da proteína pGSK-3b, enquanto que em 24 horas, observamos aumento da translocação nuclear da b-CATENINA. Além disso, pode-se verificar aumento de BDNF ao longo de todo o decurso temporal.
Title in English
Intrahippocampal injection of Ouabain activates NF-kB and WNT signaling pathways in rats.
Keywords in English
Cellular signal transduction
CNS
Enzyme activation
Neuropharmacology
Ouabain
Rats
Abstract in English
The enzyme Na+,K+-ATPase is an integral membrane protein, highly conserved in eukaryotes, that establishes the electrochemical gradient across the plasma membrane, which is essential to maintain the osmotic balance of cells, the resting membrane potential and the excitatory property of nerve and muscle cells. Besides its role in ion homeostasis, several recent studies suggest that this pump may also act as a signal transducer and transcription activator involved in cell growth, differentiation and programmed cell death. Ouabain (OUA), the ligand of Na+,K+-ATPase, is a steroid derivative that is produced by the adrenal cortex and hypothalamus. After OUA binding, the Na+,K+-ATPase signaling seems to activate pathways such as Src, MAPK, NF-kB and Ca2+. Some evidences indicate a possible crosstalk between the NF-kB signaling pathway and the canonical WNT pathway, however the molecular mechanisms are still unknown. The canonical WNT play important roles during embryogenesis and in adult tissue homeostasis. The aim of this project is to verify if the intrahipocampal administration of OUA is able to modulate the activity of the canonical pathways of NF-kB and WNT. Both pathways were studied after 1 and 2 hours, and after 10, 24 and 48 hours by methods such Western blot, RT-PCR and Electrophoretic mobility shift assays. The results show that the OUA (10 nM) was able to activate the signaling pathway NF-kB after 1, 10, 24 and 48 hours. The OUA was also able to activate the canonical WNT pathway, since after 10 hours there was an increased in pGSK-3b protein, whereas in 24 hours, we observed increased nuclear translocation of b-CATENIN. Moreover, we found increased levels of BDNF throughout the time course.
 
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Publishing Date
2012-06-04
 
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