Banca de DEFESA: GABRIELI BOVI DOS SANTOS

Uma banca de DEFESA de MESTRADO foi cadastrada pelo programa.
DISCENTE : GABRIELI BOVI DOS SANTOS
DATA : 11/11/2024
HORA: 13:00
LOCAL: São Bernardo do Campo
TÍTULO:

ESTABLISHMENT OF A 3D IN VITRO MODEL OF RETINITIS PIGMENTOSA

PÁGINAS: 80
RESUMO:

The retina can be affected by different types of diseases, with or without origin in genetic changes, which can compromise its morphological structure and the survival of specific cells. Among these diseases, retinitis pigmentosa (RP) is a hereditary disease related to around 100 mutations, especially in genes that encode proteins necessary for phototransduction in rod photoreceptors. Mutation in phosphodiesterase 6 β (PDE6β) alters cellular activity that results in apoptosis, but a new cell death pathway has recently been related to RP, necroptosis. Necroptosis can be considered as a 'programmed necrosis', can influence the transcription of inflammatory genes, with literature on necroptosis in RP being scarce. This work aims to establish a 2D and 3D model of RP in vitro, which can recapitulate the loss of photoreceptors that occurs during the progression of RP. Obtaining this model will help test new molecules and gene manipulations to establish therapeutic strategies for treating RP. As a starting point, we standardized the formation of neurospheres, composed of retinal progenitor cells. In a second step, we tested protocols for differentiation into rods, an arrangement called photosphere. In parallel, we performed 2D mixed retinal cultures of WT (C57BL/6J) and PDE6β knockout (RD1) to standardize experiments with light stimulation. We used LEDs in the cell incubator to stimulate mixed retinal cultures exposed to 200 lux and 12,000 lux for 2 and 5 hours. Cell cultures performed from WT and RD1 animals had no change in cell viability by MTT and PrestoBlue at 200 lux. In cell cultures stimulated with 12,000 lux, we observed a significant decrease in cell viability with PrestoBlue and increased TUNEL labeling. Double-labeling experiments showed that the TUNEL-positive cells were predominantly rods. LDH, RIPK3 markers and activated capase-3 were increased. In conclusion, to accelerate the induction of cell death in the photoreceptors of the RD1 model in vitro, a 2D light assay was standardized in addition to the 3D cell culture with differentiation of retinal precursors into a predominantly photoreceptor sphere, which we call the photosphere.

MEMBROS DA BANCA:
Presidente - Interno ao Programa - 1676367 - ALEXANDRE HIROAKI KIHARA
Membro Titular - Examinador(a) Interno ao Programa - 1762353 - MARIA CAMILA ALMEIDA
Membro Titular - Examinador(a) Externo à Instituição - CAROLINA BELTRAME DEL DEBBIO - USP
Membro Suplente - Examinador(a) Interno ao Programa - 1669152 - DANIEL CARNEIRO CARRETTIERO
Membro Suplente - Examinador(a) Interno ao Programa - 1887027 - FERNANDO AUGUSTO DE OLIVEIRA RIBEIRO
Membro Suplente - Examinador(a) Externo à Instituição - GUILHERME SHIGUETO VILAR HIGA