Banca de QUALIFICAÇÃO: MARILIA INES MOVIO
Uma banca de QUALIFICAÇÃO de MESTRADO foi cadastrada pelo programa.DISCENTE : MARILIA INES MOVIO
DATA : 17/07/2019
HORA: 13:00
LOCAL: sala S17,Térreo, Bl. Delta,Campus SBC, Al .da Universidade. s/n, B.Anchieta-S.Bernardo do Campo-SP
TÍTULO:
Functional expression of AGO2: study of the ‘core of RISC complex’ role in retinal development
PÁGINAS: 76
GRANDE ÁREA: Ciências Biológicas
ÁREA: Fisiologia
SUBÁREA: Fisiologia de Órgãos e Sistemas
ESPECIALIDADE: Neurofisiologia
RESUMO:
MicroRNAs (miRNAs) are small non-coding RNAs that control protein levels in a post-transcriptional manner. The action of miRNAs depends on well-orchestrated machinery that includes several elements. In this regard, it is particularly interesting the role of Argonaute-2 (AGO2), an essential protein in RNA-induced silencing complex (RISC). The main aim of this work was to characterize the role of AGO2 in retinal development. Long Evans rats in early post-natal (P0) and adult (P60) ages were provided by UFABC vivarium, kept in 12:12h dark-light cycle. Animals were euthanized using intraperitoneal urethane (25%) and decapitation. Retinas were harvested to describe AGO2 in the developing and adult retina using real-time PCR (n=6), ii) western blotting (WB, n=5) and immunofluorescence (IF) (n=6). We also employed Manders’ and Spearman coefficient analyses (n=6) for AGO2-
nucleus colocalization. To induce AGO2 knockdown, morpholino oligo (MO) or scramble control oligo (Ctl) were injected in P0 subretinal space, and retinas were harvested after 7 days for WB (n=8), IF (n=6), and hematoxilin-eosin staining (HE, n=6). All procedures were approved by UFABC Animal Care Ethics Committee (16/2014) and results were measured using descriptive statistics and compared by paired t-test. PCR and WB revealed that both
gene expression and protein levels of AGO2 are lower at P0 (PCR: 2^-1=0.5-fold expression; P<0.05; WB: P0: 0.57±0.07 vs P60: 1.18 ± 0.09 normalized optical density, P<0.01). IF and fractionated protein quantification showed no changes of AGO2 in the cytosol, while in P60 AGO2 cumulates in the nucleus (P0: 15.63±1.77 vs P60: 23.95±2.32, P<0.05). When AGO2+ cells were analyzed, results showed that AGO2 localization depends on cell differentiation state. In P0, Spearman analysis showed lower nuclear AGO2 in immature cells than in mature
(-0.04±0.22 vs 0.25±0.18, p<0.05), while Manders’ revealed no changes in nuclear AGO2 in differentiated ganglion cells. MO treatment caused reduction of 52% in AGO2 protein levels, which induced several changes in the retina, as reduction in the thickness of the inner nuclear layer (Ctl:17.37±1.25 vs MO:13.69±1.38, P<0.05), increase in PKCα+ bipolar cells (Ctl: 14.83 ± 1.39 vs MO: 18.58 ± 0.67, P<0.01), CR+ amacrine cells (Ctl: 4.58 ± 1.56 vs MO: 11.17 ± 2.19, P<0.05), and decrease in ChAT+ amacrine cells (Ctl: 5.50 ± 0.58 vs
MO: 6.00 ± 0.71, P<0.05). Taking together, our results revealed that AGO2 translocates to the nucleus during retinal development, and its presence is essential for coordinated formation of retinal cell layers and differentiation of retinal subtypes.
MEMBROS DA BANCA:
Presidente - Interno ao Programa - 1887027 - FERNANDO AUGUSTO DE OLIVEIRA RIBEIRO
Membro Titular - Examinador(a) Interno ao Programa - 1872537 - MARCELA BERMUDEZ ECHEVERRY
Membro Titular - Examinador(a) Externo à Instituição - CLARISSA ROCHA - UNIFESP
Membro Suplente - Examinador(a) Interno ao Programa - 1955999 - ANDRE MASCIOLI CRAVO