Navegando por Palavras-chave "Caspase-3"
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- ItemSomente MetadadadosCaracterísticas do tecido ósseo e cartilagíneo do fêmur de um modelo experimental para Distrofia Muscular de Duchenne(Universidade Federal de São Paulo (UNIFESP), 2019-07-08) Santos, José Fontes dos [UNIFESP]; De Oliveira, Flavia [UNIFESP]; http://lattes.cnpq.br/3387760393535776; http://lattes.cnpq.br/4452664613699869; Universidade Federal de São Paulo (UNIFESP)Duchenne muscular dystrophy (DMD) is induced by genetic mutation in the protein dystrophy. Patients suffering DMD have muscle weakness, loss of mobility and, greater bone resorption, reduced bone mass and greater susceptibility to fracture. The experimental MDX murine model, as in dystrophic humans, has a genetic mutation that causes the lack of dystrophin. However, unlike dystrophic humans, these animals have muscle regeneration and non-progressive disease phenotype. Thus, the study aims to evaluate bone and cartilage from femur of murine models DMD, C57BL/10-dmdmdx (MDX group, n=3), and their respective controls (Control group, n=3). Both animal groups were euthanized at 16 weeks-old. After that, the proximal epiphysis was separated and processed through histological techniques for obtaining some tissue that were subsequently submitted to staining HE and Sirius red staining for structural, cellular analysis such as, collagen and immunohistochemistry for RUNX-2/RANK-L; MMP-2 e MMP-9; Caspase-3 and KI-67. Statistical analyzes were performed by the unpaired Student t test. The results showed that both articular cartilage and bone tissue underwent severe morphological changes due to muscular dystrophy. In addition, histopathology analysis showed a predominance of type I collagen fibers, with the pantographic mesh compacted in the articular cartilage from dystrophic animals. In the immunohistochemical analyzes, the MDX group showed expression for all proteins used in this study, particularly in the regions of subchondral bone, medulla and diaphysis. These results indicate that the changes present in bone and cartilage tissue are not exclusively related to muscle weakness, but also due to action of the osteogenic regulation proteins and remodeling bone process.
- ItemSomente MetadadadosEfeitos Do Exercício Aeróbio Prévio Em Ratos Wistar Submetidos À Lesão De Isquemia/Reperfusão Renal(Universidade Federal de São Paulo (UNIFESP), 2017-02-28) Lima, Weslei Vicente De [UNIFESP]; Schor, Nestor [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)The relationship between previous aerobic exercise (PAE) and acute kidney injury (AKI) has been poorly investigated in Wistar rats without pathologies. We evaluate the influence of previous aerobic training on the renal function by analyzing the blood biochemistry (creatinine and urea), urine (proteinuria) in Wistar rats which underwent the AKI by I/R. Furthermore, we also evaluate the stages of acute tubular necrosis (ATN) by histological examination and the levels of caspase-3 activity by immunohistochemistry. Male Wistar rats were divided into 3 groups: sham (S), ischemic and reperfusion (I/R) and ischemic and reperfusion exercise (I/R Ex). The group I/R Ex went through progressive aerobic training with moderate intensity for 4 weeks. After that, each group underwent an AKI induction surgery by means of clamping the bilateral renal arteries for 45 minutes. Serum creatinine (sCr), urea (sUr) and proteinuria levels were evaluated after 48 hour of reperfusion. In separate experiments, the kidneys of the animals were removed, fixed in 10% formalin and stained with hematoxylin and eosin (H&E) and PAS (Schiff) for histological analysis. Tubule interstitial changes were evaluated by measuring tissue necrosis graded on a 0 to 4 scale in relation to the extent of kidney damage. The PAE produced significant improvement in the levels of creatinine and urea (p <0.05). Agreeing with the histopathologic analysis, the group I/R Ex showed a lesser degree of renal tubular injury and immunohistochemistry showed reduced activated caspase-3 levels compared to the other groups. Our results suggest that PAE attenuates renal damage induce by I/R. Further studies are needed in order to identify the mechanisms involved in this phenomenon.
- ItemAcesso aberto (Open Access)Papel da caspase-3 na quiescência dos gonócitos de ratos(Universidade Federal de São Paulo (UNIFESP), 2019-01-31) Santos, Marina Nunes Dos [UNIFESP]; Teixeira, Taiza Stumpp [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)The germ cell lineage arises from a progenitor cell population in the epiblast called primordial germ cells (PGC). These cells erase their somatic program and enter a germ cell program after what they migrate to the gonads through the gut endoderm and their dorsal mesentery. During their migration and just after they enter the gonadal ridges, the male PGC proliferate for a short period and then enter in quiescence. These cells are then called gonocytes. Because gonocytes are quiescent cells, very few studies about them are available, so that the mechanisms of the quiescence establishment and regulation are not well known. Studies by our group showed that rat gonocytes are mitotic until 18 days post coitum (dpc) and enter in quiescence at 19dpc. During this period, they stay arrested in G0/G1 and no death or proliferation is observed. Our group also showed very abundant and intense caspase-3 (Casp3) labeling in the cytoplasm of all gonocytes, suggesting that this enzyme may play a non-apoptotic role during rat gonocyte quiescence. In addition, there is evidence that Casp3 cleaves the pluripotent marker NANOG in embryonic stem cells, which is also expressed by the male germ cells. Aim: The aim of this study was to investigate if Casp3 and NANOG interact and whether the inhibition of Casp3 activity leads to alterations of the expression of cell cycle genes in the quiescent gonocyte. Methods: For this, 19dpc testis were collected, dissociated and the isolated gonocytes were incubated in vitro with Casp3 inhibitor for 24h. The expression of cell cycle, apoptosis and pluripotency markers was analyzed by qPCR. Gonocyte viability was investigated by propidium iodide and flow cytometry. Protein extraction was performed from whole 19dpc testes for the investigation of Casp3 and NANOG protein expression. Results: The analysis of gonocyte viability showed that the cultures treated with Casp3 inhibitor presented more dead gonocytes than the control cultures. The inhibition of Casp3 also lead to an increase of Pcna expression as well as a decrease of p21cip, p27kip, Bcl2 and Kras expression. When compared with gonocytes that were not maintained in culture for 24h, the cultured gonocytes showed an increase of p21cip expression in control and treated cultures and of p27kip in the control cultures. The expression of Sox2 and Nanog was not detected in the cultured gonocytes, although Sox2 have been detected in non-cultured gonocytes. Casp3 protein was detected in 19dpc testis extract, although NANOG protein was not detected. Conclusion: The results obtained here lead us to conclude that Casp3 inhibition affects the expression of classical cell cycle markers, suggesting that this enzyme plays a non-apoptotic role in male germ cell development by controlling the cell cycle.
- ItemAcesso aberto (Open Access)Papel de oct4, sox2 e caspase-3 na quiescência dos gonócitos de rato(Universidade Federal de São Paulo (UNIFESP), 2014-10-29) Arantes, Anelise Diniz [UNIFESP]; Teixeira, Taiza Stumpp Teixeira [UNIFESP]; Oliva, Samara Urban de [UNIFESP]; http://lattes.cnpq.br/5886968082690915; http://lattes.cnpq.br/1024329256080770; http://lattes.cnpq.br/0559069185165585; Universidade Federal de São Paulo (UNIFESP)Introduction: The germ cells emerge from the epiblast and migrate to the gonads, where they become gonocytes. The gonocytes are the precursors of the spermatogonial stem cells, but little is known about their differentiation. It is seems that the rigid control of gonocyte proliferation, quiescence and pluripotent marker expression is crucial for germ cell development. Objective: Identify the pre-natal period of male germ cell quiescence in the rat and whether caspase-3 (Casp3) is involved in the process. Methods: Rat embryonic gonads were collected at 15, 17 and 19 days post-coitum (dpc). The expression of pluripotency (SOX2, OCT4) and proliferation (Ki67, phosphorylated Retinoblastoma 1 (pRb1)) markers as well as of cleaved Casp3 was analysed. To address Casp3 role in rat gonocyte quiescence, testes from 19dpc embryos were incubated with Casp3 inhibitor and submitted to the immunolabelling of Ki67, PCNA, Casp3 and NANOG. Results: The number of Ki67 and pRB1-labelled gonocytes reduced from 15dpc to 17dpc reaching zero at 19dpc, indicating that quiescence starts around 15dpc and that they are quiescent at 19dpc. OCT4 labelling followed the same detection pattern, whereas SOX2 was present only at 15dpc. These data suggest that the establishment of the quiescence period involves the downregulation of these pluripotent markers. Casp3 labelling was opposite to OCT4, pRB1 and Ki67 labelling, suggesting that it has a role in rat gonocyte quiescence. Casp3 labelling was maintained after the incubation with Casp3 inhibitor, what was expected since the inhibitor does not induce Casp3 degradation. Ki67 was not detected in the gonads incubated with Casp3 inhibitor, suggesting that Caps3 inhibition does not reactivate the cell cycle. NANOG was detected in the gonocyte cytoplasm at 19dpc and its labelling was identical to that of Casp3, suggesting that it has a role in the control of germ cell cycle in the embryo and may interact with Casp3. PCNA-positive and negative gonocytes were observed in 19dpc gonads before and after culture. However, the number of PCNA-negative gonocytes increased in the gonads incubated with Casp3-inhibitor, suggesting that Casp3 is a positive regulator of PCNA. Conclusion: These results suggest that OCT4 and SOX2 downregulation as well as Casp3 and NANOG expression are involved in rat gonocyte quiescence and that PCNA depend on Casp3 activity in these cells.