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Yes-associated protein 1 YAP1 is a transcription factor-like protein, which belongs to the Hippo pathway. YAP1 plays an important role in growth control and organ renewal [ 11 — 13 ]. The YAP1 gene is overexpressed in some of the most frequent human tumors such as colon, lung and ovarian cancers [ 14 ].

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In addition, YAP1 overexpression is associated with a poor prognosis in medulloblastomas, colon and ovarian cancer [ 11 , 15 , 16 ]. In vitro , YAP1 nuclear accumulation resulted in worsening of the malignant phenotype and induced chemotherapy resistance in ovarian cancer cell lines [ 16 ]. YAP1 cooperates with beta-catenin to activate genes involved in stem cell proliferation for epithelial repair [ 11 ].

There are no studies investigating the role of YAP1 in adrenal development and tumorigenesis. For this reason, herein we investigated YAP1 expression in fetal adrenals and in postnatal normal adrenal cortices as well as in pediatric ACT and its relationship with disease stage and outcome.

We studied 42 children with ACT, being 34 girls Median follow-up was 3. Most of the clinical and laboratory data of this cohort had been previously described [ 5 , 20 ]. YAP1 cytoplasmic and nuclear staining in normal fetal adrenal. Weak YAP1 staining in normal post-natal adrenal cortex. Magnification: x and x. In order to evaluate the role of YAP1 in adrenal development, we investigated YAP1 protein expression in fetal and postnatal normal adrenals.

P53 p. In addition to poor outcome, YAP1 overexpression was associated with advanced disease stage according to Sandrini's classification in pediatric ACT Estimation: 4. These results were independent of the P53 p. RH mutation. The half-maximal inhibitory concentration IC 50 of PNU for NCI-H cells 48 hours after treatment as well as the effect of PNU on cell viability, apoptosis and steroidogenesis had been previously described by our group [ 21 ]. Immunofluorescence demonstrated increased beta-catenin nuclear expression 48 hours after silencing of YAP1. Green: beta-catenin mouse anti-beta-catenin , BD Biosciences ; blue: nuclear staining DAPi , Cell Signaling Technology ; light green: beta-catenin nuclear staining merged.

The oncogene YAP1 plays a role in tissue renewal, cell proliferation and embryogenesis [ 11 — 13 , 22 ]. The overexpression of YAP1 is associated with tumor progression in cancer cell lines [ 23 ]. Furthermore, YAP1 is overexpressed in common human malignancies such as colon, lung, ovary, esophagus and bladder cancer [ 14 , 16 , 22 , 24 ]. In the present study, we evaluated, the expression of the oncogene YAP1 in pediatric ACT as well as in fetal and postnatal normal adrenal cortices at the mRNA and protein levels.

Immunohistochemistry revealed strong YAP1 nuclear staining in fetal adrenals, whereas postnatal normal adrenals showed weak YAP1 staining.

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The strong YAP1 accumulation in both nucleus and cytoplasm of fetal adrenal cells, together with decreased YAP1 expression in postnatal adrenal cortices, reinforces the role of YAP1 in promoting cell dedifferentiation and proliferation [ 12 , 13 ]. Therefore, YAP1 overexpression in tumors was associated with lower survival in pediatric patients, reinforcing the association between YAP1 overexpression and worse prognosis. Similar to our findings, a meta-analysis study [ 25 ] confirmed that YAP1 overexpression was associated with lower overall survival and lower disease-free survival in other cancers.

An association between worse prognosis and YAP1 overexpression has been previously reported in colon cancer, ovarian tumors and medulloblastoma [ 11 , 15 , 16 ]. Additionally, YAP1 overexpression may be linked to tumor progression resulting in a worse prognosis, as observed in pediatric ACT. YAP1 plays an important role in cell mechanotransduction, in which its expression represents biochemical signals triggered by mechanical inputs [ 26 , 27 ]. YAP1 interacts with alpha-catenin binding to cell adhesion molecules and is essential to promote cellular reactions in response to changes in extracellular matrix ECM [ 28 ].

The increasing ECM rigidity causes a loss of cell junctions leading to epithelial-to-mesenchymal transition EMT and metastasis [ 29 ]. Additionally, cell-cell junctions are lost in the EMT process, which triggers the Hippo signaling blocking and, consequently, YAP1 activation [ 26 ]. In the present study, YAP1 knockdown resulted in a decrease in cell migration and cell viability, demonstrating the potential involvement of YAP1 in adrenocortical cell growth and metastasis.

Other pathways besides the Hippo signaling can regulate YAP1.

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Consistent with conserved mechanisms between brain tumorigenesis and development, SHH induces YAP1 expression and its nuclear localization in cerebellar granule neuron precursors, whose proliferation can be driven by YAP1 [ 15 ]. The NCI-H cell line harbors the p. Similar findings were previously observed in colonic tumorigenesis and tissue renewal.

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Cai et al reported increased YAP1 protein expression in colonic regeneration models, which was not related to increased gene transcription, since YAP1 mRNA expression was decreased in the intestinal crypts during the regeneration process [ 32 ]. Moreover, it is known that depending on its availability and phosphorylation state, YAP1 can either retain beta-catenin in the cytoplasm or facilitate its transport into the nucleus [ 33 ]. Other abnormalities in the Hippo pathway could also lead to its activation [ 34 ].

In pediatric ACT, we have not deliberately assessed whether there is a difference in the expression of YAP1 regarding histology adenoma and carcinoma due to the difficulty for histological differentiation in pediatric ACT [ 35 ]. YAP1 plays an important role in adrenocortical tumorigenesis and it is a candidate to be a prognostic marker for pediatric ACT.

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Disease stage was classified according to the Sandrini classification proposed for pediatric ACTs [ 9 ]. Ten adrenal cortices obtained during autopsies from children with non-adrenal endocrine diseases or with Wilm's tumor submitted to surgery nephrectomy and adrenalectomy were confirmed to be normal after macroscopic evaluation and used as controls. Fetal adrenal cortices were obtained from spontaneously miscarried fetuses who underwent autopsies in the Department of Pathology, Ribeirao Preto Medical School as described previously were used in the IHQ analysis [ 20 ].

As negative controls, all specimens were incubated with no primary antibody under identical conditions. All slides were evaluated randomly in at least 10 representative high-power fields x and x by an experienced pathologist author L. These results were blind checked by the authors R. Cells were cultured as previously described [ 21 ]. For all experiments, cell lines were harvested during their third passage. NCI-H cells were seeded at 2x10 5 cells per well in well plates for gene expression and protein analysis. After 48 hours, cells were treated with vehicle 0.

After 48 hours, cells were harvested for RNA and protein isolation. At least two independent experiments were performed in triplicate. NCI-H cells were seeded in 96, 24 or 6-well cultures plates 2x10 4 , 2x10 5 or 1x10 6 cells per well, respectively. After 24 hours of incubation, the medium was replaced and the cells were harvested after 48 hours for gene expression and immunofluorescence, after 72 hours for transwell migration and after 96 hours for cell viability analysis. YAP1 silencing experiments were always performed in triplicate. Fluorescence was acquired with an Imager.

After 24 hours, the cells were transfected in triplicate with siYAP1 or Control as previously described. After another 48 hours of incubation, the cells were fixed with methanol and non-migrated cells remaining on the topside of the membrane were removed with a cotton swab. The migrated cells were stained with Gills Hematoxylin and images of four representative fields of each insert were taken using an Imager.

NCI-H cells 2x10 4 per well were seeded in 96 well plates 24 hours before siRNA transfection as previously described. Absorbance at nM was obtained using a microplate reader Bio Rad. Cell viability values were expressed as percentages of control cells. Two independent experiments were performed in triplicate. Survival analysis was carried out by Kaplan-Meier curves and compared by the log-rank test, considering death as the unfavorable event and patients who were lost to follow-up were censored considering their last follow-up visit.

GraphPad Prism 6. In addition, ACT data were adjusted by linear regression based on the Student t-test focused on a Bayesian model.

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We chose this statistical method due to sample size and problems to use statistical tests based on asymptotic theory. Multiple comparisons were performed by orthogonal contrasts. We thank the statistician Davi C. Read article at publisher's site DOI : Aging Albany NY , 11 19 , 15 Oct Sci Rep , 8 1 , 13 Jul Erickson LA. Histopathology , 72 1 , 01 Jan Cited by: 5 articles PMID: Endocrinology , 11 , 01 Nov Free to read. To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation.