Navegando por Palavras-chave "Methylation of dna"

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    Busca por potenciais marcadores genéticos nas fases iniciais da esquizofrenia
    (Universidade Federal de São Paulo (UNIFESP), 2016-10-25) Santoro, Marcos Leite [UNIFESP]; Belangero, Síntia Iole Nogueira [UNIFESP]; http://lattes.cnpq.br/2623781262478620; http://lattes.cnpq.br/8899941647240638; Universidade Federal de São Paulo (UNIFESP)
    Schizophrenia is a severe and disabling psychiatric disorder that affects about 1% of the population. It is a complex disease characterized by the change in multiple genes of susceptibility. Once schizophrenia has been diagnosed, most patients have a poor prognosis, with few returning to normal executive functions after the first psychotic episode (PEP). Thus, the study of individuals at ultra-high risk to develop psychosis (EMR) is of great importance for the understanding of the pathophysiology of schizophrenia, before the establishment of the disease, as well as to find biomarkers related to the transition to schizophrenia. Equally important, the investigation of individuals in their PEP, antipsychotic-naive and before disease progression, is extremely useful for understanding the complexity of schizophrenia and its treatment. Considering that schizophrenia is a chronic disease, its progression and antipsychotic use may be confusing factors in the interpretation of the results of gene expression and DNA methylation. Our main objective was to identify genetic markers of risk, progression and response to treatment in the early stages of schizophrenia by different approaches (genomic, transcriptomic and epigenomic analyses). We investigated 22 subjects at EMR, 66 PEP patients before and after 2 months of treatment with risperidone (PEP-2M), and 67 healthy controls (with no family history or progression of severe psychiatric illness). To better understand the results, we divided this thesis into two studies. In study 1 we evaluated the expression of 12 candidate genes by the TLDA (Taqman Low Density Array) technique in EMR, PEP before treatment and controls. In study 2 we used three large-scale measures (polygenic risk score for schizophrenia, transcriptome and DNA methylome) in controls (N = 60) and in PEP patients before and after risperidone treatment (N = 60). In study 1, we found two differentially expressed genes (UFD1L and MBP), and the UFD1L gene showed an increased expression in the EMR group in relation to the PEP and the controls, suggesting a specific alteration of the individuals at EMR. In study 2, we validated the polygenic risk score (PRS) and demonstrated that this is a measure that can be used effectively in the Brazilian population. In addition, we found positive associations in PEP between PRS and clinical variables, so that the higher the PRS, higher was the impairment. However, these associations are not observed after two months of treatment with risperidone, reinforcing the importance of working with individuals in treatment-free PEP. Finally, we identified some differentially expressed genes and differentially methylated regions related to the disease (when comparing individuals in PEP and controls) and related to the response to treatment (when we followed individuals in PEP before and after risperidone treatment). Using genomic, transcriptomic and epigenomic techniques, we were able to identify genetic markers related to progression and the early stages of schizophrenia in peripheral blood.
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    Supressor tumoral ABI3 (ABI family member 3): desvendando os mecanismos moleculares nos tumores da tiroide humana
    (Universidade Federal de São Paulo (UNIFESP), 2016-08-24) Moraes, Lais de Sousa [UNIFESP]; Cerutti, Janete Maria [UNIFESP]; http://lattes.cnpq.br/1384038091754225; http://lattes.cnpq.br/5887267461766517; Universidade Federal de São Paulo (UNIFESP)
    We identified the loss of ABI3 gene expression in thyroid carcinomas. Functional analysis demonstrated that ectopic expression of ABI3 inhibits cell growth, invasion, migration and tumor growth in vivo associated with an increased in cellular senescence, suggesting ABI3 as a potential tumor suppressor. Nevertheless, there is no genetic/ epigenetic mechanisms described as associated with its loss of expression, and it is still unknown the signaling pathway by which ABI3 exerts its function. Based on these data about ABI3, our first objective was to investigate if DNA methylation could be the mechanism responsible for the loss of ABI3 expression in follicular thyroid carcinoma (FTC). The cell lines derived from follicular thyroid carcinoma (WRO, FTC 238, and FTC236 FTC133) and melanoma (NPA) were treated with demethylating agent (5-Aza) and the level of ABI3 expression was assessed. We observed the restoration of ABI3 expression in follicular carcinoma cell lines treated with 5-Aza (P<0.01). Thus, we correlated the level of ABI3 expression with degree of methylation of its promoter. Although the in silico analysis showed that ABI3 has no CpG islands, we identify the occurrence of multiple adjacent CpG sites in its promoter. These regions called R1, R2 and R3 were evaluated. The degree of methylation of R1 region was inversely correlated with ABI3 expression in these cells (P<0.05). These data were validated on FTC (n=17) and follicular thyroid adenomas (FTA) (n=11) samples. We observed a hypermethylation of R1 in CFT when compared to the AFT (P<0.001), which correlated with the expression of ABI3 (P=0.0002). Interestingly, we identified in R1 a canonical site for the thyroid transcription factor NKX2-1, flanked by the CpG sites. The analysis revealed that ABI3 was expressed in the presence of NKX2-1 and when R1 region is hipomethylated. Luciferase assays suggested that R1 might be a potential enhancer. Our data suggest that ABI3 expression in thyroid tumors appears to be regulated by R1 methylation degree in ABI3 promoter associated with expression of NKX2-1. Yet, to better understand the molecular mechanisms involved in the role of ABI3 in thyroid cancer, our second objective was to investigate the signaling pathways by which ABI3 acts exercising their suppressive effect in thyroid carcinomas. To this end, the follicular thyroid carcinoma cell lines, WRO and FTC133 were transfected with the expression vector containing the wild type cDNA of ABI3 and negative control, the empty vector. Initially, we used the Proteome Profiler Array to identify the signaling pathways, and some pathways described as having been a role in several types of cancer were identified as modulated by ABI3, particularly AKT pathway was enriched in our analysis. To better determine the location of the ABI3 in the signaling pathway, we performed immunoprecipitation assays (IP) and mass spectrometry analysis that showed ABI3 as a part of the WAVE regulatory complex (WRC) with WAVE2 and CYFIP1 proteins. To validate these data, the cells lines were treated with AKT inhibitor (LY294002). In addition, the ABI3 S342A mutant was generated by site-directed mutagenesis. The determination of S342 site was performed by in silico analysis with MotifScan. For the first time, our studies with AKT inhibitor and S342A mimetic mutant of non-phosphorylated form of ABI3 demonstrated that ABI3 is a phosphoprotein, directly phosphorylated by AKT in S432. And this phosphorylation probably interferes with the formation of the WRC complex. The results were observed in both cell lines and the correlation between ABI3, WAVE2 and CYFIP1 was validated in samples of patients (n=23) that expressed ABI3, FTA and normal thyroid tissue (P<0.05). These data are new in the literature and suggests a relationship between the ABI3 phosphorylation, AKT and the formation of WRC complex. Furthermore, these data suggest a possible pathway by which ABI3 acts exerting its suppressive effects, particularly their effects on cell growth and migration.