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Analysis of signals of route map kinase in lymphocytes CD4 + activated in short exposure periods to acquired immunodeficiency virus type 1 HIV-1

Grant number: 16/22876-3
Support Opportunities:Regular Research Grants
Duration: August 01, 2017 - July 31, 2019
Field of knowledge:Biological Sciences - Biology
Principal Investigator:Luiz Mário Ramos Janini
Grantee:Luiz Mário Ramos Janini
Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil


In Brazil, data released by the ONU (United Nations Union) recently highlighted that there are 734,000 people infected with HIV and only in 2014 there were a number of 44,000 new infections. Even with all the government support, the number of infected people is increasing, especially in the younger population. The viral latency is the one of the factors that prevents the elimination of HIV-1 in infected patients. Many factors are involved in the establishment and maintenance of HIV latency, including the organization of chromatin in regions of viral integration by preliminary epigenetic mechanism. Result changes made in master's thesis completed at the Retrovirology Laboratory, revealed an increase in gene and protein expression of RSK2 in HIV-infected CD4 cells, this protein is related to the epigenetic mechanisms that lead to transcriptional activation of HIV gene, in return this same study a decrease in the phosphorylation of serine 227 of protein RSK2 noted, this phosphorylation is important for activation kinase RSK2 function, with decreased phosphorylation of this it's activity was decreased kinase and consequently there was a decrease in phosphorylation of transcriptional factors that are related will RSK2, as CREB and H3S10 factor, leading to a decrease in the gene transcriptional activity global. Thus, we see that the function of RSK2 is being modulated in a negative way, but this modulation is directly related to via that RSK2 is inserted, via the Map-kinase, studying this pathway in more detail is needed for understanding the mechanisms of activation of RSK2 that may be related to a number of important factors also for transcription of HIV gene. Besides this, as RSK2 is involved in a pathway important intracellular activation, the analysis of the RSK2 protein may be important for the discovery of factors that can be of great relevance in viral replication and latency process and can serve as mechanisms for obtaining therapeutic strategies in the containment of viral infection and in understanding the mechanisms of establishment of latency. Objective: The overall objective of this study is to analyze the signaling pathway map-kinase pathway that RSK2 is inserted through the silencing of critical factors for activation of this pathway, thereby verifying the modulations that can occur in several related factors will RSK2 and are important in the transcription of HIV gene. Materials and methods: From a concentration of erythrocytes and leukocytes in peripheral blood mononuclear cells will be separated by density gradient using Ficoll Paque reagent, the CD4 T lymphocytes are purified from PBMCs and activated. Besides, the cells will be transfected with primers silencing of RSK2. After transfected cultures, wiil be infected with HIV-1 T-tropic. Infections wiil be stopped in 6hrs, 12h, 24h and 36h for obtaining samples of DNA, RNA and protein. The DNA samples will be used to analyze the proviral DNA and as a control of infections by quantitative PCR methodology, RNA extracted wiil be used for gene expression analysis for the RSK2 method and quantitative PCR analysis. The proteins obtained will be separated into nuclear and cytoplasmic extracts and used for the analysis of protein expression and modification by Western blot. The viral production will be the p24 ELISA analysis methodology, which analyzes the output of the viral envelope protein and the production of viral mRNA by automated analysis system mAbbot. (AU)

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