Mecanismos Moleculares envolvidos na Teratogênese do vírus Zika

Em 2015, o vírus Zika (ZIKV) foi identificado como um teratógeno, capaz de causar anomalias congênitas em embriões e fetos expostos durante a gestação, as quais compõem a atualmente chamada Síndrome Congênita associada à infecção pelo vírus Zika (SCZ). Estudos moleculares descreveram que alterações da expressão gênica e proteica eram envolvidas na teratogênese do ZIKV. Além disso, se percebeu que o ZIKV causava anomalias em menos da metade os indivíduos (até 42%), discutindo-se o papel de fatores de risco genéticos na ocorrência desta condição. Assim, o objetivo desta tese foi investigar, através de diferentes abordagens, os mecanismos moleculares envolvidos na teratogênese do ZIKV, e descrever fatores genéticos de suscetibilidade para a ocorrência da SCZ. Nós inicialmente conduzimos um estudo de revisão sistemática, descrevendo os principais mecanismos moleculares descritos como associados à teratogênese do ZIKV em modelos animais. Tais estudos descreveram genes de resposta antiviral como positivamente regulados no cérebro dos animais expostos, enquanto genes envolvidos de neurodesenvolvimento sofriam regulação negativa. Além disso, considerando que células neurais humanas são os principais alvos do ZIKV, conduzimos uma meta-análise de estudos de RNA-seq que avaliaram a expressão gênica nestes tipos de células após a exposição ao ZIKV. Identificou-se que genes envolvidos na resposta antiviral são positivamente regulados nestas células, enquanto dentre os genes negativamente regulados estavam aqueles que quando mutados causam microcefalia primária autossômica recessiva. O papel da genética na suscetibilidade à SCZ foi inicialmente investigado através de estudos de caso-controle, com 40 crianças com SCZ e 48 crianças expostas ao vírus durante a gestação, que nasceram sem anomalias congênitas. Os genes investigados são pertencentes às principais vias biológicas descritas como envolvidas na infecção e teratogênese do ZIKV, como entrada e resposta ao vírus, resposta imune e inflamatória, regulação do ciclo celular e neurodesenvolvimento. Identificou-se que variantes em NOS2 e MIR124 podem atuar como fatores de risco para a SCZ, e variantes em TNF e TP53 podem atuar como fatores de risco para microcefalia e lisencefalia, respectivamente, em casos de SCZ. Descrevemos ainda um caso de gêmeos discordantes para SCZ e, através da avaliação de seu exoma e comparação de suas variantes genéticas, identificamos variantes de possível risco para a SCZ no gêmeo afetado. Dentre estas, deleções e variantes de sentido trocado em genes atuam em processos de desenvolvimento e organização celular, os quais também são afetados pelo ZIKV, considerando a expressão gênica, sugerimos que tais variantes podem ter contribuído para a SCZ no gêmeo afetado, pois ele apresentaria uma combinação de fatores que tornariam tais processos, importantes para o desenvolvimento embrionário, menos eficientes. Em outro estudo investigamos novamente fatores de risco genéticos para a SCZ, a partir do sequenciamento do exoma completo de uma série de casos com SCZ (n=4) e casos com microcefalia devido a uma etiologia desconhecida (n=4). Relatamos, em casos de SCZ, variantes de risco para a doença em genes que quando mutados causam fenótipos semelhantes à SCZ, como microcefalia. Finalmente, considerando que os efeitos a longo prazo da exposição ao ZIKV durante a gestação ainda são pouco conhecidos, discutimos o papel que esse evento poderia ter na ocorrência de transtornos neurológicos, formulando uma hipótese de como o autismo poderia ocorrer em função desta exposição. De maneira geral, os trabalhos desenvolvidos conseguiram alcançar o objetivo principal a que esta tese se propôs, investigando e discutindo, através de diferentes abordagens, mecanismos envolvidos com a teratogenicidade do ZIKV.In 2015, the Zika virus (ZIKV) was identified as a teratogen, capable of causing congenital anomalies in embryos and fetuses exposed during pregnancy, which compose the currently called Congenital Zika Syndrome (CZS). Molecular studies described that alterations in gene and protein expression were involved in the ZIKV teratogenesis. In addition, it was reported that ZIKV caused anomalies in less than half the individuals (up to 42%), being discussed the role of genetic risk factors in the occurrence of this condition. Therefore, the aim of this thesis was to investigate, through different approaches, the molecular mechanisms involved in the ZIKV teratogenesis, and to describe genetic factors in the susceptibility to the occurrence of SCZ. We initially conducted a systematic review study describing the main molecular mechanisms described as associated with ZIKV teratogenesis in animal models. Such studies described antiviral response genes up-regulated in the brain of exposed animals, while genes involved in neurodevelopment were down- regulated. Furthermore, considering that human neural cells are the main targets of ZIKV, we conducted a meta-analysis of RNA-seq studies that evaluated gene expression in these cell types after ZIKV exposure. It was identified that genes involved in the antiviral response are up-regulated in these cells, while among the down-regulated genes were those that, when mutated, cause autosomal recessive primary microcephaly. The role of genetics in susceptibility to CZS was initially investigated through case-control studies, with 40 children with CZS and 48 children exposed to the virus during pregnancy, who were born without congenital anomalies. The investigated genes belong to the main biological pathways described as involved in ZIKV infection and teratogenesis, such as virus entry and response, immune and inflammatory response, cell cycle regulation and neurodevelopment. It was identified that variants in NOS2 and MIR124 can act as risk factors for CZS, and variants in TNF and TP53 can act as risk factors for microcephaly and lissencephaly, respectively, in CZS cases. 17 We also describe a case of discordant twins for SCZ and, by evaluating their exome and comparing their genetic variants, we identified possible risk variants for CZS in the affected twin. Among these, deletions and missense variants in genes that act in cell development and organization processes, which are also affected by ZIKV considering the gene expression. Therefore, we suggest that such variants may have contributed to CZS in the affected twin, as he would present a combination of factors that would make such processes less efficient. In another study, we again investigated genetic risk factors for CZS, through the whole-exome sequencing of a series of cases with CZS (n=4) and cases with microcephaly due to an unknown etiology (n=4). We report variants with a possible role in the occurrence of CZS in genes that, when mutated, cause phenotypes similar to CZS, such as microcephaly. Finally, considering that the long-term effects of exposure to ZIKV during pregnancy are still poorly understood, we discuss the role that this event could play in the occurrence of neurological disorders, formulating a hypothesis on how autism could occur as a result of this exposure. In general, the developed studies were able to achieve the aims proposed by this thesis, investigating and discussing, through different approaches, mechanisms involved with the teratogenicity of ZIKV

Molecular mechanisms of Zika virus teratogenesis from animal studies : a systematic review protocol

Background: Due to the diversity of studies in animal models reporting that molecular mechanisms are involved in the teratogenic effect of the Zika virus (ZIKV), the objective of the present study is to evaluate the methodological quality of these studies, as well as to demonstrate which genes and which molecular pathways are affected by ZIKV in different animal models. Methods: This search will be performed in four databases: PubMed/MEDLINE, EMBASE, Web of Science, and Scopus, as well as in the grey literature. The studies selection process will be reported through the PRISMA Statement diagram model. All studies describing the molecular mechanisms possibly involved in the development of malformations caused by embryonic/fetal ZIKV exposure in animal models with an appropriate control group and methodology will be included (including, for instance, randomized and non-randomized studies). All animals used as experimental models for ZIKV teratogenesis may be included as long as exposure to the virus occurred during the embryonic/fetal period. From the selected studies, data will be extracted using a previously prepared standard form. Bias risk evaluation will be conducted following the SYRCLE’s Risk of Bias tool. All data obtained will be tabulated and organized by outcomes (morphological and molecular). Discussion: With the proposed systematic review, we expect to present results about the methodological quality of the published studies with animal models that investigated the molecular mechanisms involved in the teratogenic effect of ZIKV, as well as to show the studies with greater reliability

The role of ESCO2, SALL4 and TBX5 genes in the susceptibility to thalidomide teratogenesis

Thalidomide is widely used for several diseases; however, it causes malformations in embryos exposed during pregnancy. The complete understanding of the mechanisms by which thalidomide afects the embryo development has not yet been obtained. The phenotypic similarity makes TE a phenocopy of syndromes caused by mutations in ESCO2, SALL4 and TBX5 genes. Recently, SALL4 and TBX5 were demonstrated to be thalidomide targets. To understand if these genes act in the TE development, we sequenced them in 27 individuals with TE; we verifed how thalidomide afect them in human pluripotent stem cells (hPSCs) through a diferential gene expression (DGE) analysis from GSE63935; and we evaluated how these genes are functionally related through an interaction network analysis. We identifed 8 variants in ESCO2, 15 in SALL4 and 15 in TBX5. We compared allelic frequencies with data from ExAC, 1000 Genomes and ABraOM databases; eight variants were signifcantly diferent (p<0.05). Eleven variants in SALL4 and TBX5 were previously associated with cardiac diseases or malformations; however, in TE sample there was no association. Variant efect prediction tools showed 97% of the variants with potential to infuence in these genes regulation. DGE analysis showed a signifcant reduction of ESCO2 in hPSCs after thalidomide exposure

PREVALÊNCIA DE MUTAÇÕES ASSOCIADAS AO DESENVOLVIMENTO DE RESISTÊNCIA À LAMIVUDINA EM PACIENTES COM HEPATITE B CRÔNICA DO MUNICÍPIO DE BENTO GONÇALVES, RS.

A infecção pelo vírus da hepatite B (HBV) é um problema de saúde pública mundial. O tratamento com lamivudinaem portadores crônicos do HBV está frequentemente associado a altas taxas de resistência viral, relacionadas a mutações nomotivo YMDD do gene da transcriptase reversa. O objetivo do presente estudo foi investigar a prevalência de mutações nomotivo YMDD em pacientes com hepatite B crônica do município de Bento Gonçalves, RS. Um estudo transversal foi realizadoentre julho e dezembro de 2010 incluindo 102 pacientes. O DNA viral foi sequenciado em amostras de 21 portadoresativos do HBV. Foi observada a presença de um HBV mutante portando a variação YMDD (rtM204I) acompanhadada substituição rtL180M. Outras mutações (rtA181V e rtQ215S) foram verificadas em dois pacientes

Comparative Genomics Identifies Putative Interspecies Mechanisms Underlying Crbn-Sall4-Linked Thalidomide Embryopathy

The identification of thalidomide–Cereblon-induced SALL4 degradation has brought new understanding for thalidomide embryopathy (TE) differences across species. Some questions, however, regarding species variability, still remain. The aim of this study was to detect sequence divergences between species, affected or not by TE, and to evaluate the regulated gene co-expression in a murine model. Here, we performed a comparative analysis of proteins experimentally established as affected by thalidomide exposure, evaluating 14 species. The comparative analysis, regarding synteny, neighborhood, and protein conservation, was performed in 42 selected genes. Differential co-expression analysis was performed, using a publicly available assay, GSE61306, which evaluated mouse embryonic stem cells (mESC) exposed to thalidomide. The comparative analyses evidenced 20 genes in the upstream neighborhood of NOS3, which are different between the species who develop, or not, the classic TE phenotype. Considering protein sequence alignments, RECQL4, SALL4, CDH5, KDR, and NOS2 proteins had the biggest number of variants reported in unaffected species. In co-expression analysis, Crbn was a gene identified as a driver of the co-expression of other genes implicated in genetic, non-teratogenic, limb reduction defects (LRD), such as Tbx5, Esco2, Recql4, and Sall4; Crbn and Sall4 were shown to have a moderate co-expression correlation, which is affected after thalidomide exposure. Hence, even though the classic TE phenotype is not identified in mice, a deregulatory Crbn-induced mechanism is suggested in this animal. Functional studies are necessary, especially evaluating the genes responsible for LRD syndromes and their interaction with thalidomide–Cereblon

Terapias de reperfusão em infarto agudo do miocárdio: uma revisão atualizada

Este artigo apresenta uma revisão atualizada sobre as terapias de reperfusão no tratamento do Infarto Agudo do Miocárdio (IAM). Inicia-se com uma explanação sobre a definição, etiologia e fisiopatologia do IAM, destacando a importância da restauração do fluxo sanguíneo para prevenir danos cardíacos irreversíveis. A revisão das diretrizes das sociedades médicas relevantes, como a American Heart Association (AHA) e a European Society of Cardiology (ESC), destaca as recomendações atuais para a prática clínica, enfatizando a importância da reperfusão precoce e a individualização do tratamento de acordo com as características do paciente e os recursos disponíveis