Functional Analysis of DNA Demethylation - During IL4/GM-CSF-driven Differentiation of Human Monocytes

Epigenetic processes, such as DNA (de)methylation and chromatin remodeling are fundamental for hematopoietic cell differentiation and lineage specification. Hence, their dysregulation is associated with many hematological malignancies. In this thesis, the analyses were focused on DNA demethylation processes during monocyte (MO) to immature dendritic cell (iDC) differentiation in the presence of the cytokines IL4 and GM-CSF. Due to the absence of cell proliferation in this system, we were able to investigate TET-mediated active demethylation events occurring in a replication-independent context. In hematopoietic cells, comprising MO and MO-derived cells, the TET2 enzyme is the main hydroxylase catalysing the initial oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and likely the remaining oxidative steps as well. In this thesis, the TET2 gene was transiently knocked-down in MO using a well-established siRNA-mediated approach and its functional consequences during iDC differentiation were assessed. Overall, TET2 depletion was associated with very mild effects on iDC differentiation, transcriptional programs and chromatin remodeling processes. Nevertheless, the data was reproducible among donors and corroborated the importance of TET2-mediated active DNA demethylation processes during MO differentiation. Based on preliminary genome-wide methylation analyses in MO and iDCs, 7610 iDC-specific differentially methylated regions (DMRs) were identified. These regions were characterized by a progressive active DNA demethylation (5hmC enrichment) and an increase in chromatin accessibility. Since we were mostly interested on differentiation-associated processes, the analyses were centered on transcription factors (TFs) enriched across DMRs and strongly induced in iDCs, such as IRF4 and EGR2. Consequently, both TFs were individually and transiently knocked-down in MO and their effects on iDC differentiation and associated epigenetic processes were determined. IRF4 depletion prevented the differentiation of iDC, and instead cells acquired a macrophage-like morphology confirming previous mouse work244. Interestingly, we found that IRF4 might be involved in open chromatin at its binding sites regardless of changes in DNA demethylation, as reflected by the reduced chromatin accessibility and the minor effects on DNA methylation upon its depletion. Remarkably, this work is the first to describe an essential role of EGR2 in IL4/GM-CSF-mediated MO differentiation. Indeed, data showed a strong impact of EGR2 depletion on cell morphology and viability as well as profound changes in iDC transcriptional programs. Besides its importance on MO biology, EGR2 is implicated in active DNA demethylation and chromatin remodeling processes as indicated by iDC DMRs that remained methylated and failed to gain accessibility upon EGR2-silencing. Notably, the presence of a methylation footprint in the EGR2 motif associated with its ability to bind methylated DNA, suggest that the epigenetic pioneer EGR2 can target de novo demethylation processes at its transient and stable binding sites. In fact, co-immunoprecipitation data showed an interaction between EGR2 and TET2, emphasizing the EGR2 role in targeting the DNA demethylation machinery. In summary, this thesis provides new insights into dynamic epigenetic changes through IL4/GM-CSF-mediated MO differentiation. Accordingly, we identified key regulators on MO biology and differentiation-associated epigenetic processes, specifically IRF4 and EGR2. Importantly, EGR2 was found to recruit TET2 to its binding sites even before chromatin opening or TF binding detection, suggesting that at certain iDC DMRs active DNA demethylation and chromatin accessibility changes are uncoupled. In addition, this work demonstrated that DNA methylation-spikes (identified in EGR2 as well as other consensus motifs of TFs) are cell type-specific and protected from demethylation by bound key epigenetic pioneer factors

Characteristics of a long-stay institution for the elderly

Objective: To evaluate the operation of a Long-Stay Institution for the Elderly (LSIE), aiming adequacy of the LSIE according to legislation and improving care. Methods: A qualitative, exploratory-descriptive, action research study was performed, in Currais Novos/RN. Routine observation, interviews and records were the used ways for data collection. Results: The institution was philanthropic, approaching type II care modality. The elderly were confined mainly by family abandonment. Health team was incomplete, with workload fewer than recommended by law, and the care team worked without coordination and supervision of a nurse. Conclusion: We hope that intervention proposal will contribute to improvements in the LSIE, improving care quality provided to the elderly

Effectiveness of plant-derived proanthocyanidins on demineralization on enamel and dentin under artificial cariogenic challenge

Dental caries is considered a disease of high prevalence and a constant problem in public health. Proanthocyanidins (PAs) are substances that have been the target of recent studies aiming to control or treat caries. Objective The aim of this in vitro study was to evaluate the efficacy of a treatment with grape seed extract, under cariogenic challenge, to minimize or even prevent the onset of caries in the enamel and dentin. Material and Methods Blocks of enamel and dentin (6.0x6.0 mm) were obtained from bovine central incisors, polished, and selected by analysis of surface microhardness (SH). The blocks were randomly divided into 3 groups (n=15), according to the following treatments: GC (control), GSE (grape seed extract), GF (fluoride – 1,000 ppm). The blocks were subjected to 6 daily pH cycles for 8 days. Within the daily cycling, the specimens were stored in buffered solution. The blocks were then analyzed for perpendicular and surface hardness and polarized light microscopy. Results The means were subjected to statistical analysis using the ANOVA and Fisher's PLSD tests (

Relato de experiência: projeto estratégias para educação e saúde da criança em atendimento durante a pandemia: Experience report: education and health strategies for childrens extension project in care during the pandemic

Este texto que ora se apresenta trata-se de um relato da experiência vivenciada pelo “Projeto de Extensão Estratégias para Educação e Saúde da Criança”, da Universidade Católica Dom Bosco, de Campo Grande/MS. Este projeto desenvolve atividades em parceria com seis locais de atendimento e/ou assistência a crianças. A pandemia causada pelo coronavírus SARS-CoV-2 trouxe interferências para toda a população, devido à suspensão das atividades presenciais. Docentes e discentes se aperfeiçoaram e adaptaram metodologias de comunicação, utilizando as redes sociais e plataformas digitais como  instrumento de continuidade das atividades desenvolvidas pelo projeto. O maior desafio desta nova estratégia tecnológica foi atingir, sensibilizar e manter o público-alvo ativo

Fenomenologia nas pesquisas em turismo: análise das dissertações dos programas de pós-graduação no Brasil

Na perspectiva de aferir como se vem procedendo metodologicamente e verificar a exata aplicação do método fenomenológico aos estudos em turismo no Brasil, foi realizada neste presente trabalho uma pesquisa de caráter exploratório-descritivo, onde foram analisadas 570 (quinhentos e setenta) dissertações de mestrados acadêmicos, depositadas entre os anos de 2008 e 2016 em todas as instituições aqui examinadas, pertencentes ao território brasileiro. Em um enfoque da busca da compreensão do caminho metodológico foi feita uma análise de dados, observando-se os processos e técnicas utilizadas pelos pesquisadores em questão, tais como entrevistas em profundidade, observação participante e relatos experienciais. Da análise da discussão dos resultados observou-se que, das 570 (quinhentos e setenta) dissertações analisadas, apenas 13 (treze) adotaram o método fenomenológico. Diante deste fato, conclui-se que, dada a necessidade de um embasamento teórico complexo de seu pesquisador, este método é de pouca aplicação nas instituições envolvidas nesta pesquisa. Ademais, a essencialidade da postura neutra de seu pesquisador dificulta muito a caracterização das pesquisas como adotantes do método