National Institute of Health Dr. Ricardo Jorge
Repositório Científico do Instituto Nacional de SaúdeNot a member yet
9086 research outputs found
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Behind the curtain: unveiling DIS3L2 role in NMD and human cancer
No full textNonsense-mediated mRNA decay (NMD) is a surveillance mechanism that targets and degrades mRNAs carrying premature translation-termination codons (PTCs), preventing the production of truncated proteins potentially harmful for the cells. In addition to this, several studies have shown that NMD regulates the levels of many physiological mRNAs that encode full-length proteins. Nevertheless, NMD is inhibited in tumor microenvironment and (de)regulates oncogenes and tumor suppressors in several types of cancer. In humans, the mRNA degradation pathways involve exonuclease proteins, such as the DIS3-like protein family (DIS3, DIS3L1 and DIS3L2); however, it is not known whether these proteins are involved in NMD. In order to unveil the role of DIS3L2 in NMD, we performed its knockdown, by RNA interference, in HeLa cells and measured, by RT-qPCR, the mRNA levels and half-lives of various natural NMD targets. Our results show that some NMD targets are highly stabilized in DIS3L2-depleted cells. In addition, mRNA half-life analysis indicate that these NMD targets are in fact direct DIS3L2 substrates. By performing DIS3L2, TUT4 and TUT7 triple knockdown, we also observed that DIS3L2-mediated decay depends on the terminal uridylyl transferases (TUTases) Zcchc6/11 (TUT7/4) activity. Among the NMD targets regulated by DIS3L2, we highlight GADD45A. GADD45A is involved in cell cycle arrest, DNA damage response and apoptotic process. Furthermore, GADD45A deregulation is associated with several types of cancer, such as, esophageal, lung, bladder and pancreatic. Together, our findings establish the role of DIS3L2 and uridylation in NMD and in the regulation of oncogenes and tumor suppressor gene expression. These results might be highly relevant for the advance in diagnosis, prognosis and treatment of many human cancers.FCT/PTDC/BIMONC/4890/2014N/
p53 mutations influence IRES-dependent expression of p53 isoforms
No full textFull-length p53 (FLp53) is a tumour suppressor protein that has been considered a master regulator of many cellular functions. Several isoforms have been described for p53 so far and some of the functions of shorter p53 isoforms have been elucidated and they are different from and complement FLp53 activity. p53 is the most commonly mutated gene in cancer and depending on its mutation status p53 may act as a tumour suppressor or a proto-oncogene. Recently, we have shown that the most common p53 cancer mutants express a larger number and higher levels of shorter p53 protein isoforms that are translated from the mutated FLp53 mRNA (Candeias et al. EMBO R. 2016). Also, we found that cells expressing these shorter p53 isoforms exhibit mutant p53 “gain-of-function” cancer phenotypes, such as enhanced cell survival, proliferation, invasion and adhesion, altered mammary tissue architecture and invasive cell structures. Here, we found that some of these mutations affect the function of an Internal Ribosome Entry Site (IRES) in p53 mRNA. Using bicistronic constructs, primer extension, FACS and other techniques , we investigated which mutations influence — by altering IRES structure and function — IRES-dependent translation of shorter p53 isoforms and to what extent this may lead to the onset or progression of some types of tumours.FCT/PTDC/BIM-ONC/4890/2014N/
Transcriptomic screen for DIS3, DIS3L1 and DIS3L2-associated functional networks in colorectal cancer
No full textThe final step of eukaryotic mRNA degradation proceeds in either a 5’-3’ direction, catalyzed by XRN1, or in a 3’-5’ direction catalyzed by DIS3, DIS3L1 (the catalytic subunits of the exosome) and/or DIS3L2 (exosome-independent). Important findings over the last years have shed a new light onto the mechanistic details of RNA degradation by these exoribonucleases. In addition, it has been shown that they are involved in growth, mitotic control and important human diseases, including cancer. In this work, we aim to analyze how DIS3, DIS3L1 and DIS3L2 regulate the human transcriptome, and how their functional interactions modulate the transcriptional reprogramming of colorectal cancer cells.
Each one of these nucleases was depleted by RNA interference in HeLa cells and levels of several endogenous targets was monitored by RT-qPCR. Our results show that these exoribonucleases are target specific and not directly involved in any known mRNA decay mechanisms such as nonsense-mediated decay (NMD). However, we do not know yet what defines such target preference.
In parallel, our bioinformatics analysis of available transcriptomic data from cells depleted of DIS3L1, DIS3L2, XRN1 or UPF1 (which has a central role in NMD) has shown some, but not full, redundancy among the transcripts regulated by these nucleases, which supports our experimental data.
Presently, we are exploring the mechanism through which DIS3L2 is involved in NMD and how it modulates the expression of NMD targets.FCT/PTDC/BIMONC/4890/2014N/
Implementation of an epidemiological survey targeting owners of backyard animal production systems in Portugal
No full textA detenção e a criação de animais em explorações caseiras podem representar um risco para a transmissão de agentes patogénicos zoonóticos e de genes de resistência a antibióticos, com impacto na saúde pública. Em Portugal, a ausência de registo de sistemas de produção caseira (SPC) dificulta a caracterização das práticas de gestão e criação de animais, assim como o perfil dos seus proprietários. Este estudo piloto pretende avaliar o método de amostragem dos detentores de SPC e desenvolver um questionário epidemiológico pioneiro para avaliar as condições de criação, biossegurança e riscos sanitários e ambientais em SPC. Estruturado em cinco seções, o questionário foi disponibilizado em i) formato eletrónico e ii) em papel, tendo-se testado duas formas de divulgação: através de divulgação digital e por aplicação presencial por pontos de contacto (veterinários, inspetores sanitários e associações do setor). Até ao momento, observou-se um maior número de respostas na versão online, pela sua maior facilidade e abrangência de divulgação. Apesar das limitações na distribuição dos questionários, a metodologia adotada permitirá caracterizar os SPC e os seus proprietários, contribuindo para a avaliação de possíveis riscos e o seu impacto na saúde pública. A continuidade da recolha de dados fortalecerá esta caracterização, apoiando estratégias de mitigação de riscos zoonóticos, segurança alimentar e promoção da saúde animal sob a abordagem One Health.Keeping and rearing animals on backyard farms can pose a risk for the transmission of zoonotic pathogens and antibiotic resistance genes, impacting public health. In Portugal, the lack of registration for backyard production systems (BPS) hinders the characterization of management and animal husbandry practices, as well as the profile of their owners. This pilot study aims to evaluate the sampling method of BPS owners and develop a pioneering epidemiological questionnaire to assess breeding conditions, biosecurity, health and environmental risks in BPS. Structured into five sections, the questionnaire was made available in i) electronic format and ii) on paper, having tested two dissemination strategies: digital distribution and face-to-face application by key contact points (veterinarians, health inspectors and sector associations). To date, the online version has received a higher number of responses due to its broader reach and ease of dissemination. Despite limitations in questionnaire distribution, the adopted methodology will enable the characterization of BPS and their owners, contributing to the assessment of potential risks and their impact on public health. Continued data collection will strengthen this characterization, supporting strategies for mitigating zoonotic risks, ensuring food safety and promoting animal health under the One Health framework
Boletim Epidemiológico Observações: Vol. 14, Nº 37, jan-abr 2025
No full textObservações é uma publicação científica do INSA, IP, que visa contribuir para o conhecimento da saúde da população, os fatores que a influenciam, a decisão e a intervenção em Saúde Pública, assim como a avaliação do seu impacte na população portuguesa. Através do acesso público e gratuito a resultados científicos gerados por atividades de observação em saúde, monitorização e vigilância epidemiológica nas áreas de atuação do Instituto - Alimentação e Nutrição, Doenças Infeciosas, Genética Humana, Saúde Ambiental, Promoção da Saúde e Prevenção de Doenças Não Transmissíveis, Epidemiologia, Investigação em Serviços e Políticas de Saúde - é dada especial atenção à disseminação rápida de informação relevante para a resposta a temas de relevo para a saúde da população portuguesa, tendo como principal alvo todos os profissionais, investigadores e decisores intervenientes na área da Saúde Pública em Portugal
Transcriptomic analysis and epigenetic regulators in human oocytes at different stages of oocyte meiotic maturation
No full textHuman oocytes are highly specialized cells with the capacity to store and regulate mRNAs during oocyte maturation, in preparation for post-fertilization steps. Here we performed single-oocyte transcriptomic analysis of human oocytes in three meitoic maturation stages - Germinal Vesicle (GV; n = 6), Metaphase I (MI; n = 6) and Metaphase II (MII; n = 7). Single-oocyte transcriptomic analysis revealed that the total number of expressed genes progressively decreased from GV to MII stages, with 9660 genes being transcribed in GV, 8734 in MI and 5889 in MII. The same tendency was observed for the number of uniquely expressed genes, with 1328 uniquely expressed genes in GV, 401 in MI and 72 in MII. GO analysis of the uniquely expressed genes showed distinct terms in GV oocytes such as transferase activity, organonitrogen compound metabolic process and ncRNA processing. Analysis of Differentially Expressed Genes (DEGs) between the three maturation stages revealed 1165 DEGs between GV and MII oocytes, with 635 being upregulated and 528 downregulated, 42 DEGs between GV and MI, with 38 being upregulated and 4 downregulated, and no significant changes in gene expression between MI and MII oocytes. Comprehensive analysis of epigenetic regulators showed high expression of several histone-modifying enzymes, namely deacetylases, acetylases, lysine demethylases and methyltransferases, and DNA methylation regulators, namely the maintenance methyltransferase DNMT1 and its co-regulators DPPA3 and UHRF1. Some of these epigenetic regulators were differentially expressed between maturation stages, namely SIRT3, SIRT6, KDM3AP1, KMT2E, DNMT1, DPPA3 and the MEST and RASGRF1 imprinted genes. Our study contributes with important information on the transcriptional landscape of human oocytes in different stages of meiotic maturation, providing important insights into candidate biomarkers of human oocyte quality.This work was funded by Fundação para a Ciência e a Tecnologia (FCT, Portugal) - through doctoral fellowships to C.C. (SFRH/141855/2018) and S.V. (SFRH/BD/147440/2019), a researcher contract to C.J.M. (CEECIND/00371/2017) and a research project grant (EXPL/MED-GEN/1261/2021)
Re‐evaluation of citric acid esters of mono‐ and diglycerides of fatty acids (E 472c) as a food additive in foods for infants below 16 weeks of age and follow‐up of its re‐evaluation
No full textCitric acid esters of mono‐ and diglycerides of fatty acids (E 472c) was re‐evaluated in 2020 by the Food Additives and Flavourings Panel (FAF Panel) along with acetic acid, lactic acid, tartaric acid, mono‐ and diacetyltartaric acid, mixed acetic and tartaric acid esters of mono‐ and diglycerides of fatty acids (E 472a,b,d,e,f). As a follow‐up to this assessment, the FAF Panel was requested to assess the safety of citric acid esters of mono‐ and diglycerides of fatty acids (E 472c) for its use as food additive in food for infants below 16 weeks of age belonging to food categories (FCs) 13.1.1 (Infant formulae as defined by Directive 2006/141/EC) and 13.1.5.1 (Dietary foods for infants for special medical purposes and special formulae for infants). In addition, the FAF Panel was requested to address the recommendation of the re‐evaluation of E 472c as a food additive to update the EU specifications in Commission Regulation (EU) No 231/2012. For this, a call for data was published to allow interested partied to provide the requested information for a risk assessment. The Panel concluded that the technical data provided by the interested business operators support an amendment of the EU specifications for E 472c. Regarding the safety of the use of E 472c in food for infants below 16 weeks of age, the Panel concluded that there is no safety concern from its use at the reported use levels and at the maximum permitted levels in food for infants below 16 weeks of age (FCs 13.1.1 and 13.1.5.1)
Re‐evaluation of neotame (E 961) as food additive
No full textThe present opinion deals with the re‐evaluation of neotame (E 961) as a food additive. Neotame is the chemically manufactured compound N‐[N‐(3,3‐dimethylbutyl)‐l‐α‐aspartyl]‐l‐phenylalanine 1‐methyl ester. The main impurity of neotame (E 961) is also a degradation product (de‐esterified form), N‐[N‐(3,3‐dimethylbutyl)‐l‐α‐aspartyl]‐l‐phenylalanine (NC‐00751) and the primary metabolite. No new data were received following the call for biological and toxicological data. A summary of the toxicological studies available in the EFSA opinion of 2007 is presented and studies gathered from the literature are summarised. Neotame is rapidly absorbed and pre‐systemically metabolised, systemic intact neotame is likely to be excreted in the urine with its metabolites. The potential aneugenic effects at the site of contact are not expected to occur; overall, there is no concern for genotoxicity of neotame (E 961) at the maximum permitted levels or reported use levels. A review of the other endpoints from the already available toxicological database did not indicate an adverse effect for neotame at the highest doses tested. The Panel established an acceptable daily intake (ADI) of 10 mg/kg bw per day for neotame based on the no observed adverse effect level (NOAEL) of 1000 mg/kg bw per day from a 52‐week chronic and 104‐week carcinogenicity studies in rats. This ADI replaces the ADI of 2 mg/kg bw per day established by EFSA in 2007. The resulting exposure to methanol and its metabolite formaldehyde from the use of neotame at the ADI of 10 mg/kg bw per day does not raise a concern. The dietary exposure estimates of neotame (E 961) for the different population groups of all exposure scenarios did not exceed the ADI. The Panel concluded that there is no safety concern for neotame (E 961) at the currently permitted and reported uses and use levels. The Panel recommended the European Commission to consider revising the EU specifications of neotame (E 961)
Harmonização dos valores de anticorpos IgG contra a proteína Spike do vírus SARS-CoV-2
No full textDissertação de mestrado em Estatística Médica, apresentada à Universidade de Aveiro, 2025Orientadora Vânia Gaio (Departamento de Epidemiologia, INSA)O surgimento da COVID-19 conduziu ao rápido desenvolvimento de vacinas e testes de diagnóstico. Para avaliar a resposta de anticorpos IgG contra a proteína Spike do vírus SARS-CoV-2 (IgG anti-S SARS-CoV-2) em profissionais de saúde do meio hospitalar, foi realizado um estudo de coorte entre 2020 e 2022 em três centros hospitalares portugueses: Centro Hospitalar de Lisboa Ocidental (CHLO), Centro Hospitalar Tondela-Viseu (CHTV) e Centro Hospitalar e Universitário de Coimbra (CHUC). Os níveis de anticorpos foram medidos em seis momentos: antes da vacinação, após vacinação completa, aos 3, 6 e 12 meses após a segunda dose, e após a dose de reforço. Cada hospital utilizou um método analítico distinto: CMIA da Abbott, ECLIA Elecsys® da Roche e ADVIA Centaur® da Siemens, o que gerou desafios na comparabilidade dos dados. O presente estudo teve como objetivo harmonizar os anticorpos IgG anti-S SARS-CoV-2 entre os hospitais para permitir uma análise conjunta e uma melhor compreensão da dinâmica da imunidade nos profissionais de saúde em Portugal. Para assegurar uma conversão adequada dos títulos de anticorpos obtidos por métodos laboratoriais diferentes, foram aplicadas e comparadas várias estratégias de harmonização, nomeadamente a conversão internacional proposta pela Organização Mundial da Saúde (WHO) e a interpolação de quantis, seguida da aplicação de regressão de Deming. A interpolação de quantis seguida de regressão revelou-se mais eficaz do que a conversão recomendada pela OMS, ao preservar as características individuais de distribuição dos dados de cada hospital e ao permitir que os valores harmonizados refletissem a escala e a magnitude do método usado como referência (CMIA da Abbott).
Após a harmonização, observou-se o padrão esperado de tendência temporal do título de anticorpos, com um aumento acentuado após a vacinação, seguido de um declínio ao longo dos meses e, por fim, um novo aumento mais pronunciado após a dose de reforço. Embora não tenha sido realizada uma validação laboratorial através da análise cruzada das amostras, uma limitação importante para a confirmação definitiva da abordagem, a metodologia demonstrou ser prática, reprodutível e com elevado potencial de aplicação em estudos multicêntricos e multinacionais que requerem a integração de dados serológicos obtidos por diferentes plataformas, nomeadamente no âmbito de colaborações europeias e internacionais. Após a harmonização, a análise estatística recorreu a modelos mistos para avaliar a evolução do título de anticorpos ao longo do tempo e a regressões lineares para analisar separadamente cada fase. Os modelos mistos evidenciaram aumentos significativos após a vacinação e o reforço, destacando diferenças entre os centros hospitalares. Nas análises por fase, além das variações entre os centros hospitalares no período pós-reforço, observou-se que indivíduos acima dos 50 anos apresentaram uma resposta imunitária superior. Estes resultados sugerem que tanto as características individuais como as diferenças institucionais influenciaram a resposta imunitária dos profissionais de saúde.The emergence of COVID-19 led to the rapid development of vaccines and diagnostic tests. To assess the IgG antibody response against the Spike protein of the SARS-CoV-2 virus (IgG anti-S SARS-CoV-2) in healthcare workers within hospital settings, a cohort study was conducted between 2020 and 2022 across three Portuguese hospital centers: Centro Hospitalar de Lisboa Ocidental (CHLO), Centro Hospitalar Tondela-Viseu (CHTV) and Centro Hospitalar e Universitário de Coimbra (CHUC). Antibody levels were measured at six-time points: before vaccination, after complete vaccination, at 3, 6, and 12 months following the second dose, and after the booster dose.
Each hospital used a distinct analytical method: Abbott’s CMIA, Roche’s Elecsys® ECLIA, and Siemens’ ADVIA Centaur®, which created challenges in data comparability. The present study aimed to harmonize IgG anti-S SARS-CoV-2 antibody measurements across the hospitals to enable joint analysis and a better understanding of the dynamics of immunity among healthcare workers in Portugal. To ensure the appropriate conversion of antibody titers obtained through different laboratory methods, several har monization strategies were applied and compared, namely the international conversion proposed by the World Health Organization (WHO) and quantile interpolation followed by Deming regression. Quantile interpolation com bined with regression proved more effective than the WHO-recommended conversion, as it preserved the individual distribution characteristics of each hospital’s data and allowed the harmonized values to reflect the scale and magnitude of the reference method (Abbott’s CMIA). After harmonization, the expected temporal pattern of antibody titers was observed, with a sharp increase after vaccination, followed by a gradual decline over the months and a more pronounced rise after the booster dose. Although no labora tory validation through cross-analysis of samples was performed, which is an important limitation for the definitive confirmation of the approach, the methodology proved practical, reproducible, and highly promising for appli cation in multicenter and multinational studies that require the integration of serological data obtained through different platforms, particularly within the scope of European and international collaborations. Following harmoni zation, statistical analysis relied on mixed models to assess the evolution of antibody titers over time and on linear regressions to analyze each phase se parately. The mixed models revealed significant increases after vaccination and booster administration, highlighting differences between the hospital centers. In the phase-specific analyses, in addition to variations between hospital centers in the post-booster period, it was observed that individuals over 50 years of age exhibited a superior immune response. These findings suggest that both individual characteristics and institutional differences in fluence the immune response of healthcare workers.The data of the study were originally collected as part of the project ‘Developing an infrastructure and performing vaccine effectiveness studies for COVID-19 vaccine in the EU/EEA’ (Contract ECD.11486 Lot3 (HCW) and amendment Nº ECD.11486), and the Enhanced laboratory support to perform assessment of vaccine effectiveness against SARS-CoV-2 infection (ECD.12175) and the ‘Vaccine Effectiveness, Burden and Impact Studies (VEBIS) of COVID-19 and Influenza’, funded by the European Centre for Disease Prevention and Control through a service contract with Epiconcept (ECD.12609)
Whole-genome sequencing-based surveillance system for Mycobacterium tuberculosis in Portugal
No full textTo improve TB surveillance and diagnosis, the Portuguese National Reference Laboratory (NRL) began implementing whole-genome sequencing (WGS) for all RR/MDR-TB cases in 2019. Since 2020, this approach has been expanded to indiscriminately include all received isolates. We describe the current WGS-based surveillance system in Portugal, framed in prospective and retrospective data (n = 1171), upgraded for antimicrobial resistance (AMR) prediction and epidemiological analysis. This system relies on three main steps: QC/QA and contamination assessment, with a novel data filtering step; genotyping and AMR prediction; and dynamic SNP-based approach, maximizing variable sites under analysis. While lineage 4 was the most prevalent (84.3 %) followed by lineage 2 (9.1 %), less common EU/EEA sub-lineages (e.g., lineages 3 and 6) showcased cross-border transmissions. Molecular clusters (n = 157) displayed distinct AMR profiles and diverse possible epidemiological contexts. Among the pipeline upgrades, we highlight: i) the novel filtering step that allowed the improvement of 123 out of 128 contaminated samples; ii) tolerating missing data per site more than doubled core variable site resolution; iii) automatic maximization of shared variable sites for in-depth cluster analysis, key for consolidating genetic links in epidemiological investigation. This study highlights the importance of sustained prospective genomic surveillance towards strengthening TB management and diagnosis in Portugal.This study was co-funded by the project “Sustainable use and integration of enhanced infrastructure into routine genome-based surveillance and outbreak investigation activities in Portugal” - GENEO (Project no. 101113460) on behalf of the EU4H programme (EU4H-2022-DGA-MS-IBA-01-02)