DGH - Relatórios científicos e técnicos
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- 1º Workshop sobre Biomonitorização Humana em Portugal: síntese do encontroPublication . Silva, Maria João; Lavinha, JoãoRealizou-se no passado dia 11 de maio de 2018 o 1st HBM-PT, tendo reunido no Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA), em Lisboa, mais de oitenta participantes da Academia, Indústria, Saúde Ocupacional e Regulamentação, entre outros. O Encontro partiu da iniciativa do conjunto de parceiros que constituem o denominado National Hub (NH-PT) para o projeto “Human Biomonitoring Initiative - HBM4EU” que inclui a Fundação para a Ciência e a Tecnologia, I.P. (FCT), Instituto Nacional de Saúde Dr. Ricardo Jorge, I.P. (INSA), Direção Geral da Saúde (DGS) e Agência Portuguesa do Ambiente, I.P. (APA), em colaboração com a Faculdade de Medicina, Universidade de Lisboa (FMUL) e Escola Superior de Tecnologia da Saúde Lisboa (ESTeSL), Instituto Politécnico de Lisboa. Este primeiro Workshop visou juntar investigadores reguladores, público em geral e outros atores-chave para discutir a contribuição da biomonitorização humana para as políticas de saúde e ambiente e para a avaliação de risco para a saúde humana. Para além disso, pretendeu informar as partes interessadas acerca do projeto HBM4EU, incluindo aspetos relevantes, tais como a sua estrutura e as atividades já desenvolvidas durante o primeiro ano do projeto ou a desenvolver futuramente. Nesta síntese os autores pretenderam oferecer uma visão geral do evento, através de um breve resumo das apresentações dos oradores convidados e dos temas abordados pelo painel de discussão tecendo ainda algumas considerações finais sobre o evento.
- 3-Oxo-β-Sultams and 4-Oxo-β-Lactams as Chemical Tools for Activity-based Protein Profiling of Serine HydrolasesPublication . Carvalho, Luis Miguel Afonso; Moreira, Rui; Lucas, Susana; Penque, DeborahActivity-based protein profiling (ABPP) is a technique that analyzes the dynamics in enzymatic activity in complex proteomes by using small molecular probes, deemed activity-based probes (ABPs), containing a reactive group to covalently bind enzyme catalytic residues, a tag for detection of labeled targets and a linker as a spacer and also specificity-enhancing. Different warheads have been developed to engage a wide range of enzymatic families but there is a constant need to create new chemical tools to expand the pool of engageable targets. In this work we evaluated two 4-membered ring warheads as new warheads for ABPP of serine hydrolases. The 3-Oxo-β-Sultam warhead was revealed to be a highly reactive chemotype which labels a wide range of proteins with limited target occupancy. A crystallographic analysis of the reaction of 3-Oxo-β-Sultams with elastase enzymes revealed a previously unknown mechanism of inhibition of these enzymes by sulfonylation, suggesting 3-Oxo-β-Sultam compounds could be used to expand the pool of available sulfonylating tools in chemical biology. 4-Oxo-β-Lactams were shown to potently hit a selected group of serine hydrolase with high target occupancy, including human neutrophil elastase (HNE) and members of the ABHD and DPP families of enzymes. A competitive-ABPP approach revealed high potency of a library of 4-Oxo-β-Lactams to target these enzymes. 4-Oxo-β-Lactams were identified used as a new chemotype for DPP8 and DPP9 inhibition. Crystallography experiments revealed a new binding mode of these enzymes with 4-Oxo-β-Lactams, highlighting that these compounds could be used to pursue selective DPP8 or DPP9 inhibitors, a highly pursued field in current medicinal chemistry.
- AD6.2 WP6 - Sustainability and capacity building. Results of the interaction and surveys with the Environment Protection Agency network and the National Reference Centre on Environment & HealthPublication . Lobo Vicente, Joana; Katsonouri, Andromachi; Hans, Reynders; Campenhout, Karen Van; Tarroja, Elena; Louro, Henriqueta; Isidro, Glória; Silva, Maria João; Bourqui, Martine; von Goetz, NatalieThe aim of this additional deliverable is to analyse the result of the interaction and surveys with the Environment Protection Agency network and the National Reference Centre on Environment & Health. The consultation to both the EPA network and the NRCs was done through a similar targeted survey, with the aim of understanding their perception of the current HBM4EU and their ideas for a future Human Biomonitoring initiative. The online survey also explored if and how their institution would use HBM4EU results in their work and it also explored the focus that their institution considered a future initiative might take. This could be in terms of exploring exposure to new emerging risks or understanding exposure to known chemical risks, such as heavy metals. In addition, it explored interest in participating in a new initiative and openness towards sharing of best practice. This included questions regarding the possible role that the EPAs and NRCs might play in such an initiative. The invitation was sent out to 29 EPA countries and 15 NRCs, from which 19 answers were obtained in total with 4 EPAs answered and 13 NRCs. One of the EPAs is also a HBM4EU partner, whereas from the 15 NRCs, 13 of those provided their countries/institution from which 7 are HBM4EU partners. The survey produced quite interesting results, where it was pointed out that HBM is the only instrument that can assess human exposure in an integrated and reliable way. From the institutions that are not part of HBM4EU, most of them were aware of the project. The ones that were already part of it would like to carry on as such if a future initiative is to take place. Despite the fact that a slightly higher percentage of institutions do not use HBM at the moment in their work (53 % vs 47 %), all of the institutions recognised the value of HBM and plan on using it in the future. All the NRCs and 75 % of the EPAs are interested in being part of a future initiative with a possible role in: positioning Human Biomonitoring in the strategic agenda of implementation of environmental policy and state of the environment in Europe, supporting the activities of the HBM initiative through an existing interest group under the NRC Network, creating joint working initiatives with other relevant networks. It was clear from the answers given that HBM has triggered an interest and there is a willingness to be part of the future initiative.
- Analysis of the translatome by ribosome profiling in colorectal cancerPublication . Silva, Joana; Romão, Luísa; Luchessi, AugustoColorectal cancer (CRC) is a serious health problem due to its high incidence and mortality rates despite the major advances in cancer therapeutic approaches [1]. CRC carcinogenesis progression is based in a continuous accumulation of genetic alterations that leads to variations in the overall gene expression profiles [2]. This creates the need for deep analysis of cancer gene expression patterns and, thus, a more reliable understanding of the human proteome to disclose the molecular and cellular pathways as well as the regulatory mechanisms involved in cancer progression [2-4]. Genome wide analyses of gene expression have so far focused on the abundance of mRNA species as measured either by microarray or, more recently, by RNA deep sequencing [5,6]. However, neither approach provides information on protein synthesis, an important end point of gene expression [5,7]. Ribosome profiling is an emerging technique that uses deep sequencing to monitor in vivo translation and provide global and quantitative measurements of translation [7,8]. It can also reveal unexpected complexity in translation, including the presence of ribosomes outside of classical protein-coding regions of the transcriptome [5]. In this approach, translation is profiled by nuclease footprinting of ribosomes on RNA templates and high-throughput sequencing in order to determine the precise positions of ribosomes on a transcript and its overall density [8]. Ribosome profiling studies have been performed in cancer cell lines, where they showed an increase in overall protein identification and new proteins not yet annotated that possibly were originated from N-terminal extensions or upstream open reading frames (uORFs) [9-12]. The main goal of this project is to determine the changes between the translatome of CRC and normal colorectal cells and the role of such alterations in the tumorigenesis process of CRC cells. For this purpose, we will perform ribosome profiling in normal (NCM460) and CRC (HCT116) cell lines. Bioinformatics and gene ontology analysis of the translated mRNAs will elucidate the main cellular pathways through which the corresponding proteins are involved in CRC progression. Then, we will dissect which of these proteins can interfere and induce cell survival of CRC cells. Furthermore, we aim to analyze the potential contribution of translatable short alternative ORFs (AltORFs) and/or the corresponding peptides towards CRC progression. This information will be crucial to the development of new therapeutic strategies for CRC.
- Analysis of the translatome by ribosome profiling in colorectal cancerPublication . Silva, Joana; Santos, Hugo; Romão, LuísaColorectal cancer (CRC) has a high incidence and mortality rates worldwide [1]. CRC carcinogenesis is a continuous accumulation of genetic alterations with concomitant variations in gene expression profiles [2]. To study the variations of gene expression profiles involved in cancer progression, genome-wide analyses have so far focused on the abundance of mRNA as measured either by microarray or RNA sequencing [3,4]. However, neither approach provides information on protein synthesis, which is the true end-point of gene expression [3-5]. Ribosome profiling (Ribo-Seq) emerges to monitor in vivo translation, providing global and quantitative measurements of translation by deep sequencing of ribosome-protected mRNA fragments (RPFs) [5,6]. This technique has revealed unexpected complexity in translation, including the presence of ribosomes outside of classical protein-coding regions of the transcriptome [3]. The main goal of this project is to determine the changes between the translatome of CRC and normal colorectal cells and their role in CRC tumorigenesis. For that, we aim to analyze ribosome profiling data already available for the CRC cell line HCT116, and eventually data from non-neoplasic colorectal cells (if available). Gene ontology and network interaction analysis of the differentially translated mRNAs will elucidate the main molecular pathways through which the corresponding proteins are involved in CRC progression. Furthermore, we aim to analyze the function of translatable small open reading frames (sORFs), such as the upstream ORFs (uORFs), and/or the corresponding encoded peptides in the regulation of CRC progression. We have performed a computational analysis of HCT116 Ribo-Seq data to detect potential translatable uORFs. For that we are currently determining the number of RPFs in the 5’UTR of transcripts. Meanwhile, and based on previously published data about the prediction/detection of translatable alternative ORFs (AltORFs) in CRC cells [7], ABCE1, ABCF1, ABCF2 and ABCF3 mRNAs were chosen for further studies. To analyze their mRNA expression levels, we performed semi-quantitative RT-PCR analysis using RNA from HCT116, Caco-2 and SW480 CRC cells, as well as from non-neoplasic colorectal NCM460 cells. Our results show that these mRNAs are down-regulated in HCT116 cells comparing to their expression in the other three cell lines. In addition, we have been involved in mapping, by circular rapid amplification of cDNA ends (cRACE), cloning and sequencing, the exact 5’ end of the ABCE1 5’UTR. After getting this information, we will clone this 5’UTRs in a reporter construct that will allow us to test the ABCE1 uORF potential function in CRC progression.
- Annual Scientific Report of the Center for Toxicogenomics and Human HealthPublication . Penque, Deborah; Vieira, Luis; Gonçalves, João; Louro, Henriqueta; Silva, Maria João
- Consultation on a sustainable HBM initiative in Europe - Deliverable Report D6.3 WP6 Sustainability and Capacity building.Publication . Ganzleben, Catherine; Vicente, Joana Lobo; Barouki, Robert; Tarroja, Elena; Silva, Maria João; Isidro, Glória; Louro, Henriqueta; Katsonouri, Andromachi; Reynders, Hans; Campenhout, Karen Van; Mampaey, Maja; Sepai, Ovnair; Virgolino, AnaThis deliverable was produced under Task 6.3 of Work Package 6 on “Longer-term needs and expectations of stakeholders (2021-2030)”. The aim of this task was to gather information on the needs and expectations of both the National Hubs (NH) and a broad range of stakeholders regarding a long-term Human Biomonitoring (HBM) programme for Europe, to be establish in follow up to the current project, HBM4EU. Firstly and with a focus on options for financing a future initiative, information was collected on a range of available funding mechanisms at national, regional and international level. In terms of national funding, the results presented rely on responses to a survey with the NH. Unfortunately, the response from NHs was very limited, and as such the report only captures funding mechanisms from a limited number of countries, including Portugal, Spain, France, Germany and Cyprus. In order to gain insight into the status of the National Hubs and their capacities for HBM, a survey was conducted. The results provide an overview of the current situation across the NHs with regards to a range of aspects, including the level of activity of the NH, status of political support, availability of funding, ongoing HBM studies and willingness to align studies with HBM4EU. The results suggest that HBM4EU has raised the political profile of HBM in partner countries. Regarding ongoing HBM studies, only six countries have national HBM programmes, with most countries having only hot spot studies. Despite this, there was support for the alignment of studies to achieve European geographical coverage. Regarding funding, access to European funding is seen as important to leverage funds at national level. A second survey was targeted at a wide range of stakeholders, aiming to better understand their expectations for a long-term sustainable HBM initiative. The survey was followed by a workshop, where a more limited number of stakeholders had the opportunity to discuss the survey results and address such aspects as the scope of a future initiative, how to achieve financial sustainability, how to involve and how a future initiative might contribute to chemical policies. Concerning the needs and expectations of stakeholders, there is a strong interest in and support for a future HBM initiative at European level. The involvement of a European Union (EU) institution as part of a future steering committee was seen as crucial. According to this stakeholders’ consultation, the initiative should focus on protecting human health and the environment in Europe from hazardous chemical exposures by producing harmonised, high quality, transparent and inclusive data for effective risk assessment and management.
- Deliverable 6: Characterisation of manufactured nanomaterials for their clastogenic/aneugenic effects or DNA damage potentials and correlation analysis. Final report.Publication . NANOGENOTOX Joint Action; WP6 partners; Fessard, V.Objectives: To generate data from selected in vivo genotoxicity tests, and to assess the correlation between in vivo and in vitro results taking into account the kinetic results. Schedule: The WP6 lasted for 16 months from October 2011 until the end of the Joint Action (February 2013). Few months at the beginning were devoted to trainings and trials before performing the experiments on Manufactured Nanomaterials (MNs). Even though, some last results are not yet available and will be collected after the end of the Joint Action, especially on the micronucleus on colon as well as on histology.
- Diagnóstico e Tratamento de Doenças Lisossomais: relatório 2011Publication . Grupo de Trabalho Coordenador do Diagnóstico e Tratamento de Doenças Lisossomais; Fortuna, Ana MariaAs Doenças Lisossomais de Sobrecarga (DLS) constituem um grupo de doenças hereditárias do metabolismo que se caracterizam por uma acumulação intralisossomal quer de substratos não degradados, quer de produtos do catabolismo. Estão descritas, atualmente, cerca de 50 DLS e em Portugal a prevalência deste grupo de patologias em recém-nascidos é de 25:100.000 nados vivos. As DLS apresentam uma grande variedade de sintomas clínicos que vão desde a presença de doença neurológica grave, a casos menos graves ou até mesmo assintomáticos. Os sintomas surgem geralmente nos primeiros meses/anos de vida e os sinais mais frequentes são alterações neurológicas, ósseas, cardiovasculares, cutâneas, oculares, hematológicas, organomelia, dismorfia facial e hidrópsia fetal. Nos últimos anos, várias terapêuticas de substituição enzimática e de redução do substrato têm sido desenvolvidas possibilitando o tratamento dos doentes com algumas destas patologias, nomeadamente, a Doença de Gaucher de tipo I, a Doença de Fabry, a Doença de Pompe, as Mucopolissacaridoses tipo I, II e VI e a Doença de Niemann Pick tipo C. Em 2011, o Grupo de Trabalho Coordenador do Diagnóstico e Tratamento de Doenças Lisossomais (GTCDTDL), prosseguiu a sua missão garantindo a gestão e a coordenação a nível nacional do Diagnóstico e Tratamento das Doenças Lisossomais de Sobrecarga, no Centro de Genética Médica Doutor Jacinto Magalhães (CGMJM), então um serviço desconcentrado do Instituto Nacional de Saúde Doutor Ricardo Jorge, no Porto. Do relatório anual do GTCDTDL, relativo ao ano 2011, agora disponibilizado pelo INSA, destaca-se a realização das seguintes atividades: - Análise e emissão de 27 pareceres relativos a pedidos de tratamento, ajuste de dose ou alteração do tratamento. Foram emitidos dez pareceres para a Doença de Gaucher, cinco para a Doença de Fabry, 2 para a Doença de Hunter e 10 para a Doença de Pompe. - Compilação da documentação relativa aos doentes com a finalidade de constituir de uma base de dados. - Avaliação atualizada e envio regular dos encargos mensais por doença e entidade prestadora dos cuidados de saúde, bem como a data de início e/ou suspensão das terapêuticas à Administração Central do Sistema de Saúde (ACSS). - Consolidação da constituição do grupo de trabalho e formulação de convite a consultores científicos, quando pertinente. - Estabelecimento de contactos com instituições ou organismos relevantes (INFARMED, ACSS, DGS). - Estabelecimento de contactos com as associações de doentes (APL, Raríssimas, Eurordis). - Estabelecimento de contactos com a indústria farmacêutica (Genzyme, Actelion, Biomarin, Shire). - Filiação no grupo europeu de estudo das doenças lisossomais (ESGLD, European Study Group on Lysosomal Diseases). - Organização logística interna e externa e de documentos de suporte. - Compilação de documentação científica e informação de entidades europeias. - Participação da Unidade de Bioquímica Genética (UBQ) do CGMJM em programas europeus de controlo de qualidade qualitativo e quantitativo – ERNDIM (European Research Network of Disorders of Inherited Metabolism), nomeadamente no “Special Assays in Urine”, “Special Assays in Serum” e o “Diagnostic Proficiency Testing. A UBQ como membro do grupo europeu de estudo das doenças lisossomais (European Studying Group on Lysossomal Disorders, ESGLD) estabeleceu também colaboração com diversos centros de referência internacionais.