National Institute of Health Dr. Ricardo Jorge
Repositório Científico do Instituto Nacional de SaúdeNot a member yet
9086 research outputs found
Sort by
17.ª Reunião Anual PortFIR - Segurança dos Alimentos: Governança, Ciência e Novos Modelos de Produção Face aos Desafios Globais: Resumo da reunião
No full textResumo da 17.ª Reunião Anual PortFIR subordinada ao tema "Segurança dos Alimentos: Governança, Ciência e Novos Modelos de Produção Face aos Desafios Globais". A publicação apresenta as comunicações e abstracts/posters submetidos, bem como os resultados da avaliação ao grau de satisfação dos participantes no evento e algumas fotos do evento
Hazard identification and characterization of leachable chemicals from plastic products – a new PARC project
No full textThis article is part of the Research TopicEuropean Partnership on the Assessment of Risks from Chemicals (PARC): Continued Focus on New Approach Methodologies (NAMs) and Data Gap Filling.A recent study has suggested that plastics may contain more than 16,000 chemicals, including additives, processing aids, starting substances, intermediates and Non-Intentionally Added Substances. Plastic chemicals are released throughout the plastic life cycle, from production, use, disposal and recycling. Most of these chemicals have not been studied for potential hazardous properties for humans and in the environment. To refine the risk assessment of these leachable chemicals, additional hazard data are needed. The PlasticLeach project within the EU co-funded Partnership for the Assessment of Risks from Chemicals (PARC) aims to address this data gap by screening several plastic products in daily use. Leachates will be prepared from a number of these plastic items, and these chemical mixtures will be further tested using several test guideline compliant assays and New Approach Methodologies covering both human health and environmental endpoints. The most toxic leachates will be characterized using a non-targeted analysis pipeline to identify chemicals in the leachate. When single chemicals of concern are identified, these will be further tested to determine hazardous properties and identify the respective potency factors to better understand their specific hazard profiles. A tiered approach for hazard testing will be followed. The experimental work will be complemented by toxicological profiling, using publicly available toxicity databases and tools, including Artificial Intelligence tools that cover both human and environmental endpoints. A comprehensive array of endpoints, including cytotoxicity, endocrine disruption, genotoxicity, immunotoxicity, reproductive toxicity and effects related to ecotoxicity will be evaluated. In this paper, we outline the plastic products to be tested and the battery of assays that will be used to identify hazards relevant to both human health and the environment. Data generated from approaches will be reported using standardized formats, stored within a centralized repository, and harmonized to adhere to the FAIR data principles (Findable, Accessible, Interoperable, and Reusable). This integrated strategy will not only advance our understanding of the risks associated with plastic-derived chemicals but will also provide critical support for regulatory decision-making and facilitate the development of safer, and more ecofriendly plastic materials in the future.This work was supported by the author’s institutions and by the Partnership for the Assessment of Risks from Chemicals (PARC) that has received funding from the European Union’s Horizon Europe Research and Innovation Programme under Grant Agreement No. 101057014. The UAVR partner acknowledges national funds through FCT–Fundação para a Ciência e a Tecnologia I.P., under the project CESAM-Centro de Estudos do Ambiente e do Mar, references UID/50017/2025 (doi.org/10.54499/UID/50017/2025) and LA/P/0094/2020 (doi.org/10.54499/LA/P/0094/2020). NIVA acknowledges cofunding from the Research Council of Norway project EXPECT (RCN-315969) and NIVA’s Computational Toxicology Program, NCTP (RCN-342628). NIB is cofinancing the studies by ARIS P1-0245. KRICT is funded by Korea Research Institute of Chemical Technology (KRICT) through Development of Chemical Safety Platform Technologies (Project No. KK2552-10) and the Korea Environment Industry & Technology Institute (KEITI) through the Technology Development Project for Safety Management of Household Chemical Products (RS-2022-KE002056)
Genomic diseases at the Department of Human Genetics of INSA: from physical map resolution to personalized genomic medicine
No full textNutritional and Bioactive Profiling of Cucumis melo L. By-Products: Towards a Circular Food Economy
No full text(This article belongs to the Special Issue Food Sustainability: Promising By-Products for Valorization—2nd Edition)Food waste, due to the high quantities produced, becomes a significant environmental, economic, and social challenge worldwide. Simultaneously, the rising prevalence of chronic diseases has intensified the demand for healthier food options. A promising approach to address these issues involves the valorisation of food by-products for the development of innovative and healthier food products. Cucumis melo L., commonly consumed as a fruit, generates peels and seeds that are typically discarded. In the present study, the nutritional composition and antioxidant potential of pulp, peel, and seeds of C. melo L. (yellow and green melon) were comprehensively evaluated. The seeds were identified as a rich source of dietary fibre (39.0 and 39.7 g/100 g dw; p > 0.05) and protein (21.0 and 21.3 g/100 g dw; p > 0.05), exhibiting an appealing fatty acid profile. The peel contains high levels of dietary fibre (39.7 and 47.1 g/100 g dw; p > 0.05) and total phenolic compounds (1976 and 2212 mg GAE/100 g dw; p > 0.05), suggesting significant bioactive potential. The peels showed a high antioxidant capacity for both methods used, DPPH• (120 and 144 mg TE/100 g dw; p > 0.05) and FRAP (6146 and 7408 mg TE/100 g dw; p > 0.05) assays. Potassium emerged as the predominant mineral in the seeds (799 and 805 mg/100 dw; p > 0.05), while glutamic acid was the most abundant amino acid (4161 and 4327 mg/100 g dw; p > 0.05). These findings emphasise the antioxidant and nutritional properties of C. melo L. by-products, highlighting their potential for inclusion in novel food formulations. This study not only advances the understanding of C. melo L. properties but also supports the reduction of food waste and promotes sustainability within the food supply chain.Funding: This work was financially supported by the Foundation for Science and Technology (FCT/MES) under the project Food4DIAB (EXPL/BAA-AGR/1382/2021) and MELON4FOOD (2018DAN1492).
Acknowledgments: Rita C. Alves gives thanks to FCT/MES under the CEECIND/01120/2017 contract
Analysis of the translatome by ribosome profiling in colorectal cancer
No full textColorectal 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.N/
The effect of G418 and PTC124 as suppression therapy for beta-thalassemia
No full textDissertação de mestrado em Biologia Molecular e Genética, apresentada à Faculdade de Ciências da Universidade de Lisboa, 2016Orientadora: Luísa Romão, Investigadora do Instituto Nacional de Saúde Doutor Ricardo JorgeNonsense mutations are alterations that introduce, prematurely, in the coding region of the messenger RNA (mRNA), a translational termination codon. Nonsense mutations or premature termination codons (PTCs) can arise from various types of mutations in germ or somatic cells. PTCs promote premature translational termination and, in most cases, the induction of nonsense-mediated mRNA decay (NMD). NMD is a quality control pathway that recognizes and rapidly degrades mRNAs that contain PTCs, preventing the synthesis of C-terminally truncated proteins, possibly toxic for the cell. In recent years, a therapeutic approach called suppression therapy is being developed using low molecular weight compounds to induce the translation machinery to recode a PTC into a sense codon. Beta thalassemia is one of the most common genetic diseases worldwide, being characterized by reduced or absent beta globin chain synthesis, resulting in diminished hemoglobin in red blood cells, increased red blood cells production and anemia. Some studies have shown that aminoglycosides and non-aminoglycosides can suppress PTCs in cystic fibrosis and Duchenne’s muscular dystrophy, but it remains unclear whether beta thalassemia would also be responsive to a similar drug treatment. Preliminary results obtained in our lab have shown that the aminoglycoside G418 (geneticin) can suppress a nonsense mutation at codon 39 of the human beta globin mRNA, although at low levels in cultured erythroid cells. To investigate if suppression therapy can restore enough beta globin protein to possibly correct the disease manifestations of beta thalassemia, HeLa cells were transfected with plasmids containing the human beta globin wild type gene (BN) or the counterpart carrying a nonsense mutation at codon 39 (B39). HeLa cells were then treated with G418 or PTC124 (ataluren) to ascertain if these compounds are able to induce efficient levels of suppression, in a dose-dependent manner. Our results showed that G418 produces slight differences between the treated and untreated samples, and so it is not clear if the compound is capable of restoring beta globin. Results from PTC124 treatment were somehow inconsistent because the increase of the mRNA levels was not identical throughout the experiments, although it is quite normal considering a testing phase. On the other hand, PTC124 increased nearly 4-fold the mRNA levels of the B39 transcript when comparing with the B39 without treatment, which may indicate a possible effect of the drug, although the observed differences are not statistically significant. To obtain more clarifying results we suggest testing more drug concentration ranges. Due to the fact that we were unable to test the effect of the drug on the protein level, in the future it would be of great interest to evaluate the effect of these two compounds on protein expression levels. Additionally, since suppression therapy is an extremely difficult approach to achieve efficient results, future methodologies should combine this therapy with others, for example, NMD inhibition.A beta talassémia é uma doença genética que se caracteriza pela redução ou ausência de
síntese de beta globina, um dos constituintes da hemoglobina, resultando na escassez de glóbulos
vermelhos e, consequentemente, em anemia. Uma das causas desta doença tem origem na alteração
de um nucleótido, levando à introdução de um codão de terminação prematura (na sigla inglesa PTC
ou codão nonsense) nos transcritos de beta globina. A presença de um PTC não permite que a
tradução progrida normalmente, ocorrendo, na maior parte dos casos, a formação de péptidos
truncados que são, possivelmente, não funcionais ou, até, tóxicos para a célula.
No sentido de devolver a homeostase, a célula desenvolveu mecanismos de controlo de
qualidade para assegurar a fidelidade da expressão genética. Entre estes mecanismos está o
decaimento do RNA mensageiro (mRNA) mediado por mutações nonsense (na sigla inglesa NMD –
nonsense mediated mRNA decay) que permite identificar e degradar transcritos que contenham um
PTC, reduzindo a sua abundância. Deste modo, a eliminação destes transcritos anómalos evita a
formação e acumulação de proteínas truncadas que, caso contrário, danificariam a célula. Assim, o
NMD tem, na maioria dos casos, um papel protetor contra erros genéticos. A degradação do
transcrito que contém o PTC inicia-se logo após a exportação da ribonucleoproteína mensageira (na
sigla inglesa mRNP) para o citoplasma. Durante a ronda pioneira de tradução, se o transcrito não
possuir um PTC, o ribossoma remove da mRNP todos os complexos de junção exão-exão (na sigla
inglesa EJC) até chegar ao codão de terminação. Os EJC são complexos multiproteicos, depositados
20 a 24 nucleótidos (nt) de distância a montante das junções exão-exão (local de excisão dos intrões)
durante o splicing. Se, por ventura, o transcrito possuir um PTC e este se localizar a, pelo menos, 50 a
55 nt de distância a montante da última junção exão-exão, a tradução é terminada prematuramente
e pelo menos um EJC fica associado ao mRNA. Nestas condições, o EJC facilita o recrutamento e a
interacção dos factores do NMD com o complexo de terminação da tradução, activando o NMD. Este
é o caso mais comum, no qual o transcrito é degradado, reduzindo os seus níveis drasticamente
(entre 0 a 20% dos níveis normais). Como exemplo para esta situação temos o caso do transcrito de
beta globina com uma mutação nonsense no codão 39. Contudo, esta regra posicional dos EJC não é
tão linear como aparenta, dado que existem transcritos que, mesmo estando nas condições previstas
pelo modelo, conseguem resistir à acção do NMD, como é o caso de mRNAs que contenham PTCs
próximos do codão de iniciação. Como exemplo de um mRNA que escapa à degradação pelo NMD,
temos o transcrito de beta globina que possui uma mutação nonsense no codão 15, sendo que os
seus níveis de expressão são semelhantes aos do gene da beta globina normal. De facto, verificou-se
que existem outras características do mRNA, para além dos EJC, que influenciam a activação do NMD, tais como distância física entre um PTC e a proteína citoplasmática de ligação à cauda poli-A do
mRNA (na sigla inglesa PABPC1). Para além disso, o tamanho da região não traduzida a 3’ (na sigla
inglesa 3’UTR) é outro dos fatores que influencia a ativação do NMD. Apesar de, na maioria dos
casos, o NMD assumir um papel protector, este pode também pode agravar o fenótipo da doença
pelo facto de eliminar mRNAs que originam proteínas parcialmente funcionais ou com actividade
residual.
O processo de terminação da tradução ocorre quando o codão stop é reconhecido por
proteínas que formam o complexo de terminação, promovendo a libertação da cadeia polipeptídica
formada até então. Em certos casos, ao invés de ser reconhecido pelo complexo de terminação, o
codão stop é identificado por um aminoacil tRNA, complementar a duas das três bases (na
terminologia inglesa near cognate aminoacyl tRNA). Ocorre, assim, a recodificação do codão de
terminação prematura, permitindo que a elongação prossiga em fase, até que o ribossoma atinja um
codão stop normal, possibilitando a formação da proteína completa e potencialmente funcional. Este
processo de readthrough de um PTC ocorre espontaneamente na célula, embora não seja frequente
(cerca de 1%). No entanto, certas moléculas de baixo peso molecular demonstram elevar a
frequência destes acontecimentos, uma vez que favorecem a competição entre os near cognate
aminoacyl tRNAs e o complexo de terminação. Desta forma, nas últimas décadas tem sido
desenvolvida uma terapia, designada como terapia de supressão, que utiliza estes compostos com
vista a aliviar os sintomas de diversas doenças causadas por mutações nonsense, devido ao aumento
da quantidade de proteína funcional disponível. Os compostos mais estudados e utilizados na terapia
de supressão são os aminoglicosídeos, um grupo de antibióticos cujo potencial de supressão provém
da sua capacidade de reduzir a fidelidade na incorporação dos aminoácidos (na terminologia inglesa
misincorporation), promovendo a introdução de near cognate aminoacyl tRNAs. À parte dos
aminoglicosídeos, existe um composto, o PTC124, que tem vindo a ser alvo de grande interesse
devido ao facto de não apresentar actividade antibacteriana nem toxicidade, assumindo um papel
promissor no desenvolvimento da terapia de supressão.
Este trabalho teve como objectivo investigar se os dois compostos a testar, o aminoglicosídeo
G418 (geneticina) e o não-aminoglicosídeo PTC124 (atalureno), possuem a capacidade de aumentar
os níveis de beta globina normal para, possivelmente, atenuar as manifestações da beta talassémia
devido a mutações nonsense. Para tal, foi delineada uma estratégia experimental, que consiste em
transfectar células HeLa com plasmídeos que contêm o gene da beta globina humana normal (BN) ou
o equivalente com uma mutação nonsense no codão 39 (B39), expondo-as aos dois compostos. No
final, foram analisadas as diferenças nos níveis de mRNA, consoante as concentrações utlizadas. Os
nossos resultados mostraram que o G418 produz diferenças subtis entre as amostras tratadas e não tratadas, não sendo claro se o composto é capaz de aumentar os níveis de mRNA da beta globina, no
o modelo experimental usado. Os resultados relativamente ao composto PTC124 foram, de certa
forma, inconsistentes, uma vez que o aumento dos níveis de mRNA não foi idêntico ao longo das
experiências, embora seja bastante normal aquando de uma fase de testes. Ainda assim, o PTC124
induziu o aumentou de 4 vezes os níveis de mRNA do transcrito B39 na concentração de 100µM,
quando comparamos com o B39 sem tratamento, o que pode indicar um possível efeito da droga,
embora as diferenças observadas não sejam estatisticamente significativas.
De modo a obter resultados mais esclarecedores, é aconselhável testar diferentes
concentrações para ambas as drogas. Devido ao facto de não ter sido possível testar o efeito das
drogas relativamente à sua expressão proteica, no futuro seria de grande interesse avaliar o efeito
destes dois compostos a este nível. Para além disso, a utilização de outro modelo celular, como as
células MEL (murine erythroleukemia), seria de grande interesse, devido ao facto de se tratar de
células eritróides. Uma vez que a terapia de supressão é uma abordagem na qual é extremamente
difícil alcançar resultados consistentes, reprodutíveis e satisfatórios, futuramente é recomendável
acoplar esta terapia com outras metodologias, por exemplo, a inibição NMD.
Apesar da investigação no ramo da terapia de supressão ser bastante complexa e exigente, é
de recordar que este tratamento não se foca nos sintomas, mas sim na origem molecular de muitas
doenças genéticas.N/
Probiotic-loaded microcapsule system for human in situ folate production: Encapsulation and system validation
No full textThis study focused on the use of a new system, an alginate|Ɛ-poly-L-lysine| alginate|chitosan microcapsule
(APACM), able to immobilize a folate-producing probiotic, Lactococcus lactis ssp. cremoris (LLC), which provides
a new approach to the utilization of capsules and probiotics for in situ production of vitamins. LLC is able to produce
95.25±26 μg·L−1 of folate, during 10 h, andwas encapsulated in the APACM. APACMproved its capacity to
protect LLC against the harsh conditions of a simulated digestion maintaining a viable concentration of
6 log CFU·mL−1of LLC. A nutrients exchange capacity test, was performed using Lactobacillus plantarum UM7,
a high lactic acid producer was used here to avoid false negative results. The production and release of 2 g·L−1
of lactic acidwas achieved through encapsulation of L. plantarum, after 20 h. The adhesion of APACM to epithelial
cells was also quantified, yielding 38% and 33% of capsules adhered to HT-29 cells and Caco-2 cells, respectively.The authors Philippe E. Ramos and Ana Pinheiro, are recipient of
fellowships from the Fundacão para a Ciência e Tecnologia, POPHQREN
and FSE (FCT, Portugal) through grants, SFRH/BD/80800/
2012 and SFRH/BPD/101181/2014, respectively. The authors thank
the FCT Strategic Project PEst-OE/EQB/LA0023/2013 and the project
“BioInd—Biotechnology and Bioengineering for Improved Industrial
and Agro-Food Processes”, ref. NORTE-07-0124-FEDER-000028 cofunded
by the Programa Operacional Regional do Norte (ON.2–O
Novo Norte), QREN, FEDER.info:eu-repo/semantics/publishedVersio
Limited Knowledge About Hydatidosis Among Farmers in Northwest Portugal: A Pressing Need for a One Health Approach
No full textHydatidosis is a re-emerging disease. Farmers are a vulnerable population; however, little is known about their awareness of this disease. The purpose of this study was two-fold: (1) to assess sheep and goat farmers' awareness of, perceptions of, and attitudes towards parasitic zoonoses and hydatidosis and (2) to identify the preferred means for promotion of information about hydatidosis. A cross-sectional study was conducted. An in-person questionnaire was constructed and administered to 279 individuals. A coprological survey in shepherd dogs was performed using 88 faecal samples. SPSS version 18.0 was used for statistical analysis. Farmers reported several risk practices (69% practice home slaughtering, 46% do not deworm the dogs, 58% of these dogs have contact with other animals) and very little knowledge about hydatidosis (97% have never heard about it). Nevertheless, 75% of the farmers demonstrated interest in receiving information, mainly from a veterinarian. A wide diversity of potentially zoonotic parasites (Trichuris spp., Ancylostomatidae, Toxocara spp., Taeniidae) was found in 61% of the dogs. This survey revealed farmers' lack of knowledge in relation to hydatidosis and a high prevalence of potentially zoonotic parasites in dogs, thus pointing to the need for health education and a closer collaboration between veterinarian and public health professionals.This work was supported by the Portuguese Science and Technology
Foundation (FCT) under the Project UID/CVT/00772/2013.info:eu-repo/semantics/publishedVersio
Evidence for a role of nonsense-mediated mRNA decay pathway genes in Autism Spectrum Disorder
No full textIntroduction: Autism Spectrum Disorder (ASD) is a highly heterogeneous neurodevelopmental disorder with an unclear etiology. Genetic factors are estimated to account for 50 to 80% of the familial ASD risk, but most of the genetic determinants are still not known and a role for other regulatory mechanisms is likely. The nonsense-mediated decay (NMD) pathway controls mRNA quality and plays an important role in the regulation of the transcriptome. Mutations in genes involved in the NMD pathway have been linked to neurodevelopmental disorders, with intriguing evidence for an involvement of mutations in the UPF3B gene, a core component of the NMD pathway, in ASD.Support for this work was provided by Fundação para a Ciência e a Tecnologia (grant PD/BD/113773/2015 to Ana Rita Marques).N/
Integrative network approach to predict new NMD-associated proteins
No full textIn our study, we apply such approach to predict new players involved in nonsense-mediated mRNA decay (NMD), a regulatory pathway of mRNA translation that recognizes and selectively degrades specific mRNAs. NMD is a complex process that relies on the involvement of numerous players, however the interactions and connectivity among these components is only partly elucidated.N/