Browsing by Issue Date, starting with "2017-05-08"
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- Gene-environment interactions in Autism Spectrum Disorder (ASD): excess copy number variants (CNVs) in genes regulating the effect of environmental exposure to toxicantsPublication . Xavier Santos, JoãoASD is an heterogeneous neurodevelopmental disorder, with an estimated heritability of 50% and a complex genetic etiology. Recent studies also implicate exposure to environmental factors in this disorder. We thus hypothesize that ASD is the result of the interaction of a genetic susceptibility with exposure to environmental toxicants early in development. Here we tested whether affected individuals have an excess of CNVs targeting genes involved in control of toxicant effects, including detoxification or regulation of barrier permeability (blood-brain barrier, placenta and respiratory cilia).
- Brand New World of TranslationPublication . Neves, Ana Rita; Lacerda, Rafaela; Romão, Luísa; Candeias, MarcoIn recent years, non-canonical translation initiation mechanisms have been recognized as key factors in the development of different diseases such as cancer, as they present a survival answer during stress conditions by ensuring the expression of vital proteins. Internal Ribosome Entry Sites (IRESes) were first discovered on viruses, and later in eukaryotes, as mRNA secondary structures capable of recruiting the ribosome to the vicinities of an initiation codon. One of the most studied cancer-related genes, the p53 tumor suppressor gene, was found to possess on its mRNA an IRES capable of regulating the expression of the full length isoform, p53FL, and one of its isoforms, Δ40p53 differently by the interaction with MDM2 protein, an IRES trans-acting factor (ITAF) of p53. Our aim is to study a shorter p53 protein isoform that lacks tumor suppressor behaviour acting instead as a cancer promoter (Candeias et al., 2016). One of our goals is to characterize the IRES associated with its expression. For that we will try to perform a sequencing reaction variant where the fragments to be sequenced result from uncomplete reverse transcription due to nucleotide-specific modification. Additionally, we will try to unveil new ITAFs by pulling-down the IRES and, consequently, associated factors, using two different methods: IRES biotinylation and MS2 tagging. Furthermore, we intend to find new IRESes by pulling-down MDM2 and possible bounded mRNAs followed by RNA-sequencing in order to identify them.
- The DIS3 proteins family: role in the human transcriptome regulation and CRC tumorigenesisPublication . Costa, Paulo; Santos, Hugo; Gama-Carvalho, Margarida; Romão, LuísaThe 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. With the aim of analyzing how DIS3, DIS3L1 and DIS3L2 regulate the human transcriptome, each one of these nucleases was depleted by RNA interference in HeLa cells and levels of several reporter mRNAs was monitored by RT-qPCR. Our results show that these exoribonucleases are target specific and not directly involved in a particular mRNA surveillance mechanism. In parallel, our bioinformatics analysis of available transcriptomic data from cells depleted of DIS3L1, DIS3L2, XRN1, or UPF1 (which has a central role in nonsense-mediated mRNA decay) has shown some, but not full, redundancy among the transcripts regulated by these nucleases, which supports our experimental data. Presently, we are exploring the molecular mechanisms underlying our observations and looking for relationships between their specific targets and their potential role in tumorigenesis.
- Decephring the toxicity of polycyclic aromatic hydrocarbons in HepG2 cell linePublication . Morgado, Patrícia I.; Jordao, LuisaPolycyclic Aromatic Hydrocarbons (PAHs) are persistent pollutants present in the environment with known mutagenic and carcinogenic properties. In the present study the effect of exposure to single or multiple doses of benzo[a]anthracene (BaA), pyrene (Pyr) and three HPAHs (1-ClPyr; 1-BrPyr and 7-ClBaA) were evaluated in a liver-derived human cell line (HepG2). Cytotoxicity as accessed by the classic MTT and neutral red showed a mild toxic effect in response to single or multiple dose exposure for up to 72h; except for multiple dose exposure to BaA and 7-ClBaA (cumulative concentration of 4 µM) and single exposure to 10 µM BaA. Furthermore, a selective mitochondrial and lysosomal toxicity was observed for Pyr and BaA series, respectively. In order to understand the underlying molecular mechanisms responsible for this effect, ROS production, mitochondrial membrane depolarization, lysosomal pH, DNA fragmentation and apoptosis mediators were evaluated after exposure to single PAHs doses. All compounds were able to trigger oxidative stress after 24h as measured by catalase activity and a good correlation was found between mitochondrial membrane depolarization, lysosomal pH increase and MTT and neutral red assays, respectively. The evaluation of cell death mediators showed that caspase-3/7 but not anexin-V pathways were involved in toxicity triggered by the studied compounds. In conclusion, the studied PAHs, especially 1-BrPyr and BaA, exhibit cytotoxic effects when accumulated, and may have adverse effects to humans after long periods of exposure.
- Less is More: an overview on the use of RNAi as a tool to achieve Substrate Reduction in MucopolysaccharidosesPublication . Coutinho, Maria Francisca; Santos, Juliana Inês; Alves, SandraMucopolysaccharidoses (MPSs) are a subgroup of Lysosomal Storage Diseases (LSDs) caused by dysfunction in enzymes responsible for the intralysosomal degradation of glycosaminoglycans (GAGs). Given their complex nature and the limitations of available therapies, the shift towards the development of combination treatments to counteract more effectively the pathological burden of these disorders is in the agenda of current research. We consider that treatment strategies relying on RNA interference (RNAi), as well as in other RNA-based methodologies, may be feasible and particularly promising in the context of a synergistic combinatorial therapeutic approach. Therefore, we have designed an RNAi-dependent strategy based upon the selective downregulation of genes involved in the biosynthesis of GAGs, which is currently under evaluation. Our goal is to promote an effective reduction of the accumulating substrate, ultimately decreasing or delaying MPSs’ symptoms. Taking advantage of the RNAi technology potential, we have designed and assayed specific siRNAs targeting genes on those biosynthetic cascades to decrease the levels of production of each one of the four substrates: dermatan sulphate (DS), heparan sulphate (HS), keratan sulphate (KS), and chondroitin sulphate (CS). Their efficiency is currently being evaluated in vitro. Here we present an overview of the preliminary results of this project and unveil its next steps towards a full characterization/evaluation of its potential therapeutic effect.
- Qualidade do ar interiorPublication . Cano, ManuelaPalestra sobre a qualidade do ar Interior e a avaliação do risco para a saúde humana.
- House dust fungal communities’ characterization: a double take on the six by sixty by six project (6x60x6)Publication . Amaro, Raquel; Coelho, Sónia; Pastorinho, M Ramiro; Taborda-Barata, Luís; Vaz-Pato, Maria Assunção; Monteiro, Marisa; Nepomuceno, Miguel; Lanzinha, João C. G.; Teixeira, João Paulo; Pereira, Cristiana C.; Sousa, Ana C. A.Background and objective(s): Fungi are a group microbes that are found with particular incidence in the indoor environment. Their direct toxicity or capability of generating toxic compounds has been associated with a large number of adverse health effects, such as infectious diseases, allergies and other toxic effects. House dust is a time integrative matrix easy to obtain and have been recommended for epidemiological studies on human exposure to environmental contaminants. This study aims to quantify and identify the fungal community on house dust samples collected using two different methodologies: active and passive sampling.
- Expression of human UPF1 is regulated by a cap-independent translation initiation mechanismPublication . Lacerda, Rafaela; Menezes, Juliane; Romão, LuísaGene expression comprises several intertwined steps. Translation initiation, which, under normal circumstances, is mostly cap-dependent, can also occur via a cap-independent mechanism, which drives protein synthesis under stress conditions impairing canonical translation initiation. Human up-frameshift 1 (UPF1) is a key-protein involved in nonsense-mediated mRNA decay, telomere replication and homeostasis, and cell cycle progression. These crucial UPF1 functions suggest its tight gene expression regulation. To test whether UPF1 5’ untranslated region (5’UTR) mediates cap-independent translation, we cloned the UPF1 5’UTR in a bicistronic luciferase vector upstream the downstream cistron (Firefly luciferase [FLuc]), and transfected cervical and colorectal cancer cell lines with this construct. We observed a significant increase in FLuc expression levels compared to those from Renilla luciferase (upstream cistron) in cells transfected with the UPF1 5’UTR-containing constructs compared to those transfected with the empty transcript. To find which sequence segments are required for mediating cap-independent translation, we performed a deletional and mutational analysis of the sequence and verified that cap-independent translation was ceased when the first 100 nucleotides, or the last 125, were absent or altered. Also, such activity is maintained under canonical translation initiation-impairing conditions, such as hypoxia or endoplasmic reticulum stress. We also produced in vitro cap-lacking monocistronic UPF1 5’UTR-containing transcripts and observed a significant increase in relative FLuc expression levels in cells transfected with them. These results indicate that UPF1 5’UTR mediates cap-independent translation initiation. Understanding this mechanism and its biological relevance might provide tools for developing new therapies for UPF1 deregulation-associated diseases, such as cancer.
- Stimulation of RAC1/PAK1 signalling upregulates DNA damage repair genes via STAT5 stimulation of BCL6 repressed lociPublication . Barros, Patrícia; Amaral, Andreia; Abrantes, Leonor; Oliveira, Tiago; Louro, Henriqueta; Silva, Maria João; Jordan, Peter; Gama-Carvalho, Margarida; Matos, PauloColorectal cancer is one of the most prevalent types of cancer worldwide. The GTPase RAC1 and its effector PAK1 have been found overexpressed or hyperactivated in colorectal cancers, particularly those with more aggressive and invasive features, leading to unfavourable clinical prognosis, often resulting from chemoresistance.
- Health literacy: a tool for health promotionPublication . Costa, A.; Saboga-Nunes, L.; Costa, L.Objective: Assess the HL level of a Portuguese sample using the HLS-EU methodology, and compare it with the results published by the HLS-EU Consortium.