Browsing by Issue Date, starting with "2019-03-01"
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- Study of human Argonaute 1 cap-independent translationPublication . Carvalho, Manuel; Romão, Luísa; Menezes, JulianeTranslation of mRNA into protein is one of the most important and costly processes of the cell, since it supports and regulates several cellular events. Under normal conditions, translation initiation takes place through the canonical mechanism, in which first occurs the recognition of the cap structure at the 5'end of the transcript, followed by the scan of the 5'UTR (untranslated region) until the identification of the initiation codon in a favourable context. However, during high energy cost cell events, such as mitosis, or during some stresses such as hypoxia, mechanisms are activated that decrease canonical translation. Thus, under conditions of inhibition of canonical translation, there are proteins whose synthesis is not affected and is maintained by alternative mechanisms. These include those in which ribosomal recruitment occurs without cap recognition and/or 5'UTR scanning. In these cases, ribosomal recruitment occurs through secondary structures present in mRNA 5'UTR, which interact with the 40S ribosomal subunit, and with or without the help of initiation factors, position it near to the initiation codon. Therefore, it was hypothesized that Argonaute proteins, more properly AGO1 (human Argonaute RNA-induced silencing complex catalytic component 1) could be translated by non-canonical mechanisms due to its importance in the biogenesis of the microRNAs and in the gene silencing. Previous results obtained in our laboratory using a bicistronic system, demonstrated that AGO1 5'UTR is able to mediate cap-independent translation of a reporter gene, even under stress conditions that inhibit canonical translation. Thus, the purpose of this work was to complete this research project, continuing to study the cap-independent translation mediated by the 5'UTR of AGO1. In this way, the first objective was to reinforce the previously obtained results. For this it was intended to show that the AGO1 5´UTR can mediate an alternative initiation mechanism in a bicistronic vector different from the one previously used. For this, the proposed bicistronic system contains two reporter genes, one corresponding to enhanced green fluorescent protein (EGFP) and another corresponding to a red fluorescent protein (mCherry). The characteristic of this system is that in the first cistron is the sequence of EGFP, which is translated by the canonical mechanism. The second cistron is the mCherry sequence that can only be translated in cap-independent manner. With this in mind, the strategy was to clone the sequence corresponding to AGO1 5´UTR, upstream of the mCherry and to verify if it was able to potentiate cap-independent translation of this fluorophore. In this thesis we present the cloning strategy developed to obtain this construct, although we were not able to complete this task. However, we present some possible solutions to complete these clonings. Other objective was to evaluate how the expression of Argonaute proteins, more properly the endogenous AGO1, varies during endoplasmic reticulum stress, which inhibits the canonical translation. For this purpose, we treated HCT116 cells (colorectal cancer-derived cell line) with thapsigargin, which is a drug that induces the stress of the endoplasmic reticulum and, consequently, the inhibition of the canonical translation. Our results demonstrated that treatment with the drug induces a decrease in the AGO1 and AGO2 mRNA levels, a decrease in the total protein amount of Argonautes (AGO1, AGO2, AGO3 and AGO4) and a decrease in the AGO1 protein. In a way, this is an inconclusive result for the study of non-canonical translation of this protein during stress conditions. However, we think that this result is mainly due to two factors. One has to do with the fact that the treatment with the drug decreases the AGO1 mRNA level, thus having less transcript available for translation, which may lead to less protein synthesis, albeit in an alternative way. The other factor is related to the possibility that the non-canonical translation mechanism is not as efficient in as the canonical way, which explains the observed decrease. Thus, in future experiments it will be important to test other stresses that inhibit canonical translation, but that do not induce changes in the amount of mRNA and test this experiment in other cell lines. Finally, we aim to predict what structural features make the AGO1 5´UTR succeed in promoting a non-canonical translation initiation mechanism and thus try to estimate the secondary structure adopted by this sequence. For this we submited AGO1 5'UTR sequence to an in silico analysis. Among other characteristics, we found that it has a percentage of guanines/cytosines of 72.3% and it is organized into a stable secondary structure, which has four stem loops. As a future perspective, we aim to experimentally validate this structural prediction through circular dichroism. Next was performed an in silico deletion analysis in which nucleotides were removed from the sequence in order to predict the structural dynamics and to check if there was any minimal sequence/structure required to maintain stable the spatial conformation of AGO1 5'UTR. By our analysis it was verified that when stem loop I or stem loop IV is removed, the structure does not seem to change much in relation to the original, indicating that the conformation remains stable when small deletions occur. To complement this in silico deletion analysis it is intended in the future to validate experimentally, in a bicistronic vector, the effect of this deletions. We believe that once these research lines have been completed, we will better understand the relevance of alternative translation mechanisms in the maintenance of synthesis of several proteins, including AGO1, during stress situations, and even during the tumorigenesis process.
- Influência do processamento no perfil lipídico de alimentos processados: aspetos nutricionais e toxicológicosPublication . Albuquerque, T.G.; Oliveira, M.B.P.P.; Costa, H.S.Aspetos nutricionais e toxicológicos na perspectiva da influência do processamento no perfil lipídico de alimentos processados.
- Targeted next generation sequencing identifies novel pathogenic variants and provides molecular diagnoses in a cohort of pediatric and adult patients with unexplained mitochondrial dysfunctionPublication . Nogueira, Célia; Silva, Lisbeth; Pereira, Cristina; Vieira, Luís; Leão Teles, Elisa; Rodrigues, Esmeralda; Campos, Teresa; Janeiro, Patrícia; Gaspar, Ana; Dupont, Juliette; Bandeira, Anabela; Martins, Esmeralda; Magalhães, Marina; Sequeira, Sílvia; Vieira, José Pedro; Santos, Helena; Vilarinho, Sílvia; Vilarinho, LauraMitochondrial diseases (MD) are a group of rare inherited disorders, characterized by phenotypic heterogeneity, with hitherto no effective therapeutic options. The aim of this study was to develop a next generation sequencing (NGS) strategy, by using a custom gene panel and whole mitochondrial genome, to identify the disease causing pathogenic variants in 146 patients suspicious of MD. The molecular analysis of this cohort revealed six novel and 15 described pathogenic variants, as well as 26 variants of unknown significance. Our findings are expanding the mutational landscape of these disorders and support the use of a NGS strategy for a higher diagnostic yield.
- Goji berries superfood – contributions for the characterisation of proteome and IgE-binding proteinsPublication . Teixeira, Sandrina; Luis, Inês; Oliveira, M.; Abreu, Isabel; Batista, RitaGoji berries’ bioactive compounds, which allowed classifying them as superfruits, led to an enormous increase of its consumption in western countries. However, the potential risk of allergy is a concern. In this study, we aimed to characterise the proteome of goji berries (Lycium barbarum) and identify proteins with putative role in the allergic reaction (IgE-binding proteins). We firstly used twodimensional (2D) gel electrophoresis followed by mass spectrometry (MS) to characterise goji berries’ proteome, and then Immunoblot reactivity with plasma from tomato and potato (same botanical family, Solanaceae) allergic individuals was assessed to characterise goji berries IgE-binding proteins. An inhibition assay was further performed to evaluate cross-reactivity among potato, tomato and goji berries. We significantly identified 93 out of the 180 MS analysed spots, corresponding to 29 protein functions. From these, 11 could be identified as goji berries IgE-binding proteins. We further demonstratedcross-reactivity between goji berries, tomato and potato.