Browsing by Issue Date, starting with "2018-11-23"
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- Whole genome analysis of influenza A(H3) viruses detected between 2016-2018 in the scope of EuroEVA/I-MOVE vaccine effectiveness studyPublication . Pechirra, Pedro; Borges, Vítor; Cristóvão, Paula; Costa, Inês; Conde, Patrícia; Machado, Ausenda; Rodrigues, Ana Paula; Gomez, Verónica; Kislaya, Irina; Nunes, Baltazar; Gomes, João Paulo; Guiomar, RaquelBackground: NGS techniques, allow a much deeper genetic analysis of influenza viruses, compared to traditional Sanger sequencing of hemagglutinin gene. The present study aims to perform phylogenetic and mutational analysis at whole-genome level in order to search for genetic features related to vaccine failure. Methods: Nasopharyngeal swabs were collected during 2016/17 and 2017/18 winter seasons, from ILI patients participating in EuroEVA/I-MOVE study. Whole genome sequences were obtained for 179 influenza A(H3) viruses by NGS in a MiSeq platform and subsequent bioinformatics analysis using the web-based platform INSaFLU (https://insaflu.insa.pt/). Additional fine-tune sequence analysis was performed using MEGA-7. Results: All sequenced viruses clustered in 2 HA-based genetic groups: 58 (32.4%) in 3C.2a group and 121 (67.6%) in 3C.2a1. Vaccine failure cases were detected in a higher proportion in 3C.2a1 group (20/121, 16.5%) than in 3C.2a (8/58, 13.8%). WGS analysis further revealed intra-subtype reassortments based on the closest genetic relatedness of each viral segment to the representative virus of seasonal A(H3) genetic (sub-)groups, with viruses being distributed in 6 different patterns of genome constellation. The group with all genomic segments most closely related to A/Singapore/INFIMH-16-0019/2016 harboured a higher number of vaccine failure cases (14/69, 20.3%). Despite 16 viruses (from 28 detected in vaccinated cases) presented amino acid substitutions not found in unvaccinated cases, these substitutions revealed a sporadic pattern. Conclusions: Vaccine failure cases were not exclusive of any genetic group or reassortment pattern, although they were found in slightly higher proportion among 3C.2a1 viruses and in viruses with all genetic segments mostly similar to A/Singapore/INFIMH-16-0019/2016. The further use of WGS in flu surveillance is essential to better understand genetic determinants of infection and evolutionary dynamics of influenza virus.
- Study on the regulation of the expression of alternative protein isoforms involved in carcinogenesisPublication . Pereira, Bruna; Loison, Luísa Romão; Candeias, Marco MarquesIn eukaryotes, gene expression is a highly complex process composed of several steps. One of those is translation, the step that converts the genetic information contained in the messenger ribonucleic acid (mRNA) into functional proteins. Under normal conditions, most proteins are translated through the canonical translation initiation mechanism, which starts with cap structure recognition at the 5’-end of mRNAs, followed by 5’ UTR (untranslated region) scanning until the appearance of an initiation codon in a favorable context. Yet, under unfavorable or energy-depriving conditions, such as endoplasmic reticulum (ER) stress, hypoxia, nutrient starvation, mitosis and cell differentiation, canonical translation is impaired and protein synthesis globally decreases. Nevertheless, some mRNAs, usually related to stress-responses, cell growth and cell death control, continue to be translated through alternative mechanisms. One of them involves internal ribosome entry sites (IRES), in which the ribosome is directly recruited to the vicinity of the initiation codon, without requiring the cap structure. This mechanism relies on mRNA secondary structures and can be assisted by some canonical factors and other auxiliary proteins named ITAFs (IRES trans-acting factors). Tumor cells take advantage of this mechanism to cope with the unfavorable conditions that characterize tumor microenvironment and proliferate. Indeed, many mRNAs containing IRES elements are found deregulated in cancer. One example is the tumor suppressor p53. The TP53 gene is the most commonly mutated gene in cancer and surprisingly, p53 mutations usually lead to the production of a mutant protein with oncogenic functions. The presence of three promoters on TP53 gene leads to the expression of different transcripts expressing different alternative translation products: the full-length transcripts allow the expression of FL-p53, Δ40p53 and Δ160p53, and a shorter transcript produces Δ133p53 and also Δ160p53. While FL-p53 and Δ40p53 protein isoforms have been widely studied in terms of internal initiation mechanisms, the fact that Δ160p53 expression is mediated through an IRES element was not known until recently. Since this shorter p53 isoform was already associated with survival, proliferation and invasion of tumor cells, the recently identified Δ160p53 IRES may have an important role in tumorigenic functions of Δ160p53. Thus, considering the aforementioned data, we proposed to study the regulation of the expression of alternative protein isoforms involved in carcinogenesis, more specifically, the regulation of Δ160p53 expression through its IRES element, aiming to understand the role of IRES-mediated translation in cancer development. Knowing that Δ160p53 IRES is located within the first 432 nucleotides of Δ160p53 coding sequence and that its activity is inhibited by Δ160p53 5’ UTR, we evaluated the effect of hotspot p53 missense mutations (R175H, R248Q, R273H e R282W) in reverting the inhibitory effect of Δ160p53 5’ UTR on Δ160p53 IRES activity. To do that, we used a bicistronic system containing two reporter genes: Renilla Luciferase (RLuc) and firefly Luciferase (FLuc). RLuc is the first cistron and its expression is driven by cap-dependent mechanisms, while FLuc is the second cistron and is only translated if there is an upstream sequence capable of promoting its translation through cap-independent mechanisms. In our experiments, we were able to see the inhibitory effect of Δ160p53 5’ UTR on Δ160p53 IRES activity, in HeLa cells, corroborating the previous reported results. Moreover, from all tested p53 missense mutations (R175H, R248Q, R273H e R282W), only R175H was capable of reverting some of the 5’ UTR inhibitory effect on Δ160p53 IRES activity. This was observed for cells under 2 μM Thapsigargin-induced ER stress, which is known to impair cap-dependent translation. The obtained results seem to indicate that R175H oncogenic functions go beyond the alteration or loss of protein function, since this mutation also appears to act through an mRNA-dependent manner by inducing the expression of Δ160p53, an isoform that has already been shown to have importance in promoting tumorigenesis. Furthermore, in this thesis, we also aimed the identification of Δ160p53 IRES auxiliary proteins, using a system that takes advantage of MS2 RNA–MS2 coat protein interaction. Cloning p53 sequences of interest, such as the Δ160p53 IRES, upstream of MS2 RNA repeats, followed by co-transfection of these constructs with that expressing the MS2 coat protein and co-immunoprecipitation, will allow the identification of p53 mRNA-interacting proteins, through mass spectrometry. Here, we describe some of the cloning strategies used, though unsuccessfully, to attempt to clone p53 sequences of interest upstream MS2 repeats as well assome possible solutions. Moreover, knowing that Hdm2 (murine double minute 2 human homolog) interacts with several mRNAs commonly deregulated in cancer cells, such as p53 and XIAP (X-linked inhibitor of apoptosis protein), regulating their non-canonical translation, we also performed Hdm2 immunoprecipitation optimizations, so that, in the future, co-immunoprecipitation of Hdm2-bound mRNAs can be performed. Then, new possible IRES-containing mRNAs regulated by Hdm2 that may also have a preponderant role in cancer progression will be identified by RNA sequencing. At the end, concluding all these lines of research, we hope to unveil new insights regarding IRES-mediated translation of cancer-related mRNAs. In fact, understanding how IRES-containing mRNAs are regulated under different stress conditions and how the switch between cell homeostasis and cell neoplastic transformation is triggered, will provide important knowledge for the development of new therapeutic strategies.