DGH - Apresentações orais em encontros internacionais
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- 3- Methylcrotonyl-coa Carboxylase Deficiency: Biochemical and Molecular Studies in 36 PatientsPublication . Fonseca, Helena; Sousa, Carmen; Marcão, Ana; Rocha, Hugo; Lopes, Lurdes; Vilarinho, Laura3-methylcrotonylglycinuria (MCG) is a disease included in the expanded newborn screening that until recently was considered a rare inherited disorder of the metabolism. In the catabolism of leucine, MCG is blocked in the fourth step due to deficiency of the enzyme 3-methylcrotonyl-CoA carboxylase (3-MCC) (Dantas et al). The biochemical diagnosis of disability in 3-MCC is characterized by marked increase of acid 3-hydroxyisovaleric (3-HIVA) and 3-methylcrotonylglycine (3-MCG) in urine and high concentrations of 3-hydroxyisovalerylcarnitine (C5-OH) in the blood. The molecular characterization is the study of genes MCCA and MCCB that encodes the enzyme 3-MCC. The authors report biochemical and mutation data of 36 MCC deficient individuals, one diagnosed due to clinical symptoms, 25 identified by newborn screening and 10 mothers identified following the positive newborn screening of their sons. All patients had an increased value of C5-OH, primary biochemical marker screening for this condition. In this cohort of 36 patients the genetic study intended to identify the pathogenic mutations using an analysis of 19 exons in the MCCA gene and 17 exons in the MCCB gene. A total of 32 mutations were detected of which 24 (75%) have, neither been described in the literature nor in the Human Gene Mutation Database. The results described show that the genotype cannot predict the phenotype or metabolic risk of these cases, but it is capable to confirm the diagnosis in doubtful cases. The clinical phenotype is very heterogeneous, most patients showing different mutations making the phenotype-genotype correlation difficult. The 3-MCC deficiency is a pathology not completely understood described as a genetic condition with low clinic penetrance. However, it can lead to a severe clinic phenotype resembling classic organic acidurias, has it was recently demonstrated by Grunert et al. Dantas, M. F., T. Suormala, et al. (2005). 3-Methylcrotonyl-CoA carboxylase deficiency: mutation analysis in 28 probands, 9 symptomatic and 19 detected by newborn screening.
- Aflatoxins and ochratoxin A in baby foods and analysis of interactive cyto- and genotoxic effects in a human intestinal cell linePublication . Tavares, Ana; Alvito, Paula; Loureiro, Susana; Louro, Henriqueta; Silva, Maria JoãoMycotoxins are natural fungal metabolites and food contaminants with potential to cause severe acute and chronic conditions. Food contamination with mycotoxins such as aflatoxins (AF) and ochratoxin A (OTA) have been causing great concern, especially due to their potential mutagenic and carcinogenic effects. Children are especially vulnerable to the deleterious effects of these mycotoxins due to their physiological immaturity and high metabolic rate. Previous studies showed the co-occurrence of low concentrations of aflatoxins and OTA in baby foods. However, studies addressing potential interactive cyto- and genotoxic effects between these toxins are still scarce. In the present study we aimed to develop and validate a method for detection and quantification of total aflatoxins (B1, B2, G1, G2), AFM1 and OTA, and to evaluate the cytotoxic and genotoxic effects of mixtures of AFM1 and OTA, comparatively to their individual effects, in a human-derived intestinal cell line. A method based on immunoaffinity column cleanup and High Performance Liquid Chromatography with fluorescence detection (HPLC-FD), was applied and validated for total aflatoxins, AFM1 and OTA. The method was adequate for the analysis of these mycotoxins in baby foods and met the requirements of validation and quality control. The application of the method to a small set of baby foods marketed in Portugal showed an absence of quantifiable amounts of these mycotoxins. The individual and combined cytotoxic and genotoxic effects of AFM1 and OTA were characterized in Caco-2 cells using the Neutral Red and the Comet assays, respectively. A dose-dependent cytotoxicity was observed after individual exposure to OTA and AFM1, and the IC50 values were determined. The cytotoxic effect observed for several AFM1 and OTA mixtures was compared to the expected effect predicted by concentration addition (CA) and independent action (IA) conceptual models, using the MIXTOX model. A preliminary approach regarding the total data pool and considering the CA model as the most conservative model, pointed to an antagonistic cytotoxic effect caused by the mixture of both mycotoxins. However, a dose level deviation was observed after IA modelling, reflecting antagonism at low dose levels and synergism at higher dose levels. To better support data modelling, further cytotoxicity results from mixtures will be obtained and analyzed. To which respects the genotoxic effects, no induction of DNA damage was observed for the tested low doses, neither for individual toxins nor for their mixtures. The present study reinforces the relevance of exploring possible interactive adverse effects of mycotoxins that can contaminate foodstuff and thus having impact in human health. Future studies will face the challenge of understanding the mode of action of such mycotoxins when in mixture, in order to try predicting their effects.
- Alternative splicing of tumour-related Rac1b is regulated by upstream signalling pathwaysPublication . Gonçalves, Vânia; Matos, Paulo; Jordan, PeterThe small GTPase Rac1 regulates signalling pathways controlling actin-dependent cell motility as well as gene transcription. An alternative splicing variant Rac1b is overexpressed in a subset of colorectal tumours and cooperates with mutant B-Raf to sustain tumour cell viability. The alternative splicing mechanism regulating Rac1b expression involves two antagonistic splicing factors, ASF/SF2 and SRp20. Using a Rac1 minigene approach and siRNA-mediated depletion, we identified ASF/SF2 as an enhancer of endogenous Rac1b splicing whereas SRp20 acts as a silencer. Inhibition of the PI3-kinase pathway increased protein levels of ASF/SF2 and promoted Rac1b generation. By contrast, depletion of endogenous protein kinase SRPK1 led to decreased Rac1b expression. Together, these data indicate that altered upstream signaling pathways in colorectal cancer cells will target splicing factors that regulate alternative splicing of the small GTPase Rac1.
- An account of the portuguese experience: advances and challenges in diagnosis, therapy and research in lysosomal disordersPublication . Amaral, OlgaIntroductory background: Inborn errors of metabolism constitute an important group of rare hereditary diseases. Such diseases started to be studied in Portugal in the early-mid 1980s. Among them, Lysosomal Storage Disorders (LSDs) constitute a challenging group of disorders. In Portugal, Gaucher disease and Tay Sachs variant B1 were the first to be studied and were the subject of intricate laboratory diagnosis and careful research. Aim: Provide an account of the experience with lysosomal storage disorders in Portugal.
- An unexpected role for DIS3L2 over human NMD targetsPublication . Costa, Paulo; Saramago, Margarida; Viegas, Sandra; Arraiano, Cecília; Romão, LuísaThe nonsense-mediated mRNA decay (NMD) pathway selectively degrades mRNAs carrying a premature translation-termination codon but also regulates the abundance of a large number of physiological RNAs that encode full-length proteins. In human cells, NMD-targeted mRNAs are degraded by endonucleolytic cleavage and exonucleolytic degradation from both 5’ and 3’ ends. This is done by a process not yet completely understood that recruits decapping and 5’-to-3’ exonuclease activities, as well as deadenylating and 3’-to-5’ exonuclease exosome activities. In yeast, DIS3/Rrp44 protein is the catalytic subunit of the exosome, but in humans, there are three known paralogues of this enzyme: DIS3, DIS3L1, and DIS3L2. However, DIS3L2 exoribonuclease activity is independent of the exosome. DIS3L1 and DIS3L2 exoribonucleases localize in the same compartment where NMD occurs, however nothing is known about their role in this process. In order to unveil the role of DIS3L2 in NMD, we performed its knockdown in HeLa cells and measured the mRNA levels of various natural NMD targets. Our results show that some NMD targets are highly stabilized in DIS3L2-depleted cells. In addition, mRNA half-life analysis indicated that these NMD targets are in fact direct DIS3L2 substrates. We also observed that DIS3L2-mediated decay depends on the activity of the terminal uridylyl transferases (TUTases) Zcchc6/11 (TUT7/4). Together, our findings establish the role of DIS3L2 and uridylation in NMD.
- Analysis of the divergences in the nonsense-mediated mRNA decay mechanism among two globin transcripts highly conservedPublication . Pereira, Francisco JC; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature termination codons (PTCs, or nonsense codons), which has been highly conserved during evolution. Knowing that human a- and b-globin genes descend from a common ancestral, the aim of this work was to understand if both encoded mRNAs carrying a nonsense mutation, share parallel NMD profiles. We have previously shown that human b-globin mRNAs carrying a PTC located in close proximity to the translation initiation AUG codon escape NMD. This was called the “AUG-proximity effect”. The present work illustrates that the extension of the AUG-proximity effect, i.e. to what position in the open reading frame (ORF) an AUG-proximal PTC does not trigger NMD, is different between human a- and b-globin mRNAs. Remarkably, our data also demonstrate that, contrary to what occurs in the b-globin transcripts, a-globin mRNAs carrying an AUG-proximal PTC allow for efficient translation re-initiation, although it only partially explains their NMD resistance. In addition, our results reveal that in the a- and b-globin transcripts, the extension of the AUG-proximity effect is determined by the ORF sequence. Furthermore, we show how the mRNA secondary structure, which is affected by the ORF sequence, determines the AUG-proximity effect extension. Our data point out that the time taken to translate the short ORF, affected by its sequence and stability, besides being involved in modulating translation re-initiation, also plays an important role in establishing the extension of the AUG-proximity effect. Thus, although both mRNAs are evolutionarily related they show different features in their NMD response.
- Anchoring HBM4EU data to chemicals risk assessmentPublication . Silva, Maria JoãoA major goal of the European Human Biomonitoring Initiative, HBM4EU, is to coordinate and advance human biomonitoring across Europe and reinforce its application in different regulatory frameworks for chemicals. To illustrate the development of state-of-the-art approaches and the potential of human biomonitoring (HBM) data to refine exposure assessment and, thereby, risk assessment, this presentation will be focused on the work carried out on hexavalent chromium, [Cr(VI)], an important occupational lung carcinogen, which is currently authorized in Europe for several industrial activities. Cr(VI) was considered a priority substance under the HBM4EU Project, indicating the need for generating and analyzing data on human exposure, despite the recently agreed binding limit value for occupational exposure established in the Eu-ropean Union. The anchoring of HBM to risk assessment and management practices will be evidenced through the infor-mation generated from three different studies, namely: i) a critical review on effect biomarkers to link Cr(VI) exposure to health outcomes, ii) a multinational, collaborative study to support management of oc-cupational exposure to Cr(VI), and iii) the development of a case study on co-exposure to Cr (VI), nickel and polycyclic aromatic hydrocarbons, to advance the identification of mixture health effects and to pro-gress towards a more refined risk assessment. These research efforts to integrate HBM into new risk assessment approaches need to be supported by mechanistic knowledge obtained from in vitro/in vivo studies, toxicokinetic data and the development of adverse outcome pathways, as well.
- Antisense oligonucleotide exon-skipping as a therapeutic approach for Mucolipidosis type II a/b: in vitro and in vivo studiesPublication . Matos, Liliana; Gonçalves, Mariana; Santos, Juliana Inês; Coutinho, Maria Francisca; Prata, Maria João; Pires, Maria João; Oliveira, Paula; Alves, SandraMucolipidosis type II alpha/beta (ML II alpha/beta) is one of the most severe Lysosomal Storage Disorders and is caused by the deficiency of the enzyme GlcNAc-1-phosphotransferase. This enzyme is responsible for the addition of the mannose 6-phosphate marker to lysosomal enzymes, which allow their targeting to lysosomes. Of the several mutations that occur in ML II alpha/beta, the deletion of 2 nucleotides from GNPTAB exon19 (c.3503_3504del) is the most frequent, making it a good target for a specific mutation therapy as there is no therapy for this disease. In this study, we explored the possibility of an innovative therapeutic strategy based on the use of antisense oligonucleotides (AOs) for ML II alpha/beta. In a previous in vitro study in ML II alpha/beta patient fibroblasts, AOs were used to promote the exon 19 skipping from the GNPTAB pre-mRNA, resulting successfully in the production of an in-frame mRNA1. Currently, our objective is to evaluate the therapeutic potential of this approach, both in vitro in C57BL/6 fibroblasts and in vivo in C57BL/6 mice. For this, 18 animals were used, divided into 6 groups: groups 1 and 4 were injected with saline solution, groups 2 and 5 were injected with AO at 25 mg/kg and groups 3 and 6 were injected with AO at 50 mg/kg. All animals were injected by intraperitoneal route and were sacrificed after 4 days (groups 1, 2, 3) or 7 days (groups 4, 5, 6) post-treatment. At the end of the experiment, the organs were collected and frozen at -80ºC, for later RNA extraction, cDNA synthesis and RT-PCR. After results analysis, the exon 19 skipping was not observed using any of the tested doses or incubation periods. So, we can theorize that the doses administered were not sufficient to achieve a response or the AO might have had a high clearance rate. As for the in vitro experience, the C57BL/6 fibroblasts were seeded in 6-well plates and subsequently transfected with concentrations of AO ranging from 10nM to 600nM. After 24h or 48h of incubation, cells were collected and cDNA analysis revealed a full length transcript but also another one of lower molecular weight compatible with exon-skipping. These are preliminary data, so in the near future more experiments will be done. 1. Matos L, Vilela R, Rocha M, et al. Development of an antisense oligonucleotide-mediated exon skipping therapeutic strategy for Mucolipidosis II: validation at RNA level. Hum Gene Ther, 2020, 31(13-14):775-783.
- Aplicação de biomarcadores de efeito na biomonitorização humana: teste do cometa e teste do micronúcleoPublication . Louro, HenriquetaSobre a utilização de biomarcadores de efeito em estudos de biomonitorização humana.
- Applications of iPSCs in Gaucher DiseasePublication . Amaral, Olga; duarte, ana; Ribeiro, Diogo; Santos, Renato; Bragança, JoséIn recent years, human induced pluripotent cell (hiPSC) models have slowly become a trend in experimental modelling of disease, following and complementing animal based models. Human iPSCs provide an innovative manner for modelling Gaucher Disease (GD). Since 2008 several groups have created iPSCs models from GD patients, with various genotypes, and differentiated iPSCs to neural precursors and macrophages among many other types of cells. hiPSC models have been developed from multiple GD donors, recapitulating the disease phenotypic hallmarks. These models have provided a new platform for pathophysiology studies and for the testing of small molecules with therapeutic goals.