http://hdl.handle.net/10400.18/46 Wed, 12 Jun 2013 14:10:36 GMT 2013-06-12T14:10:36Z http://hdl.handle.net/10400.18/1126 Title: Genotoxicity evaluation of three classes of manufactured nanomaterials on primary human lymphocytes Authors: Tavares, Ana; Louro, Henriqueta; Antunes, Susana; Quarré, Stéphanie; Simar, Sophie; Nesslany, Fabrice; Silva, Maria João Abstract: Human exposure to manufactured nanomaterials (MNMs) through consumers’ goods has increased worldwide, including titanium dioxide (TiO2), multiwalled carbon-nanotubes (MWCNT) and silicon dioxides (SiO2). Specific properties of MNMs, such as size and high surface area/mass, render them attractive for many applications, but may also be associated to higher toxicity in biological systems and adverse effects in humans. In the context of an EU Joint Action (NANOGENOTOX), aimed at establishing a robust methodology for evaluation of the MNMs genotoxicity, the objective of the present work was to characterize the potential genotoxic effects of a panel of well-characterized MNMs in human lymphocytes. Cultures of lymphocytes from healthy volunteers were exposed to several non-cytotoxic concentrations of four TiO2, six MWCNT and four SiO2 MNMs, dispersed according to a previously established procedure, for 30h; concurrent positive controls included a chemical (mitomycin C) and a reference nanosized ZnO. The cytokinesis-block micronucleus assay was performed according to the OECD guideline 487 and the frequencies of micronucleated binucleate (MNBNC) and mononucleate cells were determined. The results showed that slight but significant increases in the frequencies of MNBNC were observed for some TiO2 NMs and MWCNTs, as compared to controls, while all SiO2 NMs were negative in the micronucleus assay; a single dose of ZnO was able to significantly induce MNBNC. However, no clear monotonic dose-response relationships could be disclosed, not enabling clear conclusions about the genotoxicity of the tested MNMs. No influence on cell cycle progression and cell viability was noted by the cytokinesis-block proliferation index. In summary, although the micronucleus assay in primary human lymphocytes is suitable to evaluate the genotoxicity of MNMs following standardized dispersion and assay procedures, data from other cell lines or from other endpoints (e.g. comet assay) have to be gathered in order to achieve a robust genotoxicity evaluation. Thu, 18 Oct 2012 00:00:00 GMT http://hdl.handle.net/10400.18/1126 2012-10-18T00:00:00Z http://hdl.handle.net/10400.18/1080 Title: Assessing the involvement of Dis3L1 in mammalian quality control pathways Authors: Reis, Filipa Pereira; Teixeira, Alexandre; Cruz, David; Morgado, Ana; Arraiano, Cecília Maria; Romão, Luísa Abstract: The exosome is an evolutionarily conserved protein complex that is involved in all aspects of RNA metabolism, namely, RNA decay, processing and quality control. The only catalytic subunit of the core exosome is a 3´end exoribonuclease from the RNase II family of enzymes. In humans, two different homologues of this protein were identified, Dis3 and Dis3L1. While Dis3 mainly localizes in the nucleoplasm and has endonucleolytic activity, Dis3L1 is strictly cytoplasmic and has no endonucleolytic activity. The rapid decay of aberrant transcripts is not completely understood, but it is known that involves both 5’ to 3’ and 3’ to 5’ degradation. Despite that it localizes in the same compartment where NMD generally occurs, nothing is known about the role of Dis3L1 in quality control processes. In this work, we assessed the involvement of Dis3L1 in the 3’ to 5’ degradation of reporter human b-globin transcripts with premature termination and nonstop codons. Sat, 01 Sep 2012 00:00:00 GMT http://hdl.handle.net/10400.18/1080 2012-09-01T00:00:00Z http://hdl.handle.net/10400.18/1042 Title: Nonsense-mediated decay resistance of AUG-proximal nonsense-mutated transcripts relies on the interaction of PABPC1 with the translation initiation complex Authors: Peixeiro, Isabel; Barbosa, Cristina; Romão, Luísa Abstract: Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing a premature termination codon (PTC). The unified model for NMD proposes that the decision of NMD triggering is the outcome of the competition between the cytoplasmatic poly(A)-binding protein 1 (PABPC1) and the NMD effector UPF1 for the termination complex. Consequently, PTCs located far, in a linear sense, from the poly(A) tail and associated PABPC1, in mRNAs containing downstream exon junction complexes (EJCs), are expected to elicit NMD. Nevertheless, we have reported that human b-globin mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD. We have reported that translation termination at an AUG-proximal PTC lacks the ribosome stalling that is evident in an NMD-sensitive PTC. In fact, we have shown that the establishment of an efficient translation termination reaction at the AUG-proximal PTC is dependent on PABPC1 interaction with the initiation factor eIF4G and with the release factor eRF3 at the terminating ribosome. These interactions underlie critical 3’-5’ linkage of translation initiation with efficient termination at the AUG-proximal PTC and contribute to an NMD-resistant PTC definition at an early phase of translation elongation. Furthermore, we provide strong evidence that the eIF3 is involved in delivering eIF4G-associated PABPC1 into the vicinity of the AUG-proximal PTC. This work corroborates a role for PABPC1 on NMD evasion of transcripts carrying an AUG-proximal PTC and provides further insights into the mechanistic details of PTC definition and translation initiation. Description: Apresentação oral por convite. Sat, 01 Sep 2012 00:00:00 GMT http://hdl.handle.net/10400.18/1042 2012-09-01T00:00:00Z http://hdl.handle.net/10400.18/973 Title: A Rare Disease Patient Manager Authors: Lopes, Pedro; Mendonça, Rafael; Rocha, Hugo; Oliveira, Jorge; Vilarinho, Laura; Santos, Rosário; Oliveira, José Abstract: The personal health implications behind rare diseases are seldom considered in widespread medical care. The low incidence rate and complex treatment process makes rare disease research an underrated field in the life sciences. However, it is in these particular conditions that the strongest relations between genotypes and phenotypes are identified. The rare disease patient manager, detailed in this manuscript, presents an innovative perspective for a patient-centric portal integrating genetic and medical data. With this strategy, patient’s digital records are transparently integrated and connected to wet-lab genetics research in a seamless working environment. The resulting knowledge base offers multiple data views, geared towards medical staff, with patient treatment and monitoring data; genetics researchers, through a custom locus-specific database; and patients, who for once play an active role in their treatment and rare diseases research. Description: Artigo publicado em: Advances in Intelligent and Soft Computing. 2012;154:173-180. Wed, 28 Mar 2012 00:00:00 GMT http://hdl.handle.net/10400.18/973 2012-03-28T00:00:00Z