DGH - Apresentações orais em encontros nacionais
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- Abordagens terapêuticas de exon-skipping em doenças raras – aplicação à Neurofibromatose tipo 1Publication . Alves, SandraA Neurofibromatose tipo 1 (NF1) é uma das doenças neurocutâneas mais frequentes.
- Adverse Outcome Pathways (AOPs) development, a tool for predictive nanotoxicologyPublication . Rolo, Dora; Louro, HenriquetaNanomaterials (NMs) have the potential to improve novel and useful wide applications in electronics, chemicals, environmental protection, biological medicine, food and others. Therefore, NMs rapid proliferation presents a dilemma to regulators regarding hazard identification, with increased concerns for public health. Predictive nanotoxicology describes a multidisciplinary approach to NMs evaluation that uses a set of in vitro and in silico methods to forecast the effects on biological systems. This approach offers advantages to traditional hazard assessment methods, such as reducing the reliance on animal studies, associated costs and ethical issues. It may be used with several applications in environmental and human health risk assessment and NMs hazard identification, as well as for regulation. The Adverse Outcome Pathways (AOPs) are the central element of a toxicological knowledge framework, promoted by member countries through OECD, built to support chemical risk assessment based on mechanistic reasoning. AOPs describes a logical sequence of causally linked events at different levels of biological organisation, which follows exposure and leads to an adverse health effect in humans or wildlife. The integrative analysis of the cellular and molecular mechanisms of nanotoxicity towards a definition of key events, may lead to adverse outcomes, driving a sequential line and defining an AOP landscape. Each defined AOP is available for crossing data, linking known and unknown landscapes. Since the biological effects that relate to possible genotoxicity and increased risk of cancer due to NMs exposure are under analysis, the development and assessment of AOPs are important novel strategic tools for predictive nanotoxicology.
- Alterações do perfil bioquímico e testes diagnósticosPublication . Rocha, HugoApresentação sobre as alterações do perfil bioquímico e testes diagnósticos em contexto de formação pós-graduada em Doenças Hereditárias do Metabolismo do Adulto.
- Ambiente, genoma e cancroPublication . Silva, Maria JoãoComunicação sobre factores ambientais e o impacto na saúde, nomeaamente relativo à exposição humana a agentes potencialmente mutagénicos e/ou carcinogénicos.
- Anemia das Células Falciformes: hemólise crónica e vasculopatia cerebralPublication . Faustino, PaulaSão apresentados os resultados obtidos no âmbito de dois projetos de investigação no âmbito dos fatores genéticos moduladores da hemólise crónica e da vasculopatia cerebral (AVC e enfartes silenciosos) na Anemia das Células Falciformes. São discutidos os mecanismos subjacentes e hipóteses de uso como alvos terapêuticos.
- Antisense oligonucleotide exon-skipping as a therapeutic approach for Mucolipidosis type II α/β: in vitro and in vivo studiesPublication . Gonçalves, Mariana; Matos, Liliana; Santos, Juliana Inês; Coutinho, Maria Francisca; Prata, Maria João; Pires, Maria João; Oliveira, Paula; Alves, SandraGenetic therapy directed towards the correction of RNA mis-splicing is being investigated at basic research and in late-stage clinical trials. Many mutations that change the normal splicing pattern and lead to aberrant mRNA production have been identified in Lysosomal Storage Disorders (LSDs). Mucopolysaccharidosis IIIC (MPS IIIC) is one of those LSDs caused by mutations in the HGSNAT gene that encodes an enzyme involved in heparan sulphate degradation. Splicing mutations are one of the most frequent (~20%) genetic defects in MPS IIIC. Approximately 55% correspond to 5' splice-site (ss) mutations thus constituting a good target for mutation-specific therapeutic approaches. Recently, we have demonstrated in fibroblast cells that a modified U1 snRNA vector designed to improve the definition of exon 2 5’ss of the HGSNAT can restore splicing impaired by the mutation c.234+1G>A. Currently, our goal is to evaluate in vivo the therapeutic potential of that modified U1 snRNA by testing it in mice expressing the human splicing defect. For this purpose, two full-length constructs were generated by cloning the wild-type (wt) or the mutated HGSNAT splicing-competent cassettes in the pcDNA 3.1 vector. Then, in an in vitro assay, the wt or mutated construct was transfected in Hep3B and COS-7 cells. After molecular analysis it was observed that both minigenes reproduce the healthy control and patient cDNA’s splicing pattern. Therefore, both constructs were used to generate mice of the C57BL/6 strain expressing the human mutation c.234+1G>A in the liver and test its modified U1-mediated rescue in vivo. Wt or mutant minigenes were administrated in mice by hydrodynamic injection following a reported protocol(1). After 48 hours animals were sacrificed, the liver was collected and molecular analysis was performed. Preliminary results showed expression of the HGSNAT cDNA from the mutant construct in the liver of at least one animal. Thus, further tests will be carried out to optimize some limiting points, such as the administration of the minigenes (e.g. increase of injection volume from 7% to 8-9% of mice body weight; inclusion of an in vivo transfection reagent to enhance delivery efficiency) and the use of other mice strain. 1. Balestra D, et al. (2014) J Thromb Haemost 12(2):177–185.
- Aplicação da sequenciação de nova geração ao diagnóstico genético do cancro da mama hereditárioPublication . Theisen, Patrícia I.; Silva, Catarina; Pereira-Caetano, Iris; Rodrigues, Pedro; Isidro, Glória; Vieira, Luís; Gonçalves, JoãoIntrodução: O cancro da mama hereditário (CMH) constitui 5-10% dos casos de cancro da mama, dos quais cerca de 30% se devem a mutações germinais de elevada penetrância nos genes BRCA1 e BRCA2. Embora tenham sido identificadas mutações noutros genes de suscetibilidade para cancro da mama, estas são raras, pelo que a análise sequencial de múltiplos genes se torna ineficaz e dispendiosa pelos métodos convencionais(1,2). A determinação da causa genética subjacente ao CMH permite não só identificar os indivíduos com risco aumentado de cancro da mama, como oferecer uma medicina personalizada, mais eficaz na redução da incidência de cancro da mama assim como da morbilidade e mortalidade associadas(3). A sequenciação de nova geração (NGS) veio possibilitar a pesquisa de mutações em múltiplos genes em simultâneo, permitindo um diagnóstico molecular rápido, eficaz e com custo inferior ao da sequenciação de Sanger. Objetivos: Otimizar o diagnóstico molecular do CMH através da implementação de um protocolo de NGS baseado num painel abrangente de genes de suscetibilidade para cancro da mama. A 1ª fase de concretização deste objetivo consistiu em validar a utilização da NGS na deteção de mutações nos genes BRCA1, BRCA2 e TP53. Material e métodos: Foram analisadas por NGS 12 amostras de doentes com cancro da mama, previamente sequenciadas pelo método de Sanger para os genes BRCA1, BRCA2 e TP53. As bibliotecas de sequências-alvo foram preparadas a partir de DNA genómico pelo método de captura por hibridação, num protocolo que integrou o Trusight Cancer Sequencing Panel e o kit TruSight Rapid Capture (Illumina), e sequenciadas numa plataforma MiSeq com leituras paired-end de 150 bp. A análise bioinformática incluiu os softwares MiSeq Reporter, VariantStudio e Isaac Enrichment (Illumina). Resultados: Foram detetadas por NGS um total de 97 variantes de sequência nos genes BRCA1, BRCA2 e TP53, das quais 35 são variantes únicas (33 single nucleotide variants e 2 deleções), numa concordância de 100% com a sequenciação de Sanger. Conclusões: Estes resultados preliminares comprovam a eficiência da NGS na deteção de variantes em 3 genes de elevada penetrância para cancro da mama e abrem caminho para a oferta de um painel de genes de suscetibilidade para cancro da mama que permitirá um diagnóstico molecular do CMH mais abrangente, rápido e com custos reduzidos relativamente à sequenciação de Sanger. Bibiografia 1. Tung N, Batteli C, Allen B et al. (2015). Frequency of mutations in individuals with breast cancer referred for BRCA1 and BRCA2 testing using next-generation sequencing with a 25-gene panel. Cancer, 121: 25-33. 2. Apostolou P, Fostira F (2013). Hereditary breast cancer: the era of new susceptibility genes. BioMed Res Int, 2013: 747318. 3. Ellsworth R, Decewicz D, Shriver C, Ellsworth D (2010). Breast cancer in the personal genomics era. Curr Genomics, 11: 146-161.
- Are standard genotoxicity tests useful for the safety evaluation of nanomaterials?Publication . Louro, Henriqueta; Tavares, Ana; Vital, Nádia; Antunes, Susana; Costa, Pedro; Alverca, Elsa; Lavinha, João; Silva, Maria JoãoNanomaterials (NMs) are widely used in a diversity of consumer products, despite uncertainties surrounding the potential health risks that they pose to humans and the environment. One of the major concerns is the potential to induce cancer. To analyze in a short term the carcinogenic properties of a compound, genotoxicity assays in mammalian cell lines or animal models are frequently used. In the context of an EU Joint Action, in the present work we have used standard genotoxicity assays (comet, micronucleus and mutation assays) to investigate the effects associated with the exposure to titanium dioxide nanomaterials (TiO2), following standardized dispersion and assay procedures, in three types of human cells and in a mouse model. The results showed slight but significant increases in the frequencies of micronuclei after exposure to some of the NMs, as compared to controls. No clear dose-response relationships could be disclosed. One of the tested TiO2 yielded equivocal results in vitro micronucleus assay and was positive in the comet assay in pulmonary cells. In view of the inconclusive results,it was further analyzed in vivo, using the lacZ transgenic mouse model. It did not induce genotoxic effects in mice, 28 days after injection, despite the accumulation of the NM observed in the mouse liver. Regarding safety assessment, the different genotoxicity observed for closely related NMs, but that differ in some physicochemical characteristics, highlights the importance of investigating the toxic potential of each NM individually, instead of assuming a common mechanism and equal genotoxic effects for a set of similar NMs. The equivocal genotoxicity of the nanosized TiO2 in human cells in vitro was not confirmed in vivo, and therefore the predictive value of these in vitro tests for identifying genotoxic (and potentially carcinogenic) NMs in vivo should be clarified, before extrapolating the conclusions for human health.
- Assessment of ecologic and human health risks from a contaminated estuarine environmentPublication . Silva, M.J.; Pinto, M.; Caeiro, S.Due to their particular geographic characteristics, estuaries are often used for diverse human and industrial activities that may generate and release potentially harmful chemical contaminants. Thus, estuaries may be reservoirs of a wide variety of chemicals, including mutagenic and carcinogenic ones (e.g., metals, polycyclic aromatic hydrocarbons and pesticides), particularly when considering the potential for aquatic sediments to accumulate and continuously re-suspend contaminants into the water column. Importantly, besides being available to the local biota, the proximity/use of these contaminated sites might also be unsafe to the local population, provided that they are directly exposed (e.g. fishing, agricultural or recreational activities) or indirectly, through their diet. This work was focused on the river Sado Estuary, where the northern banks include urban areas and a heavy-industry park while the southern banks are mainly impacted by extensive agriculture activities. Sizable amounts of hazardous contaminants (e.g., metals, pesticides and polycyclic aromatic hydrocarbons) were identified in the estuarine sediments raising concern to the local populations because estuarine organisms and locally produced vegetables constitute the basis of their diet. The study intended to assess the adverse effects of estuary contaminants on human and ecosystem health through an integrative methodology based on several lines of evidence. An epidemiological survey was conducted to assess the exposure of a local population to the estuary environment as well as their health status. Furthermore, analyses of sediments, soil and wells water contamination were performed. Biological effects were assessed in major estuarine species with commercial value (clams, sole and cuttlefish); the cytotoxic and genotoxic potential of sediments were characterized in a human cell line. The results showed that the sediments’ contaminants were capable of inducing concentration-dependent cytotoxic and genotoxic damage in vitro and also allowed the distinction between two ecogeographical areas (urban/industrial and rural) with respect to type and intensity of the genetic damage, likely reflecting different patterns of toxicants mixtures. The integration of data from all study components indicated that the estuary is moderately impacted by complex mixtures of toxicants, affecting the aquatic biota and potentially able of inducing adverse effects on human cells. Although no clear evidence of contaminant-related adverse health effects in the studied population was found, the dose-related genotoxicity observed in the in vitro approach together with the evidences of direct and indirect human exposure indicates that those toxicants might constitute a risk factor to the development of chronic-degenerative diseases.
- Atrofias Musculares Espinhais: do estudo genético ao registo de doentesPublication . Oliveira, Jorge; Rodrigues, Luisa; Maia, Nuno; Oliveira, Marcia E.; Santos, RosárioA Atrofia Muscular Espinhal (AME) caracteriza-se pela degeneração das células do corno anterior da medula espinhal, resultando em fraqueza e atrofia muscular progressiva. O espectro clínico da doença é variável, desde formas graves de inicio neonatal (tipo I/Werdnig-Hoffman, MIM#253400) a fenótipos mais ligeiros com apresentação na idade adulta (tipo IV, MIM#271150). Considerando a frequência de portadores de AME na nossa população (1/52)1, estima-se que a incidência da doença em Portugal seja ~1/10800. A AME resulta de mutações no gene SMN1, no entanto foram já descritos outros genes (nomeadamente IGHMBP2, ATP7A, GARS, TRPV4 e PLEKHG5) associados a atrofias musculares espinhais mais raras. Desde 1994 foram estudados, na unidade de Genética Molecular, 549 doentes com suspeita clínica de AME. Foi confirmado o envolvimento do gene SMN1 em 223 doentes (40,6%) pertencentes a 219 famílias. Nestes doentes, o defeito genético mais frequente consiste na delecção em homozigotia do gene SMN1 (91,9%). Em 18 doentes (8,1%) foram identificadas mutações pontuais: c.346A>T (n=1), c.524delC (n=1) c.734dupC (n=1) e c.770_780dup (n=15). De forma a possibilitar o acesso de destes doentes a futuros ensaios clínicos, encontra-se em implementação o registo nacional de doentes com AME como previamente acordado com a SPEDNM e a rede internacional TREAT-NMD. Considerando o número de doentes com suspeita de AME sem confirmação molecular e a possível sobreposição fenotípica com outras patologias (distrofias, CMT e ALS), torna-se pertinente a re-avaliação clínica destes doentes. Esta permitirá o alargamento do estudo molecular a outros genes candidatos e eventualmente a identificação de novas causas genéticas para a AME recorrendo à análise genómica em larga escala.