Tratamento de dados de NGS para pesquisa de novas mutações associadas à miocardiopatia hipertrófica

Tese de mestrado em Bioestatística, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2012A Sequenciação de Nova Geração (NGS - Next Generation Sequencing) está a revolucionar a investigação na área da biomédica, contribuindo significativamente para o avanço da medicina personalizada. A NGS, apoiando-se nos conhecimentos da Estatística Bayesiana e da Bioinformática para a análise e tratamento dos dados que esta técnica origina, torna-se num excelente exemplo dos novos campos multidisciplinares emergentes da Ciência. As novas potencialidades da NGS que a tornam tão atractiva para tantos novos projectos de investigação são a sua capacidade de misturar diversas amostras numa só leitura, com recurso a um sistema de código de barras, diminuindo custos e aumentando a rapidez da obtenção das amostras. Com o intuito de descobrir potenciais novas mutações associadas à Miocardiopatia Hipertrófica, estudou-se uma coorte de indivíduos que possuiam o diagnóstico clínico da doença, mas que, no entanto, não apresentavam qualquer mutação exónica patogénica. Tornam-se claras as vantagens desta nova metodologia, pois trazendo mais rapidez e permitindo uma análise a um maior número de genes, com custos reduzidos, torna possível a análise de todos os genes com associação conhecida e descrita na literatura. Neste projecto, obtiveram-se resultados positivos em relação a muitos genes já conhecidos que pela sequenciação padrão não tinham sido sequenciados ou detectados. A NGS demonstrou ter todo o potencial para tornar-se o novo método padrão de diagnóstico, sendo, por isso, necessário continuar aperfeiçoar e a melhorar a metodologias de tratamento e análise de dados provenientes desta nova técnica. Foram descobertos sete genes (CAV3, GLA, LDB3, MYLK2, MYOZ2, PRKAG2 e VCL) que ainda não foram oficialmente associados à patologia, sendo que na literatura apenas são referidas mutações a nível exónico. Apenas o gene ANKRD1 detectado com alterações já foi descrito com mutações exónicas e intrónicas. O gene CAV3 surge como associado à doença oficialmente em 2010, num artigo de revisão.The Next-Generation Sequencing (NGS) is revolutionizing biomedical research, signi ficantly contributing to the enrichment of health sciences' field, inclusively towards a personalized medicine. NGS takes advantage of Bayesian Statistics and Bioinformatics knowledge, in order to analyse and process data, which is originated by this technique. NGS is an excellent example of the new multidisciplinary fields of Sciences. The innovative NGS's features, which make this technique so attractive for so many different research projects , are the possibility of mix several samples in just one read, using an associated barcode, which can reduce costs. Obtaining samples is quicker than with the standard method. With the purpose of discover unknown mutations associated with Hypertrophic Cardiomyopathy, we studied a group of individuals, which, in spite of presenting the disease's symptoms, did not have any pathogenic exonic mutation with the standard method of sequentiation. The advantages of this new methodology are quite clear. Accomplishing faster results and allowing analysis of a larger number of genes, with reduced costs, which allows to diagnose the patient concerning all genes described in the literature. In this project, we obtained positive results for many genes already known that were not sequenced or not detected by standard sequencing method. The NGS shown to have the necessary potential to become the new standard diagnostic method, and is therefore imperative to further re- fine and improve methods of treatment and analysis of data originated by this technique. In our project, we found seven genes (CAV3, GLA, LDB3, MYLK2, MYOZ2, PRKAG2 and VCL) which were not officially associated with the disease yet and in the literature only exonic mutations were described. Only ANKRD1 gene was detected with both exonic and intronic mutations. CAV3 gene appears in 2010 as officially associated with thedisease, in a review article

Development of a stream flood susceptibility index at the municipal level in mainland Portugal

The growing computational capabilities and data availability promotes the development of flood susceptibility methodologies, although there is still field for improvement regarding small-scale flood susceptibility assessment. In this research, after assessing a national stream flood susceptibility (SFS) on a cell-by-cell basis, a municipal representation of SFS was performed to rank the 278 municipalities in mainland Portugal.info:eu-repo/semantics/publishedVersio

A flood susceptibility model at the national scale based on multicriteria analysis

River flooding is a specific worldwide type of flooding responsible for considerable human and material losses. An extensive knowledge about flood conditioning factors and a diverse set of methodologies for flood susceptibility evaluations are available, although there is still field for improvement regarding methodologies for small-scale flood susceptibility assessment, particularly relevant in data-scarce contexts. This research applied to mainland Portugal, introduces a multicriteria methodology to assess flood susceptibility at national scale considering three flood-conditioning factors: flow accumulation, average slope angle and average relative permeability. These three factors resume other factors usually considered in literature, related to morphology and potential runoff. This work includes data from the flood conditioning factors considering the cumulative role of the entire contributive area and not only the on-site characteristics. The weight of each factor was assigned based on expert opinion and validated using available flood damages databases with >150 years of records. From the several tested flood susceptibility models, the one that best fits the historical records was chosen, which corresponds also to a more valued role of flow accumulation factor. Results provide an accurate differentiation of transboundary, regional and local rivers. The scores of stream flood susceptibility were later transformed to a single value per each of the 278 municipalities of mainland Portugal. Representing the natural susceptibility to river flooding, these results can be cross-analyzed with structural mitigation measures, spatial planning instruments, exposure and vulnerability data along the respective floodplains, in order to identify water streams that require a more detailed and concerned future intervention and an exhaustive susceptibility study at the local scale.info:eu-repo/semantics/publishedVersio

Sistema de promoção e proteção de crianças e jovens expostos à violência interparental: perceções dos técnicos das CPCJ

A exposição de crianças e jovens à violência interparental é uma problemática que tem merecido cada vez mais atenção tanto por parte da literatura, perdendo-se a conta aos estudos já realizados sobre as consequências biopsicossociais a curto, médio e longo prazo, tanto pelo Legislador, com a inclusão dos menores que testemunham um contexto familiar violento no Estatuto de Vítima, como ainda, mais recentemente, por parte do Estado Português, através da aprovação de procedimentos específicos de intervenção das Comissões de Proteção de Crianças e Jovens (CPCJ) e da Rede Nacional de Apoio às Vítimas de Violência Doméstica com menores. Não obstante, parece haver ainda muitas cifras negras que envolvem este crime público, bem como constrangimentos na organização, recursos humanos, intervenção sistémica e apoios num Sistema que devia ser protetor e contentor do impacto da violência interparental. Por este motivo, a presente investigação pretende conhecer o Sistema de Promoção e Proteção a partir de dentro, dando voz aos profissionais que aplicam a Lei e gerem os processos de cada menor desde a sinalização, avaliação do risco ou perigo, deliberação das medidas de promoção e proteção, acompanhamento do cumprimento do acordo até ao seu arquivamento. Trata-se, portanto, de um estudo qualitativo e exploratório, com dados recolhidos através de entrevistas semiestruturadas aplicadas a dez técnicos de diferentes CPCJ, de Norte a Sul de Portugal, selecionadas aleatoriamente, com vista a responder à questão-problema: Serão os procedimentos de sinalização, aplicação das medidas de promoção e proteção e recursos no acompanhamento da sua execução suficientes e eficazes na proteção de crianças e jovens expostos à violência interparental? Através da análise do conteúdo das entrevistas realizadas, é possível perceber que o tempo de afetação de cada técnico à CPCJ, o parco número de técnicos em Comissões com grande volume processual, a carência de formação especializada na área, as dificuldades do trabalho em rede com entidades de primeira linha e a diminuta prevenção por parte das Entidades com Competência em Matéria de Infância e Juventude, demonstram ser um obstáculo na intervenção. Tais resultados podem explicar eventuais falhas na proteção e promoção do interesse superior da criança e dos seus direitos, especificamente nos instrumentos e periodicidade da supervisão do cumprimento do Acordo estabelecido entre o técnico gestor de caso e os progenitores e/ou menor, nas sucessivas revisões das medidas de promoção e proteção que arrastam a intervenção devido à incapacidade do técnico acompanhar múltiplos processos ao mesmo tempo e nas dificuldades de articulação com as entidades que devem dar continuidade à intervenção da CPCJ.The exposure of children and young people to interparental violence is an issue that has received increasing attention from literature given by, the numerous studies already carried out on the biopsychosocial consequences in the short, medium and long term, the legislator, through the inclusion of the minors who witness a violent family context in the Victim Statute, and, more recently, the Portuguese State, by approving specific intervention procedures by the Commissions for the Protection of Children and Youth (CPCJ) and by the National Support Network for Victims of Domestic Violence with Minors. Nevertheless, there still seem to be many obstacles that involve this specific public crime, as well as constraints on organization, human resources, systemic intervention and support, in a System that should protect and contain the impact of interparental violence. For this reason, the present research aims to understand the Promotion and Protection System from within, giving voice to the professionals who apply the Law and manage the processes of each minor, from signaling, risk or danger assessment, deliberation of promotion and protection measures, and monitoring the agreement made between the family and the organization, to check if it is being maintained until its archiving. Therefore, this is a qualitative and exploratory study, with data collected through semi-structured interviews applied to ten technicians from different CPCJ, from North to South of Portugal, randomly selected, in order to answer the question: Are the signaling procedures, the application of promotion and protection measures and the resources in monitoring their execution, sufficient and effective in protecting children and young people exposed to interparental violence? Through the analysis of the content of the interviews carried out, it is possible to understand that the time of assignment of each technician to the CPCJ, the small number of technicians in Commissions with large cases volume, the lack of specialized training in the area, the difficulties of networking with first-line entities and the low level of prevention by the Entities with Competence in the Matter of Children and Youth, prove to be an obstacle regarding the intervention. Results may explain any failures in the protection and promotion of the best interests of the child and their rights, specifically in the instruments and frequency of the supervision of the compliance with the Agreement established between the technical case manager and the parents and/or minor, in the successive revisions of the promotion and protection measures that drag the intervention due to the technician's inability to follow multiple processes at the same time, and the difficulties of articulation with the entities that must continue the intervention of the CPCJ

Avaliação da suscetibilidade das linhas de água a cheias utilizando análise multicritério

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Aplicação exploratória de análise multicritério para representação municipal da susceptibilidade a cheias rápidas em Portugal continental

[...] Este estudo descreve uma metodologia exploratória de avaliação de susceptibilidade a cheias rápidas para Portugal Continental, adotando uma abordagem multicritério, a partir de um conjunto de 9 fatores condicionantes, definidos ao município: escoamento acumulado, hierarquia fluvial de Strahler, rugosidade, declive, depósitos aluvionares, grau de impermeabilização, litologia de elevada permeabilidade, Landform Classification e escoamento anual. A atribuição do peso de cada fator baseou-se em Análise Hierárquica de Processos (AHP) [...]info:eu-repo/semantics/publishedVersio

A new ensemble coevolution system for detecting HIV-1 protein coevolution

Background: A key challenge in the field of HIV-1 protein evolution is the identification of coevolving amino acids at the molecular level. In the past decades, many sequence-based methods have been designed to detect position-specific coevolution within and between different proteins. However, an ensemble coevolution system that integrates different methods to improve the detection of HIV-1 protein coevolution has not been developed. Results: We integrated 27 sequence-based prediction methods published between 2004 and 2013 into an ensemble coevolution system. This system allowed combinations of different sequence-based methods for coevolution predictions. Using HIV-1 protein structures and experimental data, we evaluated the performance of individual and combined sequence-based methods in the prediction of HIV-1 intra- and inter-protein coevolution. We showed that sequence-based methods clustered according to their methodology, and a combination of four methods outperformed any of the 27 individual methods. This four-method combination estimated that HIV-1 intra-protein coevolving positions were mainly located in functional domains and physically contacted with each other in the protein tertiary structures. In the analysis of HIV-1 inter-protein coevolving positions between Gag and protease, protease drug resistance positions near the active site mostly coevolved with Gag cleavage positions (V128, S373-T375, A431, F448-P453) and Gag C-terminal positions (S489-Q500) under selective pressure of protease inhibitors. Conclusions: This study presents a new ensemble coevolution system which detects position-specific coevolution using combinations of 27 different sequence-based methods. Our findings highlight key coevolving residues within HIV-1 structural proteins and between Gag and protease, shedding light on HIV-1 intra- and inter-protein coevolution. Reviewers: This article was reviewed by Dr. Zoltán Gáspári. © Li et al

Assessing transmissibility of HIV-1 drug resistance mutations from treated and from drug-naive individuals (AIDS (2015) 29 (2045-2052))

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Low Serum Levels of DKK2 Predict Incident Low-Impact Fracture in Older Women

There are currently no robust noninvasive markers of fragility fractures. Secreted frizzled related protein-1 (sFRP-1), dickkopf-related protein 1 (DKK1) and DKK2, and sclerostin (SOST) inhibit Wnt signaling and interfere with osteoblast-mediated bone formation. We evaluated associations of serum levels of sFRP-1, DKK1, DKK2, and SOST with incident low-impact fracture and BMD in 828 women aged ≥65 years from EpiDoC, a longitudinal population-based cohort. A structured questionnaire during a baseline clinical appointment assessed prevalent fragility fractures and clinical risk factors (CRFs) for fracture. Blood was collected to measure serum levels of bone turnover markers and Wnt regulators. Lumbar spine and hip BMD were determined by DXA scanning. Follow-up assessment was performed through a phone interview; incident fragility fracture was defined by any new self-reported low-impact fracture. Multivariate Cox proportional hazard models were used to analyze fracture risk adjusted for CRFs and BMD. During a mean follow-up of 2.3 ± 1.0 years, 62 low-impact fractures were sustained in 58 women. A low serum DKK2 level (per 1 SD decrease) was associated with a 1.5-fold increase in fracture risk independently of BMD and CRFs. Women in the two lowest DKK2 quartiles had a fracture incidence rate of 32 per 1000 person-years, whereas women in the two highest quartiles had 14 fragility fractures per 1000 person-years. A high serum sFRP1 level was associated with a 1.6-fold increase in fracture risk adjusted for CRFs, but not independently of BMD. Serum levels of SOST (r = 0.191; p = 0.0025) and DKK1(r = -0.1725; p = 0.011) were correlated with hip BMD, but not with incident fragility fracture. These results indicate that serum DKK2 and sFRP1 may predict low-impact fracture. The low number of incident fractures recorded is a limitation and serum levels of Wnt regulators should be further studied in other populations as potential noninvasive markers of fragility fractures. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.publishersversionpublishe

Perfis municipais de risco a cheias e inundações para aplicação ao ordenamento do território em Portugal continental

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