Nanopore sequencing in human genetic studies: application to structural variant detection

Background: Nanopore sequencing is a recent technology which allows direct real-time sequencing of DNA or RNA molecules and production of read lengths as long as the size of the original fragments. It is based on the application of an ionic current to move DNA strands through an array of nanopores embedded in an electrically-resistant membrane. An ASIC chip placed below this membrane is responsible for recording signal changes in the nanopore, which are then translated into a specific base sequence. Although major improvements have been made in the genomic short-read sequencing technologies, structural variants (SV) detection still stands as a challenge. The characteristics of nanopore sequencing make it well suited for whole genome sequencing and as a preferential tool to identify SV, namely those associated with tumours. In this work we present a complete workflow to detect SV in tumour samples using nanopore sequencing. Methods: We used a test tumour sample to prepare DNA libraries using the rapid sequencing kit (Oxford Nanopore Technologies-ONT) and sequenced those in a single R9.4 flow cell on the MinION device (ONT). We implemented a pipeline for SV detection using multiple bioinformatics tools: MinKNOW software (ONT) (run set up and data acquisition), Albacore pipeline (ONT) (basecalling), LAST aligner (mapping) and Picky (SV detection and analysis). Results: The use of the rapid sequencing kit allowed a fast preparation of sequencing libraries when compared with standard library preparation procedures. The MinION generated a total of 2.34 Gb of DNA. The highest number of reads were obtained during the first 8-10h of the sequencing run. Overall, 87.8% of reads had a quality (Q) value >7 (quality threshold) and 54.2% had a Q value >10. The longest read obtained had 74369 bases and the mean read length was of 4508 bases. All reads with a length >50 kb had Q values >= 7. A total of 3470 SV were identified, including deletions, duplications, translocations, insertions and inversions. Conclusions: Long-read sequencing technology is becoming an important tool as it poses itself as a powerful complementary method that suppresses the inherent limitations of other technologies. Nanopore sequencing is a fast and sensitive approach of great potential for human genomics research and for use in a clinical perspective, namely in the detection of SV in the human genome. A critical issue in nanopore sequencing is DNA quality and integrity because longer size reads make it easier to detect large SV.This work was supported by Toxomics (Centre for Toxicogenomics and Human Health), Nova Medical School and is a result of the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT).info:eu-repo/semantics/publishedVersio

Study and development of custom “serious games” for patients and users

Video games are part of our culture and permeate several segments of society, from casual entertainment to serious purposes. This research work focuses on the latter, more specifically on the use of Serious Games in the healthcare context. In this sense, Serious Games have been researched through clinical studies, implemented to improve several conditions and have already proved to be efficient and valid, especially due to their ability to captivate and motivate the patient. With this in mind, this research explored the use of Serious Games for physical rehabilitation of stroke impaired patients and how this approach could be implemented in the Portuguese healthcare system. The research started with a thorough literature review of the validity and effectiveness of the use of Serious Games for this type of patients. Then, through surveys and interviews with healthcare professionals and game developers, the main barriers to the implementation and development of Serious Games for physical rehabilitation were accessed. Posteriorly, the feasibility of implementing a business model for this specific niche (in Portugal) was evaluated through a market analysis, abridging social, economic, political and technological aspects that culminated in the design of several hypothetical scenarios that envisioned the possibility of making this approach work. This research allowed to achieve an understanding of what needs to be overcome in order for this technique to be successful: Serious Games are still widely unknown among healthcare professionals and there is an underlying stigma against video games that clouds the possible benefits of using Serious Games. Moreover, the social context of future users will also determine their acceptance and compliance. Nevertheless, the research has evidenced that the implementation of this paradigm is economically viable as it would indirectly allow the monetization of rehabilitation therapies by making them available to a larger slice of the disabled population, which is of utmost importance, mainly during the times we currently live in.Os videojogos fazem parte da nossa cultura e permeiam vários segmentos da sociedade, desde entretenimento casual até propósitos mais sérios. Este trabalho de investigação concentra-se na segunda opção – mais especificamente, no uso de Serious Games no contexto da saúde. Neste sentido, os Serious Games têm sido investigados através de estudos clínicos, implementados para melhorar diversas condições e patologias e já provaram ser eficientes e válidos, principalmente graças à sua capacidade de motivar e cativar o paciente. Tendo isto em conta, esta investigação explorou o uso dos Serious Games para reabilitação física de pacientes pós-AVC e de que forma é que esta abordagem poderia ser implementada no Serviço Nacional de Saúde. A investigação começou com uma revisão bibliográfica completa da validade e eficácia do uso de Serious Games para este tipo de pacientes. De seguida, através de questionários e entrevistas a profissionais de saúde e desenvolvedores de videojogos, as principais barreiras para implementação e desenvolvimento de Serious Games para reabilitação física foram levantadas. Posteriormente, a viabilidade de executar um business plan para esse nicho específico (em Portugal) foi avaliada através de uma análise de mercado, abrangendo aspetos sociais, económicos, políticos e tecnológicos, que culminou no desenho de vários cenários hipotéticos que previam a possibilidade de fazer com que este paradigma se tornasse possível e funcional. Este trabalho de investigação permitiu atingir uma compreensão daquilo que necessita de ser ultrapassado para que esta abordagem tenha sucesso: os Serious Games ainda são amplamente desconhecidos entre os profissionais de saúde e existe um estigma subjacente aos videojogos que obscurece os possíveis benefícios do seu uso. Para além disso, o contexto social dos futuros utilizadores também irá determinar a sua aceitação e adesão. No entanto, esta investigação evidenciou que a implementação deste modelo é economicamente viável, pois iria permitir, de uma forma indireta, a rentabilização de terapias de reabilitação ao fazer com que estas fossem acessíveis a uma fatia mais larga da população debilitada, o que é de extrema importância, principalmente nos dias de hoje

EU Case Law Developments on Age Discrimination

The principle of non-discrimination on grounds of age has been declared an autonomous EU law principle by the European Court of Justice. This principle has been specified in a Directive, but its scope of application is currently limited to employment and occupational activities. The Directive protects both younger and older workers from being directly or indirectly discriminated due to their age. However, given the specificity of age as a factor of discrimination, the Directive allows the Member States to apply some derogations to this principle if, within the context of national law, they are objectively and reasonably justified by a legitimate aim. In the present contribution, we intend to analyse the Court’s application and interpretation of the principle of non-discrimination on grounds of age as established in the Directive and comment on some of the most relevant preliminary rulings. In many of these rulings, the Court was asked whether the Directive precludes national norms which establish a mandatory retirement age or foresee the termination of the employment contract when the worker reaches a certain age. The Court’s interpretation of the principle of non-discrimination on grounds of age, as established in the Directive, could contribute to easing some of the EU´s current economic, social and demographic challenges and to the improvement of European workers’ lives; however, the Court seems to accept Member States’ derogations to this principle too easily

The Cogeco Case: The First Preliminary Ruling on the Private Enforcement Directive

In March this year, the European Court of Justice (hereinafter “CJ”) answered the first preliminary question regarding the Private Enforcement Directive (“Directive”).1 One might expect this decision2 to remain relevant for the next few years, as it sheds some light on the rather intricate issue of the Directive’s temporal application. The CJ explains what rules are applicable to actions for damages regarding infringements which occurred prior either to the Directive’s adoption or to its implementation in the respective Member States. The case is also of major interest since it illustrates the role that the principle of effectiveness can play when applied alongside Articles 101 or 102 of the Treaty on the Functioning of the European Union (“TFEU”).3 Finally, albeit not expressly addressed, the case is also of interest regarding the controversial issue of parent company liability in private enforcement, where it represents a novelty in the Portuguese legal order

Determinants of households´ consumption in Portugal - a machine learning approach

Machine Learning has been widely adopted by researchers in several academic fields.Although at a slow pace, the field of economics has also started to acknowledge the pos-sibilities of these algorithm based methods for complementing or even replace traditionalEconometric approaches. This research aims to apply Machine Learning data-driven variable selection models for accessing the determinants of Portuguese households’ consumption using the Household Finance and Consumption Survey. I found that LASSO Regression and Elastic Net have the best performance in this setting and that wealth related variables have the highest impact on households’ consumption levels, followed by income, household’s characteristics and debt and consumption credit

Review: Playing at a Distance - Borderlands of Videogame Aesthetic

Sonia Fizek's Playing at Distance: Borderlands of Video Game Aesthetic navigates the intricate intersection of digital technology, aesthetics, and play in the context of video games, challenging conventional ideas of player agency. This critical recension delves into the book's contributions, highlighting its innovative exploration of mediated distance, interactivity, delegation, automation, ambience, spectacle, and the evolving nature of player engagement. While acknowledging its theoretical depth, this review emphasizes the book's significance in reshaping our understanding of video game aesthetics and the complex relationship between humans, machines, and digital play

O impacto das exigências emocionais no engagement : o papel do coping social como variável mediadora

Tese de mestrado integrado em Psicologia (Psicologia dos Recursos Humanos, do Trabalho e das Organizações), apresentada à Universidade de Lisboa através da Faculdade de Psicologia e de Ciências da Educação, 2008A presente investigação tem como objectivo estudar o trabalho emocional envolvido na relação dos bombeiros com os seus colegas de turno e o impacto deste no engagement destes profissionais, e ainda o papel do coping social como variável mediadora desta relação. Para isto, recolheram-se dados sobre as exigências emocionais da função, as estratégias de coping e o engagement através da aplicação de questionários a 417 bombeiros sapadores, num total de 29 turnos. Os resultados permitem-nos concluir que o engagement se associa positivamente com o recurso a estratégias de coping activoprosocial e com a expressão de emoções positivas; por outro lado, relaciona-se negativamente com a expressão de emoções negativas e a utilização de estratégias de coping activo antisocial e passivo. Para alem disto, verificou-se que o coping activo prosocial medeia totalmente a relação entre exigências emocionais negativas e o engagement, e possui um efeito de mediação parcial da relação deste com as exigências emocionais positivas.The present investigation claims to study the emotional work involved in the relationship between the firemen and their team mates and its impact on the engagement of these workers, and the role of social coping as a mediation variable in this relation. To do so, we collected data about the emotional demands of the function, coping strategies and engagement data from a sample of 417 professional firemen, in a total of 29 teams. The results obtained allow us to conclude that engagement is positively related to the use of active-prosocial coping strategies and the expression of positive emotions; on the other hand, it is negatively related to the expression of negative emotions and, active antisocial and passive coping strategies. In addition to these results, it was also found that the active-prosocial coping entirely mediates the relationship between negative emotional demands and engagement, and has a partial mediation effect between engagement and positive emotional demands

Mães e pais em reclusão: Diferenças e comunalidades no contacto com descendentes menores

Dissertação de mestrado integrado em Psicologia (área de especialização em Psicologia da Justiça)O aumento da população prisional, a nível mundial, traduziu-se num inequívoco acréscimo do número de pais e mães encarcerados. Comprovada a importância do contacto entre progenitores recluídos e seus filhos/as para o ajustamento psicológico de ambos, e dada a escassez de investigações nacionais a este nível, o presente estudo visou avaliar, primordialmente, a frequência de contacto estabelecida entre detentos e seus filhos menores, a qualidade da relação entre estes e o sentido de competência parental dos primeiros. A presença de diferenças entre os sexos, ao nível dos aspetos avaliados, foi igualmente aferida. Desta forma, administrou-se um questionário, constituído para os efeitos deste estudo, a 200 reclusos/as. Os resultados sugerem algumas diferenças entre homens e mulheres, bem como aspetos semelhantes para ambos os sexos. No que concerne às diferenças, talvez essas sejam o reflexo da sociedade atual em que a mãe, apesar de tudo, continua a ter o papel de cuidadora emocional e o pai, mais distante emocionalmente, terá encargos relacionados com a subsistência financeira da família.The increase in worldwide population in prison has resulted in an unequivocal growth of the number of incarcerated parents. Given the importance of the contact between incarcerated parents and their children to the psychological adjustment of both of them, and in face of the scarcity of national studies at this level, the present study aimed to assess the frequency of the contact established between prisoners and their minor children, the quality of the relationship between them, and the sense of parental competence of the incarcerated mothers and fathers. This study also assessed the presence of differences between sexes regarding the studied issues. A questionnaire composed for the purposes of this study was administered to 200 inmates. Results suggest some differences between men and women, as well as similar aspects for both sexes. Those differences are, probably, the reflection of today's society in which the mother continues to interpret the role of the emotional caregiver and the father, more emotionally distant, has occupations related to the financial maintenance of the family

Sistemas compartimentais

Mestrado em Matemática e AplicaçõesNesta tese estuda-se sistemas dinâmicos compartimentais contínuos e discretos. Partindo do modelo utilizado para os sistemas compartimentais contínuos, aplica-se dois processos distintos de discretização (exacta e aproximada) de forma a obter os sistemas compartimentais discretos correspondentes, comparando-se o comportamento destes sistemas em relação ao do sistema contínuo inicial. Estuda-se ainda o problema do controlo da massa tanto no caso contínuo como no discreto, assim como no contexto da discretização de sistemas contínuos.In this work we study continuous- and discrete-time compartmental systems. Starting from a continuous-time compartmental model, we apply two different discretization methods, namely exact and approximate discretization, and compare the behaviour of the two discretized models when compared to the original continuous-time model. A study of the mass control problem both in the continuous- and the discrete-time case, as well as in the context of discretization of continuous-time systems, is also performed

NGS Panels applied to Hereditary Cancer Syndromes

Cancer is among the leading causes of morbidity and mortality worldwide (Okur et al, 2017). Germline pathogenic variants for monogenic, highly penetrant cancer susceptibility genes are observed in 5%–10% of all cancers (Lu et al, 2014). Hereditary cancers due to monogenic causes are characterized by earlier age of onset, other associated cancers, and often a family history of specific cancers. From the clinical perspective, it is important to recognize the affected individuals to provide them the best clinical management (Hennessy et al, 2010; Ledermann et al, 2014; Pennington et al, 2014) and to identify at-risk family members who will benefit from predictive genetic testing and enhanced surveillance, including early detection and/or risk reduction measures (Kurian et al, 2010; Okur et al, 2017). Germline variants identified in major cancer susceptibility genes associated with hereditary breast or ovarian cancer (HBOC) or hereditary colorectal cancer (HCRC), also account for 5-10% of the patients with these cancers. In the last years, new susceptibility genes, with different penetrance degrees, have been identified. Variants in any of those genes are rare and classical methodologies (e.g. Sanger sequencing - SS) are time consuming and expensive. Next-generation sequencing (NGS) has several advantages compared to SS, including the simultaneous analysis of many samples and sequencing of a large set of genes, higher sensitivity (down to 1% vs 15-20% in SS), lower cost and faster turnaround time, reasons that make NGS the best approach for molecular diagnosis. It is possible nowadays to choose between whole-genome sequencing (WGS), whole-exome sequencing (WES) and NGS limited to a set of genes (NGS-Panel). In cases where a suspected genetic disease or condition has been identified, targeted sequencing of specific genes or genomic regions is preferred (Grada et al, 2013). For that reason, we use NGS-Panel approach using TruSight Cancer (Illumina) to sequence DNA extracted from blood samples of patients with personal and/or familiar history of cancer. This hereditary cancer gene panel sequences 94 genes associated with both common (e.g., breast, colorectal) and rare hereditary cancers and allows the creation of virtual gene panels according to each phenotype or disease under study. NGS workflow analysis (Figure 1) includes five steps: quality assessment of raw data, read alignment to a reference genome, variant identification/calling, variant annotation and data visualization (Pabinger et al, 2013). The establishment of the most appropriate bioinformatics pipeline is crucial in order to achieve the best results. NGS data allows the identification of several types of variants like single nucleotide variants (SNVs), small insertions/deletions, inversions and also copy number variants (CNVs).FCT - UID/BIM/0009/2016info:eu-repo/semantics/publishedVersio