Protein targeting to the plasma membrane of adult skeletal muscle fiber: An organized mosaic of functional domains

The plasma membrane of differentiated skeletal muscle fibers comprises the sarcolemma, the transverse (T) tubule network, and the neuromuscular and muscle-tendon junctions. We analyzed the organization of these domains in relation to defined surface markers, beta -dystroglycan, dystrophin, and caveolin-3, These markers were shown to exhibit highly organized arrays along the length of the fiber. Caveolin-3 and beta -dystroglycan/dystrophin showed distinct, but to some extent overlapping, labeling patterns and both markers left transverse tubule openings clear. This labeling pattern revealed microdomains over the entire plasma membrane with the exception of the neuromuscular and muscle-tendon junctions which formed distinct demarcated macrodomains. Our results suggest that the entire plasma membrane of mature muscle comprises a mosaic of T tubule domains together with sareolemmal caveolae and beta -dystroglycan domains. The domains identified with these markers were examined with respect to targeting of viral proteins and other expressed domain-specific markers, We found that each marker protein was targeted to distinct microdomains, The macrodomains were intensely labeled with all our markers. Replacing the cytoplasmic tail of the vesicular stomatitis virus glycoprotein with that of CD4 resulted in retargeting from one domain to another. The domain-specific protein distribution at the muscle cell surface may be generated by targeting pathways requiring specific sorting information but this trafficking is different from the conventional apical-basolateral division. (C) 2001 Academic Press.

Reabilitação energética de um edifício de serviços

Trabalho Final de Mestrado para obtenção do grau de Mestre em Engenharia Civil na Área de Especialização de Edificações

Quality in Hospital Administrative Databases

The clinical content of administrative databases includes, among others, patient demographic characteristics, and codes for diagnoses and procedures. The data in these databases is standardized, clearly defined, readily available, less expensive than collected by other means, and normally covers hospitalizations in entire geographic areas. Although with some limitations, this data is often used to evaluate the quality of healthcare. Under these circumstances, the quality of the data, for instance, errors, or it completeness, is of central importance and should never be ignored. Both the minimization of data quality problems and a deep knowledge about this data (e.g., how to select a patient group) are important for users in order to trust and to correctly interpret results. In this paper we present, discuss and give some recommendations for some problems found in these administrative databases. We also present a simple tool that can be used to screen the quality of data through the use of domain specific data quality indicators. These indicators can significantly contribute to better data, to give steps towards a continuous increase of data quality and, certainly, to better informed decision-making.

Software languages engineering: experimental evaluation

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Engenharia Informática

Automatização de testes à framework myMIS

Atualmente, verifica-se um aumento na necessidade de software feito à medida do cliente, que se consiga adaptar de forma rápida as constantes mudanças da sua área de negócio. Cada cliente tem os seus problemas concretos que precisa de resolver, não lhe sendo muitas vezes possível dispensar uma elevada quantidade de recursos para atingir os fins pretendidos. De forma a dar resposta a estes problemas surgiram várias arquiteturas e metodologias de desenvolvimento de software, que permitem o desenvolvimento ágil de aplicações altamente configuráveis, que podem ser personalizadas por qualquer utilizador das mesmas. Este dinamismo, trazido para as aplicações sobre a forma de modelos que são personalizados pelos utilizadores e interpretados por uma plataforma genérica, cria maiores desafios no momento de realizar testes, visto existir um número de variáveis consideravelmente maior que numa aplicação com uma arquitetura tradicional. É necessário, em todos os momentos, garantir a integridade de todos os modelos, bem como da plataforma responsável pela sua interpretação, sem ser necessário o desenvolvimento constante de aplicações para suportar os testes sobre os diferentes modelos. Esta tese debruça-se sobre uma aplicação, a plataforma myMIS, que permite a interpretação de modelos orientados à gestão, escritos numa linguagem específica de domínio, sendo realizada a avaliação do estado atual e definida uma proposta de práticas de testes a aplicar no desenvolvimento da mesma. A proposta resultante desta tese permitiu verificar que, apesar das dificuldades inerentes à arquitetura da aplicação, o desenvolvimento de testes de uma forma genérica é possível, podendo as mesmas lógicas ser utilizadas para o teste de diversos modelos distintos.

Supercondensadores

Uma nova tecnologia, os EDLC, também denominados por supercondensadores, tem-se tornado numa importante e aliciante área de interesse. Estes regem-se pelos mesmos princípios fundamentais dos condensadores clássicos, no entanto possibilitam receber capacidades superiores, devido a uma maior área de superfície e a um dielétrico menos espesso. Esta particularidade permite obter uma maior densidade energética, comparativamente com os condensadores clássicos e uma maior densidade de potência, comparativamente com as baterias. Consequentemente a utilização de supercondensadores tem aumentado, representando já uma alternativa fiável, segura e amiga do ambiente, em detrimento das baterias comuns. Assim, este projeto tem como principais objetivos, identificar os diferentes tipos de supercondensadores, apresentar as vantagens de cada tipo e explorar a sua resposta, quer no domínio das frequências quer no domínio dos tempos, e por fim modelá-los recorrendo a componentes elétricos clássicos, nomeadamente resistências e condensadores. A modelação foi realizada recorrendo ao MALTAB, através da função de minimização fminunc e foram construídos quatro modelos equivalentes, com o objetivo de modelar a resposta dos vários EDLC analisados. Por escassez de tempo o principal foco de análise recaiu sobre o EDLC de 0,022 F.

A framework for supporting knowledge representation – an ontological based approach

Dissertação para obtenção do Grau de Mestre em Engenharia Electrotécnica e de Computadores

Linked Data based Health Information Representation, Visualization and Retrieval System on the Semantic Web

Dissertation submitted in partial fulfillment of the requirements for the Degree of Master of Science in Geospatial Technologies.

Derivation and consistency checking of models in early software product line engineering

Dissertação para obtenção do Grau de Doutor em Engenharia Informática

Expressiveness improvements of OutSystems DSL query primitives

Dissertação para obtenção do Grau de Mestre em Engenharia Informática

Família de DSLs para integrated modular avionics

Na atualidade existe a necessidade de produzir novos aviões de forma rápida, eficiente e económica com o objetivo de abrir novas rotas de voo, expansão das já existentes e substituição de aviões em fim de vida útil. Neste contexto, e sem nunca deixar de cumprir os apertados requisitos do domínio que incluem a exigência de elevada qualidade, a indústria adotou a arquitetura IMA que permite executar várias aplicações aviónicas num único sistema de computação partilhado. Com esta arquitetura, o desenvolvimento do software para as aeronaves ganhou uma elevada relevância, sendo necessário gerar código automaticamente, reutilizar código já testado proveniente de outras aplicações anteriormente desenvolvidas e garantir o mais cedo possível que o software desenvolvido se encontra de acordo com os standards. Apesar da complexidade do domínio, desenvolveu-se uma ferramenta que permite produzir a estrutura do código de novas aplicações para a aviónica. Aferramenta lida com a variabilidade das diversas linhas de produto e reduz o tempo de desenvolvimento. Uma DSL poderia ser uma solução apropriada, pois permite cobrir diversos requisitos exigidos, no entanto, esta solução não é exequível porque seria necessário produzir uma linguagem para cada configuração de software aviónico diferente se pretendêssemos tirar partido da especificidade. Com esta dissertação, solucionou-se esta dificuldade com recurso à noção de família de DSLs. Trata-se de um conjunto de linguagens para um domínio específico, que apresentam um conjunto comum de conceitos chave, mas que adaptam alguns desses conceitos para cumprir a variabilidade dos requisitos. Utilizou-se a abordagem MDD para desenvolver um gerador automático de DSLs que é capaz de produzir a linguagem desejada de acordo com a configuração de software pretendida para a partição pertencente a um módulo aviónico. As linguagens geradas apresentam um nível de usabilidade adequada para o domínio, bem como têm a capacidade de validar as construções efetuadas usando a DSL e produzir os artefactos pretendidos.

Incremental compilation and deployment for OutSystems Platform

OutSystems Platform is used to develop, deploy, and maintain enterprise web an mobile web applications. Applications are developed through a visual domain specific language, in an integrated development environment, and compiled to a standard stack of web technologies. In the platform’s core, there is a compiler and a deployment service that transform the visual model into a running web application. As applications grow, compilation and deployment times increase as well, impacting the developer’s productivity. In the previous model, a full application was the only compilation and deployment unit. When the developer published an application, even if he only changed a very small aspect of it, the application would be fully compiled and deployed. Our goal is to reduce compilation and deployment times for the most common use case, in which the developer performs small changes to an application before compiling and deploying it. We modified the OutSystems Platform to support a new incremental compilation and deployment model that reuses previous computations as much as possible in order to improve performance. In our approach, the full application is broken down into smaller compilation and deployment units, increasing what can be cached and reused. We also observed that this finer model would benefit from a parallel execution model. Hereby, we created a task driven Scheduler that executes compilation and deployment tasks in parallel. Our benchmarks show a substantial improvement of the compilation and deployment process times for the aforementioned development scenario.

Knowledge management for systems biology a general and visually driven framework applied to translational medicine

Background: To enhance our understanding of complex biological systems like diseases we need to put all of the available data into context and use this to detect relations, pattern and rules which allow predictive hypotheses to be defined. Life science has become a data rich science with information about the behaviour of millions of entities like genes, chemical compounds, diseases, cell types and organs, which are organised in many different databases and/or spread throughout the literature. Existing knowledge such as genotype - phenotype relations or signal transduction pathways must be semantically integrated and dynamically organised into structured networks that are connected with clinical and experimental data. Different approaches to this challenge exist but so far none has proven entirely satisfactory. Results: To address this challenge we previously developed a generic knowledge management framework, BioXM™, which allows the dynamic, graphic generation of domain specific knowledge representation models based on specific objects and their relations supporting annotations and ontologies. Here we demonstrate the utility of BioXM for knowledge management in systems biology as part of the EU FP6 BioBridge project on translational approaches to chronic diseases. From clinical and experimental data, text-mining results and public databases we generate a chronic obstructive pulmonary disease (COPD) knowledge base and demonstrate its use by mining specific molecular networks together with integrated clinical and experimental data. Conclusions: We generate the first semantically integrated COPD specific public knowledge base and find that for the integration of clinical and experimental data with pre-existing knowledge the configuration based set-up enabled by BioXM reduced implementation time and effort for the knowledge base compared to similar systems implemented as classical software development projects. The knowledgebase enables the retrieval of sub-networks including protein-protein interaction, pathway, gene - disease and gene - compound data which are used for subsequent data analysis, modelling and simulation. Pre-structured queries and reports enhance usability; establishing their use in everyday clinical settings requires further simplification with a browser based interface which is currently under development.

Summarization of specialized discourse: the case of medical articles in Spanish

In this article, we present the current state of our work on a linguistically-motivated model for automatic summarization of medical articles in Spanish. The model takes into account the results of an empirical study which reveals that, on the one hand, domain-specific summarization criteria can often be derived from the summaries of domain specialists, and, on the other hand, adequate summarization strategies must be multidimensional, i.e., cover various types of linguistic clues. We take into account the textual, lexical, discursive, syntactic and communicative dimensions. This is novel in the field of summarization. The experiments carried out so far indicate that our model is suitable to provide high quality summarizations.

Lats2, a novel regulator of Elongin BC ubiquitin ligase

Résumé : Le Large tumor suppressor, Lats2, est une protéine humaine homologue au suppresseur de tumeur Warts (Lats) de Drosophila melanogaster, qui réprime la prolifération des cellules en altérant leur cycle au niveau des transitions Gl/S et G2/M, et en induisant l'apoptose. Pourtant, la voie moléculaire par laquelle Lats2, une sériase-thréonine kinase, déclenche l'arrêt du cycle cellulaire, est toujours inconnue. Notre équipe a d'abord déterminé que Lats2 était un gène de réponse à la protéine p53 (Kostic et al., 2000). Par la suite, nous avons identifié des protéines interagissant avec Lats2, notamment les modules de reconnaissance du substrat des ligases Colline E3 (des protéines contenant Socs box ou F box) ainsi que deux Bous-unités du Signalosome CSN: CSN4 et CSNS. En outre, Lats2 est connue pour s'associer au Super-complexe composé de CSN et des ligases Colline E3 (Rongere, thesis, 2004; Rongere, unpublished results, 2005). Le travail présenté ici sur Lats2 a confirmé que cette protéine est une kinase associée à CSN. Nous avons caractérisé les interactions spécifiques de domaines de Lats2 avec hSocs3, hWsb 1 (des protéines Socs box) et hFBX-7 (une protéine F box), ainsi que les conséquences physiologiques des interactions avec hSocs3, hWsb1 et hSocs1. Des expériences de GST pull-down ont montré que les deux domaines, N-terminal et kinase, de Lats2 interagissent avec hSocs3, hWsb1 et hFBX-7, ce qui suggère aussi que l'ensemble de la protéine Lats2 est impliqué dans ces interactions. Une étude approfondie des interactions entre Lats2 et hSocs3 indique que le domaine kinase de Lats2 interagit avec la région de hSocs3 contenant un domaine SH2, situé en amont du domaine Socs box de hSocs3. Par ailleurs, Lats2 phosphoryle des régions spécifiques entre les domaines N-terminal et SH2 (Sl), et, entre les domaines SH2 et Socs box (S3) de la protéine hSocs3. Ces résultats révèlent que hSocs3 est un.nouveau substrat de Lats2. Des modifications de l'activité kinase ont aussi révélé que la protéine sauvage Lats2 (wt Lats2) était capable de phosphoryler hSocs3, alors qu'un mutant dead du domaine kinase Lats (poche ATP délétée, Lats2OATP) non. L'analyse des mutations a permis d'identifier deux résidus sériase situés aux positions 1441145 (S3), spécifiquement phosphorylés par wt Lats2. La phosphorylation des protéines représentant un signal de dégradation protéolytique, nous avons envisagé que Lats2 pouvait cibler hSocs3 pour une dégradation protéasomale. Lorsque wt Lats2 est surexprimée dans des cellules HEK293T et COS7, la demi-vie de hSocs3, un élément de la ligase Elongine BC-Colline É3 (ligase EBC), diminue significativement, effet que n'a pas la surexpression de Lats2OATP. De plus, la stabilité de hSocs3 dépend de la phosphorylation des résidus sériase aux positions 144/145 par wt Lats2. Bien que les sites de phosphorylation ne soient pas définis pour les deux autres modules de reconnaissance du substrat de la ligase EBC: hWsb 1 et hSocsl, leurs demi-vies diminuent également quand wt Lats2 est surexprimée. Pour les tests in vivo, nous avons synthétisé des esiRNA pour diminuer l'expression du gène endogène lats2, ce qui a entraîné une augmentation d'un facteur 2 de la demi-vie de hSocs3 et de hWsbl dans les cellules HEK293T. En conclusion, nos résultats suggérent que Lats2, une kinase associée au CSN, est un nouveau régulateur de la fonction des ligases EBC, agissant sur le renouvellement des protéines hSocs3, hSocs1 et hWsb1. Ainsi, Lats2 altère la spécificité et la capacité des ligases EBC, régulant par là même la stabilité de nombreuses protéines, ciblées par les ligases EBC pour une dégradation protéasomale. D'autres études devraient révéler si la modification observée de la fonction de la ligase EBC par Lats2, associée au Super-complexe, est également responsable du renouvellement des régulateurs du cycle cellulaire et des changements dans ce même cycle observés lors de la surexpression de Lats2. Summary : The Large tumor suppressor 2 (Lats2) is a human homologue of the Drosophila melanogaster tumor suppressor Warts (Cats) who negatively regulates cell proliferation by altering cell cycle Gl/S and G2/M transition and inducing apoptosis. However, the molecular pathway by which Lats2, a serine-threonine kinase, mediates cell cycle arrest is still unknown. Lats2 was initially identified to be a p53 response gene by our group (Kostic et al., 2000). Subsequently, our group identified interacting candidates of Lats2, including substrate recognition modules of Cullin-based E3 ligases (Socs box or F-box containing proteins) as well as two subunits of the Signalosome (CSN), CSN4 and CSNS. Additionally, Lats2 was shown to associate with a Super-complex, composed of CSN and Cullin-based E3 ligases (Rongere, thesis, 2004; Rongere, unpublished results, 2005) We hypothesized that Lats2 may perform its physiological function through interaction with CSN and Cullin-based E3 ligases. The present work on Lats2 has confirmed that Lats2 is a CSN associated kinase. We defined the domain specific interactions of Lats2 with hSocs3, hWsb1 (Sots box proteins) and hFBX-7 (F box protein), as well as the physiological consequences of interaction with hSocs3, hWsb1 and hSocs1. Both the N-terminal and the kinase domains of Lats2 interact with full-length hSocs3, hWsb1 and hFBX-7, determined in GST pull-down assays suggesting that full-length Lats2 protein is involved in interactions. Refinement of the Lats2 interaction with hSocs3 indicated that the kinase domain of Lats2 interacts with a region of hSocs3 containing a SH2 domain located upstream of the Socs box domain of the hSocs3. Moreover, Lats2 phosphorylated specific regions between the N-terminal and SH2 domain (S l) as well as between the SH2 domain and Socs box domain of hSocs3 (S3).These results indicate that hSocs3 is a novel Lats2 substrate. The kinase assay has also demonstrated that wt Lats2 was able to phosphorylate hSocs3, but not Lats2 kinase dead mutant (deleted ATP pocket, Lats20ATP). Mutational analysis identified two serine residues located at positions 144/145 (S3) to be specifically phosphorylated by wt Lats2. Phosphorylation of proteins has been shown to be a signal for proteolytic degradation of many characterized proteins. Thus we hypothesized that Lats2 could target hSocs3 for proteasomal degradation. When wt Lats2 was over-expressed in HEK293T cells and COST cells, the half-life of hSocs3, as a component of Elongin BC Cullin-based E3 ubiquitin ligase (EBC ligase), decreased significantly. In contrast, aver-expression of the Lats2OATP did not alter the half-life of hSocs3. Furthermore, the stability of hSocs3 depended on phosphorylation of serine residues at positions 144/145 by wt Lats2. Although the sites of phosphorylation were not defined for two other substrate recognition modules of EBC ligasehWsbl and hSocsl, their half-lives also decreased when wt Lats2 was over-expressed. To test in vivo, we synthesized esiRNA to knock-down endogenous Lats2 and subsequently we measured the half-lives of hSocs3 and hVVsb l . Here we demonstrated that the half-lives of hSocs3 and hWsbl were increased by the factor of two in Lats2-depleted HEK293T cells. In conclusion, our findings suggest that Lats2, a CSN associated kinase, is a novel regulator of EBC ligase function by regulating the turn-over of hSocs3, hSocs1 and hWsb1. Thus, Lats2 alters the specificity and capacity of EBC ligases regulating thereby the stability of numerous proteins which are targeted by EBC ligases for proteasomal degradation. Further studies should reveal whether the observed modulation of EBC ligase function by Lats2 associated with a Super-complex is also responsible for the turn-over of cell cycle regulators and the observed alteration in cell cycle by Lats2 over-expression.