Changing the ligand-binding specificity of E. coli periplasmic binding protein RbsB by rational design and screening

Periplasmic binding proteins (PBPs) form a superfamily of bacterial proteins with a conserved bilobal structure, which are involved in substrate scavenging for bacterial cells. A wide variety of natural ligand-binding domains has evolved. PBPs are composed of two domains connected by a hinge region, which form a binding pocket between the two domains. They can be found in two stable conformations; in absence of ligand the PBP adopts an open conformation, where the binding pocket is exposed. In presence of the ligand, the protein changes to the closed conformation where the ligand is buried in the middle of the protein. This project focused on the ribose-binding protein of Escherichia coli (RbsB). Ribose binding to RbsB stabilizes the closed state. RbsB-bound ribose is presented to a cytoplasmic transport channels (RbsAC), from where it is imported into the cell, or interacts to membrane receptors (i.e., Trg) and can elicit a chemotactic signal. Due to their unique ligand-binding characteristics and wide variety of natural binding pockets PBPs have been of interest for the development of biosensors and bioreporter systems. PBP bioreporters were initiated over 20 years ago by a development in the group of Hazelbauer, who fused the C-terminal part of the E. coli EnvZ osmoregulation histidine kinase to the N-terminal part of the Trg methyl-accepting chemotaxis receptor protein, creating a hybrid receptor Trz1. Ligand bound galactose-binding protein (GBP) and ribose-bound RbsB interact with Trz1, which eventually leads to phosphorylation of the response regulator OmpR, activating transcription from the ompC promotor (and any reporter gene fused to this). In 2003, Hellinga’s group proposed that based on crystal structure information of ligand-bound PBPs variants with new ligand recognition specificities could be designed by computational approaches. Notably, they claimed the design of a RbsB-variant with nM affinity for recognition of 2,4,6-trinitrotoluene (TNT). This idea inspired the scientific community, because it could easily extend PBP-binding to a tremendous variety of compounds, including non- natural molecules, and would thus permit a wide variety of biosensor and bioreporter systems based on RbsB/GBP and Trz1. Unfortunately, independent engineering of some of the most promising published mutants failed to reproduce the reported in vivo and in vitro results. These studies further concluded that the published variants were actually misfolded proteins and/or impaired in stability as result of the introduced ligand-pocket mutations. This fact was largely ignored by Hellinga’s publications. Still inspired by the concept and trying to understand the reason of such limited success, our group raised the hypothesis that changing from ribose to TNT in a single step was likely unfeasible, but given the wide range of naturally evolved PBP ligand binding pockets, a step by step change of ribose binding to a non-natural analogue should be possible. To test this, we selected compounds with distinct differences but still chemically similar to ribose: 1,3-cyclohexanediol (13CHD) and cyclohexanol (CH). Mutant ligand binding pockets that might accommodate 13CHD and/or CH were computationally simulated and calculated using Rosetta, from which a list of critical amino acid residues to mutate in RbsB was selected. These were then synthesized and cloned into E. coli; a resulting set of 2 million mutants containing one of five possible substitutions at each of 9 selected critical amino acid positions. The library was introduced into an E. coli bioreporter strain, which carries the Trz1 hybrid signaling pathway coupled to GFP production when the (new) ligand would bind the (mutant) RbsB. The main goals of this work were to screen and characterize mutants from this first library, and potentially improve mutants for the new ligand binding in further rounds of mutagenesis. In the first part of this work a precise and user-friendly high-throughput strategy to screen the mutant library was developed. Clones were grown as individual microcolonies in alginate beads, to reduce single cell GFP expression variability, which were screened by fluorescence activated cell sorting (FACS) for gain-of-function GFP expression in presence of 13CHD. Six mutants with modest (1.5- fold) but consistent induction with 1 mM 13CHD were isolated. Moreover, these mutants completely lost the capacity to react to ribose. The RbsB mutants were characterized in terms of periplasmic space abundance, stability, secondary structure and ligand affinity. Isothermal microcalorimetry confirmed 13CHD binding, although only two mutants were sufficiently stable upon purification. Circular dichroism and quantification of periplasmic space abundance suggested the mutants to be prone to misfolding and/or defects in translocation. In the second part of this work, we used random and semi-random mutagenesis to improve the affinity and/or stability of the six isolated mutants with 13CHD binding capacity. Several mutant libraries were produced and screened with the previous described strategy. Variants displaying higher expression levels of GFP in presence of 13CHD were collected by FACS, and were used as starting point for the next round of evolution. This mutagenesis and rigorous screening strategy allowed us to isolate 7 mutants with improved (3.2-fold) GFP induction in presence of 13CHD and in a concentration- dependent manner. Several variants were observed that displayed open and closed conformations simultaneously, suggesting they were impaired in transition dynamics. Moreover, our screening strategy largely ignores potential variants with improved binding and closed conformation stability, but that are unable to interact with Trz1 receptor (e.i., trigger the signaling cascade). Finally in the third part of this work, we developed and tested an in vivo system to characterize the quality of the translocation process and receptor interactions. Wild-type- and mutant-RbsB proteins were fused to mCherry reporter protein to study protein abundance and subcellular localization. Whereas RbsB-mCherry proteins clearly localized to the periplasmic space and centered in polar regions depending on chemoreceptor availability, mutant-RbsB-mCherry expression resulted in high proportions of cells devoid of clear foci and low proportions of cells with multiple fluorescent foci, suggesting poorer translocation and mislocalisation. In addition, polar foci of mutants were less fluorescent, suggesting poorer chemoreceptor binding. By spiking further derivative mutant libraries generated by error-prone PCR without or with different proportions of E. coli expressing wild-type RbsB-mCherry we could estimate the potential improvement and deterioration of mutants with wild- type-like periplasmic localisation. The in vivo translocation system may thus be used to detect mutants with better signal transduction capacity. In conclusion, we firmly showed that design of PBP receptor proteins with new binding capacities for non-natural compounds is feasible, but still largely a matter of trial and error. The combination of computational simulations, random mutagenesis and rigorous screening allowed us to isolate variants with new recognition for 13CHD and loss of ribose binding. However, our results also showed that most predicted ligand-binding pocket mutations lead to poorly folding and functioning proteins, and it is likely that the dynamic transition needed between open and closed conformations of (here) RbsB is insufficiently understood and currently predictable to allow rational expansion to a wide range of new ligands. -- Les protéines de liaison périplasmiques (PLP) constituent une superfamille de protéines bactériennes avec une structure bilobée. Elles sont impliquées dans la captation de substrats pour les cellules bactériennes, et montrent grande diversité de domaines de liaison à des composés naturels. Les PLP sont composées de deux domaines connectés par une région charnière, ce qui forme une poche de liaison au substrat entre les deux domaines. Les PLP montrent deux états stables : ouverte en l’absence de ligand, conformation dans laquelle la poche de liaison est exposée, et fermée quand le ligand est séquestré dans la poche de liaison. Ce projet a porté sur l’étude de la PLP RbsB liant le ribose chez Escherichia coli. La liaison du ribose stabilise l’état fermé de RbsB et permet l’interaction avec le transporteur cytoplasmique RbsAC et son passage dans le cytoplasme de la cellule, ou son interaction avec des récepteurs membranaires tels que Trg permettant en une réponse chimiotactique. Étant données leurs caractéristiques uniques de liaison aux ligands et la grande variété de poches de liaison naturellement observée chez les PLP, elles présentent un grand intérêt pour le développement de biosenseurs et de systèmes biorapporteurs. Les premiers biorapporteurs basés sur des PLP ont été développés 20 ans auparavant par le groupe de Hazelbauer. Cette équipe a fusionné la partie C-terminale de la protéine kinase à histidine impliquée dans l’osmorégulation (EnvZ) et l’extrémité N-terminale du récepteur chimiotactique accepteur de groupement méthyle (Trg), pour créer le récepteur hybride Trz1. Les PLP liant le galactose (GBP) et le ribose (RbsB) interagissent avec Trz1, ce qui entraine la phosphorylation du régulateur réponse OmpR qui lui-même va activer la transcription à partir du promoteur du gène ompC (ou n’importe quel système rapporteur placé en aval de ce promoteur). En 2003, le groupe de Hellinga proposait que, sur la base de la structure cristallographique de différents PLP liées à leur ligand, des variants reconnaissant de nouveaux ligands pourraient être générés sur la base d’une approche informatique. En particulier, cette équipe se targue d’avoir générer un variant de RbsB permettant de lier le 2,4,6-trinitrotoluène (TNT) avec une affinité de l’ordre du nanomolaire. Cette idée a inspiré la communauté scientifique car cette approche pourrait s’étendre à une diversité incroyable de composés naturels ou non, ce qui permettrait le développement de biosenseurs et biorapporteurs variés basés sur ce système. Malheureusement, la construction des mutants les plus prometteurs par des équipes indépendantes n’ont pas permis de rapporter de l’activité in vivo et/ou in vitro. Cela a été ignoré dans les publications du groupe Hellinga. Inspirés par ce concept et voulant savoir quelles étaient les raisons de ce succès quelque peu limité, notre groupe a émis l’hypothèse que le changement de spécificité de RbsB du ribose au TNT en une étape était probablement infaisable mais, étant donnée la grande diversité de poches de liaisons naturellement observées chez les LPL, un changement pas à pas du ribose vers un composé analogue non naturel devrait être possible. Pour tester cela, nous avons sélectionné des composés distincts du ribose mais présentant tout de même des similarités : 1,3-cyclohexanediol (13CHD) and cyclohexanol (CH). Des mutants qui pourraient accueillir le 13CHD et/ou CH ont été générés par simulation informatique en utilisant le programme Rosetta, lequel a fourni une liste d’acides aminés critiques à muter. Une librairie de mutant a été synthétisée, celle-ci contenant 2 millions de variants de RbsB avec 1 substition parmi 5 possibles à 9 positions sélectionnées pour leur aspect critique dans la reconnaissance du substrat. La librairie a été introduite et criblée chez une souche reportrice d’E. coli contenant la chaine de signalisation hybride Trz1 couplée à la production de la protéine fluorescente verte (GFP) lorsque le (nouveau) ligand se liera à la protéine RbsB (sauvage ou mutante). Le but principal de ce travail était de caractériser cette librairie de mutants, et éventuellement d’améliorer la capacité de ces mutants à lier un autre composant par des cycles de mutagénèses additionnels. Dans la première partie de ce travail, une stratégie simple et efficace pour cribler la librairie de mutant a été développée. Les différents clones/variants ont été cultivés individuellement en microcolonies dans des billes d’alginate afin de réduire la variabilité du signal GFP observé au niveau de la cellule unique. Les billes ont été analysées par trieur de cellules reposant sur la fluorescence (FACS) afin de détecter des mutants présentant une activité GFP accrue en présence de 13CHD. Six mutants ont été isolés pour leur modeste mais significative induction (1,5 fois) en présence de 1 mM de 13CHD. De plus, ces mutants avaient totalement perdu leur capacité à réagir au ribose. Les mutants RbsB ont été caractérisés plus en détails pour leur localisation dans périplasme, leur stabilité, leur abondance et leur affinité pour le ligand. La technique de microcalorimétrie isotherme a confirmé que ces mutants lient le 13CHD, bien que seulement 2 de ces protéines mutantes se soient révélées suffisamment stables après purification. L’analyse par dichroïsme circulaire et la quantification de l’abondance des protéines dans l’espace périplasmique suggèrent que les protéines mutantes sont sujettes à un mauvais repliement et/ou un problème dans la translocation du cytoplasme au périplasme. Dans une seconde partie, nous avons muté les six mutants isolés précédemment de façon aléatoire ou semi-aléatoire afin d’améliorer leur affinité pour le 13CHD et/ou leur stabilité. Plusieurs librairies de mutants ont été produites et analysées selon la méthode décrite plus tôt. Les variants montrant une plus forte expression du système rapporteur GFP en présence de 13CHD ont été isolés par FACS, et utilisés comme point de départ pour la prochaine étape d’évolution. Cette mutagénèse et l’analyse rigoureuse des librairies nous ont permis d’isoler 7 mutants avec une augmentation de 3,2 fois du signal GFP en présence de 13CHD, et d’une façon dose-dépendante. Plusieurs variants ont montré qu’ils adoptaient la conformation ouverte et fermées au sein de la population bactérienne. Cette dernière observation suggère que ces mutants sont affectés dans leur capacité à passer d’une conformation à l’autre. De plus, notre stratégie de crible ne tient pas compte les variants qui montreraient une liaison accrue et une bonne stabilité de la conformation fermée, mais qui seraient incapables d’interagir avec le récepteur Trz1 (et donc de déclencher la cascade de signalisation du rapporteur). Finalement, dans la troisième partie de ce travail, nous avons développé et testé un système in vivo permettant de caractériser la qualité du processus de translocation dans l’espace périplasmique et l’interaction avec les récepteurs. Les protéines RbsB sauvage et mutantes ont été fusionnées à la protéine fluorescente rouge mCherry afin de visualiser l’abondance et la localisation sub-cellulaire des protéines au niveau de la cellule unique en utilisant la microscopy à épifluorescence et le traitement des images obtenues. Alors que la protéine de fusion RbsB sauvage montre une localisation périplasmique centrées au niveau des pôles de la cellule dépendamment de la disponibilité des chimiorécepteurs, les fusions avec les variants de RbsB montraient une forte proportion de cellules dépourvues de foci, et une faible proportion de cellules avec de multiples foci, suggérant une plus faible liaison aux chimiorécepteurs. En analysant plus en détails des librairies de mutants générées par PCR mutagène, en mélangeant ou non avec des cellules contenant la protéine de fusion RbsB sauvage, nous avons pu estimer l’amélioration potentielle ou la détérioration des qualités des mutants RbsB par rapport au sauvage en terme de localisation périplasmique. Ce système de translocation in vivo pourrait être utilisé afin de détecter des mutants permettant une meilleure transduction du signal. En conclusion, nous avons montré que la conception de protéines réceptrices PLP présentant de nouvelles capacités de liaison pour des composés non naturels est bien faisable, mais repose encore sur une stratégie d’essais et erreurs. La combinaison de simulations informatiques, de mutagénèses aléatoires et de crible rigoureux nous a permis d’isoler des variants de RbsB avec une capacité à reconnaitre le 13CHD, tout en ne liant plus le ribose. Néanmoins, nos résultats ont également montré que la plupart des prédictions de mutations au niveau de la poche de liaison ont mené à un mauvais repliement ou fonctionnement des protéines. Il est très probable que la dynamique de transition entre la conformation ouverte et fermée (de RbsB pour cette étude) ne soit pas encore assez bien comprise, et donc actuellement non prédictable pour permettre le test d’une grande variété de nouveaux ligands

Facilitação de Gamificação em Contextos de Seguros

A Gamificação é um método amplamente utilizado para aumentar o investimento de utilizadores, que tem evoluído constantemente na última década. As suas áreas de aplicação são diversas e, no caso específico dos produtos de seguro, a Gamificação começou a ser adotada para resolver vários problemas, sendo os mais relevantes a necessidade de aumentar as interações entre a empresa e o segurado e a redução da quantidade de dados não relatados à Companhia de Seguros, que causam cálculos de prémio menos exatos. A aplicação da Gamificação na área de seguros em geral foi analisada para fornecer uma solução de software que permita a sua implementação numa ampla variedade de áreas e produtos, para isso, analisaram-se Frameworks para Gamificação, assim como as possíveis áreas de aplicação no setor de seguros. Um design para um conjunto de aplicações é então apresentado, seguido por uma descrição detalhada da sua implementação. O trabalho foi desenvolvido na i2S de meados de janeiro a setembro de 2018 e tentou fornecer uma solução, com uma abordagem ampla, que fosse adaptável a vários tipos de apólices, utilizando seguro automóvel de tipo telemático, para fornecer um exemplo de uso. Este projeto foi utilizado para avaliar a viabilidade do desenvolvimento de um módulo independente para permitir a Gamificação de produtos de seguro, para comercialização pela i2S. Este módulo diferenciaria a empresa no mercado, já que as seguradoras estão a considerar começar a utilizar a Gamificação a médio prazo, para enriquecer os seus produtos.Gamification is a widely utilised method to increase of user engagement that has been steadily evolving over the last decade. It’s areas of application are diverse and in the specific case of insurance products Gamification has started to be adopted to solve various problems, the most prominent of which is the need to increase interactions between company and policy holder, reducing the amount of data unreported to the insurance company causing less exact premium calculations. The application of Gamification and the insurance area in general were analysed in order to provide a software solution to enable its implementation in a wide variety of areas and products. Frameworks for Gamification were analysed as are the possible areas of application in the insurance sector. A design for a set of applications is then presented followed by a detailed description of their implementation. The work was developed in i2S from mid-January to September 2018 and it attempted to provide a solution with a broad approach, being adaptable to many types of policy, utilizing auto insurance of a telematic kind to provide an example of usage. This project was utilized to assess the viability of developing an independent module to enable Gamification of insurance products to be commercialized by i2S. this would differentiate the company in the market as insurance companies are looking to utilize Gamification in the medium term to enhance their products

ARTICULANDO VANTAGENS POSICIONAIS: UMA PROPOSTA DE MEDIDA DE CONTROLE PRESIDENCIAL DO PROCESSO LEGISLATIVO

Este trabalho propõe um índice para medir as vantagens posicionais do poder Executivo no processo legislativo da Câmara dos Deputados. Chamaremos de Índice de Controle Presidencial do Processo Legislativo (ICPPL). Para essa medida, utilizaremos indicadores posicionais e de distância ideológica entre parlamentares que ocupam funções chaves no processo legislativo e o chefe do Executivo. Foram definidas dezesseis funções legislativas como chaves no processo de definição da agenda. Dentre as funções, temos a presidência da Câmara, presidência e relatoria da Comissão Mista de Orçamento (CMO), e a presidência da Comissão de Constituição Justiça e Cidadania (CCJC) e mais treze comissões temáticas permanentes da Câmara dos Deputados. Dedicaremos uma sessão para explicar a composição do índice, e como cada posto chave compõe um eixo temático e contribui com pesos diferentes no cálculo final. O processo legislativo brasileiro oferece vantagens ao Executivo por meio de prerrogativas constitucionais, e de regras internas da Câmara, que, mesmo assim, não são suficientes para garantir ao Presidente total controle do processo legislativo impondo sua agenda. Assim, o ICPPL nos permitirá verificar como os Presidentes brasileiros se articulam posicionalmente no processo legislativo, empilhando políticos fiéis ao governo em postos chave no legislativo. Neste artigo, apresentaremos o índice, e faremos uma aplicação com dados exploratórios em uma análise observacional de linha do tempo, abrangendo seis legislaturas completas e mais o primeiro ano da 56ª legislatura, compreendendo assim o período entre 1995 e 2019. Foi possível verificar, por exemplo, diferenças no domínio presidencial sobre os postos chave entre governos

O impacto do Enterprise 2.0 nas PME's de excelência em Portugal

Mestrado em Ciências EmpresariaisO uso generalizado das tecnologias Web 2.0 (como blogues, redes sociais ou wikis) pelos utilizadores da Internet, abrange, cada vez mais, a área de negócios. Este fenómeno deu origem ao conceito do Enterprise 2.0 (E2.0), ou seja, à exploração de tecnologias Web 2.0 e aplicativos para empresas com o objetivo de alcançar fortes vantagens competitivas. O Impacto do E2.0 pode ser sentido, principalmente, em duas áreas: dentro das organizações, uma vez que as tecnologias Web 2.0 permitem uma comunicação mais dinâmica e interativa que conduz ao aumento da eficiência e da produtividade; como, também, das empresas para os clientes, fornecedores e parceiros, com a finalidade de aumentar as receitas e a satisfação do cliente, bem como promover a cooperação e a cocriação de serviços/produtos. No entanto, a integração destas tecnologias pode ser um desafio. Através de um questionário aplicado a Pequenas e Médias Empresas (PME) portuguesas de Excelência, este estudo procura avaliar o nível de implementação do E2.0 nas organizações e compreender o seu impacto nas dinâmicas internas e externas das PMEs. Os resultados mostram a tendência de adoção das tecnologias Web 2.0 nas PMEs e identificam não só as tecnologias mais adotadas, mas, também, os fatores críticos de sucesso (FCS) para implementar o E2.0.The widespread use of Web 2.0 technologies (as blogs, social networks or wikis) by internet users is being increasingly extended to the business arena. This phenomenon led to the concept of Enterprise 2.0 (E2.0), i.e., the exploitation of Web 2.0 technologies and applications to enterprises in order to achieve strong competitive advantages. The impact of E2.0 can be felt mainly in two areas: within organizations, once they enable communication through new dynamic manners of communicate and interact, in order to enhance efficiency and productivity; from companies to customers, suppliers and partners, increasing revenues and customer satisfaction, and promoting cooperation and cocreation to improve services/products. However to implement these technologies within enterprises can be a challenge. Through a survey applied to Portuguese Small and Medium Enterprises (SMEs) of Excelence this study intent to evaluate the level of implementation of E2.0 on organizations and understand the impact of it on SMEs' internal and external dynamics. The results show the adoption trend of Web 2.0 technologies on SMEs and identify not only the most adopted technologies but also critical success factors (CSF) to implement E2.0

Espiritualidade: cuidados espirituais em fim de vida

Introdução: A espiritualidade é o aspeto da humanidade que se refere à forma como os indivíduos procuram e expressam sentido e propósito e está frequentemente relacionada com o significante ou sagrado. A espiritualidade torna-se particularmente importante nos cuidados em fim de vida já que, neste contexto, os indivíduos podem experimentar os limites da sua existência. Objetivos: Os objetivos deste trabalho são perceber de que forma os cuidados espirituais podem afetar a vida dos doentes em fim de vida, bem como perceber de que forma os profissionais de saúde podem avaliar as necessidades espirituais dos pacientes e quais são as barreiras à prestação deste tipo de cuidados. Desenho: Este trabalho consiste numa revisão narrativa a partir de uma pesquisa de revisões sistemáticas e artigos de revisão sobre o tema em questão em bases de dados online. Após a pesquisa e seleção dos artigos, estes foram agrupados e analisados qualitativamente segundo as seguintes abordagens temáticas: Espiritualidade; Religião; Sofrimento espiritual; Cuidados espirituais; Instrumentos de avaliação da espiritualidade; Impacto da espiritualidade nos doentes em fim de vida; Barreiras à prestação de cuidados espirituais; Espiritualidade em doentes com cancro; Outros aspetos da espiritualidade. Conclusões: É importante avaliar a espiritualidade e as necessidades espirituais dos pacientes, havendo vários instrumentos que o permitem fazer. A prestação de cuidados espirituais em pacientes em fim de vida tem um impacto positivo no bem-estar espiritual, na qualidade de vida e na satisfação dos pacientes com os cuidados de saúde. Existem várias barreiras que impedem a prestação adequada de cuidados espirituais, sendo a falta de formação particularmente importante. Os pacientes com cancro são uma população particularmente vulnerável no que toca a necessidades espirituais, muito por causa da forte associação desta doença a situações de terminalidade.Introduction: Spirituality is the aspect of humanity that refers to the way individuals seek and express meaning and purpose and is often related to the meaning or sacred. Spirituality is particularly important in end-of-life care since, in this context, individuals can experience the limits of their existence. Aims: The aim of this review is to understand how spiritual care can affect the lives of patients at the end of their lives, as well as to understand how health professionals can assess patients' spiritual needs and what the barriers are to provide this type of care. Design: This is a narrative review based on systematic reviews and review articles on the topic through online databases. After researching and selecting the articles, they were grouped and analyzed qualitatively according to the following thematic approaches: Spirituality; Religion; Spiritual suffering; Spiritual care; Spirituality assessment instruments; Impact of spirituality on end-of-life patients; Barriers to the provision of spiritual care; Spirituality in cancer patients; Other aspects of spirituality. Conclusion: It is important to assess patients' spirituality and spiritual needs and there are several instruments to do so. Providing spiritual care to end-of-life patients has a positive impact on spiritual well-being, quality of life and patient satisfaction with health care. There are several barriers that prevent adequate spiritual care from being provided, and lack of training is a particularly importante one. Cancer patients is a particularly vulnerable population in respect to spiritual needs, largely because of the strong association of this disease with terminallit

Gestão de Afluências Indevidas em Redes de Águas Residuais Urbanas

As afluências indevidas resultantes direta ou indiretamente da precipitação são um dos principais fatores que provocam graves problemas de carácter ambiental, estrutural e económico, no âmbito de um bom funcionamento dos sistemas separativos de saneamento e tratamento de águas residuais urbanas. Ao longo dos anos, com a expansão do serviço de saneamento à população, tem sido reconhecido que os caudais excedentes às redes de drenagem de águas residuais e estações de tratamento são um problema grave e para isso estão a ser criados critérios cada vez mais exigentes no domínio da gestão e operação destes sistemas de saneamento. Para uma melhor compreensão sobre a incidência das afluências indevidas nos sistemas de saneamento e nas estações de tratamento de águas residuais, é necessário realizar estudos de quantificação e caracterização no sistema de esgotos. Com a necessidade de compreender a plenitude e natureza deste problema, tornou-se necessário desenvolver metodologias com o objetivo de melhorar a eficiência e eficácia hidráulica dos sistemas de drenagem e de tratamento. Este trabalho tem como objetivo a análise quantitativa das afluências indevidas, recorrendo a um caso de estudo de uma bacia de drenagem de águas residuais. Para adquirir mais conhecimento sobre o tema foi necessário recolher várias metodologias existentes de maneira a selecionar, estudar e aplicar o método mais vantajoso. Nesta dissertação, foram utilizados dados disponibilizados pela SIMRIA, no que respeita aos caudais de bombagem da estação elevatória a que afluiu a rede em estudo, e pela INDAQUA Feira, no que respeita aos dados de precipitação.The improper inflows resulting of direct or indirect precipitation is one of the main factors which cause major malfunctions of environmental, structural and economical character, in the ambit of a good behaviour in separative sanitation wastewater and urban water treatment systems. Throughout the years, with the expansion of sanitation services to the population, it has been noticed that the overflows in draining networks and wastewater treatment plants are a serious issue, therefore are being created standards more and more demanding in the management and operation of these sanitation systems. For a better understanding about the improper inflows incidence in sanitation systems and wastewater treatment plants, it is necessary to conduct studies of quantification and characterization of the sewer systems. With the necessity to comprehend the plenitude and nature of this problem it has become necessary to develop methodologies with the objective of improving the hydraulic efficiency and effectiveness of draining and treatment systems. This paper main purpose is the quantitative analysis of improper inflows, resorting to a case study of a drainage basin wastewater. To acquire more knowledge on the matter it was necessary to retrieve an array of existing methodologies in view of sorting, studying and application of the most advantageous method. The used data in this dissertation was provided by SIMRIA, in the case of the pumping rate flow of the loadinglift station that surged to the network case study, and by INDAQUA Feira, for the precipitation data

Framework de gestão de notícias regionais em canais de televisão

Nos últimos anos tem-se verificado uma tendência de hiperlocalização de notícias, por parte dos canais televisivos de caráter nacional, que visa uma aproximação mais rápida e eficaz do espetador às notícias que lhe são relevantes. No entanto, os jornalistas na maior parte das vezes estão direcionados para a captação de som e imagem, descurando da potencialidade dos conteúdos audiovisuais.A evolução ao nível da performance do hardware informático veio possibilitar a virtualização dos ambientes de produção de conteúdo televisivo, e ao usar as novas tecnologias abertas baseadas em nuvem permite a interligação de todos os jornalistas espalhados pelo território nacional, possibilitando a criação de uma rede de contribuições mais próximas dos locais das notícias.Esta dissertação procura desenvolver uma plataforma em ambiente virtual capaz de conceber as fases de contribuição, produção, gestão e distribuição de conteúdo, permitindo a interligação dos vários jornalistas localizados em diferentes pontos do país. Assim, torna-se possível a redução de custos e a carga logística deste canais televisivos, para além de usar os recursos disponíveis de uma forma mais intuitiva.O objetivo passa também por descentralizar o poder computacional da nuvem e executar parte do processamento dos dados mais próximo da fonte destes, sendo possível otimizar aspetos como a latência, custos de largura de banda ao enviar para a nuvem e ainda a privacidade dos dados. Trata-se de uma tendência registada neste últimos anos visto que a nuvem tornou-se não eficiente o suficiente face à quantidade de dados que hoje em dia são produzidos e transportados para esta.In recent years, it has been a news hyper localization trend by national news TV channels aiming a more rapid and effective approach by the viewer to the news that is relevant to him. However, journalists are mostly directed towards capturing sound and image, neglecting the potential of audiovisual content.The computer hardware performance evolution has made possible the television content production environments virtualization and the use of new open-source and cloud based technologies allows the interconnection of all the journalists spread across the national territory, making it possible to create a network of contributions that are closer to the news locations.Thus, this paper proposes to develop a platform in a virtual environment capable of integrating the content contribution, production, management and distribution phases, allowing the interconnection of the various journalists located in different parts of the country. In this way, it becomes possible to reduce costs and logistic load of this television system, in addition to using available resources in a more intuitive way.The goal is also to decentralize the computing power of the cloud and to perform some of the data processing closer to the source, which can optimize aspects such as latency, bandwidth costs when sending to the cloud and data privacy. This is a trend seen in recent years since the cloud has become not efficient enough given the amount of data that is now produced and transported to it

Learning to Ask Questions for Zero-shot Dialogue State Tracking

CMU Portugal project iFetch (LISBOA-01-0247-FEDER-045920). Publisher Copyright: © 2023 Copyright held by the owner/author(s).We present a method for performing zero-shot Dialogue State Tracking (DST) by casting the task as a learning-to-ask-questions framework. The framework learns to pair the best question generation (QG) strategy with in-domain question answering (QA) methods to extract slot values from a dialogue without any human intervention. A novel self-supervised QA pretraining step using in-domain data is essential to learn the structure without requiring any slot-filling annotations. Moreover, we show that QG methods need to be aligned with the same grammatical person used in the dialogue. Empirical evaluation on the MultiWOZ 2.1 dataset demonstrates that our approach, when used alongside robust QA models, outperforms existing zero-shot methods in the challenging task of zero-shot cross domain adaptation-given a comparable amount of domain knowledge during data creation. Finally, we analyze the impact of the types of questions used, and demonstrate that the algorithmic approach outperforms template-based question generation.publishersversionpublishe

Impact of motivations to buy and offer gifts in consumerism at Christmas

This article aims to analyze a conceptual model composed of the variables motivation to buy, motivation to offer gifts and compulsive consumerism at Christmas time. In order to achieve the proposed objective, a quantitative methodology based on a questionnaire survey was used. The sample consists of 1086 Portuguese individuals, over 18 years old. To evaluate the model, structural equation modeling with partial least squares was used. The results show that the motivations to buy and to offer gifts positively influence compulsive consumerism at Christmas time, and that the motivation to buy positively influences the motivation to offer gifts at Christmas time. The model also demonstrates that the motivation to offer gifts plays a mediating role in the relationship between the motivation to buy and compulsive consumerism. In Portugal there are no known studies on the motivations for offering gifts at Christmas. The present study can help academics, researchers and professionals (managers of consumer goods companies, commercial managers and advertising campaign managers) to better understand the motivations for buying and offering gifts, and the compulsive consumerism of the Portuguese population at Christmas time.info:eu-repo/semantics/publishedVersio