Plasma membrane-specific interactome analysis reveals calpain 1 as a druggable modulator of rescued Phe508del-CFTR cell surface stability

Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), a chloride channel normally expressed at the surface of epithelial cells. The most frequent mutation, resulting in Phe-508 deletion, causes CFTR misfolding and its premature degradation. Low temperature or pharmacological correctors can partly rescue the Phe508del-CFTR processing defect and enhance trafficking of this channel variant to the plasma membrane (PM). Nevertheless, the rescued channels have an increased endocytosis rate, being quickly removed from the PM by the peripheral protein quality-control pathway. We previously reported that rescued Phe508del-CFTR (rPhe508del) can be retained at the cell surface by stimulating signaling pathways that coax the adaptor molecule ezrin (EZR) to tether rPhe508del–Na+/H+-exchange regulatory factor-1 (NHERF1) complexes to the actin cytoskeleton, thereby averting the rapid internalization of this channel variant. However, the molecular basis for why rPhe508del fails to recruit active EZR to the PM remains elusive. Here, using a proteomics approach, we characterized and compared the core components of wt-CFTR– or rPhe508del–containing macromolecular complexes at the surface of human bronchial epithelial cells. We identified calpain 1 (CAPN1) as an exclusive rPhe508del interactor that prevents active EZR recruitment, impairs rPhe508del anchoring to actin, and reduces its stability in the PM. We show that either CAPN1 downregulation or its chemical inhibition dramatically improves the functional rescue of Phe508del-CFTR in airway cells. These observations suggest that CAPN1 constitutes an attractive target for pharmacological intervention, as part of CF combination therapies restoring Phe508del-CFTR function.This work was supported by a center grant UID/MULTI/04046/2019 to BioISI and project PTDC/BIA-CEL/28408/2017 and IF2012 to PM, both from FCT, Portugal. AMM was recipient of fellowship SFRH/BD/52490/2014 from BioSYS PhD programme PD65-2012, and PB of fellowship SFRH/BPD/94322/2013.N/

Serviços de descanso ao cuidador : soluções de internamento temporário na região EDV

Mestrado em GerontologiaObjectivos: Enquanto medida de apoio aos cuidadores, os serviços de descanso visam proporcionar uma pausa efectiva e temporária da responsabilidade de cuidar, para que a pessoa que cuida repouse ou realize outras actividades, restituindo desta forma as suas estratégias de coping. Este estudo visa conhecer a estruturação e funcionamento dos Serviços de Descanso ao Cuidador – Soluções de Internamento Temporário na região Entre Douro e Vouga (região EDV) tendo como ponto de partida o entendimento daqueles serviços por profissionais de diferentes âmbitos de intervenção (social e saúde), a elaboração de um diagnóstico das respostas existentes na região a este nível e a aferição da satisfação de cuidadores informais que já beneficiaram desses serviços. Metodologia: Foram desenvolvidos três estudos complementares. O primeiro estudo, de base qualitativa, consta de dois focus groups com representantes, do sector da saúde e do social, de cada um dos municípios; o segundo estudo desenvolveuse a partir da aplicação de um breve questionário a todas as instituições pertencentes à Rede Social da região EDV; o terceiro estudo, de carácter exploratório, consiste na realização de entrevistas a alguns cuidadores que já beneficiaram dos serviços de descanso ao cuidador na região. Resultados: Os principais resultados apontam para (i) uma indefinição conceptual associada aos serviços de descanso ao cuidador entre sectores distintos (social e saúde) mas, também, dentro do mesmo sector; (ii) um elevado desconhecimento relativamente a esta resposta por parte dos técnicos e dos cuidadores; (iii) uma reduzida oferta do serviço na região EDV que leva algumas instituições a organizarem o seu espaço e funcionamento no sentido de responder de modo informal a esta carência; (iv) a indispensabilidade de ampliação da oferta justificada pela procura. Discussão: Estes resultados apoiam a necessidade de uma reflexão acerca da pertinência da diversificação e flexibilização dos serviços que visam o descanso do cuidador, pois revelam uma frequência de procura elevada na região. No futuro perspectivam-se novas investigações justificadas pelas dúvidas nocionais existentes e, também, pela necessidade de perceber melhor a viabilidade de um aumento da sua oferta e as condições em que pode ocorrer. ABSTRACT: Objective: Respite care services aim to give the caregiver an effective and temporary break from the responsibility of caring, in order to promote the opportunity to rest or to perform other activities, thus restoring coping strategies. This study aims to specifically meet the current definition, structure and functioning Respite Care Services – Residential Care in the “Entre Douro e Vouga Region” (EDV region), taking as its starting point the understanding of those services by professionals from different fields of intervention (social and health), the elaboration of a diagnosis of this type of response in the region and measuring the satisfaction of informal caregivers that have already benefited such services. Methodology: Three complementary studies were developed. The first study, based on a qualitative approach, consists of two focus groups with representatives of health and social sectors from each municipality; the second study was developed from the application of a short questionnaire to all institutions belonging to Social Network of the EDV region; the third one, with an exploratory focus, involved carrying out interviews with caregivers who have already benefited respite care services in this region. Results: Main results indicate (i) a conceptual ambiguity associated with respite care services not only between different sectors (social and health), but also within the same sector; (ii) a high unawareness regarding this service in both technicians and caregivers; (iii) a reduced supply of service in the EDV region leading some institutions to organize their space and their dynamics in order to address this deficiency in an informal manner; (iv) the need of expanding the supply, which is justified by its demanding. Discussion: These results draw attention to the relevance of services diversification and flexibility aimed at respite care, since they reveal a frequency of high demand. In the future, there is a prospect of further investigations warranted not only by the existing notional doubts, but also by the need of a better understanding on the viability of increasing this service’s offer in the region and on the circumstances it may occur

Construção de ferramentas moleculares para o estudo das proteínas arabinogalactânicas AGP6 e AGP11 de Arabidopsis thaliana

Dissertação de mestrado em Fisiologia Molecular de PlantasUm dos mais significativos marcos na evolução das plantas vasculares é o surgimento de plantas com flor, que trouxe consigo estratégias evolutivamente favoráveis: o óvulo protegido por dois tegumentos e encerrado num pistilo que atrai, retém e direcciona os gâmetas masculinos. No entanto, a reprodução sexuada das Angiospérmicas apresenta ainda alguns mistérios que só recentemente começaram a ser desvendados. O grão de pólen, ao longo do seu desenvolvimento, apresenta notáveis diferenças no transcriptoma das diferentes fases. No estado maduro, o transcriptoma do grão de pólen apresenta elevada expressão maioritariamente dos genes envolvidos em sinalização e alongamento celular, genes que serão importantes na germinação e desenvolvimento do tubo polínico, e direccionamento para o gametófito feminino. As proteínas arabinogalactânicas (AGPs) são glicoproteínas ricas em hidroxiprolina altamente glicosiladas por cadeias de arabinogalactano tipo II implicadas em vários estudos realizados ao longo das últimas duas décadas em vários processos importantes do desenvolvimento vegetal. Têm suscitado particular interesse pela sua presença marcante e específica nos tecidos dos órgãos florais, observada em estudos de imunocitoquímica e análise de transcriptomas, levantando suspeitas sobre um possível papel relevante na reprodução sexuada. Neste estudo pretendeu-se colocar em foco as proteínas arabinogalactânicas clássicas AGP6 e AGP11, expressas exclusivamente no grão de pólen, introduzindo em cada uma destas proteínas uma sequência c-myc que permita efectuar a sua localização específica nas células em plantas de Arabidopsis thaliana selvagem e mutante nulo agp6agp11, para futuramente proceder a estudos de análise fenotípica e funcional. Pretendeu-se também fundir cada uma das AGPs com o promotor CaMV 35S para obter uma sobrexpressão de tal ordem que seja possível obter proteína em quantidades manipuláveis para estudos bioquímicos. O estudo destas linhas de plantas transgénicas ajudará a compreender a função destas duas proteínas no grão de pólen, e a sua importância no processo de dupla fecundação. A capacidade de purificar individualmente cada proteina será igualmente um importante avanço para o estudo bioquímico não só da AGP6 e da AGP11, mas também de outros membros da família das proteínas arabinogalactânicas.One of the most significant achievements in tracheophyte evolution is the development of flowers, which brought along several beneficial evolutionary strategies: the ovule is protected by two integuments and enclosed in a pistil that attracts, retains and directs the male gametes. However, the sexual reproduction of Angiosperms still presents a few enigmas that to this day are being slowly uncovered. The pollen grain, throughout its development, shows significant differences in the transcriptome of the different stages. In the mature state of the pollen grain, the transcriptome shows an outstanding up-regulation of the genes associated with signaling and cell elongation, genes which will be important later on, for the germination of the pollen tube and guidance into the female gametophyte. Arabinogalactan proteins (AGPs) are hydroxiproline-rich glycoproteins highly glycosylated by chains of type II arabinogalactan, which have been implicated in several studies performed in the past two decades as participants of numerous major processes of plant development. They are intriguingly present in a very specific manner during the development of floral tissues, both in immunocytochemistry assays and transcriptome analysis, having thus given rise to suspicions about a putative relevant role in sexual reproduction. The present work intended to put in to focus the classic arabinogalactan proteins AGP6 and AGP11, exclusively expressed in the pollen grain, introducing in each one a c-myc tag that will allow for its specific subcellular localization in wild type Arabidopsis thaliana, as well as in double null agp6agp11 mutants, for studies of phenotype and function. It was also proposed to fuse each of these AGPs with the CaMV 35S promoter in order to obtain overexpression in such a way that the amount of extractable protein will allow further biochemical assays. Future study of these transgenic lines will aid in understanding the role of these proteins in the pollen grain, and their importance in the double fertilization process. The ability to purify each individually will also be an important advancement for the biochemical study of not only AGP6 and AGP11, but also of the other members of the AGP family

Anti-Social" Strategy and Brand Equity: BOTTEGA VENETA´S case

Social media poses a dilemma for luxury brands, while luxury represents exclusivity, uniqueness, and outstanding customer experience; social media is accessible, characterized by the lack of physical contact and created for the masses. Despite this conceptual paradox, research shows that social media is a powerful addition to the marketing strategy of luxury brands and has a positive impact on brand equity. However, scant evidence is available on what happens to the image and awareness of a luxury brand when it stops creating content and interacting with consumers on social media platforms. This research aims to analyse Bottega Veneta’s brand equity after adopting an “anti-social” strategy (i.e., deleting their social media accounts). An online questionnaire was used to evaluate the current state of Bottega Veneta’s brand equity and perform a comparison with a similar brand present on social media, Balenciaga. Findings based on 147 participants suggest that Bottega Veneta has low levels of brand awareness and brand image, leading to reduced overall brand equity. When compared to Balenciaga, Bottega Veneta also presents a lower brand awareness and brand image. Overall, this research concluded that reducing brand communication and relying only on one-way communication could be negative for brand awareness since customers are not frequently exposed to the brand elements, consequently resulting in low levels of brand image since potential consumers cannot form solid brand associations.As redes sociais constituem um dilema para as marcas de luxo, enquanto luxo representa exclusividade, singularidade e uma experiência excecional para o cliente; as redes sociais são acessíveis, caracterizadas pela falta de contacto físico e criadas para o público em geral. Apesar deste paradoxo conceptual, a investigação mostra que as redes sociais são uma adição preponderante à estratégia de marketing das marcas de luxo e têm um impacto positivo no brand equity. No entanto, as evidências existentes são ainda escassas sobre o que acontece à imagem e à awareness de uma marca de luxo quando esta deixa de criar conteúdo e de interagir com os consumidores nas redes sociais. Esta investigação tem como objetivo analisar o brand equity da marca Bottega Veneta após adotar uma estratégia "anti-social" (ou seja, marcas que apagam as suas contas nas redes socias). Neste sentido, foi divulgado um questionário online para avaliar o estado atual do brand equity referente à marca Bottega Veneta e realizar uma comparação com uma marca semelhante presente nas redes sociais, Balenciaga. Os resultados baseados em 147 participantes sugerem que a marca Bottega Veneta apresenta baixos níveis de awareness e imagem de marca, levando a uma redução do overall brand equity. Quando comparado com a marca Balenciaga, a Bottega Veneta também apresenta uma menor awareness e imagem de marca. Em geral, esta investigação concluiu que a redução da comunicação da marca e o recurso apenas à comunicação unidirecional pode ser negativo para a awareness da marca, uma vez que os clientes não são expostos frequentemente aos elementos da mesma, resultando consequentemente em baixos níveis de imagem de marca, dado que os potenciais consumidores não podem formar associações sólidas referentes à marca

Facebook as an educational artefact?: the Reviver na Rede project

A presente comunicação, em que se assume o registo de ensaio, propõe-se explorar a possibilidade do Facebook constituir um artefacto promovedor de aprendizagens (in)formais no contexto da Educação Aberta, tendo como exemplo o caso prático do projeto REviver na Rede. A revolução digital impulsionou o paradigma da sociedade rede com impacto nos estilos de vida, cada vez mais virtuais e online, estimulando novas formas de aprendizagem. Por exemplo, já não somos apenas meros consumidores de informação, mas tornamo-nos também produtores de conhecimento. Estas dinâmicas estão, progressivamente, a transformar as instituições e os processos educacionais, fazendo emergir novas metodologias de ensino/aprendizagem, como o movimento dos Recursos Educacionais Abertos (REA) e fortalecendo outras já existentes, no âmbito da Educação Aberta. Por isso, consideramos que a relação entre as redes sociais e a educação em geral é inevitável. Atualmente o Facebook tornou-se um gigantesco e global palco virtual, no qual se promovem comportamentos e novas possibilidades de interação, configurando-se como um recurso significativo para o processo de ensino e aprendizagem; simultaneamente, também se tornou num disseminador de práticas e experiências educativas. As novas ferramentas digitais trouxeram à educação a possibilidade de construir novos e diferentes artefactos educacionais, tanto no seu processo de construção como na forma como são disponibilizados, manuseados e apreendidos. São objetos de aprendizagem, muitos assumindo as características definidoras dos REA, utilizados como meio para adquirir um determinado saber nele contido, permitindo construir conhecimentos novos de forma imersiva e interativa. Embora o tradicional conceito de artefacto nos remeta para um objeto físico, no atual contexto tecnológico pode deixar de ser apenas uma peça de hardware e assim entrar no campo do software. Assume, pois, uma dimensão digital, conquistando uma nova vida e possibilidades próprias, advindas deste ambiente virtual. Recuperando a pergunta inicial, “O Facebook como Artefacto Educacional?”, concluímos que o Facebook tem vindo a emergir na Educação, assumindo várias valências. Para além de instrumento (artefacto) educacional, digital e multimédia, também é visto como tendo um papel muito relevante na disseminação e construção do conhecimento.This communication, in which we assume the essay format, proposes to explore the possibility of Facebook being a contributing artefact of formal learning (in) in the context of Open Education, taking as an example the practical case of the REviver na Rede project. The digital revolution has boosted the paradigm of network society with an impact on lifestyles, increasingly virtual and online, stimulating new forms of learning. For example, we are not only mere consumers of information, but we also become knowledge producers. These dynamics are progressively transforming educational institutions and processes, making new teaching/learning methodologies emerge, such as the movement of Open Educational Resources (OER) and strengthening existing ones in the open education. Therefore, we consider that the relation between social networks and education in general is inevitable. Currently Facebook has become a gigantic and global virtual stage, in which behaviors and new possibilities of interaction are promoted, configuring as a significant resource for the teaching and learning process; simultaneously, it has also become a disseminator of educational practices and experiences. The new digital tools have brought to education the possibility of building new and different educational artefacts, both in its construction process and in the way, they are made available, handled and seized. They are learning objects, many assuming the defining characteristics of the OER, used as means to acquire a certain knowledge contained therein, allowing to build new knowledge in an immersive and interactive way. Although the traditional concept of artefact refers us to a physical object, in the current technological context can cease to be just a piece of hardware and thus enter the field of the software. It therefore assumes a digital dimension, conquering a new life and its own possibilities, arising from this virtual environment. Recovering the initial question, "Facebook as Educational Artefact?" we conclude that Facebook has been emerging in Education, assuming several valences. In addition to educational, digital and multimedia instrument (artefact), it is also seen as having a very relevant role in the dissemination and construction of knowledge.info:eu-repo/semantics/publishedVersio

Complete Sequence of a blaOXA-48-Harboring IncL Plasmid from an Enterobacter cloacae Clinical Isolate

Free PMC Article: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4574381/We report a 63,584-bp conjugative IncL plasmid (pUR17313-1) from an Enterobacter cloacae clinical isolate, containing a blaOXA-48 gene. The plasmid sequence also carried important mobile genetic elements involved in the spread of antibiotic resistance, namely, the Tn1999.2 composite transposon, which enclosed blaOXA-48-, integrase-, and transposase-encoding genes.V.M. was supported by grant number SFRH/BPD/77486/2011 from Fundação para a Ciência e a Tecnologia, Lisbon, Portugal. This study was supported financially by the 2015DDI1228 project from National Institute of Health, Portugal

Caracterização dos hábitos de brincar, sono e comportamento em crianças em idade pré-escolar

Introduction: Playing is a fundamental activity of childhood. The primary goal of this study was to characterize the playing habits of Portuguese children aged between three and five years old. The secondary goals were to investigate children’s sleeping habits, audiovisual media exposure, and behavior, as well as caregivers ́ perception of these.Material and methods: Observational study based on the application of an online questionnaire to parents and caregivers about children’s sociodemographic features, playing habits, exposure to screens, sleep, and behavior.Results: A total of 240 questionnaires were retrieved, 58.3% (n=140) of which regarding male children. Overall, 93.9% (n=225) of children attended or had previously attended kindergarten, 56.9% (n=137) practiced at least one extracurricular activity, 64.4% (n=155) played outdoors in most days, and 80.9% (n=194) played with their peers. Most caregivers (68.3%, n=164) daily played with their children, but 95% (n=228) would like to do it more often. Free play was preferred among children (64%, n=154). Most children (77.4%, n=186) had contacted with audiovisual media before the age of 18 months. Most caregivers (89.6%, n=215) considered that their children slept well, with 27.4% (n=66) acknowledging that they threw frequent tantrums and 21.3% (n=51) that they were impulsive or aggressive. Most caregivers recognized the importance of playing for children. Regarding sleep habits and behavior, lower focus in quiet activities (odds ratio [OR] 4.638, 95% confidence interval [CI] 1.902-11.314) and more regular tantrums (OR 2.317, 95% CI 1.022-5.250) were independent predictors of sleeping problems. Conclusion: Free playing, frequent outdoor playing, and playing with other children stood out as protective factors of children’s physical and mental health. However, inadequate screen exposure and schedule overload with structured activities represent concerns that should be addressed in Pediatric appointments. Family-centered playful learning should be encouraged.info:eu-repo/semantics/publishedVersio

Higher Education in Lifelong Learning

ABSTRACT In the behalf of CNAVES – the Portuguese agency for HE evaluation –we developed this study with the main purpose of assessing the role played by Higher Education (HE) in the usually called lifewide learning process. Likewise six Portuguese HE institutions and four among the main economic groups, together with ten small and medium enterprises have been addressed a specific survey. Professional associations have been called to participate as well but only one of them adhered. In order to obtain a more acute characterization of the Portuguese situation we run via internet a complementary research on the articulation between foreign HE institutions and enterprises leading to educational community networks and/or corporate universities. Among the main results we could perceive the weakness of the Portuguese HE institutions * enterprises network and the conclusion that the main reasons for articulation consist in participating in the advisory boards. Universities are not adopting so far any meaningful organization’s or syllabuses’ innovation in order to specifically address “new public” needs and only a few among them systematically develop graduate follow up after they enter into labor market.Higher Education; Lifewide learning; University Entreprises Networking; Portugal