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/

High Stake Exams With Byod At The University Of Porto: Bypassing Constraints

University of Porto (U.PORTO) is a traditional higher education institution with more than 30 000 students enrolled. Nine years ago, the first final exam using computer was conducted with quite success through Moodle's quiz activity. Since 2009 the technical conditions and infrastructure were improved in such a way that we've found difficulties increasing the number of exams taking place. This year alone more than 15 000 quiz submissions were made on the first semester. Actually, these constraints - an indirect indicator of our success - were a result of mainly two factors: on the one hand a policy decision to shorten the assessment calendar, on the other hand a low ratio of computers per student, especially at some Schools. Therefore, facing the uncertainty of a considerable investment on computers but also watching the rising number of students owning a laptop, the Education Technologies unit decided to go on the path of BYOD (Bring Your Own Device) concerning computer-based assessment. So in a careful and sustainable way - this is after all a moment of extreme importance for all stakeholders, especially for the students involved - four courses from two different Schools embraced this methodology. This paper will discuss the requirements and implementation of this solution along with the methodology followed. Besides the logistics - clearly one of the most relevant aspects, despite non tech related - we will also show the results of one of the last exams on BYOD, the first at Sports School, which had some specificities, including two shifts and some students submitting on paper

High Stake Exams With Byod At The University Of Porto: Bypassing Constraints

University of Porto (U.PORTO) is a traditional higher education institution with more than 30 000 students enrolled. Nine years ago, the first final exam using computer was conducted with quite success through Moodle's quiz activity. Since 2009 the technical conditions and infrastructure were improved in such a way that we've found difficulties increasing the number of exams taking place. This year alone more than 15 000 quiz submissions were made on the first semester. Actually, these constraints - an indirect indicator of our success - were a result of mainly two factors: on the one hand a policy decision to shorten the assessment calendar, on the other hand a low ratio of computers per student, especially at some Schools. Therefore, facing the uncertainty of a considerable investment on computers but also watching the rising number of students owning a laptop, the Education Technologies unit decided to go on the path of BYOD (Bring Your Own Device) concerning computer-based assessment. So in a careful and sustainable way - this is after all a moment of extreme importance for all stakeholders, especially for the students involved - four courses from two different Schools embraced this methodology. This paper will discuss the requirements and implementation of this solution along with the methodology followed. Besides the logistics - clearly one of the most relevant aspects, despite non tech related - we will also show the results of one of the last exams on BYOD, the first at Sports School, which had some specificities, including two shifts and some students submitting on paper

Estrogen receptor dependent genetic and epigenetic factors of tamoxifen resistance

Resumo: A decisão da terapêutica hormonal no tratamento do cancro da mama baseiase na determinação do receptor de estrogénio alfa por imunohistoquímica (IHC). Contudo, a presença deste receptor não prediz a resposta em todas as situações, em parte devido a limitações do método IHC. Investigámos se a expressão dos genes ESR1 e ESR2, bem como a metilação dos respectivos promotores, pode estar relacionada com a evolução desfavorável de uma proporção de doentes tratados com tamoxifeno assim como com a perda dos receptores de estrogénio alfa (ERα) e beta (ERß). Amostras de 211 doentes com cancro da mama diagnosticado entre 1988 e 2004, fixadas em formalina e preservadas em parafina, foram utilizadas para a determinação por IHC da presença dos receptores ERα e ERß. O mRNA total do gene ESR1 e os níveis específicos do transcrito derivado do promotor C (ESR1_C), bem como dos transcritos ESR2_ß1, ESR2_ß2/cx, and ESR2_ß5 foram avaliados por Real-time PCR. Os promotores A e C do gene ESR1 e os promotores 0K e 0N do gene ESR2 foram investigados por análise de metilação dos dinucleotidos CpG usando bisulfite-PCR para análise com enzimas de restrição, ou para methylation specific PCR. Atendendo aos resultados promissores relacionados com a metilação do promotor do gene ESR1, complementamos o estudo com um método quantitativo por matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) suportado pelo software Epityper para a medição da metilação nos promotores A e C. Fez-se a avaliação da estabilidade do mRNA nas linhas celulares de cancro da mama MCF-7 e MDA-MB-231 tratadas com actinomicina D. Baixos níveis do transcrito ESR1_C associaram-se a uma melhor sobrevivência global (p = 0.017). Níveis elevados do transcrito ESR1_C associaram-se a uma resposta inferior ao tamoxifeno (HR = 2.48; CI 95% 1.24-4.99), um efeito mais pronunciado em doentes com tumores de fenótipo ERα/PgR duplamente positivo (HR = 3.41; CI 95% 1.45-8.04). A isoforma ESR1_C mostrou ter uma semi-vida prolongada, bem como uma estrutura secundária da região 5’UTR muito mais relaxada em comparação com a isoforma ESR1_A. A análise por Western-blot mostrou que ao nível da 21 proteína, a selectividade de promotores é indistinguivel. Não se detectou qualquer correlação entre os níveis das isoformas do gene ESR2 ou entre a metilação dos promotores do gene ESR2, e a detecção da proteína ERß. A metilação do promotor C do gene ESR1, e não do promotor A, foi responsável pela perda do receptor ERα. Estes resultados sugerem que os níveis do transcrito ESR1_C sejam usados como um novo potencial marcador para o prognóstico e predição de resposta ao tratamento com tamoxifeno em doentes com cancro da mama. Abstract: The decision of endocrine breast cancer treatment relies on ERα IHC-based assessment. However, ER positivity does not predict response in all cases in part due to IHC methodological limitations. We investigated whether ESR1 and ESR2 gene expression and respective promoter methylation may be related to non-favorable outcome of a proportion of tamoxifen treated patients as well as to ERα and ERß loss. Formalin-fixed paraffin-embedded breast cancer samples from 211 patients diagnosed between 1988 and 2004 were submitted to IHC-based ERα and ERß protein determination. ESR1 whole mRNA and promoter C specific transcript levels, as well as ESR2_ß1, ESR2_ß2/cx, and ESR2_ß5 transcripts were assessed by real-time PCR. ESR1 promoters A and C, and ESR2 promoters 0N and 0K were investigated by CpG methylation analysis using bisulfite-PCR for restriction analysis, or methylation specific PCR. Due to the promising results related to ESR1 promoter methylation, we have used a quantification method by matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF MS) together with Epityper software to measure methylation at promoters A and C. mRNA stability was assessed in actinomycin D treated MCF-7 and MDA-MB-231 cells. ERα protein was quantified using transiently transfected breast cancer cells. Low ESR1_C transcript levels were associated with better overall survival (p = 0.017). High levels of ESR1_C transcript were associated with non-favorable response in tamoxifen treated patients (HR = 2.48; CI 95% 1.24-4.99), an effect that was more pronounced in patients with ERα/PgR double-positive tumors (HR = 3.41; CI 95% 1.45-8.04). The ESR1_C isoform had a prolonged mRNA half-life and a more relaxed 5’UTR structure compared to ESR1_A isoform. Western-blot analysis showed that at protein level, the promoter selectivity is undistinguishable. There was no correlation between levels of ESR2 isoforms or ESR2 promoter methylation and ERß protein staining. ESR1 promoter C CpG methylation and not promoter A was responsible for ERα loss. We propose ESR1_C levels as a putative novel marker for breast cancer prognosis and prediction of tamoxifen response

Modelo de crescimento para árvores em povoamentos juvenis de sobreiro em Portugal

The juvenile and adult stages in cork oak are distinct because of the periodic debarking of the stem and branches that characterizes the adult stage. This fact implies the use of diameter under bark for the adult stage while diameter over bark is the natural variable for juvenile stands in growth and yield studies. Tree growth in the adult stage may also be affected by the periodic debarking. The differences between the two stages justify the development of different models for each of them. The objective of this paper is to develop an individual tree growth and yield model for juvenile cork oak stands for general application on the cork oak distribution area in Southern Portugal. The most important modules of this growth model were developed using data collected from a large number of trees in the juvenile stage, from several plots distributed around the South of Portugal. These modules were: an individual tree diameter growth model, a height-diameter model and a model for crown diameter prediction

Why do people favor informal giving? : the effect of social pressure, efficiency concerns and social norms : evidence from Zakat in Yemen

Social ties play a crucial role on informal giving, which includes giving to friends, family, or neighbors, but the actual reasons why individuals are more generous towards socially close people are still to be investigated. Using data from Zakat, a religious obligation of Islam in Yemen, this thesis aims to identify how social pressure, efficiency concerns and social norms impact the probability and the amount of donating Zakat to family, friends, and neighbors. We confirm the importance of this group of recipients, with around half of the givers donating, on average, 42% of their Zakat to family, friends, and neighbors. While wealth is the most important economic resource to make households more prone to give to socially close people, income seems the most important resource when it comes to the amount given. We found that the wish to control the use of their gift is the only relevant channel for the probability of giving to this group of people, as when the efficiency concerns index increases by one unit, the probability of a household to give Zakat to family, friends and neighbors increases by 10.3 percentual points. None of the channels considered impact the amount of Zakat given to family, friends, and neighbors. Additionally, we find that givers give less Zakat than the social norm demands and that, while social pressure increases the probability of giving Zakat to institutions, the opposite happens for social norms and efficiency concerns. None of these channels impact the amount of Zakat given to institutions.Os vínculos sociais são cruciais para as doações informais, que incluem doações à família, amigos e vizinhos, apesar das verdadeiras razões pelas quais os indivíduos são mais generosos com pessoas mais próximas estarem a ser investigadas. Usando dados sobre o Zakat, uma obrigação religiosa do Islamismo no Yemen, esta tese pretende verificar se a pressão social, as preocupações com eficiência e as normas sociais afetam a probabilidade de dar Zakat à família, amigos e vizinhos e o montante dado. Confirmamos a importância deste grupo de beneficiários, verificando que 51% dos doadores os escolhem e lhes dão, em média, 42% do total do Zakat doado. Enquanto a riqueza é o recurso económico mais importante para a decisão de dar Zakat às pessoas com relações de maior proximidade, o rendimento parece ser mais importante para decidir quanto dar. Concluímos que o desejo de controlar o uso da doação é o único fator relevante para a probabilidade de doar a este grupo de pessoas, visto que quando o índice de preocupações com a eficiência da doação aumenta uma unidade, a probabilidade do agregado familiar dar Zakat a familiares, amigos e vizinhos aumenta 10.3 pontos percentuais. Nenhum destes fatores é importante para o montante que lhes é dado. Também constatamos que os doadores dão menos Zakat do que deveriam. Enquanto a pressão social aumenta a probabilidade de dar Zakat às instituições, a norma social e as preocupações com eficiência têm o efeito contrário. Nenhum destes fatores afeta o montante doado a instituições

STEAP1 exepression in prostate cancer and its regulation by androgens

Six transmembrane epithelial antigen of the prostate 1 (STEAP1) was identified as a gene overexpressed in human prostate cancer and spontaneous transgenic mouse model of prostate cancer. It is localized in cell junctions of epithelial cells, and its structure with six transmembrane domains, suggests that it may act as a membrane channel or transporter protein in tight junctions, gap junctions or in cell adhesion, helping in intercellular communication in a way that allows growth of cancer cells. Although STEAP1 expression seems to be up-regulated in all stages of prostate cancer, its clinical significance remains to be clarified. Moreover, STEAP1 is more expressed in LNCaP than in PC3, suggesting that androgens may regulate its expression. Therefore, the goals of this experimental work were: i) to evaluate if STEAP1 expression correlates with clinical reports from patients; ii) to analyze if STEAP1 is regulated by 5α- dihydrotestosterone (DHT) in vitro and in vivo, by Real-time PCR and Western blot. Immunohistochemical analysis revealed that STEAP1 expression is principally associated with epithelial cells, but it is also present in some stromal cells. Analysis of STEAP1 immunoreactivity in prostate cancer is underway. In vitro results demonstrated that both STEAP1 mRNA and protein expression are down-regulated in the presence of 1nM or 10nM DHT after 24h of stimulation. However, at least one more experimental assay is required to confirm these results. In vivo results demonstrated that castration visibly increases STEAP1 protein expression when compared to intact rats, and treatment with DHT abrogates the effect of castration in STEAP1 expression, suggesting that STEAP1 protein is down-regulated by DHT. However, these results do not correlate with STEAP1 mRNA expression, suggesting that mechanisms at the translation level may be involve

Perceções da Motivação e da Satisfação para uma melhor qualidade no Trabalho: O caso do Hotel Convento do Espinheiro

Diversos autores (Day e Peters, 1994; Evans e Lindsay, 2002) têm-se confrontado com o conceito de qualidade nos serviços, como um meio que tende a divulgar a eficácia e eficiência dos mesmos. A noção de qualidade denota, atualmente, uma estratégia de competitividade e um agente para o desenvolvimento sustentado, de modo a responder às expetativas dos consumidores. No setor turístico, esse desenvolvimento ambiciona-se que seja em harmonia com um turismo melhor e de mais qualidade (Cândido, 2005). O objetivo do presente trabalho consiste em avaliar a qualidade de uma organização do setor turístico na região do Alentejo (Évora), do ponto de vista dos colaboradores internos e superiores hierárquicos, através da aplicação de um inquérito por questionário, com vista à obtenção de melhorias contínuas em todas as áreas organizacionais. A importância que a satisfação e a motivação têm no ambiente de trabalho faz com que todas as práticas de gestão formem uma estrutura coerente, que é aperfeiçoada constantemente, e que sustenta o desempenho da estratégia pretendida para a organização

Gastronomy and Wines in the Alentejo Portuguese Region: Motivation and Satisfaction of Tourists from Évora

Abstract: Food and winemaking are a recognized tangible and intangible cultural heritage of Portugal. From the relationship between these two components, a strategic product emerged with a considerable potential for tourism industry, which is not ignored by many of tourism organizations. This chapter intends to analyze food and winemaking from a tourism demand perspective. Particularly, this study describes visitors’ profile, including, their motivations, their knowledge about the enological and gastronomic resources and the degree of satisfaction. A total of 308 questionnaires were collected between February and May of 2012, from the visitors that visited the historic center of Évora (Alentejo-Portugal). Results reveal a visitor profile associated with regional cuisine and wine products from Portugal. Moreover, visitors’ evidenced a high level of knowledge regarding the Portuguese cuisine and regional wines; although this not matches with their primary motivation for visit the city of Évora. R. Amara