Universidade de Lisboa: Repositório.UL

    Do predators always win? Starfish versus limpets. A hands-on activity examining predator-prey interaction

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    In this article we propose a hands-on experimental activity about predator–prey interactions that can be performed both in a research laboratory and in the classroom. The activity, which engages students in a real scientific experiment, can be explored not only to improve students’ understanding about the diversity of anti-predator behaviours but also to promote their understanding about the various stages of experimental scientific procedures, such as the definition of a research problem, the statement of testable hypotheses, designing the experiments and drawing conclusions based on the evidence

    Chronic obstructive pulmonary disease : a proteomics approach

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    Tese de doutoramento, Biologia (Biologia Molecular), Universidade de Lisboa, Faculdade de Ciências, 2012Chronic obstructive pulmonary disease (COPD) is characterized by chronic airflow limitation that is not fully reversible even under bronchodilators effect, caused by a mixture of small airway disease – obstructive bronchiolitis – and parenchymal destruction – emphysema. At the present time, COPD is the fourth leading cause of death and its prevalence and mortality are expected to continue increasing in next decade. Spirometry is the most reproducible way to measure lung function and is nowadays the best tool to diagnose airflow limitation and, consequently, diagnose COPD itself. Biomarkers for diagnosis and/or prognosis as well as novel targets for the development of more effective therapies for COPD are still needed. Proteomics has the capacity to provide large-scale information and consequently it has the potential to expand previous knowledge on COPD. Surprisingly, given the need for new biomarkers in COPD and the power of proteomics, proteomics have been quite neglected so far. Up to date only 50 reports (14 are reviews) match the search at Pubmed (http://www.ncbi.nlm.nih.gov/pubmed, accessed June 23, 2011) for COPD proteomics. Hence, there is a clear need to engage clinically valuable proteomics studies in order to match the need for new biomarkers in COPD. In last decade, the shotgun proteomics approach has become the method of choice for identifying and quantifying proteins in most large-scale studies. Compared with 2DE, shotgun proteomics allows higher data throughput and better protein detection sensitivity. This strategy is based on trypsin digestion of proteins into peptides. This produces a complex peptide mixture that is then separated by one- or multiple dimensional liquid chromatography (LC) and subjected to peptide sequencing using tandem mass spectrometry (MS/MS) before automated database searching. In the present work we have employed different methodologies within shotgun proteomics to generate solid and comprehensive data in COPD. There are quite a lot biological materials that can be used to investigate biomarkers for this disease. Although COPD is now known to possess a systemic inflammation xii component which is responsible for affecting other organs, it is in the lung that the events that lead to breathless take place. Investigating to the lung directly is therefore an optimal strategy to be able to identify proteins that may not be detectable elsewhere either because they are not present or diluted into undetectable concentrations. But this means that lung tissue has to be collected by biopsy which is an extremely invasive technique. But besides tissue biospecimens, other sources of biological materials used to study COPD is biofluids which includes sputum, bronchoalveolar and nasal lavage fluid, exhaled breath condensate, and blood. It had been observed before by means of microscopy that red blood cells (RBCs) from COPD patients showed deformations in their shape. RBCs are crucial to the uptake of oxygen from the lungs to the cells and this transport is dependent on their ability to change shapes rapidly while navigating through blood vessels. In addition, RBCs play a crucial role in antioxidant defense when fighting against oxidative stress, which has long been recognized as feature of COPD. In this work we made use of a RBC membrane fractionation procedure, stable isotope labeling and bidimensional liquid chromatography (strong cation exchange / reverse phase) before sample acquisition using a high-resolution fourier transform - ion cyclotron resonance (FT-ICR) mass spectrometer (Chapter III). A total of 4697 peptides were quantified as present in both COPD and control spectra corresponding to 1083 proteins. Three-hundred and fourteen proteins possessing at least two peptides were identified, 46% of which were annotated as membrane proteins. Golgin-245/p230 (GOLGA4), was identified as overexpressed in COPD, a protein which is reported to be essential for intracellular trafficking and cell surface delivery of tumor necrosis factor-α (TNF), the main proinflammatory cytokine made and secreted by inflammatory macrophages enhancing activation and recruitment of T-cells and ensuring robust innate and acquired immune responses. Chorein or Vacuolar protein sorting-associated protein 13A (VPS13A) is reported to play a role in the cytoskeleton organization has been associated with thorny deformations of circulating erythrocytes, possibly due to red cell membranes deformation. This protein was found to be underexpressed in COPD patients when compared to controls by MS and this underexpression was confirmed by WB. Consequently, underexpression of chorein may play an important role in the deformation of COPD RBCs. Many other interesting proteins were identified in the xiii context of COPD and, additionally, there were a considerable number of proteins described in RBC for the first time (Chapter III). To overcome the difficulty of acquiring fresh biopsies of well characterized patients, we have established in our laboratory a procedure to collect human fresh nasal epithelial cells. We have shown previously that these cells presented similar proteome of epithelial cells presented in the lower airway. Here, two different types of studies were presented using these cells: a study performed on the effects of cigarette smoke, which is the main risk factor for developing COPD (Chapter IV) and a comparative proteomic study between COPD patients and healthy individuals (Chapter V). Both were pioneer studies by investigating the proteome of fresh nasal epithelial cells from cigarette smoker subjects (Chapter IV) or COPD patients (Chapter V). In both studies a high-resolution mass analyzer, the orbitrap, was employed increasing the number of confident peptide/protein identifications. In Chapter IV, ninety-six proteins were found to be differentially expressed between the proteomes of healthy smokers and nonsmokers. These proteins were related to processes of antigen presentation, cell-to-cell signaling and interaction, cell morphology, drug metabolism, DNA repair, energy production or mitochondrial dysfunction. Although requiring further orthogonal validation, our data was consistent with previous evidences showing CD44, MUC5AC or SOD2 differential modulation in smokers due to inflammatory response pathways. In Chapter V, 89968 peptides and 1475 proteins were identified in total, of which 1173 proteins were identified by at least two peptides. We were able to confirm previous evidences that UPR is activated in COPD patients since we were able to observe overexpression in a considerable number of proteins involved in different protein complexes involved in UPR. This includes overexpression of VCP, both components of the Hsp10/Hsp60 chaperone complex (HSPD1 and HSPE1), CALR and two members of a large ER-localized multiprotein complex of at least 11 proteins, PPIB and ERP29. We also observed an increase in expression of proteins related to Nrf2-mediated oxidative stress response such as GSTP1, TXNRD1 and GSR. Additionally, we also report an increase in drug metabolism, as all significantly differentially expressed proteins related to this biofunction were overexpressed in COPD: GSTP1, GSR, AKR1C3 and ANXA2. Further validation by orthogonal methods is needed so that the activation of xiv UPR and Nrf2-mediated oxidative stress response and the increase in drug metabolism on the nasal epithelial cells of COPD patients is fully confirmed. In Chapter VI, serum collected from COPD patients was divided into 4 different groups in all different combinations of presence/absence of the two main features of COPD, chronic bronchitis and emphysema, to study their impact in the serum proteome. Due to its complex protein mixture, serum was first immunodepleted from its most abundant proteins, comprising about 94% of total protein content, before being analyzed by 1D-PAGE – LC-MS/MS (GeLC-MS/MS) in a linear ion trap mass spectrometer. This powerful strategy was able to identify as many as 2856 proteins, of which 929 were identified by two or more peptides. Plasminogen was found to be underexpressed in COPD patients that suffer simultaneously from emphysema and chronic bronchitis, while it maintained about the same expression level over the three other groups of COPD patients and this differential expression was successfully validated by ELISA. It was possible to identify other interesting proteins as TRAF3IP2, which is associated with innate immunity in response to pathogens, inflammatory signals and stress and has also been implicated in airway hyperresponsiveness or Isoform 1 of phosphatidylinositol-glycan-specific phospholipase D (GPLD1), which is GPI degrading enzyme that was described to be responsible for secretion of prostasin, which was the first of several membrane serine peptidases found to activate the epithelium-sodium channel (ENaC). Prostasin was also reported to have a critical role in regulating epithelial sodium transport in normal and pathological conditions in the lung. The work herein presented confirmed a few findings that had already been reported and more important revealed new insights into COPD disease mechanisms as well as provided new candidate biomarkers for these diseases. Further validation and integration of all data obtained into a systems biology approach will certainly contribute to increase knowledge of COPD and ultimately bringing the well being of patients.A Doença Pulmonar Obstrutiva Crónica (DPOC) é caracterizada por uma limitação obstrutiva do fluxo aéreo do exterior para os alvéolos e destes para o exterior. Esta limitação ventilatória não é completamente reversível mesmo após a administração de um broncodilatador e tende a ser progressiva. As causas que conduzem a esta limitação são um conjunto de patologias respiratórias do qual fazem parte a bronquite crónica e o enfisema. Para a obstrução do débito de ar na bronquite crónica, contribuem a inflamação das vias aéreas inferiores, a cicatrização das suas paredes, o edema do seu revestimento, o muco e o espasmo do músculo liso. A bronquite crónica manifesta-se por tosse frequente e produção aumentada de expectoração. No entanto nem todos os doentes portadores de bronquite crónica têm ou irão desenvolver uma limitação crónica do fluxo aéreo. No caso do enfisema, as paredes alveolares estão destruidas, pelo que os bronquíolos perdem o seu apoio estrutural e, por isso, entram em colapso quando expiram o ar. Por conseguinte, no enfisema a redução do fluxo de ar é permanente e de origem estrutural. Actualmente, nenhum tratamento tem a capacidade de reduzir a progressão da DPOC ou suprimir a inflamação das vias aéreas de pequeno calibre e do parênquima pulmonar. Nos dias de hoje, a DPOC é a quarta causa de morte no mundo e prevê-se que a sua prevalência e mortalidade continuem a aumentar ao longo da próxima década. Em Portugal e de acordo com um relatório divulgado pelo Observatório Nacional das Doenças Respiratórias (ONDR), cerca de 540 000 pessoas sofriam de DPOC em 2007, o que significa que 5,2% da população padece desta condição. Recentemente, um relatório divulgado pela iniciativa Burden of Obstructive Lung Disease (BOLD), revelou que a taxa de prevalência de DPOC em Portugal é de 14,2%. A espirometria é fundamental no diagnóstico e na avaliação da DPOC por ser o meio mais objectivo, padronizado e facilmente reprodutivel de medir o grau de obstrução das vias aéreas. Considera-se que existe obstrução brônquica e, portanto, DPOC, quando após a administração de um broncodilatador a relação FEV1/FVC é menor do xvi que 70% (FEV1 – Volume Expiratório Máximo no 1.º segundo; FVC – Capacidade Vital Forçada) A Proteómica tem a capacidade de provedenciar informação em larga escala e, consequentemente, de alargar o actual conhecimento da DPOC. Surpreendentemente dada a necessidade de encontrar novos biomarcadores em DPOC e do potencial da proteómica, esta tem sido negligenciada na investigação da doença até aos dias de hoje. Actualmente, existem apenas 50 publicações científicas, dos quais 14 são artigos de revisão, correspondentes a uma pesquisa no site da PubMed (http://www.ncbi.nlm.nih.gov/pubmed, acedido em 23 de Junho de 2011) com o termo “COPD Proteomics”. No entanto, quando pesquisados em separado o número total de artigos com os termos “COPD” e “Proteomics” é de, aproximadamente, 30 000. Portanto, existe uma clara necessidade de produzir novos estudos de proteómica para colmatar esta lacuna e encontrar novos biomarcadores para a DPOC. Na última década, a abordagem designada por shotgun proteomics tornou-se a abordagem de eleição para a identificação e a quantificação de proteínas em estudos de larga escala. Comparando com a electroforese bidimensional (2DE), esta abordagem permite obter um maior número de identificações de péptidos e proteínas por ter uma maior sensibilidade. A estratégia de shotgun proteomics tem como base a digestão tríptica de proteínas para péptidos que vai conduzir a uma mistura complexa de péptidos que são separados através de uma ou múltiplas dimensões cromatográficas. Posteriormente sofrem uma sequenciação peptídica através de um espectómetro de massa e pesquisa automática contra uma base de dados. No presente trabalho utilizaram-se diferentes metodologias dentro da abordagem shotgun proteomics com o objectivo de gerar resultados contundentes em DPOC. Existe uma vasta gama de materiais biológicos que podem ser usados para procurar biomarcadores para esta doença. Embora a DPOC possua um componente de inflamação sistémica que é responsável por afectar outros orgãos, é no pulmão que os mecanismos que originam a falta de ar estão presentes. Assim sendo, investigar directamente o pulmão é a estratégia ideal para poder encontrar as proteínas responsáveis por esses mecanismos que noutros materiais biológicos podem não estar presentes ou, quando presentes, se encontram em concentrações diminutas conduzindo à sua não detecção. Isto significa ter acesso a tecido pulmonar que é xvii obtido através de biopsia, uma técnica extremamente invasiva. Para além de tecido pulmonar, existem outras fontes de material biológico têm sido utilizadas para o estudo da DPOC como são os casos de expectoração, lavado nasal e lavado broncoalveolar, condensado do ar expirado e sangue e seus componentes. No presente trabalho foram utilizados eritrócitos, células do epitélio nasal e soro para o estudo da doença. Foi reportado por meios de microscopia que os eritrócitos de doentes com DPOC exibiam alterações na sua morfologia. Os eritrócitos são fundamentais no transporte de oxigénio dos pulmões para as células e este transporte depende da sua capacidade para mudar rapidamente a sua forma ao navegar pelos capilares sanguíneos. Os eritrócitos desempenham um papel fundamental no combate ao stress oxidativo, que é uma característica da DPOC. Devido à sua hipotética importância para a doença, eritrócitos foram isolados a partir de sangue total de doentes com DPOC e indivíduos saudáveis. Neste trabalho (Chapter III), foi utilizada uma técnica de fraccionamento membranar em conjunto com uma marcação isotópica com 18O/16O e posterior separação através de cromatografia bidimensional (cromatografia de troca catiónica e de fase inversa). A cromatografia de fase inversa estava directamente acoplada a um espectómetro de massa de alta resolução (espectómetro de massa de ressonância de ião-ciclotrão com transformada de Fourier – FT-ICR). Foi possível quantificar um total de 4697 péptidos presentes em ambos os grupos estudados, que corresponderam a 1083 proteínas. 314 proteínas foram identificadas por dois ou mais péptidos, 46% das quais se encontram anotadas como proteínas de membrana. A proteína GOLGA4 foi identificada como estando sobreexpressa em DPOC. Esta proteína está descrita como sendo essencial para o tráfego intracelular e pela colocação à superfície da célula da proteína tumor necrosis factor-α (TNF), a principal citocina pró-inflamatória produzida e secretada por macrófagos para activação e recrutamento de células T. Foi possível também identificar a proteína VPS13A que está associada a deformações em eritrócitos circulantes, possivelmente devido a deformações na membrana. Como resultado do estudo concluiu-se que em doentes com DPOC esta proteína se encontra subexpressa, resultado que foi confirmado por Western Blot. Devido à dificuldade em adquirir amostras de biópsias pulmonares em doentes com DPOC, foi desenvolvido no nosso laboratório um procedimento para obter células do xviii epitélio nasal. Em trabalhos anteriores foi demonstrado que estas células apresentam um comportamento semelhante às células epiteliais das vias respiratórias inferiores. Dois diferentes tipos de estudo foram realizados com estas células: um estudo sobre os efeitos do fumo do tabaco, que é o principal factor de risco para o desenvolvimento de DPOC (Chapter IV) e um estudo comparativo entre doentes com DPOC e indivíduos saudáveis (Chapter V). Ambos os estudos são estudos pioneiros uma vez que o proteoma de células do epitélio nasal nunca tinha sido descrito quer em indivíduos saudáveis fumadores, quer em doentes com DPOC. No primeiro estudo, 96 proteínas foram identificadas como diferencialmente expressas entre fumadores e não fumadores saudáveis. Estas proteínas estão relacionadas com processos de apresentação de antigénios, sinalização e interacção celular, morfologia celular, metabolismo de xenobióticos, reparação de DNA, produção de energia e disfunção mitocondrial. Os resultados obtidos foram consistentes com anteriores evidências que mostram uma diferente modulação de CD44, MUC5AC ou SOD2 em fumadores devido a processos de resposta inflamatória. No segundo estudo, foram identificados um total de 89968 péptidos correspondentes a 1475 proteínas das quais 1173 foram identificadas por dois ou mais péptidos. Foi possível confirmar resultados obtidos em estudos anteriores que referiam que mecanismos de Unfolded Protein Response (UPR) se encontravam activados em doentes com DPOC, uma vez que foi observada a sobreexpressão de um númeo considerável de proteínas envolvidas em diferentes complexos de proteínas relacionados com UPR. Exemplos dessas proteínas incluem a VCP, os dois componentes do complexo Hsp10/Hsp60, a CALR e dois membros de um grande complexo composto por, pelo menos, 11 proteínas, localizado no retículo endoplasmático: PPIB e ERP29. Foi também possível observar um aumento na expressão de proteínas relacionadas com os mecanismos de resposta ao stress oxidativo mediados por Nrf2, como GSTP1, TXNRD1 e GSR. Relativamente ao estudo efectuado com o soro (Chapter VI), quatro grupos foram constituídos para estudar as diferentes combinações de presença/ausência dos dois principais componentes da doença: a bronquite crónica e o enfisema. Uma vez que o soro é uma mistura complexa de proteínas, o soro de doentes de DPOC foi primeiramente imunodepletado das suas proteínas mais abundantes, que xix representam cerca de 94% do seu conteúdo total de proteína. As amostras de soro imunodepletado foram analisadas através de 1D-PAGE – LC-MS/MS (GeLC-MS/MS). Esta poderosa estratégia permitiu a identificação de 33049 péptidos correspondentes a 2856 proteínas, das quais 929 foram identificadas por dois ou mais péptidos. Foi possível observar uma subexpressão de PLG em doentes com DPOC que sofrem simultaneamente de enfisema e bronquite crónica. Esta subexpressão foi validada através de ELISA. Outras proteínas de interesse foram encontradas diferencialmente expressas em cada um dos grupos de doentes com DPOC, entre as quais TRAF3IP2, que está associada à resposta imunitária inata a patogéneos e à resposta exacerbada das vias respiratórias. O trabalho aqui descrito confirmou evidências anteriormente reportadas e ao mesmo tempo formulou novas hipóteses para os mecanismos da doença e revelou potenciais novos biomarcadores. Embora alguns resultados necessitem de posterior validação através de técnicas ortogonais, o presente trabalho tornou possível a descoberta de proteínas e, inclusivamente, vias metabólicas que não tinham sido anteriormente associadas à DPOC. Este trabalho enfatiza também o uso de células epiteliais nasais na investigação da patogénese da doença, uma vez que pode conduzir/conduz à identificação de novos biomarcadores específicos desta doença.Fundação para a Ciência e a Tecnologia (FCT, SFRH/BD/2006/31415); POCI/SAU-MMO/56163/2004; FCT/Poly-Annual Funding Program e FEDER/Saúde XXI Program (Portugal

    Designing an e-tutoring system for large classes: mixed-method research

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    This study aimed at assessing the perceptions of 167 teachers about the tutoring system adopted in an online training course involving teachers from 20 Schools of Sesimbra, Setúbal and Palmela counties. The course, called “Distributed Knowledge with Web 2.0”, was officially certified as a blended learning modality, with the duration of 50 hours, 41 of which occurred online in two editions, the first in February and the second in July of 2012, each one of them involving respectively 82 and 85 teachers, divided in four classes with about 20 trainees each. This blended learning course was designed at producing educational materials in digital format, and included autonomous and group activities, knowledge sharing and reflection. A learning environment, supported by the Ning platform, was set up. At the end of the course, the trainees answered to a pencil and paper survey, in order to evaluate the adopted online tutoring strategy. Additionally the trainees’ final reports contained evidence of how the trainees assessed the tutoring model component of the course; both the survey and the reports were the basis for this research. The results show that the teachers who attended the two course editions disclosed very positive perceptions about online learning, a modality they consider adequate to their current professional status and conditions. The trainees also showed their intention of, in the future, opting for blended training arrangements. Future developments of this study involve a content analysis of the tutor’s posts, in order to understand more accurately the tutor’s messages characteristics, in their social and cognitive dimensions

    Identification of a cytokine network sustaining neutrophil and Th17 activation in untreated early rheumatoid arthritis

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    © 2010 Cascão et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by sustained synovitis. Recently, several studies have proposed neutrophils and Th17 cells as key players in the onset and perpetuation of this disease. The main goal of this work was to determine whether cytokines driving neutrophil and Th17 activation are dysregulated in very early rheumatoid arthritis patients with less than 6 weeks of disease duration and before treatment (VERA). Methods: Cytokines related to neutrophil and Th17 activation were quantified in the serum of VERA and established RA patients and compared with other very early arthritis (VEA) and healthy controls. Synovial fluid (SF) from RA and osteoarthritis (OA) patients was also analyzed. Results: VERA patients had increased serum levels of cytokines promoting Th17 polarization (IL-1b and IL-6), as well as IL-8 and Th17-derived cytokines (IL-17A and IL-22) known to induce neutrophil-mediated inflammation. In established RA this pattern is more evident within the SF. Early treatment with methotrexate or corticosteroids led to clinical improvement but without an impact on the cytokine pattern. Conclusions: VERA patients already display increased levels of cytokines related with Th17 polarization and neutrophil recruitment and activation, a dysregulation also found in SF of established RA. 0 Thus, our data suggest that a cytokine-milieu favoring Th17 and neutrophil activity is an early event in RA pathogenesis.This work was supported by a grant from Sociedade Portuguesa de Reumatologia/Schering-Plough 2005. RAM and RC were funded by Fundação para a Ciência e a Tecnologia (FCT) SFRH/BD/30247/2006 and SFRH/BD/40513/2007, respectively. MMS-C was funded by Marie Curie Intra-European Fellowship PERG-2008-239422 and a EULAR Young Investigator Award

    Prokofiev’s “Romeo and Juliet” and Socialist Realism: a Case-Study in Intersemiotic Translation

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    This paper is concerned with Prokofiev’s ballet score, Romeo and Juliet, as an inter-semiotic translation of Shakespeare’s play. My aim is to consider Prokofiev’s distinct interpretation of the play in the light of the political climate of the Soviet Union in the 1930s and to attempt to determine the extent to which it conforms to the norms of Socialist Realism. This is by no means an easy matter, for the composer was at different times lauded by the authorities and vilified by them, while the work itself has been appropriated by both sides of the ideological divide. Nevertheless, close examination of the musical discourse reveals important alterations to Shakespeare’s original concept, shedding some light upon the composer’s own ideology and consequently upon his relationship to the regime under which he lived

    Best-Sellers in Portugal: the case of Bridget Jones

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    Portugal continua a consumir em grande quantidade ficção traduzida. Em 2001, um dos maiores êxitos foi O Diário de Bridget Jones, de Helen Fielding, obra que fez rir milhares de pessoas mesmo antes do filme, que saiu no mesmo ano. Contudo, o humor desta obra é culturalmente muito específico, baseado numa rede semiótica que só pode ser apreciada por quem vive dentro da cultura de origem. Qual foi a política da tradutora relativa a estes elementos tão dificilmente traduzíveis? Conseguiu transmitir as complexidades da sociedade contemporânea britânica? Ou será que houve outro motivo menos definido por detrás desta apropriação e que teria a ver com o modo de representar o Outro neste vasto mundo globalizado

    Using zeta-potential measurements to quantify peptide partition to lipid membranes

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    © The Author(s) 2011. This article is published with open access at Springerlink.com.Open Access: This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.Many cellular phenomena occur on the biomembranes. There are plenty of molecules (natural or xenobiotics) that interact directly or partially with the cell membrane. Biomolecules, such as several peptides (e.g., antimicrobial peptides) and proteins, exert their effects at the cell membrane level. This feature makes necessary investigating their interactions with lipids to clarify their mechanisms of action and side effects necessary. The determination of molecular lipid/water partition constants (Kp) is frequently used to quantify the extension of the interaction. The determination of this parameter has been achieved by using different methodologies, such as UV-Vis absorption spectrophotometry, fluorescence spectroscopy and ζ-potential measurements. In this work, we derived and tested a mathematical model to determine the Kp from ζ-potential data. The values obtained with this method were compared with those obtained by fluorescence spectroscopy, which is a regular technique used to quantify the interaction of intrinsically fluorescent peptides with selected biomembrane model systems. Two antimicrobial peptides (BP100 and pepR) were evaluated by this new method. The results obtained by this new methodology show that ζ-potential is a powerful technique to quantify peptide/lipid interactions of a wide variety of charged molecules, overcoming some of the limitations inherent to other techniques, such as the need for fluorescent labeling.This work was partially supported by project PTDC/QUI/ 69937/2006 from Fundação para a Ciência e Tecnologia-Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES, Portugal), and by Fundação Calouste Gulbenkian (Portugal). JMF and MMD also thank FCT-MCTES for grants IMM/BT/37-2010 and SFRH/BD/41750/2007, respectively

    Differential mesenteric fat deposition in bovines fed on silage or concentrate is independent of glycerol membrane permeability

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    © The Animal Consortium 2011In the meat industry, the manipulation of fat deposition in cattle is of pivotal importance to improve production efficiency, carcass composition and ultimately meat quality. There is an increasing interest in the identification of key factors and molecular mechanisms responsible for the development of specific fat depots. This study aimed at elucidating the influence of breed and diet on adipose tissue membrane permeability and fluidity and their interplay on fat deposition in bovines. Two Portuguese autochthonous breeds, Alentejana and Barrosã, recognized as late- and early-maturing breeds, respectively, were chosen to examine the effects of breed and diet on fat deposition and on adipose membrane composition and permeability. Twenty-four male bovines from these breeds were fed on silage-based or concentrate-based diets for 11 months. Animals were slaughtered to determine their live slaughter and hot carcass weights, as well as weights of subcutaneous and visceral adipose depots. Mesenteric fat depots were excised and used to isolate adipocyte membrane vesicles where cholesterol content, fatty acid profile as well as permeability and fluidity were determined. Total accumulation of neither subcutaneous nor visceral fat was influenced by breed. In contrast, mesenteric and omental fat depots weights were higher in concentrate-fed bulls relative to silage-fed animals. Membrane fluidity and permeability to water and glycerol in mesenteric adipose tissue were found to be independent of breed and diet. Moreover, the deposition of cholesterol and unsaturated fatty acids, which may influence membrane properties, were unchanged among experimental groups. Adipose membrane lipids from the mesenteric fat depot of ruminants were rich in saturated fatty acids, and unaffected by polyunsaturated fatty acids dietary levels. Our results provide evidence against the involvement of cellular membrane permeability to glycerol on fat accumulation in mesenteric fat tissue of concentrate-fed bovines, which is consistent with the unchanged membrane lipid profile found among experimental groups.This study was supported by Fundação para a Ciência e a Tecnologia (FCT) through grant PTDC/CVT/2006/66114 and individual fellowships to Ana P. Martins (SFRH/BD/2009/65046), Ana S. H. Costa (SFRH/BD/2009/61068) and Susana V. Martins (SFRH/BPD/2009/63019). Paula A. Lopes is a researcher from the program ‘‘Ciência 2008’’ from FC

    Lack of Aquaporin 3 in bovine erythrocyte membranes correlates with low glycerol permeation

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    NOTICE: this is the author’s version of a work that was accepted for publication in Biochemical and Biophysical Research Communications. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Biochemical and Biophysical Research Communications. May 2011; 408 (3): 477-481.In general, erythrocytes are highly permeable to water, urea and glycerol. However, expression of aquaporin isoforms in erythrocytes appears to be species characteristic. In the present study, human (hRBC) and bovine (bRBC) erythrocytes were chosen for comparative studies due to their significant difference in membrane glycerol permeability. Osmotic water permeability (Pf) at 23 ºC was (2.89 ± 0.37) × 10-2 and (5.12 ± 0.61) × 10-2 cm s-1 for human and bovine cells respectively, with similar activation energies for water transport. Glycerol permeability (Pgly) for human ((1.37 ± 0.26) × 10-5 cm s-1) differed in three orders of magnitude from bovine erythrocytes ((5.82 ± 0.37) ×10-8 cm s-1) that also showed higher activation energy for glycerol transport. When compared to human, bovine erythrocytes showed a similar expression pattern of AQP1 glycosylated forms on immunoblot analysis, though in slight higher levels, which could be correlated with the 1.5-fold larger Pf found. However, AQP3 expression was not detectable. Immunofluorescence analysis confirmed the absence of AQP3 expression in bovine erythrocyte membranes. In conclusion, lack of AQP3 in bovine erythrocytes points to the lipid pathway as responsible for glycerol permeation and explains the low glycerol permeability and high Ea for transport observed in ruminants

    A Prelude to Joyce’s Chamber Music

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    It seems appropriate not to overlook the documentary value of Chamber Music in so far as it might be profitably approached as a kind of aesthetic laboratory where Joyce experimented with various styles, forms, and modes of expression. In particular, the book documents an early Joycean attempt to explore the interaction between sound and meaning, with a view to the ideal blending of two com plementary discourses, i.e. poetry and music. Such a revaluation would redress the balance in establishing the literary interest of Chamber Music, which might then emerge as a significant step both in the progress of Joyce’s art and in the development of the poetry and poetics of the pre-modernist generation.Fundação para a Ciência e a Tecnologi
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