Nanopartículas multifuncionais para a entrega intracelular de um anticorpo a células cancerígenas com expressão de CD44v6

Colorectal cancer (CRC) is one of the most incident and mortal cancers in the world, mainly due to its metastatic ability. CD44 receptor isoform containing exon 6 (CD44v6) in colorectal cells have been implied in many cancers associated process including metastasis. CD44v6 is a co-receptor for hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF), along with c-Met and VEGF receptor 2 (VEGFR-2), respectively. Standard chemotherapy regimens recommend for metastatic stages of CRC are commonly associated with severe side effects because they act non-selectively. Therapies that act specifically against a molecular target enhance efficacy of the drugs, resulting in lower toxicity. One example is bevacizumab (Avastin®), an anti-angiogenic monoclonal antibody (mAb) that inhibits VEGF action. Drug delivery systems (DDS), as nanoparticles (NPs) can be conjugated with ligands in order to be directed to a specific molecular target. One type of ligand with high affinity to cell receptors are antibodies. In this work, it was developed an innovate nanosystem, with polymeric NPs of poly(lactic-co-glycolic acid)-polyethylene glycol (PLGA-PEG) loaded with bevacizumab and decorated with an antibody fragment (Fab) specific for CD44v6-expressing human cancer cells, the AbD15179 (v6 Fab). Bare and (-) Fab PLGA-PEG NPs were also developed to evaluate the specificity of v6 Fab. The NPs produced displayed sizes in the range of 200-300 nm and a negative surface electric charge between -5 and -10 mV, with an association efficiency (AE) of bevacizumab around 85%. Overall, the formulations at a concentration of 5 and 50 μg/mL did not show toxicity in cancer cells. The v6 Fab-PLGA-PEG NPs specifically bonded to the surface of cells overexpressing CD44v6 and this affinity was maintained with the encapsulation of bevacizumab. Also, PLGA-PEG NPs functionalized with v6 Fab were more internalized in cells than the bare and (-) Fab-PLGA-PEG NPs. To confirm the specificity of bevacizumab-loaded v6Fab-PLGA-PEG NPs, intracellular levels of bevacizumab and VEGF were evaluated after incubation of PLGA-PEG NPs. Intracellular levels of bevacizumab were significantly higher in cells incubated with v6 Fab-PLGA-PEG NPs. The percentage of intracellular VEGF levels did not show significance between the groups but was possible to observe a tendency in the decrease of VEGF levels after incubation with v6 Fab-PLGA-PEG NPs. In this work it was demonstrated that it is possible to encapsulate a mAb in PLGA-PEG NPs and then functionalize with a Fab for a specific and effective intracellular delivery of the drug. Though, their use in drug delivery requires further investigation and optimization.O cancro colorretal (CCR) é um dos cancros mais incidentes e mortais no mundo, maioritariamente devido à sua capacidade de metastização. A isoforma do recetor CD44 com o exão 6 (CD44v6) nas células colorretais tem sido envolvida em diversos processos cancerígenos, incluindo metástases. O CD44v6 é coreceptor para o fator de crescimento de hepatócitos (HGF) e fator de crescimento endotelial vascular (VEGF), junto com o c-Met e o recetor VEGF 2 (VEGFR-2), respetivamente. Os regimes padrão de quimioterapia recomendada para os estádios metastáticos do CCR estão frequentemente associados a efeitos secundários graves, uma vez que não atuam seletivamente. As terapias que atuam seletivamente contra um alvo molecular melhoram a eficácia dos fármacos, resultando em menor toxicidade. Um exemplo é o bevacizumab (Avastin®), um anticorpo monoclonal (mAb) que atua inibindo a ação do VEGF. Sistemas de entrega de fármacos (DDS), como nanopartículas (NPs), podem ser conjugados com ligandos de modo a serem direcionados para um alvo molecular específico. Um tipo de ligando com elevada afinidade para recetores celulares são os anticorpos. Neste trabalho foi desenvolvido um nanosistema inovador, com NPs poliméricas de poli(ácido láctico-co-glicólico)-polietilenoglicol (PLGA-PEG) encapsuladas com bevacizumab e conjugadas com um fragmento de anticorpo (Fab) específico para células cancerígenas humanas com expressão de CD44v6, o AbD15179 (Fab v6). Foram também produzidas NPs não funcionalizadas e com um Fab (-) para avaliar a especificidade do Fab v6. As NPs produzidas apresentaram um tamanho entre 200 a 300 nm e uma carga elétrica superficial negativa entre -5 e -10 mV, com uma eficiência de associação (EA) do bevacizumab de cerca de 85%. No geral, as formulações com uma concentração de 5 e 50 μg/mL não demonstraram toxicidade em células cancerígenas. As NPs de PLGA-PEG Fab v6 ligaram-se especificamente à superfície das células que sobreexpressam o CD44v6 e essa afinidade foi mantida com a encapsulação de bevacizumab. Além disso, as NPs PLGA-PEG Fab v6 mostraram maior internalização celular do que as NPs não funcionalizadas e Fab (-). De modo a confirmar a especificidade das NPs PLGA-PEG Fab v6 com bevacizumab, os níveis intracelulares de bevacizumab e VEGF foram avaliados. Os níveis intracelulares de bevacizumab foram significativamente mais elevados nas células incubadas com NPs PLGA-PEG Fab v6. Os níveis intracelulares de VEGF não mostraram significância entre os grupos, mas foi possível observar uma tendência na diminuição dos níveis de VEGF após a incubação das NPs PLGA-PEG Fab v6. Neste trabalho foi demonstrado que é possível encapsular um mAb em NPs PLGA-PEG e funcionalizar com um Fab para uma entrega intracelular do fármaco específica efetiva. No entanto, o seu uso na entrega de fármacos requer mais investigação e otimização.Mestrado em Biomedicina Molecula

In vitro infection of lymphoid tissues by HIV-2

Tese de mestrado, Ciências Biofarmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2016Lymphoid organs constitute an optimal environment for the defense against the invasion of pathogens by promoting immune responses. They are the major reservoirs of human immunodeficiency virus (HIV) infection, however their infection has only been focused in the context of HIV-1. HIV-2 represents a more attenuated form of HIV disease, characterized by slower CD4+ T cell decline and progression to AIDS, that doesn’t have the same impact worldwide as HIV-1. Nevertheless, HIV-2 constitutes a unique naturally occurring model of attenuated HIV disease valuable for the study of HIV pathogenesis. Understanding the impact of HIV-2 infection on lymphoid organs will provide new insights regarding the ability of the tissue to promote immune responses. Here we investigated the impact of HIV-2 in human tonsillar tissue, a secondary lymphoid organ (SLO). Tonsil organ cultures (TOCs) were infected with HIV-2 or HIV-1 primary isolates using either CCR5 (R5) or CXCR4 (X4) coreceptors. All viruses were able to replicate in the tissue, as revealed by immunohistochemistry. In addition, we found that HIV-2-infected lymphocytes from TOCs presented similar levels of total HIV DNA as HIV-1. Surprisingly, Gag mRNA and protein levels were significantly higher in tissue infection by HIV-1 X4 as compared to R5-tropic HIV-1 or HIV-2, suggesting that the viral production observed in TOCs may be due to the infection of other cells in the tissue, and that viral transcriptional regulation may be altered in X4-tropic HIV-2. Interestingly, despite the lower lymphocyte-associated viral replication observed with X4-tropic HIV-2, the impact on the viability of CD4+ T cells and on the memory CD4+ T cell compartment was similar to that observed with HIV-1 X4. Moreover, we found that infection of TOCs with X4-tropic viruses resulted in a higher frequency of cells expressing Foxp3, a molecule related with the regulatory phenotype. This was particularly observed for X4-tropic HIV-2 infection and was not related with cell proliferation. In addition, we observed a significantly higher depletion of follicular cells within Foxp3+ cells in TOCs infected with X4-tropic HIV-2, indicating higher persistence of Foxp3+ Tregs. Finally, we observed that all viruses were able to deplete the T follicular helper (TFH) subset, a subset that is essential for B cell differentiation. In conclusion, our data showed that HIV-2 is able to infect lymphoid tissue in a coreceptor-dependent manner, but that the replication cycle was impaired in lymphocytes at the transcriptional level. These findings on in vitro infection of lymphoid tissues by HIV-2 will provide new insights regarding HIV immunopathogenesis.Os órgãos linfóides proporcionam respostas imunitárias contra patogénios e é onde estão a maior parte dos linfócitos existentes no corpo humano. Estes órgãos são os maiores reservatórios da infeção pelos vírus de imunodeficiência humano (VIH), no entanto o impacto da infeção é maioritariamente estudado no contexto do VIH-1. O VIH- 2 representa uma forma atenuada da doença, caracterizada pela lenta diminuição de linfócitos CD4+ e progressão para SIDA. Para além disso, o impacto na sociedade é muito menor que o do VIH-1. Contudo, o VIH-2 é um modelo único de ocorrência natural valioso para o estudo da patogénese do VIH. Por esta razão, compreender o impacto da infeção por VIH-2 em órgãos linfóides poderá levar a novas descobertas relativas à habilidade destes tecidos proporcionar respostas imunitárias. Neste projeto, investigámos o impacto do VIH-2 em tecido de amígdala humana, um órgão linfóide secundário. Culturas de amígdala foram infetadas com isolados primários de VIH-1 ou VIH-2 que usam ou o corecetor CCR5 (R5) ou o CXCR4 (X4). Demonstrámos que ambos os vírus são eficientes a infetar o tecido, tendo sido evidenciado por imunohistoquímica. Para além disso, demonstrámos que os linfócitos derivados da cultura de amígdala infetados por VIH-2 tinham uma quantidade de ADN viral integrado semelhante ao das infetadas por VIH-1. Surpreendentemente, a quantidade de mRNA e proteína Gag produzidos mostrou ser significativamente mais elevada na infeção do tecido pelo VIH-1 X4 comparativamente ao VIH-1 R5 e ao VIH-2, sugerindo que o vírus observado no tecido poderá estar relacionado com infeção de outras células, e que a transcrição do vírus X4 HIV-2 poderá estar alterada. No entanto, apesar da baixa replicação associada aos linfócitos observada no tecido infetado por VIH-2 X4, o impacto na viabilidade de linfócitos T CD4+ e no compartimento de linfócitos T CD4+ memória foi semelhante ao observado com o VIH-1 X4. No entanto, a infeção pelos vírus que usam o coreceptor CXCR4 resultou no aumento da expressão de Foxp3 nas células dos tecidos, uma molécula associada ao fenótipo regulador, e tal não se deveu a um aumento da proliferação destas células. Observámos ainda que o VIH-2 X4 depletou significativamente as células Foxp3+ foliculares, persistindo preferencialmente linfócitos T reguladores Foxp3+. Por último, observámos uma depleção dos linfócitos T foliculares por parte de todos os vírus. Em conclusão, os nossos dados mostram que o VIH-2 é capaz de infetar o tecido dependentemente do corecetor, mas o ciclo de replicação foi diminuído nos linfócitos a um nível transcricional. Estes resultados relativos à infeção de órgãos linfóides in vitro por VIH-2 permitirão novas descobertas sobre a imunopatogénese do VIH

Crowdfunding projects : what makes project creators feel successful?

Ao longo dos últimos anos, o crowdfunding tem vindo a ganhar notoriedade enquanto ferramenta de financiamento alternativa aos métodos tradicionais, numa altura em que o empreendedorismo e as ideias inovadoras são cada vez mais uma fonte de criação de negócios. As campanhas de crowdfunding permitem uma aproximação entre o criador da ideia e os consumidores finais, levando a que estes últimos possam participar na transformação da ideia em realidade através de contribuições monetárias. O método mais popularizado é aquele em que, em troca de uma contribuição para o projeto, o contribuidor recebe uma recompensa, podendo esta ser, por exemplo, um exemplar do produto final, um agradecimento, um desconto. Além da evidente aproximação criador-consumidor final, são de salientar as vantagens inerentes a este tipo de interação: o produto é testado mesmo antes de ser produzido em grande escala, obtendo-se feedback e permitindo que sejam feitas melhorias ainda numa fase inicial; e ao fazer-se a apresentação da ideia/produto para apelar à contribuição monetária, comunica-se e publicita-se o produto, podendo este ganhar notoriedade. Associados a cada campanha existem objetivos definidos do seu criador, que podem passar apenas por atingir o objetivo monetário ou beneficiar também das outras vantagens inerentes. A avaliação de cada campanha depende sempre das expetativas pelo seu criador, e é este que a pode considerar ou não um sucesso.Over the last years, crowdfunding has been gaining visibility as an alternative financing tool in relation to traditional methods, in a time where entrepreneurship and new ideas are increasingly a means to create businesses. Crowdfunding campaigns make it possible to bring project creators and final consumers closer, giving these consumers the opportunity to participate in turning an idea into reality through monetary contributions. The most popular method is the one in which, in exchange for monetary contribution to the project, contributors receive a reward, which may be for example an article of the final product, an aknowledgement, or a discount. Besides the evident connection between creators and final consumers, it is important to point out other advantages associated with this type of interaction: the product is tested even before being produced on a large scale, being possible to obtain feedback and make improvements at an early stage; and when presenting the idea/product for raising monetary contributions, the product is disclosed and advertised, thus there is a chance of increasing the visibility of the product. Associated with every campaign, there are objectives defined by each creator, which may simply be achieving a monetary goal or also taking advantage of other inherent benefits. The assessment of each campaign always depends on the expectations of its creator, the person who is best able to decide whether or not it was actually successful

Signal transduction pathways involving the hypertension-related WNK1 and WNK4 protein kinases

Dissertação apresentada para obtenção do Grau de Doutor em Biologia, na especialidade de Genética Molecular, pela Universidade Nova de Lisboa, Faculdade de Ciências e TecnologiaThe genes WNK1 and WNK4 belong to the subfamily of WNK protein kinases and their mutation causes pseudohypoaldosteronism type II, a rare familial form of hypertension with hyperkalemia and hypercalciuria. The molecular mechanisms underlying this condition involve the regulation of renal electrolyte homeostasis and the modulation of diverse ion channels and transporters via WNK kinases. Additionally, WNKs have also been reported to participate in signal transduction pathways related to cell survival and proliferation. The objective of the present thesis was to identify novel WNK1 and WNK4 interacting proteins and the underlying signal transduction pathways. First, it was found that WNK1 forms a protein complex with the Rab-GAP TBC1D4 and phosphorylates it in vitro. It was shown that the expression levels of WNK1 regulate surface expression of the constitutive glucose transporter GLUT1 in HEK293 cells. WNK1 is shown to increase the binding of TBC1D4 to regulatory 14-3-3 proteins while reducing its interaction with the exocytic small GTPase Rab8A. Moreover, these effects were kinase activity-dependent. Together, the data describe a pathway regulating constitutive glucose uptake via GLUT1, the expression level of which is related to several human diseases. Second, WNK4 was found to promote the cell surface expression of the CFTR chloride channel in mammalian cells. The mechanism by which WNK4 acts on CFTR involves interaction with the tyrosine kinase Syk, which we found to phosphorylate tyrosine 512 (Tyr512) in the first nucleotide-binding domain of CFTR. The presence of WNK4 prevents this in vitro phosphorylation in a kinase-independent manner. In BHK21 cells stably expressing CFTR, Syk reduces, while WNK4 promotes, the cell surface expression of CFTR. Mutation of Tyr512 revealed that its phosphorylation is a novel signal regulating the prevalence of CFTR at the cell surface and that WNK4 and Syk play an antagonistic role in this process. Finally, ten WNK4 variants were detected in a cohort of Portuguese patients and control individuals, which subsequently were tested for association to hypertension and/or osteoporosis. Despite none of the variants yield any significant association to hypertension, a rare missense alteration (rs56116165) in a highly conserved arginine residue showed a nominal association to osteoporosis. This finding advocates that this polymorphism is a rare allelic variant, in a candidate gene with a biological function in renal calcium homeostasis, that may contribute to a genetic predisposition to osteoporosis.Fundação para a Ciência e Tecnologia - 2006 a 2009 (SFRH/BD/23001/2005),Programa de Financiamento Plurianual do Centro de Investigação em Genética Molecular Humana e do projecto POCI/SAU-MMO/56439/200

The determinants of length of stay in the Azores : a count model approach

This paper employs count data models to estimate the determinants of length of stay, as count data models naturally lend themselves to overcome the censoring and truncation data issues associated with the non-negative, integer nature of length of stay. This paper employs a rich micro data set gathered through questionnaires ministered to a representative sample of tourists departing from the Azores: the fastest growing touristic region in Portugal. It is found that sociodemographic profiles, such as nationality and Azorean ascendancy, and trip attributes, such as repeat visitation rates and type of flight, are important determinants of length of stay. In addition, it is found that destination image and attitudes regarding environmental initiatives, constructed from a factor analysis exercise, also influence length of stay. In particular, the results suggest that marketing strategies that promote the Azores for its nature, landscape, remoteness, weather and safety may increase length of stay, whereas cultural heritage has the opposite effect.N/

Multidisciplinary in tourism: designing cultural itineraries

ABSTRACT: The aim of this article is to reflect on several tourist research experiences carried out in an interdisciplinary context by teachers and students within the scope of the Tourist course. This experience consisted in conceiving historical and cultural itineraries. The aim of these itineraries is to recuperate the heritage sites for locals and potential tourists, who are increasingly interested in knowing the local way of life based on their own self-interpretation. The planning of the itineraries should be a means to promote the journey and return of the visitor, helping to guarantee a more complete, innovative and motivating experience. After working on organizing historic-heritage information on a particular area/site, its presentation should be attractive: the art of revealing the meanings of the cultural heritage and the culture to a public making use of their leisure time.info:eu-repo/semantics/publishedVersio

Interpretação, história e turismo cultural: evidências do pórtico da catedral de Lamego

Este artigo pretende destacar a aproximação entre o ensino de História, do Turismo e da Interpretação do património. Neste sentido, uma equipa multidisciplinar tem levado a cabo uma investigação, que envolve intersecções entre estas áreas científicas, numa cidade histórica do norte de Portugal (cidade de Lamego). Foi criado um discurso interpretativo do pórtico de um dos mais emblemáticos monumentos desta cidade histórica (Sé Catedral), tendo sido realizadas entrevistas em profundidade a 15 visitantes. Concluímos que a interpretação dos recursos históricos permite a construção de um produto turístico patrimonial. Através de uma ligação mais estreita entre o discurso histórico, interpretação e turismo é possível criar uma diferenciação do produto patrimonial tornando-o num produto único e, portanto, mais atraente para os diversos segmentos. Na construção dos discursos acerca de elementos patrimoniais com vista à apresentação a potenciais visitantes, descobrimos que tanto o discurso histórico como o turístico possuem semelhanças, diferindo, sobretudo, na linguagem. O ensino de História deposita na construção de narrativas uma possibilidade de cultivar a formação histórica dos alunos para atingir consciências históricas. As narrativas recortadas pelo turismo, não negam as históricas, mas a sua intenção passa pela agregação de valores atuais e interpretações que diferenciem o Património.info:eu-repo/semantics/publishedVersio

The potential benefits of using videos in higher education

The developments of digital technology have opened new outlooks for online education which offer students the flexibility to learn at any time and any place. With all this instructional changes instructors, in all levels of the educational chain have been compelled to adapt quickly to this reality. They have a wide diversity of tools available to grab student’s attention and motivate them to embrace the knowledge in their own learning process. One of these resources is the use of videos. Through them lecturers can deliver complex information and contents to students and, if used creatively, videos can become a powerful technological tool in education. In this article we will explore some of the potential benefits and challenges associated with the use of videos in the teaching and learning process at higher education levels. We will also discuss some thoughts and examples for the use of teaching materials to enhance student’s learning and try to change ideas about the potentialities and future of video’s annotation new software resources, as incoming open tools for group work involvement

Interpretation, history and heritage tourism: Evidences from Lamego cathedrals’ pórtico

This study aims to show the approximation between the training of History and Tourism. A multidisciplinary team has researched the intersections between these scientific areas in an historic city in the north of Portugal (city of Lamego ). Was created an interpretive speech for the Pórtico of one of the most emblematic monuments of this historic city (Cathedral) and in-depth interviews were applied to 15 visitors. We conclude that the interpretation of the historic resources allows the construction of a patrimonial tourist product. Through closer link between the historical discourse, interpretation and tourism is possible to create a differentiation of the patrimonial product making it a unique product and thus more attractive to the various segments. In the construction of the discourses of the patrimonial elements, having as an end the presentation for the potential visitors, we discovered that both the historic and the touristic discourses have similarities, differing, mostly, in the language type that is used. Teaching history deposits in the narrative construction an opportunity to cultivate the history training of the students, in order to achieve historic consciousness. The narratives carved by tourism do not deny the historic ones, but their intention passes by the aggregation of modern values and interpretations that differentiate the heritage.info:eu-repo/semantics/publishedVersio

A New horizon for online teaching and learning

The Polytechnic Institute of Oporto (IPP), which has a solid history of online education and innovation through the use of technology, has been particularly interested and focused on Massive Open Online Courses (MOOC) developments. The aim of this paper is to present the whole process from initial discussions to completion of the “Mathematics Without Limits” MOOC Project that exists in IPP and also to contribute for a change in the way as teaching and learning Mathematics is seen and practiced nowadays. In 2013, IPP developed its own platform, which gave us the opportunity to explore new educational techniques as a pedagogical resource as well as to enhance students’ motivation, through a set of interactive materials at their disposal, totally adapted to their needs. Students lack of motivation is mainly justified by their weak Math preparation, poor consolidated basis on the subject and different backgrounds of the students. To tackle this issue and based on our Math online courses teaching experience, we decided to create short duration MOOC, expecting to aid retention of students and also to reverse the path of students giving up on Math by giving them a friendly way of managing their own learning commitment. We also think that this MOOC will be a good approach to level out some math skills among freshmen.IPP/ISCAP - CIC