Cavity filling mutations at the thyroxine-binding site dramatically increase transthyretin stability and prevent its aggregationres

Altres ajuts: SUDOE INTERREG IV B (SOE4/P1/E831 to S.V.) and FEDER funds through the Operational Competitiveness Programme - COMPETE [grant number FCOMP-01-0124-FEDER-022718 (PEST-c/SAU/LA0002/2011) FCT-FEDER forunit 4293 in partnership with PT2020].More than a hundred different Transthyretin (TTR) mutations are associated with fatal systemic amyloidoses. They destabilize the protein tetrameric structure and promote the extracellular deposition of TTR as pathological amyloid fibrils. So far, only mutations R104H and T119M have been shown to stabilize significantly TTR, acting as disease suppressors. We describe a novel A108V non-pathogenic mutation found in a Portuguese subject. This variant is more stable than wild type TTR both in vitro and in human plasma, a feature that prevents its aggregation. The crystal structure of A108V reveals that this stabilization comes from novel intra and inter subunit contacts involving the thyroxine (T 4) binding site. Exploiting this observation, we engineered a A108I mutation that fills the T 4 binding cavity, as evidenced in the crystal structure. This synthetic protein becomes one of the most stable TTR variants described so far, with potential application in gene and protein replacement therapies

Levels of selected pharmaceuticals and personal care products (PPCPs) in human and environmental samples collected under the framework of MultiRespira Project

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revista de Ciências da Arte

Organizando-se agora no formato de número duplo, de modo a comportar dois números por ano, a revista digital Convocarte – Revista de Ciências da Arte mantém o mesmo propósito de promover o debate e edição de questões artísticas no espaço universitário, mantendo as coordenadas dominantes: convocar um número de especialistas em torno de um tema do mundo das artes, integrar trabalhos relevantes desenvolvidos nas fases curriculares e de projecto em mestrados e doutoramentos, sobretudo da FBAUL, e publicar trabalhos desenvolvidos em linhas de investigação do CIEBA. Assim, embora de funcionamento afecto à área científica de Ciências da Arte e do Património a Convocarte está aberta a outras especialidades interessadas em contribuir para a reflexão sobre as artes em geral, incorporando ensaios de predomínio teórico enraizado nos mais predominantes modos de discurso sobre arte, tais como História da arte, Crítica de Arte, Estética, Teorias da Arte ou Curadoria. (...) O nº2 organizou-se em torno de uma homenagem a uma figura importante das teorias da arte em Portugal, estratégia que Convocarte procurará manter nos próximos números. A intenção será deixar estudos sistematizados, entre o depoimento ou o ensaio, a memória e a reflexão, que estudem figuras marcantes da cultura portuguesa. (...) Neste número essa pasta foi dedicada a Rui Mário Gonçalves. Os textos são o resultado de uma sessão especial alargada a 2 de Maio no âmbito dos 2ºs Encontros com Críticos de Arte, com organização e coordenação de Fernando Rosa Dias, Cristina Tavares e Viviane Soares Silva, e decorridos ao longo das segundas do mês de Maio de 2016 na FBAUL (http://convocarte.belasartes.ulisboa.pt/index.php/2016/04/29/2o-encontros-com-criticos-de-arte/#more-325). A partir destes trabalhos reuniu-se um conjunto de estudos em torno de Rui Mário Gonçalves, com depoimentos e estudos sobre as mais diferentes facetas desta importante figura da cultura portuguesa: crítico de arte, historiador de arte, curador artístico, pedagogo e professor, político e activista, etc. A Convocarte orgulha-se de publicar os textos dessas comunicações, acrescentado de outros estudos, agradecendo a todos os colaboradores deste evento, que consideramos uma pasta que adianta contributos dando continuidade a estudos no catálogo de homenagem e no âmbito de apresentação da colecção do crítico de arte na SNBA, realizada pouco antes na SNBA.info:eu-repo/semantics/publishedVersio

Caracterização farmacológica da contracção intestinal induzida pela metformina

Dissertação de mestrado em Bioquimica apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de CoimbraA metformina é um anti-hiperglicémico oral amplamente utilizado na terapêutica farmacológica da Diabetes Mellitus tipo 2. Esta biguanida diminui a libertação de glicose hepática e aumenta a sua captação nos tecidos periféricos (tecido adiposo e músculo esquelético). A diarreia é um dos sintomas gastrointestinais associados ao tratamento com metformina. Além disso, a diabetes tipo 2 é também responsável pela ocorrência destes sintomas per se. A diarreia diabética é, então, uma das consequências da neuropatia autonómica diabética que perturba o controlo e a função dos órgãos do tracto gastrointestinal, como o intestino. Contudo, a diarreia provocada pela metformina é independente da influência da doença. Neste sentido, para perceber os mecanismos farmacológicos associados à diarreia desencadeada pela metformina, pretendia-se caracterizar a resposta contráctil de segmentos de íleo de rato à metformina, através de estudos funcionais de contracção isométrica. Para tal, investigou-se a possível interferência do fármaco na componente neuronal, bem como nas componentes colinérgicas, nitrérgicas, histaminérgicas e serotoninérgicas. Para além disso, comparou-se o efeito contráctil da metformina no ileo isolado de ratos Wistar (controlo) com o observado no íleo isolado de ratos Goto-Kakizaki (GK), um modelo animal de Diabetes Mellitus tipo 2. As preparações biológicas de íleo foram colocadas em banhos de órgãos de 15 ml, ligadas a transdutores de força e mantidas a 37ºC, em solução fisiológica de Krebs-Henseleit arejada com carbogénio e sob a tensão basal de 29,4 mN. Inicialmente, os segmentos intestinais foram submetidos a acetilcolina exógena (100 μM) para obter directamente a contracção máxima do músculo liso do íleo e, desta forma, permitir a comparação dos resultados de experiências diferentes. Posteriormente, foram realizadas duas curvas não cumulativas frequência eléctrica-resposta e concentração-resposta à metformina (6-36 M). A primeira curva correspondeu à curva controlo onde a resposta contráctil do órgão à estimulação eléctrica de campo e/ou à metformina foi testada na ausência de qualquer composto, enquanto que na segunda curva, 15 minutos antes de cada estimulação/dose, foram adicionados os seguintes compostos: metformina (36 μM); hexametónio (300 μM), um antagonista dos receptores nicotínicos; tetrodotoxina (1 μM), uma neurotoxina bloqueadora dos canais de sódio sensíveis à voltagem; atropina (10 μM), antagonista não selectivo dos receptores muscarínicos; NG-nitro-L-arginina (250 μM), um inibidor da sintase do monóxido de azoto; mepiramina (1 μM), um antagonista/agonista inverso dos receptores histamínicos do tipo H1; metiotepina (1 μM), um antagonista não selectivo dos receptores 5-HT1, 5-HT2, 5-HT5, 5-HT6 e 5-HT7; SB 224289 (1 μM), um antagonista selectivo dos receptores 5-HT1B; ritanserina (1 μM), um antagonista não selectivo dos receptores 5-HT2; cetanserina (1 μM), um antagonista selectivo dos receptores 5-HT2A/2C; MDL 72222 (1 μM), um antagonista dos receptores 5-HT3; e reserpina (100 μM), um inibidor do transportador vesicular de monoaminas. Em todas as experiências foram utilizados segmentos controlo, aos quais se adicionaram apenas os solventes apropriados para cada composto. Relativamente às curvas frequência eléctrica-resposta, os segmentos foram sujeitos à estimulação eléctrica de campo a frequências crescentes de 1, 2, 4, 8 e 16 Hz (100 V de intensidade, 5 ms de largura do pulso) com o objectivo de activar intrinsecamente a componente nervosa do íleo. No entanto, nos estudos realizados em animais GK apenas foram efectuadas curvas concentração-resposta à metformina na ausência e na presença de ritanserina (1 μM). Paralelamente, foram executadas curvas concentração-resposta à 5-hidroxitriptamina (0,1-60 M) nos dois grupos de animais, controlo e diabéticos, na presença e na ausência de cetanserina (1 μM) ou ritanserina (1 μM). Para complementar o presente trabalho, foram realizados estudos histoquímicos e imunohistoquímicos com o intuito de determinar a micro-anatomia dos segmentos de íleo e a localização celular dos receptores serotoninérgicos dos subtipos 5-HT1B, 5-HT1D e 5-HT2A. A metformina, testada em concentrações terapêuticas (6-36 M), induziu contracções dependentes da concentração nos segmentos de íleo isolado de rato (Emáx of 12.20 ± 0.80 mN, n=48; pEC50 of 4.89 ± 0.03, n=47). A metformina (36 M) não alterou significativamente a resposta contráctil dos segmentos à estimulação eléctrica de campo, o que sugere um mecanismo de acção não neuronal. Estes resultados foram confirmados pela ausência de alterações na curva concentração-resposta da metformina na presença de hexametónio e tetrodotoxina, excluindo o envolvimento da componente nervosa autonómica colinérgica e não colinérgica. Para além disso, as curvas concentração-resposta da metformina não foram alteradas significativamente na presença de atropina ou de NG-nitro-L-arginina, excluindo o envolvimento de receptores muscarínicos ou a inibição da produção de monóxido de azoto. A mepiramina, um antagonista/agonista inverso dos receptores H1, alterou significativamente a contracção induzida pela metformina. Relativamente aos receptores serotoninérgicos, a cetanserina, o SB 224289 e o MDL 72222 não alteraram a contracção induzida pela metformina, o que sugere que os receptores 5-HT2A/2C, 5-HT1B e 5-HT3 não estão a mediar a resposta contráctil. Tanto a metiotepina como a ritanserina reduziram substancialmente a contracção intestinal induzida pela metformina, o que comprova o envolvimento de receptores serotoninérgicos presentes nas células das camadas de músculo liso, nomeadamente, os receptores 5-HT2B. Os efeitos da cetanserina e da ritanserina na curva concentração-resposta à metformina também foram observados em curvas concentração-resposta à 5-hidroxitriptamina. A acção pós-sináptica da 5-HT não pareceu alterada sob influência da doença, uma vez que o efeito da ritanserina em ratos GK foi semelhante ao observado em ratos Wistar. Contudo, no modelo animal diabético, a redução da resposta contráctil à metformina pela ritanserina apenas se revelou significativa na última dose da curva concentração-resposta. A reserpina alterou significativamente a contracção intestinal induzida pela metformina em ratos Wistar, sugerindo que o fármaco induz a libertação de 5-hidroxitriptamina a partir das células enterocromafins presentes na mucosa intestinal. Adicionalmente, os estudos imunohistoquímicos revelaram a presença de receptores 5-HT2A não nas camadas musculares mas nas células ganglionares do plexo mientérico, para além de perturbações morfológicas motivadas pela diabetes tipo 2 nos ratos GK. Em conclusão, os resultados obtidos permitem reforçar a relação entre a incidência de diarreia e a terapêutica com metformina, graças ao seu perfil farmacológico de contracção intestinal que é mediado principalmente por uma forte componente serotoninérgica.Metformin is an oral antihyperglycemic agent widely used in the treatment of type 2 Diabetes Mellitus. This biguanide decreases hepatic glucose output and increases cell glucose uptake in peripheral tissues (adipose tissue and skeletal muscle). Diarrhea is one of the gastrointestinal side effects commonly reported with metformin treatment. In addition, type 2 diabetes is also responsible for the occurrence of these symptoms per se. Diabetic diarrhea is then a consequence of diabetic autonomic neuropathy which impairs control and functioning of gastrointestinal organs such as the intestine. However, metformin-induced diarrhea is independent of the pathological status. To understand the pharmacological mechanisms associated with the metformin-induced diarrhea, we intended to characterize the contractile response of rat isolated ileum segments to metformin, through functional studies of isometric contraction. Therefore, we investigated the possible drug effect in the neuronal, cholinergic, nitrergic, histaminergic and serotonergic ileum components. Thus, we compared the contractile effect of metformin on control Wistar rats with that of Goto-Kakizaki (GK) rats, an animal model of type 2 diabetes. The ileum segments were suspended in 15 ml organ baths, attached to force transducers and maintained at 37°C in aerated Krebs-Henseleit solution at a basal tension of 29.4 mN. Initially, preparations were submitted to exogenous 100 μM acetylcholine to directly obtain the ileum smooth muscle maximal contraction in order to compare the results from different experiments. Two non-cumulative electrical frequency-response and concentration-response curves to metformin (6-36 M) were performed. The first curve corresponds to the control curve, where the contractile response to electrical field stimulation and/or metformin was tested in the absence of any compound, while in the second curve, the following compounds were added 15 min before each dose of metformin or electrical stimulation frequency: metformin (36 M); hexamethonium (300 M), a nicotinic receptor antagonist, tetrodotoxin (1 M), a selective blocker of Na+ voltage-sensitive channels which inhibits impulses propagation in neuron excitable membranes; atropine (10 M), a non-selective muscarinic receptor antagonist; NG-nitro-L-arginine (250 M), a nitric oxide synthase inhibitor; mepyramine (1 M), a histamine H1 receptor inverse agonist/antagonist; methiothepin (1 M), a non-selective antagonist of the 5-HT1, 5-HT2, 5-HT5, 5-HT6 and 5-HT7 receptors; SB 224289 (1 M), a selective 5-HT1B receptor antagonist; ritanserin (1 M), a non-selective 5-HT2 receptor antagonist; ketanserin (1 M), a selective 5 -HT2A/2C receptor antagonist; MDL 72222 (1 M), a 5-HT3 receptor antagonist and reserpine (100 M), a vesicular monoamine transporter inhibitor. In each assay, control segments were used with the appropriate solvent of each drug. Frequency-response curves were obtained through electrical field stimulation (1, 2, 4, 8 and 16 Hz, 100 V, 5 msec pulse width) in order to activate intrinsic ileum nerves. However, only concentration-response curves to metformin in the presence and in the absence of ritanserin (1 M) were performed in GK rat functional studies. Concentration-response curves to 5-hidroxytryptamine (0.1-60 M) in the presence and in the absence of ketanserin (1 M) or ritanserin (1 M) were also performed in both groups of diabetic and control animals. Histochemical and immunohistochemical studies were carried out in order to determine the micro-anatomy and the cellular localization of 5-HT1B, 5-HT1D and 5-HT2A receptors. Metformin, used in therapeutic concentrations (6-36 M), caused concentration-dependent contractions of rat isolated ileum segments (Emax of 12.20 ± 0.80 mN, n=48; pEC50 of 4.89 ± 0.03, n=47). Metformin (36 μM) did not significantly change the rat ileum frequency-dependent contractile response to electrical field stimulation suggesting a non-neuronal mechanism of action. This was confirmed by the unchanged concentration-response curve to metformin in the presence of hexamethonium and tetrodotoxin, excluding the involvement of autonomic cholinergic and non-cholinergic nerves. Moreover, the metformin concentration-response curves were not significantly altered by atropine nor by NG-nitro-L-arginine, excluding the involvement of muscarinic receptors or the inhibition of nitric oxide production alone. Mepyramine, a H1 histaminic receptor antagonist/inverse agonist, did significantly change the metformin-induced contraction. Concerning the serotonergic receptors, ketanserin, SB 224289 and MDL 72222 did not changed the metformin-induced contraction, which means that 5-HT2A/2C, 5-HT1B and 5-HT3 receptors do not mediate the contractile response. Methiothepin and ritanserin almost abolished the metformin-induced intestinal contraction, which proves the involvement of serotonergic receptors present on the smooth muscle cells layers, namely 5-HT2B receptors. The effects of ketanserin and ritanserin on the metformin concentration-response curve were also observed on the 5-hidroxytryptamine concentration-response curve. The 5-HT post-synaptic action did not seem impaired under the influence of the disease, since the effect of ritanserin in GK rats was similar to that of Wistar rats. However, in the diabetic animal model, the ritanserin reduction of the metformin-induced intestinal contraction only became significant at the last metformin dose of the concentration-response curve. Reserpine significantly changed the metformin-induced contraction in Wistar rats, indicating that metformin triggers 5-hidroxytryptamine release from enterochromaffin cells of the rat intestinal mucosa. Furthermore the immunohistochemical studies did not reveal the presence of 5-HT2A receptors in the smooth muscle layers but in the myenteric plexus ganglionic cells, in addition to morphological disturbances driven by type 2 diabetes in GK rats. In conclusion, our experiments allowed us to reinforce the gastrointestinal side effects associated with metformin treatment since it induces intestinal contraction mainly by serotoninergic ways

Learning spaces for knowledge generation

As the Internet is becoming the main point for information access, Libraries, Museums and similar Institutions are preserving their collections as digital object repositories. In that way, the important information associated with digital objects may be delivered as Internet content over portals equipped with modern interfaces and navigation features. This enables the virtualization of real information exhibition spaces rising new learning paradigms. Geny is a project aiming at defining domain-specific languages and developing tools to generate web-based learning spaces from existent digital object repositories and associated semantic. The motto for Geny is “Generating learning spaces to generate knowledge”. Our objective within this project is to use (i) ontologies—one to give semantics to the digital object repository and another to describe the information to exhibit—and (ii) special languages to define the exhibition space, to enable the automatic construction of the learning space supported by a web browser. This paper presents the proposal of the Geny project along with a review of the state of the art concerning learning spaces and their virtualization. Geny is, currently, under appreciation by Fundação para a Ciência e a Tecnologia (FCT), the main Portuguese scientific funding institution

Association between Facial Metrics and Mate Rejection for Long-Term Relationship by Heterosexual Men

Investigations on mate choice in humans frequently report preferences, but there is little knowledge about what is important for rejection by a potential mate. The present study aims to verify if facial asymmetry and facial disharmony have an influence on mate rejection by men. We hypothesized that more asymmetric and disharmonious faces would be more rejected. For this purpose, photographs of women’s faces were presented in pairs by self-declared heterosexual men. It was requested they reject one of the faces as a potential mate for a long-term relationship. Women’s faces were also analyzed to measure facial asymmetry and facial disharmony. We used a linear mixed model to evaluate the effect of the cited metrics on each face’s number of rejections. We found that the female metrics influenced mate rejection only if associated with male age and income. The older participants rejected female partners with asymmetric faces. We suggest that aging makes men more demanding in mate choices, at least considering facial asymmetry. We concluded that rejection could be a key variable in mate choice studies, but further research is needed to clarify its effects

Molecular tweezers targeting transthyretin amyloidosis.

Transthyretin (TTR) amyloidoses comprise a wide spectrum of acquired and hereditary diseases triggered by extracellular deposition of toxic TTR aggregates in various organs. Despite recent advances regarding the elucidation of the molecular mechanisms underlying TTR misfolding and pathogenic self-assembly, there is still no effective therapy for treatment of these fatal disorders. Recently, the molecular tweezers, CLR01, has been reported to inhibit self-assembly and toxicity of different amyloidogenic proteins in vitro, including TTR, by interfering with hydrophobic and electrostatic interactions known to play an important role in the aggregation process. In addition, CLR01 showed therapeutic effects in animal models of Alzheimers disease and Parkinsons disease. Here, we assessed the ability of CLR01 to modulate TTR misfolding and aggregation in cell culture and in an animal model. In cell culture assays we found that CLR01 inhibited TTR oligomerization in the conditioned medium and alleviated TTR-induced neurotoxicity by redirecting TTR aggregation into the formation of innocuous assemblies. To determine whether CLR01 was effective in vivo, we tested the compound in mice expressing TTR V30M, a model of familial amyloidotic polyneuropathy, which recapitulates the main pathological features of the human disease. Immunohistochemical and Western blot analyses showed a significant decrease in TTR burden in the gastrointestinal tract and the peripheral nervous system in mice treated with CLR01, with a concomitant reduction in aggregate-induced endoplasmic reticulum stress response, protein oxidation, and apoptosis. Taken together, our preclinical data suggest that CLR01 is a promising lead compound for development of innovative, disease-modifying therapy for TTR amyloidosis

Molecular tweezers targeting transthyretin amyloidosis.

Transthyretin (TTR) amyloidoses comprise a wide spectrum of acquired and hereditary diseases triggered by extracellular deposition of toxic TTR aggregates in various organs. Despite recent advances regarding the elucidation of the molecular mechanisms underlying TTR misfolding and pathogenic self-assembly, there is still no effective therapy for treatment of these fatal disorders. Recently, the "molecular tweezers", CLR01, has been reported to inhibit self-assembly and toxicity of different amyloidogenic proteins in vitro, including TTR, by interfering with hydrophobic and electrostatic interactions known to play an important role in the aggregation process. In addition, CLR01 showed therapeutic effects in animal models of Alzheimer's disease and Parkinson's disease. Here, we assessed the ability of CLR01 to modulate TTR misfolding and aggregation in cell culture and in an animal model. In cell culture assays we found that CLR01 inhibited TTR oligomerization in the conditioned medium and alleviated TTR-induced neurotoxicity by redirecting TTR aggregation into the formation of innocuous assemblies. To determine whether CLR01 was effective in vivo, we tested the compound in mice expressing TTR V30M, a model of familial amyloidotic polyneuropathy, which recapitulates the main pathological features of the human disease. Immunohistochemical and Western blot analyses showed a significant decrease in TTR burden in the gastrointestinal tract and the peripheral nervous system in mice treated with CLR01, with a concomitant reduction in aggregate-induced endoplasmic reticulum stress response, protein oxidation, and apoptosis. Taken together, our preclinical data suggest that CLR01 is a promising lead compound for development of innovative, disease-modifying therapy for TTR amyloidosis