EXPRESSÃO HETERÓLOGA DE ANTIGÉNIOS DO VÍRUS ZIKA EM ESCHERICHIA COLI

O vírus Zika (ZIKV) é um arbovírus que pertence ao género Flavivirus da família Flaviviridae. Atualmente surge como um agente patogénico humano altamente relevante, não só pela possibilidade que demonstrou de se dispersar rapidamente, e de forma global, como pela sua associação ao desenvolvimento de uma série de malformações congénitas (incluindo microcefalia) e a síndrome de Guillain-Barré. Contudo, ainda pouco se conhece sobre a biologia do ZIKV, sendo para tal necessário o desenvolvimento de ferramentas básicas, nomeadamente, proteínas virais recombinantes ou anticorpos específicos, que permitam a utilização de uma grande variedade de protocolos experimentais. Neste estudo realizou-se a otimização da produção/purificação de antigénios virais de ZIKV em E. coli, nomeadamente, da proteína estrutural da cápside (C) e da região C-terminal da proteína NS3, à qual está associada atividade enzimática de RNAhel (NS3hel). Para tal, as sequências virais de genótipo selvagem e versões sintéticas com codões otimizados foram clonadas no vetor pET-29a, de modo a possibilitar a sua expressão, em E. coli, como proteínas de fusão com caudas de hexa-histidina (His6) na extremidade C-terminal. A expressão proteica heteróloga foi avaliada, quer em termos quantitativos, quer na solubilidade dos produtos obtidos. A identidade das proteínas C (≈ 13kDa) e NS3hel (≈ 15kDa) fundidas com caudas de His6 (C-His6 e NS3hel-His6, respetivamente) foi verificada através da sua purificação, em condições desnaturantes, por cromatografia de afinidade (em micro-escala) usando colunas com iões níquel imobilizados, e por western blot, utilizando anticorpos monoclonais anti-His6. As proteínas C-His6 e NS3hel-His6 foram, igualmente, sujeitas a purificação em condições nativas. Contudo, nestas condições, para além das proteínas em causa foi observada uma quantidade considerável de proteínas contaminantes. Ainda assim fica como prova de conceito o sucesso do processo de purificação em condições nativas

An unusual cause of intra-abdominal calcification: A lithopedion

AbstractWe report a case of a 77-year-old female who was admitted to the emergency department complaining of diffuse abdominal pain for five days, associated with nausea, vomiting and constipation.Physical examination disclosed a large incarcerated umbilical hernia, which was readily apparent on supine abdominal plain films. These also showed a calcified heterogeneous mass in the mid-abdominal region, which was further characterized by CT as a lithopedion (calcified ectopic pregnancy). This is one of the few cases studied on a MDCT equipment, and it clearly enhances the post-processing abilities of this imaging method which allows diagnostic high-quality MIP images.Lithopedion is a rare entity, with less than 300 cases previously described in the medical literature. However, many reported cases corresponded to cases of skeletonization or collections of fetal bone fragments discovered encysted in the pelvic region at surgery or autopsy. It is thus estimated that true lithopedion is a much rarer entity.The diagnosis may be reached by a suggestive clinical history and a palpable mass on physical examination, while the value of modern cross-sectional techniques is still virtually unknown. Ultrasonography may depict an empty uterine cavity and a calcified abdominal mass of non-specific characteristics, and computed tomography or magnetic resonance imaging are able to reach a conclusive diagnosis and may additionally define the involvement of adjacent structures.The differential diagnosis includes other calcified pathologic situations, including ovarian tumors, uterine fibroids, urinary tract neoplasms, inflammatory masses or epiploic calcifications

Structure-function studies can improve binding affinity of cohesin-dockerin interactions for multi-protein assemblies

the Associate Laboratory for Animal and Veterinary Sciences (AL4AnimalS) grant LA/P/0059/2020. LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy - i4HB. National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases. Publisher Copyright: © 2022 The Author(s)The cellulosome is an elaborate multi-enzyme structure secreted by many anaerobic microorganisms for the efficient degradation of lignocellulosic substrates. It is composed of multiple catalytic and non-catalytic components that are assembled through high-affinity protein-protein interactions between the enzyme-borne dockerin (Doc) modules and the repeated cohesin (Coh) modules present in primary scaffoldins. In some cellulosomes, primary scaffoldins can interact with adaptor and cell-anchoring scaffoldins to create structures of increasing complexity. The cellulosomal system of the ruminal bacterium, Ruminococcus flavefaciens, is one of the most intricate described to date. An unprecedent number of different Doc specificities results in an elaborate architecture, assembled exclusively through single-binding-mode type-III Coh-Doc interactions. However, a set of type-III Docs exhibits certain features associated with the classic dual-binding mode Coh-Doc interaction. Here, the structure of the adaptor scaffoldin-borne ScaH Doc in complex with the Coh from anchoring scaffoldin ScaE is described. This complex, unlike previously described type-III interactions in R. flavefaciens, was found to interact in a dual-binding mode. The key residues determining Coh recognition were also identified. This information was used to perform structure-informed protein engineering to change the electrostatic profile of the binding surface and to improve the affinity between the two modules. The results show that the nature of the residues in the ligand-binding surface plays a major role in Coh recognition and that Coh-Doc affinity can be manipulated through rational design, a key feature for the creation of designer cellulosomes or other affinity-based technologies using tailored Coh-Doc interactions.publishersversionepub_ahead_of_prin

3D printed Poly(ε-caprolactone)/Hydroxyapatite scaffolds for bone tissue engineering: a comparative study on a composite preparation by melt blending or solvent casting techniques and the influence of bioceramic content on scaffold properties

Bone tissue engineering has been developed in the past decades, with the engineering of bone substitutes on the vanguard of this regenerative approach. Polycaprolactone-based scaffolds are fairly applied for bone regeneration, and several composites have been incorporated so as to improve the scaffolds’ mechanical properties and tissue in-growth. In this study, hydroxyapatite is incorporated on polycaprolactone-based scaffolds at two different proportions, 80:20 and 60:40. Scaffolds are produced with two different blending methods, solvent casting and melt blending. The prepared composites are 3D printed through an extrusion-based technique and further investigated with regard to their chemical, thermal, morphological, and mechanical characteristics. In vitro cyto-compatibility and osteogenic differentiation was also assessed with human dental pulp stem/stromal cells. The results show the melt-blending-derived scaffolds to present more promising mechanical properties, along with the incorporation of hydroxyapatite. The latter is also related to an increase in osteogenic activity and promotion. Overall, this study suggests polycaprolactone/hydroxyapatite scaffolds to be promising candidates for bone tissue engineering, particularly when produced by the MB method.info:eu-repo/semantics/publishedVersio

Dextrin hydrogel loaded with a macroporous Bonelike® scaffold and dental pulp stem cells for critical-sized defect repair

Regeneration of severe bone defects remains a challenge. A formulation of synthetic glass-reinforced hydroxyapatite bone substitute, Bonelike® Poro (BL®P), 250500 µm-diameter, with a dextrin-based hydrogel (HG), further loaded with human dental pulp stem cells (hDPSCs) with osteogenic differentiation ability, was tested for the management of critical-sized defects in an ovine model. Morphology, calcium release, and mechanical strength of HG + BL®P were analyzed. Then, BL®P, HG + BL®P, and 106 hDPSCs-loaded HG + BL®P were implanted in ovine critical-sized 14 mm-diameter calvaria defects. Bone samples were collected after 3 and 6 weeks for histological and micro-CT analysis. BL®P exhibits a suitable porous size for cell ingrowth, from the nm (>200 nm) to the µm (5 µm) range. The addition of BL®P granules to the HG resulted in increased compressive elastic modulus and ultimate tensile strength. The mildly acidic nature of the HG contributed to a faster dissolution of granules. In vivo results confirmed the HG suitability as a carrier, providing better defect filling, easy handling, and injectability of BL®P without compromising new bone formation nor biocompatibility. The HG + BL®P formulations can successfully regenerate critical-sized defects; however, addition of hDPSCs did not significantly enhance new bone formation under these conditions. Granular BL®P provides an effective alternative to autologous grafts. The HG acts as a biocompatible carrier of granular bone substitutes and cells, conferring injectability and cohesivity.Alexandra Machado and Isabel Pereira were supported by the grants SFRH/BD/132000/2017 and UMINHO/BI/131/2018 respectively, from Portuguese Foundation for Science and Technology (FCT), Portugal. The authors acknowledge the funding from FEDER and NORTE 2020 through the project no. 003262 titled “iBONE therapies: advanced solutions for bone regeneration”. This study was supported by FCT under the scope of the strategic funding of UID/BIO/04469 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte 2020 - Programa Operacional Regional do Norte. The participation of Isabel Pires, Justina Prada, Luís Maltez and José Eduardo Pereira was funded by the projects UIDB/CVT/00772/2020 and LA/P/0059/2020 supported by FCT. The participation of Rui Alvites, Ana Catarina Sousa, Mariana Branquinho, Ana Rita Caseiro, Sílvia Santos Pedrosa and Ana Colette Maurício was funded by Projects PEst-OE/AGR/UI0211/2011, UIDB/CVT/00772/2020 and LA/P/0059/2020. Mariana Vieira Branquinho (SFRH/BD/146172/2019) and Ana Catarina Sousa (SFRH/BD/146689/2019) acknowledge FCT, for financial support.info:eu-repo/semantics/publishedVersio

Estudo de conservação sob atmosfera controlada na qualidade da cereja cv. Satin

A cereja é muito apreciada e apresenta um tempo de comercialização muito curto devido a ser um fruto altamente perecível. Técnicas de conservação pós-colheita são essenciais para manter a qualidade da cereja até serem consumidas. Baixas temperaturas são utilizadas para retardar o processo de deterioração da fruta e como complemento a aplicação de atmosferas controladas permite retardar o processo de amadurecimento e envelhecimento. A diminuição de oxigénio e o aumento de dióxido de carbono e azoto inibe o amadurecimento, mantendo o sabor e a qualidade da fruta. Neste trabalho experimental, cerejas da cultivar Satin foram conservadas em câmaras de refrigeração no produtor e nas instalações do CATAA com equipamento de atmosferas controladas. Quatro atmosferas controladas com diferentes combinações de oxigénio e dióxido de carbono foram testadas e o seu efeito na qualidade das cerejas foi avaliado. Ao longo do tempo de conservação as cerejas foram analisadas a diferentes níveis: qualidade (peso, dureza, cor e sólidos solúveis totais), microbiológico e organolético. Os resultados de temperatura e humidade no produtor e no CATAA, foram comparados e indicam que ambas as situações apresentam ótimas condições de conservação. No entanto, complementar a conservação com atmosferas controladas sugere que a qualidade da cereja é mantida por mais tempo, através da minimização do envelhecimento e processo de amadurecimento.info:eu-repo/semantics/publishedVersio

Estudo de conservação sob atmosfera controlada na qualidade da cereja cv. Satin.

A cerejeira (Prunus avium L.) é uma espécie pertencente à subfamília das Prunóideas e a produção de cereja apresenta elevada importância económica na região da Beira Interior, que, embora não seja a região com maior área de produção é a principal região de produção de Portugal. A cereja apresenta um elevado teor de compostos bioativos como vitamina C, fibra, antocianinas, quercetina e carotenóides relacionados com a prevenção de doenças cardiovasculares, diabetes e cancro (McCune et al., 2011; Wang et al., 2016). No entanto, este fruto não climatérico deteriora-se rapidamente após a colheita apresentando alterações na cor da pele, acastanhamento do pedúnculo, desidratação, amolecimento da polpa, diminuição da acidez e apodrecimento (Dugan & Roberts, 1997; Wang et al., 2016). A refrigeração, combinada com a utilização de atmosferas controladas, visa o atraso da deterioração e o consequente prolongamento da vida útil alargando o período de oferta. Esta técnica consiste no armazenamento a baixa temperatura num ambiente com uma concentração elevada de CO2, uma concentração baixa de O2 e uma humidade relativa elevada (Andrade et al., 2019). Os valores indicados na bibliografia relativos à concentração de CO2 variam entre 5% e 20% (Gross et al., 2016) e, para a concentração de O2, encontram-se entre 1% (Gross et al., 2016) e 10% (Ben-Yehoshua et al., 2005)info:eu-repo/semantics/publishedVersio

Vivências do professor principiante : as duas facetas do encontro com a realidade

Tese de mestrado em Ciências da Educação (Área de especialização em Formação de Professores), apresentada à Universidade de Lisboa, através da Faculdade de Psicologia e de Ciências da Educação, 2007Como objectivo geral deste estudo, procurámos conhecer algumas dimensões do encontro dos professores principiantes com a realidade docente, nas suas duas facetas, positiva e negativa, e conhecer em que medida os professores principiantes sentem o choque com a realidade, quais os factores promotores dessa vivência e quais os seus efeitos. Ao nível metodológico, foi aplicado um questionário a cinquenta e dois professores dos 2° e 3º ciclos e do ensino secundário, o que nos permitiu conhecer as dificuldades mais sentidas e os factores a elas associados. Sobre os dados obtidos realizámos estudos de frequências, percentagens e correlações. Posteriormente realizámos um estudo de caso, recorrendo a entrevistas semi-directivas a professoras que revelaram sentir níveis de dificuldades muito distintos, com o intuito de aprofundar as suas vivências, respectivas causas e subsequente impacto. Os resultados obtidos encontram-se, de uma forma geral, em consonância com a literatura especializada, tendo a vivência do confronto com a realidade revelado uma faceta bastante positiva.The general purpose of this study was to discover some of the dimensions that new teachers face with the teaching environment in both positive and the negative aspects, as well as understand in which manner they feel the shock with daily reality, which are the key factors that promote that experience and witch are its consequences. Methodologically, it was applied a questioner to fifty two teachers from 2nd and 3rd cycles and second grade teaching, which allowed us to know what difficulties were felt the most and it's associated factors. We have done frequency studies on the gathered data, percentages and correlations. Afterwards, it was made a case study through semi-directives interviews to teachers that showed very distinct levels of difficulty, aiming to deepen its experiences, causes and consequent impact. The obtained results are, in the general from, in harmony with the specialized literature, having the experience of the meet with reality shown a very positive side

From cancer therapy to winemaking : the molecular structure and applications of beta-Glucans and beta-1, 3-Glucanases

Research Areas: Biochemistry & Molecular Biology ; Chemistry: β-glucans are a diverse group of polysaccharides composed of β-1,3 or β-(1,3-1,4) linked glucose monomers. They are mainly synthesized by fungi, plants, seaweed and bacteria, where they carry out structural, protective and energy storage roles. Because of their unique physicochemical properties, they have important applications in several industrial, biomedical and biotechnological processes. β-glucans are also major bioactive molecules with marked immunomodulatory and metabolic properties. As such, they have been the focus of many studies attesting to their ability to, among other roles, fight cancer, reduce the risk of cardiovascular diseases and control diabetes. The physicochemical and functional profiles of β-glucans are deeply influenced by their molecular structure. This structure governs β-glucan interaction with multiple β-glucan binding proteins, triggering myriad biological responses. It is then imperative to understand the structural properties of β-glucans to fully reveal their biological roles and potential applications. The deconstruction of β-glucans is a result of β-glucanase activity. In addition to being invaluable tools for the study of β-glucans, these enzymes have applications in numerous biotechnological and industrial processes, both alone and in conjunction with their natural substrates. Here, we review potential applications for β-glucans and β-glucanases, and explore how their functionalities are dictated by their structure.info:eu-repo/semantics/publishedVersio