Longitudinal effects of ketamine on cell proliferation and death in the CNS of zebrafish

Funding Information: This work was funded by FEDER funds through the Operational Competitiveness Programme – COMPETE and by National Funds through FCT – Portuguese Foundation for Science and Technology under the project FCOMP-01-0124-FEDER-028683 (PTDC/CVT-WEL/4672/2012). Publisher Copyright: © 2023 The AuthorsZebrafish is known for its widespread neurogenesis and regenerative capacity, as well as several biological advantages, which turned it into a relevant animal model in several areas of research, namely in toxicological studies. Ketamine is a well-known anesthetic used both in human as well as veterinary medicine, due to its safety, short duration and unique mode of action. However, ketamine administration is associated with neurotoxic effects and neuronal death, which renders its use on pediatric medicine problematic. Thus, the evaluation of ketamine effects administration at early stages of neurogenesis is of pivotal importance. The 1‐41–4 somites stage of zebrafish embryo development corresponds to the beginning of segmentation and formation of neural tube. In this species, as well as in other vertebrates, longitudinal studies are scarce, and the evaluation of ketamine long-term effects in adults is poorly understood. This study aimed to assess the effects of ketamine administration at the 1–4 somites stage, both in subanesthetic and anesthetic concentrations, in brain cellular proliferation, pluripotency and death mechanisms in place during early and adult neurogenesis. For that purpose, embryos at the 1–4 somites stage (10.5 h post fertilization - hpf) were distributed into study groups and exposed for 20 min to ketamine concentrations at 0.2/0.8 mg/mL. Animals were grown until defined check points, namely 50 hpf, 144 hpf and 7 months adults. The assessment of the expression and distribution patterns of proliferating cell nuclear antigen (PCNA), of sex-determining region Y-box 2 (Sox 2), apoptosis-inducing factor (AIF) and microtubule-associated protein 1 light chain 3 (LC3) was performed by Western-blot and immunohistochemistry. The results evidenced the main alterations in 144 hpf larvae, namely in autophagy and in cellular proliferation at the highest concentration of ketamine (0.8 mg/mL). Nonetheless, in adults no significant alterations were seen, pointing to a return to a homeostatic stage. This study allowed clarifying some of the aspects pertaining the longitudinal effects of ketamine administration regarding the CNS capacity to proliferate and activate the appropriate cell death and repair mechanisms leading to homeostasis in zebrafish. Moreover, the results indicate that ketamine administration at 1–4 somites stage in the subanesthetic and anesthetic concentrations despite some transitory detrimental effects at 144 hpf, is long-term safe for CNS, which are newly and promising results in this research field.publishersversionpublishe

O papel da investigação na prática pedagógica dos mestrados em ensino

Os mestrados em ensino da Universidade do Minho preveem a formação do professor/ educador como prático reflexivo e intelectual crítico, conferindo um lugar de relevo à investigação pedagógica no estágio através da construção e avaliação de um “projeto de intervenção pedagógica supervisionada” que deve enquadrar-se numa visão democrática da educação. O projeto dá origem a um relatório final, defendido em provas públicas. Com o objetivo de compreender o papel da investigação no estágio, foi analisado um corpus de 28 relatórios de 5 mestrados de diversos níveis de ensino, com base numa grelha incidente na visão de educação subjacente aos projetos, no tipo e função do conhecimento mobilizado, na articulação investigação-ensino e no valor educativo das intervenções. Os projetos evidenciam a importância da investigação no desenvolvimento de práticas educativas focadas na qualidade dos processos de ensino e de aprendizagem, embora a coexistência de diferentes modalidades de articulação investigação-ensino sinalize conceções diferenciadas de formação e da função da investigação na regulação das práticas e na (re)construção de competências profissionais. A partir das potencialidades e limitações observadas, traçam-se linhas de ação futura para a construção de uma cultura investigativa na formação inicial de professores/ educadores

Glioblastoma entities express subtle differences in molecular composition and response to treatment

Glioblastoma (GBM) is a grade IV astrocytoma. GBM patients show resistance to chemotherapy such as temozolomide (TMZ), the gold standard treatment. In order to simulate the molecular mechanisms behind the different chemotherapeutic responses in GBM patients we compared the cellular heterogeneity and chemotherapeutic resistance mechanisms in different GBM cell lines. We isolated and characterized a human GBM cell line obtained from a GBM patient, named GBM11. We studied the GBM11 behaviour when treated with Tamoxifen (TMX) that, among other functions, is a protein kinase C (PKC) inhibitor, alone and in combination with TMZ in comparison with the responses of U87 and U118 human GBM cell lines. We evaluated the cell death, cell cycle arrest and cell proliferation, mainly through PKC expression, by flow cytometry and western blot analysis and, ultimately, cell migration capability and f-actin filament disorganization by fluorescence microscopy. We demonstrated that the constitutive activation of p-PKC seems to be one of the main metabolic implicated on GBM malignancy. Despite of its higher resistance, possibly due to the overexpression of P-glycoprotein and stem-like cell markers, GBM11 cells presented a subtle different chemotherapeutic response compared to U87 and U118 cells. The GBM11, U87, U118 cell lines show subtle molecular differences, which clearly indicate the characterization of GBM heterogeneity, one of the main reasons for tumor resistance. The adding of cellular heterogeneity in molecular behaviour constitutes a step closer in the understanding of resistant molecular mechanisms in GBM, and can circumvents the eventual impaired therapy

The Expression of Connexins and SOX2 Reflects the Plasticity of Glioma Stem-Like Cells

Glioblastoma (GBM) is the most malignant primary brain tumor, with an average survival rate of 15 months. GBM is highly refractory to therapy, and such unresponsiveness is due, primarily, but not exclusively, to the glioma stem-like cells (GSCs). This subpopulation express stem-like cell markers and is responsible for the heterogeneity of GBM, generating multiple differentiated cell phenotypes. However, how GBMs maintain the balance between stem and non-stem populations is still poorly understood. We investigated the GBM ability to interconvert between stem and non-stem states through the evaluation of the expression of specific stem cell markers as well as cell communication proteins. We evaluated the molecular and phenotypic characteristics of GSCs derived from differentiated GBM cell lines by comparing their stem-like cell properties and expression of connexins. We showed that non-GSCs as well as GSCs can undergo successive cycles of gain and loss of stem properties, demonstrating a bidirectional cellular plasticity model that is accompanied by changes on connexins expression. Our findings indicate that the interconversion between non-GSCs and GSCs can be modulated by extracellular factors culminating on differential expression of stem-like cell markers and cell-cell communication proteins. Ultimately, we observed that stem markers are mostly expressed on GBMs rather than on low-grade astrocytomas, suggesting that the presence of GSCs is a feature of high-grade gliomas. Together, our data demonstrate the utmost importance of the understanding of stem cell plasticity properties in a way to a step closer to new strategic approaches to potentially eliminate GSCs and, hopefully, prevent tumor recurrence

Recommendations to policy makers and stakeholders on creativity and early years science and mathematics. National report on recommendations for policy makers and stakeholders in Portugal

The main aim of this National Report is to provide concrete recommendations to policy makers and stakeholders in Portugal for further practice grounded on the research outcomes of the Creative Little Scientists project about enabling children’s creativity through science and mathematics in preschool and first years of primary education

O caso português no projeto CLS

O projeto Creative Little Scientists (CLS) reuniu criatividade com ciência e matemática no ensino pré-escolar e nos primeiros anos do ensino básico (crianças até aos 8 anos de idade). Através do CLS foi possível obter uma imagem clara das práticas existentes, bem como as suas implicações, oportunidades e desafios. Com base nisso, o projeto propôs diretrizes, currículos e materiais exemplares para a formação de professores relevante nos vários contextos europeus. Para alcançar estes objetivos, o projeto CLS constituiu um consórcio reunindo experiência ao mais alto nível e de qualidade nas áreas de educação em ciências e matemática na infância, criatividade na educação, psicologia cognitiva, estudos comparativos de educação e formação de professores. Este consórcio realizou pesquisas numa amostra de nove países europeus (Bélgica, Finlândia, França, Alemanha, Grécia, Malta, Portugal, Roménia e Reino Unido) selecionados para representar um amplo espetro de contextos educacionais, económicos, sociais e culturais. Tendo decorrido no período de outubro de 2011 a março de 2014, o projeto CLS, financiado no âmbito do 7º Programa Quadro da União Europeia, teve na Universidade do Minho o seu parceiro português. Ao longo dos 30 meses de duração do projeto, foram realizadas várias tarefas, procurando caracterizar o contexto do ensino das ciências e da matemática nos primeiros anos do ensino básico e do ensino pré-escolar em Portugal; foi feito trabalho de campo, procurando identificar e caracterizar a criatividade em atividades de ensino de matemática e ciências, tendo originado 5 casos de estudo; com base nestes casos de estudo, foram elaborados materiais pedagógicos de apoio para a formação inicial e contínua de professores, bem como para as suas práticas pedagógicas; a realização de um fórum nacional de discussão, conjuntamente com o trabalho de pesquisa e organização da equipa da Universidade do Minho, permitiu a elaboração de um conjunto de recomendações tendo em vista alterações curriculares no ensino pré-escolar e 1º ciclo do ensino básico, assim como na formação inicial e na formação contínua de educadores e professores do 1º ciclo