Molecular effect of an OPTN common variant associated to Paget's disease of bone

Paget's disease of bone (PDB) is a chronic bone disorder and although genetic factors appear to play an important role in its pathogenesis, to date PDB causing mutations were identified only in the Sequestosome 1 (SQSTM1) gene at the PDB3 locus. PDB6 locus, also previously linked to PDB, contains several candidate genes for metabolic bone diseases. We focused our analysis in the most significantly associated variant with PDB, within the Optineurin (OPTN) gene, i.e. the common variant rs1561570. Although it was previously shown to be strongly associated with PDB in several populations, its contribution to PDB pathogenesis remains unclear. In this study we have shown that rs1561570 may contribute to PDB since its Tallele results in the loss of a methylation site in patients' DNA, leading to higher levels of OPTN gene expression and a corresponding increase in protein levels in patients' osteoclasts. This increase in OPTN expression leads to higher levels of NF-KB translocation into the nucleus and increasing expression of its target genes, which may contribute to the overactivity of osteoclasts observed in PDB. We also reported a tendency for a more severe clinical phenotype in the presence of a haplotype containing the rs1561570 T allele, which appear to be re-enforced with the presence of the SQSTM1/P392L mutation. In conclusion, our work provides novel insight towards understanding the functional effects of this variant, located in OPTN intron 7, and its implication in the contribution to PDB pathogenesis.national funds from Foundation for Science and Technology (FCT) [UID/Multi/04326/2013]; Canadian Institutes for Health Research, Canada [MOP130457]; CHU de Quebec Foundation; Canadian Foundation for Innovation; Fonds de recherche du Quebec-sante; Laval University; CHU de Quebec-Universite Laval Research Centre; FCT [SFRH/BD/77227/2011, SFRH/BPD/111898/2015]; Fonds de recherche Quebec-Sante (FRQ-S), Quebec, Canad

Matrix Gla Protein expression pattern in the early avian embryo

MGP (Matrix Gla Protein) is an extracellular matrix vitamin K dependent protein previously identified as a physiological inhibitor of calcification and shown to be well conserved among vertebrates during evolution. MGP is involved in other mechanisms such as TGF-beta and BMP activity, and a proposed modulator of cell-matrix interactions. MGP is expressed early in vertebrate development although its role has not been clarified. Previous work in the chicken embryo found MGP localization predominantly in the aorta and aortic valve base, but no data is available earlier in development. Here we examined MGP expression pattern using whole-mount in situ hybridization and histological sectioning during the initial stages of chick development. MGP was first detected at HH10 in the head and in the forming dorsal aorta. At the moment of the onset of blood circulation, MGP was expressed additionally in the venous plexus which will remodel into the vitelline arteries. By E2.25, it is clear that the vitelline arteries are MGP positive. MGP expression progresses centrifugally throughout the area vasculosa of the yolk sac. Between stages HH17 and HH19 MGP is seen in the dorsal aorta, heart, notochord, nephric duct, roof plate, vitelline arteries and in the yolk sac, beneath main arterial branches and in the vicinity of several vessels and venules. MGP expression persists in these areas at least until E4.5. These data suggest that MGP expression could be associated with cell migration and differentiation and to the onset of angiogenesis in the developing chick embryo. This data has biomedical relevance by pointing to the potential use of chick embryo explants to study molecules involved in artery calcification

Regulation of human ZNF687, a gene associated with Paget's disease of bone

Mutations in Zinc finger 687 (ZNF687) were associated with Paget's disease of bone (PDB), a disease charac-terized by increased bone resorption and excessive bone formation. It was suggested that ZNF687 plays a role in bone differentiation and development. However, the mechanisms involved in ZNF687 regulation remain un-known. This study aimed to obtain novel knowledge regarding ZNF687 transcriptional and epigenetic regulation. Through in silico analysis, we hypothesized three ZNF687 promoter regions located upstream exon 1 A, 1B, and 1 C and denominated promoter regions 1, 2, and 3, respectively. Their functionality was confirmed by luciferase activity assays and positive/negative regulatory regions were identified using promoter deletions constructs. In silico analysis revealed a high density of CpG islands in these promoter regions and in vitro methylation sup-pressed promoters' activity. Using bioinformatic approaches, bone-associated transcription factor binding sites containing CpG dinucleotides were identified, including those for NF kappa B, PU.1, DLX5, and SOX9. By co-transfection in HEK293 and hFOB cells, we found that DLX5 specifically activated ZNF687 promoter region 1, and its methylation impaired DLX5-driven promoter stimulation. NF kappa B repressed and activated promoter regions 1 and 2, respectively, and these activities were affected by methylation. PU.1 induced ZNF687 promoter region 1 which was affected by methylation. SOX9 differentially regulated ZNF687 promoters in HEK293 and hFOB cells that were impaired after methylation. In conclusion, this study provides novel insights into ZNF687 regulation by demonstrating that NF kappa B, PU.1, DLX5, and SOX9 are regulators of ZNF687 promoters, and DNA methylation influences their activity. The contribution of the dysregulation of these mechanisms in PDB should be further elucidated.info:eu-repo/semantics/publishedVersio

Autism community perspectives on autism research

Dada a sua elevada prevalência global (cerca de 1 em 100), gravidade e complexidade etiológica, o autismo é hoje uma importante área de investigação. O presente trabalho teve como objetivo compreender quais as preocupações éticas, perspetivas e expetativas de pais e profissionais da saúde e educadores ligados ao autismo, no que diz respeito à investigação desta patologia. O trabalho foi desenvolvido no âmbito de uma rede de investigação europeia sobre sinais precoces de autismo. Em paralelo em vários países da Europa, foram identificados os tópicos de maior interesse para pais e profissionais através de grupos de foco, na sequência dos quais se elaborou um inquérito online que foi distribuído por esta comunidade ligada ao autismo. Em Portugal, os inquiridos (n=331) mostraram um interesse global pela investigação em autismo, nomeadamente sobre as bases genéticas e sobre sinais precoces de autismo. Relativamente aos benefícios resultantes da participação em projetos de investigação, os pais privilegiaram o acesso a intervenção e a formação pessoal, enquanto que os profissionais deram mais importância ao acesso a mais conhecimento e a equipas especializadas. Os três grupos deram ênfase ao acesso à informação completa obtida durante a investigação e às conclusões gerais do estudo. Os resultados obtidos reforçam a importância da investigação do autismo, indicando também linhas claras de atuação que têm em observação as perspetivas da comunidade ligada ao autismo.Given its high global prevalence, clinical severity and etiological complexity, autism has become an impor tant research area. This work was developed within a European research network on early signs of autism, and aimed to understand the specific ethical concerns of parents and practitioners in health and education, with regard to research in autism, and understand their perspectives and expectations. Using focus groups, the topics of greatest interest for this community were identified and an online sur vey was developed and distributed to the community. Respondents in Por tugal (n = 331) showed a global interest in autism research, with special focus on autism genetics and early signs of autism. Concerning the par ticipation in research projects and its direct benefits, parents privileged access to inter vention and training, while professionals wanted access to more knowledge and specialized teams. The three groups favored access to the full information obtained from research and the general conclusions of the studies. Thus, the results reinforce the interest of parents and professionals in autism research and provide clear lines of conduct taking into account this community's perspectives and expectations

Conceções sobre o currículo na educação pré-escolar

O presente trabalho foca-se sobre o currículo da Educação de Infância e a forma como é perspetivado por educadores, professores do 1.º Ciclo do Ensino Básico e encarregados de educação. Primeiramente, contextualiza-se a Educação de Infância (EI), para melhor entender a necessidade de se proceder a um trabalho mais ativo no sentido de valorizar este nível de ensino. Dada a sua curta existência, acompanhada de alguns recuos e avanços, a ideia que por vezes transparece no discurso social sobre a EI é que não é tida como uma atividade cujo objetivo seja contribuir para o desenvolvimento global das crianças com idades desde os 3 anos até a entrada no 1ºCiclo do Ensino Básico (1ºCEB), persistindo conceções assistencialistas em relação a este nível de educação. Explora-se igualmente alguns instrumentos utilizados no âmbito da Educação Pré-escolar (EPE). O currículo apresenta-se como meio acerca do qual se desenvolve a prática letiva, justificando assim a intencionalidade bem como todo o envolvimento de meios para a sua construção. A intenção que orientou o estudo foi entender a conceção que EI, professores do 1ºCEB e alguns pais têm acerca deste nível de educação. Perceber se lhe reconhecem a incumbência de desenvolver competências a ter em conta na formação pessoal e social, bem com nas restantes áreas de conteúdo, que sejam o alicerce facilitador para a construção de um caminho para entrada no novo ciclo. O estudo, de natureza mista, recorreu à aplicação de inquéritos por questionário, a educadores e professores, e a alguns pais, num território no interior de Portugal, distrito de Beja; também foram realizadas entrevistas a EI dos mesmos Agrupamentos de Escolas. Os principais resultados apontam no sentido de que os EI constroem o seu currículo com base nas Orientações Curriculares para a Educação Pré-Escolar (OCEPE). Os professores do 1ºCEB tem conhecimento da existência das OCEPE, mas não demonstram ter conhecimento acerca do documento, no entanto, reconhecem que o trabalho desenvolvido no Jardim de Infância (JI) é orientado de modo a facilitar futuras aprendizagens dos seus alunos. Os pais por sua vez têm uma atitude um pouco passiva face à vida escolar dos seus educandos.This work focuses on the curriculum of preschool education and how it is seen by educators, primary school teachers and parents. Firstly, we have to contextualize education in childhood so that we can understand better the need to carry on a more active work so as to value this level of education. Considering its short existence, accompanied by some advances and setbacks, the idea that comes up in the social speech about pre-school is that it is not considered as an activity which aim is to contribute to the global development of children between the age of three and the beginning of the primary school, while misconceptions about this level of education still persist. We also explore some tools used in pre-school education. The curriculum is presented as a means to develop teaching considering the intentions as well the environment and the means to its construction. The aim of this study was to understand the conception that preschool teachers, primary school teachers and some parents have about this level of education as well as to understand if they recognize the importance of developing personal and social skills together with other content areas which might be a facilitating scafold for the entrance in the following cycle. This study, using a mixed methodology, used questionnaires to preschool educators, teachers and some parents in region of inner Portugal, the district of Beja. Some interviews were also carried out to preschool educators belonging to the same groups of schools. The main results show that preschool educators build the curriculum based on the General Curricular Orientations for Pre-school (OCEPE). Although primary school teachers know about the existence of the OCEPE, they don’t demonstrate to have much knowledge about its content. Nevertheless, they recognize that the work carried out in preschool aims to facilitate future learning processes for the children. On the other hand, parents have a more passive attitude towards their children’s school life

Inclusão e qualidade de vida em crianças com necessidades educativas especiais: representações e expetativas das famílias

Dissertação de mestrado, Educação Especial (Domínios Cognitivo e Motor), Escola Superior de Educação e Comunicação, Universidade do Algarve, 2015O presente estudo, de natureza qualitativa, aborda a temática da inclusão e qualidade de vida em crianças com necessidades educativas especiais. Trata-se de dar voz aos pais procurando compreender o que significa para eles os seus filhos terem qualidade de vida e qual o papel da inclusão educativa, de forma a desenvolver estratégias que os envolva na intervenção educativa e assim, contribuir para a sua inclusão e qualidade de vida. Como tal, centramo-nos no estudo dos testemunhos de pais de crianças com necessidades educativas especiais, tendo como objetivo geral compreender as representações sobre a qualidade de vida na promoção da inclusão dos filhos. Tendo em vista o objetivo traçado, optou-se por uma metodologia qualitativa, adotando o paradigma interpretativo, recorrendo a entrevistas semiestruturadas e a uma entrevista de grupo focal (focus group). Participaram no estudo nove famílias com crianças com necessidades educativas especiais, de ambos os sexos, com idades compreendidas entre os 3 e os 7 anos, de cinco concelhos do Algarve. Em síntese, o estudo revela que para a promoção da qualidade de vida dos seus filhos com NEE, a valorização que os pais atribuem às dimensões de bem-estar material e emocional e o acesso a serviços médicos e terapêuticos, sociais e educativos. Como principais obstáculos sobressaem as atitudes de ceticismo e discriminação, a resposta parcial ou ineficaz do sistema educativo e a manutenção da falta de intervenção do Estado

Cdkl5 mutant zebrafish shows skeletal and neuronal alterations mimicking human CDKL5 deficiency disorder

CDKL5 deficiency disorder (CDD) is a rare neurodevelopmental condition characterized primarily by seizures and impairment of cognitive and motor skills. Additional phenotypes include microcephaly, dysmorphic facial features, and scoliosis. Mutations in cyclin-dependent kinase-like 5 (CDKL5) gene, encoding a kinase essential for normal brain development and function, are responsible for CDD. Zebrafish is an accepted biomedical model for the study of several genetic diseases and has many advantages over other models. Therefore, this work aimed to characterize the phenotypic, behavioral, and molecular consequences of the Cdkl5 protein disruption in a cdkl5 mutant zebrafish line (sa21938). cdkl5(sa21938) mutants displayed a reduced head size, suggesting microcephaly, a feature frequently observed in CDD individuals. Double staining revealed shorter craniofacial cartilage structures and decrease bone mineralization in cdkl5 homozygous zebrafish indicating an abnormal craniofacial cartilage development and impaired skeletal development. Motor behavior analysis showed that cdkl5(sa21938) embryos had less frequency of double coiling suggesting impaired glutamatergic neurotransmission. Locomotor behavior analysis revealed that homozygous embryos swim shorter distances, indicative of impaired motor activity which is one of the main traits of CCD. Although no apparent spontaneous seizures were observed in these models, upon treatment with pentylenetetrazole, seizure behavior and an increase in the distance travelled were observed. Quantitative PCR showed that neuronal markers, including glutamatergic genes were dysregulated in cdkl5(sa21938) mutant embryos. In conclusion, homozygous cdkl5(sa21938) zebrafish mimic several characteristics of CDD, thus validating them as a suitable animal model to better understand the physiopathology of this disorder.info:eu-repo/semantics/publishedVersio

Keutel Syndrome, a review of 50 years of literature

Keutel syndrome (KS) is a rare autosomal recessive genetic disorder that was first identified in the beginning of the 1970s and nearly 30 years later attributed to loss-of-function mutations in the gene coding for the matrix Gla protein (MGP). Patients with KS are usually diagnosed during childhood (early onset of the disease), and the major traits include abnormal calcification of cartilaginous tissues resulting in or associated with malformations of skeletal tissues (e.g., midface hypoplasia and brachytelephalangism) and cardiovascular defects (e.g., congenital heart defect, peripheral pulmonary artery stenosis, and, in some cases, arterial calcification), and also hearing loss and mild developmental delay. While studies on Mgp-/- mouse, a faithful model of KS, show that pathologic mineral deposition (ectopic calcification) in cartilaginous and vascular tissues is the primary cause underlying many of these abnormalities, the mechanisms explaining how MGP prevents abnormal calcification remain poorly understood. This has negative implication for the development of a cure for KS. Indeed, at present, only symptomatic treatments are available to treat hypertension and respiratory complications occurring in the KS patients. In this review, we summarize the results published in the last 50 years on Keutel syndrome and present the current status of the knowledge on this rare pathology.CA-16115 - EJPRD2019-290 - UIDB/04326/2020info:eu-repo/semantics/publishedVersio

Zebrafish models to study ectopic calcification and calcium-associated pathologies

Ectopic calcification refers to the pathological accumulation of calcium ions in soft tissues and is often the result of a dysregulated action or disrupted function of proteins involved in extracellular matrix mineralization. While the mouse has traditionally been the go-to model organism for the study of pathologies associated with abnormal calcium deposition, many mouse mutants often have exacerbated phenotypes and die prematurely, limiting the understanding of the disease and the development of effective therapies. Since the mechanisms underlying ectopic calcification share some analogy with those of bone formation, the zebrafish (Danio rerio)—a well-established model for studying osteogenesis and mineralogenesis—has recently gained momentum as a model to study ectopic calcification disorders. In this review, we outline the mechanisms of ectopic mineralization in zebrafish, provide insights into zebrafish mutants that share phenotypic similarities with human pathological mineralization disorders, list the compounds capable of rescuing mutant phenotypes, and describe current methods to induce and characterize ectopic calcification in zebrafish.info:eu-repo/semantics/publishedVersio

Lab-it is taking molecular genetics to school

The Molecular Genetics Mobile Lab or “Laboratório itinerante de Genética Molecular” (Lab-it) was funded in 2008 by Leonor Cancela to promote the learning of molecular genetics which had been introduced at that time into high school biology programms. The project aimed to introduce hands-on laboratory activities in molecular genetics to complement the theoretical concepts taught in school. These included the development of experimental protocols based on theoretical scenarios focusing on themes of forensics sciences, biomedical applications, diagnostic methods, and ecological research using basic molecular biology techniques, such as DNA extraction, polymerase chain reaction (PCR), electrophoresis, and restriction enzyme application. In these scenarios, the students execute all the procedures with the help of the Lab-it instructor and using the Lab-it equipment, followed by a discussion of the results with all the participants and the school teacher. These approaches help the students to consolidate the concepts of molecular biology and simultaneously promote discussions on new advances in the area and choices for university careers. In addition to practical sessions, Lab-it also promotes seminars on topics of interest to the students and teachers. Since 2008, 18 high schools have participated in the region of Algarve, averaging each year about 400 students participating in practical activities. In 2021, despite the COVID pandemic, 9 schools and 379 students were involved in Lab-it practical sessions and 99% of them considered the activity to contribute to better understanding the molecular biology methods approached in theoretical classes and expressed high interest in those sessions.info:eu-repo/semantics/publishedVersio