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Aging is characterized by changes in gene expression that drive deleterious cellular phenotypes leading to senescence. The transcriptional activation of senescence genes has been mainly attributed to epigenetic shifts, but the changes in chromatin accessibility and its underling mechanisms remain largely elusive in natural aging. Here, we profiled chromatin accessibility in human dermal fibroblasts (HDFs) from neonatal and octogenarian individuals. We found that AP-1 binding motifs are prevalent in elderly specific accessible regions of the chromatin while neonatal-specific regions are highly enriched for TEAD binding motifs. We further show thatTEAD4andFOXM1share a conserved transcriptional regulatory landscape controlled by an age-dependent enhancer that closes with aging and drives senescence when deleted. Finally, we demonstrate thatFOXM1ectopic expression in elderly cells partially resets chromatin accessibility to a youthful state due to FOXM1 repressive function in the promoters of several members of the AP-1 complex. These results placeFOXM1at a top hierarchical level in chromatin remodeling required to prevent senescence.

Supplementary Figures S1-S7

Background The use of plaque incision and graft techniques (PIG) for the treatment of severe Peyronie’s disease (PD), may lead to erectile dysfunction (ED); graft size is 1 of the contributing factors for post-PIG ED. Recently the iGrafter software APP was introduced using a mathematical algorithm to distribute the incisions along the penile length resulting in a smaller grafting area. Aim Compare 2 PIG techniques, the Double-Y(DY) and iGrafter, in 3 main aspects: (i) Total grafting area; (ii) The variation in calculating the grafting to be used; (iii) time to perform the PIG. Methods Six urologists with expertise in sexual medicine performed both techniques twice using four 3-D validated training models for PD with a standard 60° uniplanar dorsal curvature. Outcomes The graft areas and operative partial and total time for each step of the operation were recorded for each procedure. Unpaired t-test and the coefficient of variation for graft area across surgeons was calculated comparing both techniques. Results For all surgeons, the use of iGrafter resulted in 2 grafts, for the DY technique in 1 graft. Overall, TT for the iGrafter was significantly longer than for DY technique (49.4 ± 11 vs 40.7 ± 5.7 minute; P = .02), The iGrafter grafting area was significantly smaller (11.6 ± 1.2 vs 23.3 ± 5.4 cm2; P: .01), representing a 50.2% area reduction when compared to the DY. The variation of graft area, using the iGrafter also yielded a more consistent graft across all surgeons (CV = 10.56% vs 23.28%). Clinical Significance The iGrafter, when compared to DY technique, reduced the graft area by 50%, which potentially means less erectile dysfunction. Strengths and Limitations Our study eliminates anatomical variations found in a real clinical case making it possible to compare surgical techniques with the same penile anatomy. However, the 3D-printed model cannot replicate the living human tissue property preventing a simulation close to actual surgery. Conclusion The use of the iGrafter software for PIG surgery has shown to be a promising technique for severe PD management resulting in smaller graft size (about 50% smaller when compared to the DY), although it might be more time-consuming.

Vertebrate pancreas organogenesis is a stepwise process regulated by a complex network of signaling and transcriptional events, progressively steering the early endoderm toward pancreatic fate. Many crucial players of this process have been identified, including signaling pathways, cis-regulatory elements, and transcription factors (TFs). Pancreas-associated transcription factor 1a (PTF1A) is one such TF, crucial for pancreas development. PTF1A mutations result in dramatic pancreatic phenotypes associated with severe complications, such as neonatal diabetes and impaired food digestion due to exocrine pancreatic insufficiency. Here, we present a brief overview of vertebrate pancreas development, centered on Ptf1a function and transcriptional regulation, covering similarities and divergences in three broadly studied organisms: human, mouse and zebrafish.

Inhibition of spindle microtubule (MT) dynamics has been effectively used in cancer treatment. Although the mechanisms by which MT poisons elicit mitotic arrest are fairly understood, efforts are still needed towards elucidating how cancer cells respond to antimitotic drugs owing to cytotoxicity and resistance side effects. Here, we identified the critical G2/M transcription factor Forkhead box M1 (FOXM1) as a molecular determinant of cell response to antimitotics. We found FOXM1 repression to increase death in mitosis (DiM) due to upregulation of the BCL-2 modifying factor (BMF) gene involved in anoikis, an apoptotic process induced upon cell detachment from the extracellular matrix. FOXM1 binds to a BMF intronic cis-regulatory element that interacts with both the BMF and the neighbor gene BUB1B promoter regions, to oppositely regulate their expression. This mechanism ensures that cells treated with antimitotics repress BMF and avoid DiM when FOXM1 levels are high. In addition, we show that this mechanism is partly disrupted in anoikis/antimitotics-resistant tumor cells, with resistance correlating with lower BMF expression but in a FOXM1-independent manner. These findings provide a stratification biomarker for antimitotic chemotherapy response.

Many single nucleotide polymorphisms (SNPs) associated to type 2 diabetes overlap with putative endocrine pancreatic enhancers, suggesting that these SNPs modulate enhancer activity and consequently, gene expression. We performed in vivo mosaic transgenesis assays in zebrafish to quantitatively test the enhancer activity of type 2 diabetes-associated loci. Six out of ten tested sequences are endocrine pancreatic enhancers. The risk variant of two sequences decreased enhancer activity, while in another two incremented it. One of the latter (rs13266634) locates in a SLC30A8 exon, encoding a tryptophan-to-arginine substitution that decreases SLC30A8 function, being the canonical explanation for type 2 diabetes risk association. However, other type 2 diabetes associated SNPs that truncate SLC30A8, confer protection to this disease, contradicting this explanation. Here, we clarify this incongruence showing that rs13266634 boosts the activity of an overlapping enhancer, suggesting a SLC30A8 gain-of-function as the cause for the increased risk for the disease. We further dissected the functionality of this enhancer finding a single nucleotide mutation sufficient to impair its activity. Overall, this work assesses in vivo the importance of disease-associated SNPs in the activity of endocrine pancreatic enhancers, including a poorly explored case where a coding SNP modulates the activity of an enhancer.

The pancreas is a central organ for human diseases. Most alleles uncovered by genome-wide association studies of pancreatic dysfunction traits overlap with non-coding sequences of DNA. Many contain epigenetic marks of cis-regulatory elements active in pancreatic cells, suggesting that alterations in these sequences contribute to pancreatic diseases. Animal models greatly help to understand the role of non-coding alterations in disease. However, interspecies identification of equivalent cis-regulatory elements faces fundamental challenges, including lack of sequence conservation. Here we combine epigenetic assays with reporter assays in zebrafish and human pancreatic cells to identify interspecies functionally equivalent cis-regulatory elements, regardless of sequence conservation. Among other potential disease-relevant enhancers, we identify a zebrafish ptf1a distal-enhancer whose deletion causes pancreatic agenesis, a phenotype previously found to be induced by mutations in a distal-enhancer of PTF1A in humans, further supporting the causality of this condition in vivo. This approach helps to uncover interspecies functionally equivalent cis-regulatory elements and their potential role in human disease., This study was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (ERC-2015-StG-680156-ZPR and ERC-2016-AdG-740041-EvoLand to J.L.G.-S.). J.B. is supported by an FCT CEEC grant (CEECIND/03482/2018). J.L.G.-S. is supported by the Spanish Ministerio de Economía y Competitividad (BFU2016-74961-P), the Marató TV3 Fundacion (Grant 201611) and the institutional grant Unidad de Excelencia María de Maeztu (MDM-2016-0687). R.B.C. was funded by FCT (ON2201403-CTO-BPD), IBMC (BIM/04293-UID991520-BPD) and EMBO (Short-Term Fellowship). J.Tx. (SFRH/BD/126467/2016), M.D. (SFRH/BD/135957/2018), A.E. (SFRH/BD/147762/2019), and F.J.F. (PD/BD/105745/2014) are PhD fellows from FCT. M.G. was supported by the EnvMetaGen project via the European Union’s Horizon 2020 research and innovation programme (grant 668981). This work was funded by National Funds through FCT—Fundação para a Ciência e a Tecnologia, I.P., under the project UIDB/04293/2020”.

Introductory paragraphThe pancreas is a central organ for human diseases that have a dramatic societal burden, such as pancreatic cancer and diabetes1,2. Non-coding cis-regulatory elements (CREs) of DNA control gene expression3,4, being required for proper pancreas function. Most disease-associated alleles5,6 are non-coding, often overlapping with CREs5, suggesting that alterations in these regulatory sequences contribute to human pancreatic diseases by impairing gene expression. However, functional testing of CREs in vivo is not fully explored. Here we analysed histone modifications, transcription, chromatin accessibility and interactions, to identify zebrafish pancreas CREs and their human functional equivalents, uncovering disease-associated sequences across species. We found a human pancreatic enhancer whose deletion impairs the tumour suppressor gene ARID1A expression, conferring a potential tumour suppressor role to this non-coding sequence. Additionally, we identified a zebrafish ptf1a distal enhancer which deletion generates pancreatic agenesis, demonstrating the causality of this condition in humans7 and the interspecies functional equivalency of enhancers.

Summary Since its first application for site-directed mutagenesis, the CRISPR-Cas9 system has revolutionized genome engineering. Here, we present a validated workflow for the generation of targeted genomic deletions in zebrafish, including the design, cloning, and synthesis of single-guide RNAs and Cas9 mRNA, followed by microinjection in zebrafish embryos and subsequent genotype screening for the establishment of a mutant line. The versatility and efficiency of this pipeline makes the generation of zebrafish models a widely used approach in functional genetics. For complete details on the use and execution of this protocol, please refer to Amorim et al. (2020)., Graphical Abstract, Highlights • Step-by-step details from design to production of target-specific sgRNAs • Microinjection of sgRNAs/Cas9 complexes into one-cell-stage zebrafish embryos • PCR-based genotyping of in vivo deletions • Cost- and time-effective pipeline for establishment of a zebrafish stable mutant line, Since its first application for site-directed mutagenesis, the CRISPR-Cas9 system has revolutionized genome engineering. Here, we present a validated workflow for the generation of targeted genomic deletions in zebrafish, including the design, cloning, and synthesis of single-guide RNAs and Cas9 mRNA, followed by microinjection in zebrafish embryos and subsequent genotype screening for the establishment of a mutant line. The versatility and efficiency of this pipeline makes the generation of zebrafish models a widely used approach in functional genetics.

Summary The notochord is an evolutionary novelty in vertebrates that functions as an important signaling center during development. Notochord ablation in chicken has demonstrated that it is crucial for pancreas development; however, the molecular mechanism has not been fully described. Here, we show that in zebrafish, the loss of function of nog2, a Bmp antagonist expressed in the notochord, impairs β cell differentiation, compatible with the antagonistic role of Bmp in β cell differentiation. In addition, we show that nog2 expression in the notochord is induced by at least one notochord enhancer and its loss of function reduces the number of pancreatic progenitors and impairs β cell differentiation. Tracing Nog2 diffusion, we show that Nog2 emanates from the notochord to the pancreas progenitor domain. Finally, we find a notochord enhancer in human and mice Nog genomic landscapes, suggesting that the acquisition of a Nog notochord enhancer occurred early in the vertebrate phylogeny and contributes to the development of complex organs like the pancreas., Graphical Abstract, Highlights • nog2 loss of function leads to a reduced number of pancreatic progenitor and β cells • A tissue-specific enhancer controls the expression of nog2 in the notochord • The nog2 notochord enhancer is required for establishing a correctly sized pancreas • Regulation of notochord expression of Nog might be similar in many vertebrate lineages, Amorim et al. find that Nog2 is expressed in the zebrafish notochord by the action of a tissue-specific enhancer, and it diffuses to the pancreatic domain and controls its size. The identification of Nog enhancers in other vertebrate lineages suggests a conserved mechanism for pancreas development in vertebrates.

Many single nucleotide polymorphisms (SNPs) associated with type 2 diabetes overlap with putative endocrine pancreatic enhancers, suggesting that these SNPs modulate enhancer activity and, consequently, gene expression. We performed in vivo mosaic transgenesis assays in zebrafish to quantitatively test the enhancer activity of type 2 diabetes–associated loci. Six out of 10 tested sequences are endocrine pancreatic enhancers. The risk variant of two sequences decreased enhancer activity, while in another two incremented it. One of the latter (rs13266634) locates in an SLC30A8 exon, encoding a tryptophan-to-arginine substitution that decreases SLC30A8 function, which is the canonical explanation for type 2 diabetes risk association. However, other type 2 diabetes–associated SNPs that truncate SLC30A8 confer protection from this disease, contradicting this explanation. Here, we clarify this incongruence, showing that rs13266634 boosts the activity of an overlapping enhancer and suggesting an SLC30A8 gain of function as the cause for the increased risk for the disease. We further dissected the functionality of this enhancer, finding a single nucleotide mutation sufficient to impair its activity. Overall, this work assesses in vivo the importance of disease-associated SNPs in the activity of endocrine pancreatic enhancers, including a poorly explored case where a coding SNP modulates the activity of an enhancer.

O texto objetiva compreender o trabalho informal e o apoio governamental na percepção de envolvidos, questionando se essa política incentiva a formalização. Por meio de pesquisa documental e entrevistas semiestruturadas, conclui que, atualmente, “economia informal” é a expressão mais apropriada que “setor informal” para designar as atividades analisadas.

This paper reports research that produces a prospective understanding of the university of the future. The study establishes a 20 year horizon and supports the analysis of this situation on a model composed of markers and conceived considering the classic definition of university, organizational models and predetermined scenarios. The application and validation of the model uses an empirical dimension consisting of a sample of n = 51 participants. The qualitative and quantitative information gathered comes from diverse sources of opinion / information in the context of the discussion of hypothetical scenarios, of theoretical construct. Audiovisual recording instruments and mixed techniques were used: worksheets, questionnaires, observation and valuation scales. The contributions of the application of this model are translated into an optimized systematization of opinions, validation of the model, generalization of the process to other contexts of reflection, hybrid qualitative-quantitative techniques that allow a greater valuation of the qualitative results supported by the descriptive statistics summaries of the corresponding quantitative data., Este artículo reporta investigación que tiene por objetivo prospectivar la universidad del futuro. El estudio establece un horizonte de 20 años y basa el análisis de esta situación en un modelo constituido por marcadores y concebido considerando la definición clásica de universidad, modelos organizacionales y escenarios predeterminados. La aplicación y validación del modelo recurre a una dimensión empírica constituida por una muestra de n = 51 participantes. La información cualitativa y cuantitativa recogida proviene de diversas fuentes de opinión / información en el contexto de la discusión de escenarios hipotéticos, de constructo teórico. Se utilizaron instrumentos de registro audiovisual y técnicas mixtas: fichas de trabajo, cuestionarios, observación y escalas valorativas de opinión. Las contribuciones de la aplicación de este modelo se traducen en una sistematización optimizada de opiniones, validación del modelo, generalización del proceso a otros contextos de reflexión, técnicas híbridas cualitativas-cuantitativas que permiten una mayor valoración de los resultados cualitativos soportada por los resúmenes de estadística descriptiva de los correspondientes datos cuantitativos., Este artigo repor ta investigação que tem por objetivo prospetivar a universidade do futuro. O estudo estabelece um horizonte de 20 anos e baseia a análise desta situação num modelo constituído por marcadores e concebido considerando a definição clássica de universidade, modelos organizacionais e cenários pré-determinados. A aplicação e validação do modelo recorre a uma dimensão empírica constituída por uma amostra de n=51 participantes. A informação qualitativa e quantitativa recolhida provem de diversas fontes de opinião/informação no contexto da discussão de cenários hipotéticos, de constructo teórico. Utilizaram-se instrumentos de registo audiovisual e técnicas mistas: fichas de trabalho, questionários, observação e escalas valorativas de opinião. Os contributos da aplicação deste modelo traduzem-se numa sistematização otimizada de opiniões, validação do modelo, generalização do processo a outros contextos de reflexão, técnicas híbridas qualitativas-quantitativas que permitem uma maior valoração dos resultados qualitativos suportada pelos sumários de estatística descritiva dos correspondentes dados quantitativos.

Este artigo apresenta os resultados de uma investigação sobre uma proposta de modelo de marcadores para a universidade dos próximos 20 anos, com um enquadramento teórico fundamentado na essência da universidade, nos modelos organizacionais e na projeção de cenários. Privilegia-se a vertente de uma componente empírica e relata-se o processo de análise de dados qualitativos e quantitativos referentes à validação do modelo, obtidos em sessões de grupos focais com 51 participantes (estudantes, investigadores e professores), solicitados a discutirem cenários prováveis teoricamente construídos. A recolha de dados, com registo audiovisual, utilizou técnicas mistas: fichas de trabalho, questionários, observação, escalas valorativas de opinião. Como contributos, salienta-se a sistematização das reflexões, a validação do modelo, a transferabilidade para outros contextos e técnicas mistas inovadoras para processar e representar dados qualitativos e quantitativos, suportadas em informação de estatística descritiva e representação visual infográfica. Apresentam-se ainda alguns resultados cruzando informação de índole mais qualitativa com outra mais quantitativa que é destacada também nas conclusões.

Advances in human genetics in recent years have largely been driven by next-generation sequencing (NGS); however, the discovery of disease-related gene mutations has been biased toward the exome because the large and very repetitive regions that characterize the non-coding genome remain difficult to reach by that technology. For autosomal-dominant spinocerebellar ataxias (SCAs), 28 genes have been identified, but only five SCAs originate from non-coding mutations. Over half of SCA-affected families, however, remain without a genetic diagnosis. We used genome-wide linkage analysis, NGS, and repeat analysis to identify an (ATTTC)n insertion in a polymorphic ATTTT repeat in DAB1 in chromosomal region 1p32.2 as the cause of autosomal-dominant SCA; this region has been previously linked to SCA37. The non-pathogenic and pathogenic alleles have the configurations [(ATTTT)7-400] and [(ATTTT)60-79(ATTTC)31-75(ATTTT)58-90], respectively. (ATTTC)n insertions are present on a distinct haplotype and show an inverse correlation between size and age of onset. In the DAB1-oriented strand, (ATTTC)n is located in 5' UTR introns of cerebellar-specific transcripts arising mostly during human fetal brain development from the usage of alternative promoters, but it is maintained in the adult cerebellum. Overexpression of the transfected (ATTTC)58 insertion, but not (ATTTT)n, leads to abnormal nuclear RNA accumulation. Zebrafish embryos injected with RNA of the (AUUUC)58 insertion, but not (AUUUU)n, showed lethal developmental malformations. Together, these results establish an unstable repeat insertion in DAB1 as a cause of cerebellar degeneration; on the basis of the genetic and phenotypic evidence, we propose this mutation as the molecular basis for SCA37. We thank the families who participated in this study. We are grateful to Goncalo Abecasis, Miguel Costa, Tito Vieira, and Andre Torres for help with MERLIN analysis; Beatriz Sobrino, Jorge Amigo, and Pilar Cacheiro for next-generation sequencing analysis, performed at the Santiago de Compostela node of the Spanish National Genotyping Center; Nuno Santarem and Anabela Cordeiro-da-Silva for assistance with cloning; Antonio Amorim, Laura Vilarinho, and Paula Jorge for samples from the Portuguese population; and Paula Magalhaes from the Institute for Molecular and Cell Biology Cell Culture and Genotyping Core for DNA extraction. This work was financed by Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020 Operational Program for Competitiveness and Internationalization (POCI) of Portugal 2020 and by Portuguese funds through the Fundacao para a Ciencia e a Tecnologia (FCT) and Ministerio da Ciencia, Tecnologia, e Inovacao in the framework of the project "Institute for Research and Innovation in Health Sciences" (POCI-01-0145-FEDER-007274); and by FCT grant PTDC/SAU-GMG/098305/2008 to I.S. A. I.S. was the recipient of an FCT scholarship (SFRH/BD/30702/2006). J.R.L. was supported by scholarships from PEst-C/SAU/LA0002/2013 and the European Molecular Biology Organization (ASTF494-2015). C.L.O. was supported by a scholarship from PEst-C/SAU/LA0002/2013. This work was also financed by the Porto Neurosciences and Neurologic Disease Research Initiative at the Instituto de Investigacao e Inovacao em Saude (Norte-01-0145-FEDER-000008), supported by Norte Portugal Regional Operational Programme (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement through FEDER, and by the Fondo de Investigacion Sanitaria of the Instituto de Salud Carlos III (grant PI12/00742).

O presente estudo enquadra-se no domínio do Autocuidado e das Tecnologias de Informação e Comunicação (TIC) e, na sua relação com o processo de ensino-aprendizagem dos estudantes de enfermagem. Atualmente, nesta era digital, é essencial proporcionar aos estudantes do Ensino Superior ferramentas de ensino progressivamente mais interativas e dinâmicas. A criação de um objeto virtual de aprendizagem (OVA) como ferramenta educacional interativa pode constituir-se uma mais-valia para o processo de ensino-aprendizagem. Trata-se de um estudo de cariz quantitativo, observacional, descritivo, com dois momentos de avaliação de referência cruzada. Insere-se no âmbito de um projeto de investigação da Unidade Científico-Pedagógica (UCP) Autocuidado, da Unidade de Investigação da Escola Superior de Enfermagem do Porto (UNIESEP), que tem como intuito o desenvolvimento e validação de uma ferramenta tecnológica interativa denominada Plataforma de Procedimentos de Enfermagem (PoPE), contendo procedimentos de Enfermagem ilustrados e vídeos explicativos que facilitam a compreensão das etapas dos procedimentos, bem como um Quiz que permite testar os conhecimentos sobre a temática posicionar pessoa. Na primeira fase do estudo, fase de diagnóstico, procedeu-se à aplicação de um questionário, ao qual responderam 122 estudantes do 2º ano do Curso de Licenciatura em Enfermagem (CLE), da Escola Superior de Enfermagem do Porto (ESEP), inscritos na Unidade Curricular (UC) A Pessoa Dependente e os Familiares Cuidadores, no primeiro semestre do ano letivo 2016-2017. Esta fase teve como objetivo avaliar o grau de satisfação e eficácia do método atual em que são disponibilizados os procedimentos aos estudantes (em suporte de papel). Numa segunda fase foi desenvolvido o OVA, através do aplicativo WordPress que inclui os procedimentos de enfermagem ilustrados, com vídeos narrados e um Quiz para avaliação de conhecimentos. Na fase final foi aplicado um questionário, ao qual responderam 78 estudantes a frequentar o segundo semestre da mesma UC, com o intuito de avaliar a satisfação dos mesmos face à ferramenta interativa, verificar a aplicabilidade e navegabilidade da ferramenta, bem como a adequação dos conteúdos. Verificou-se que os estudantes consideram a PoPE uma ferramenta muito útil e de grande interesse para o processo de ensino-aprendizagem, obtendo um valor médio de avaliação global da mesma de 8,01, numa escala de 0 a 10 (em que 0 é o score mínimo e 10 o máximo). A par deste contributo, mostra ser um recurso potencialmente facilitador do dinamismo e interatividade das aulas.

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The development of organs composed by repeated functional units is, in many cases, accomplished by the transition of cells from a progenitor to a differentiation domain, triggering a reiterated developmental program. Yet, how these discrete fields are formed during development is still a largely unresolved question. The posterior lateral line (pLL), a sensory organ present in fish and amphibians, develops from a primordium that migrates along the flanks of the animal periodically depositing neuromasts, the pLL functional units. In zebrafish (Danio rerio), the developmental program of the pLL is triggered by the transit of progenitor cells from a Wnt to a Fgf signaling domain. It has been proposed that these two fields are defined by the antagonistic activity of these two signaling pathways, but how they are formed and maintained is still an open question in the development of the pLL. In this work, we show that sox21a, an HMG -box transcription factor, is expressed within the Fgf domain. We demonstrate that, while the Fgf signaling pathway do not control sox21a, knockdown of sox21a causes impairment of Fgf signaling, expansion of the Wnt signaling domain and disruption of neuromast development. These results suggest that sox21a is a key player in the pLL primordium patterning, fine-tuning the border of the Fgf and Wnt signaling domains.

Neste artigo apresenta-se a pequena história de um projeto, ainda no seu início, mas em que o caminho do seu desenvolvimento fica traçado. As ideias que lhe serviram de suporte levaram à identificação de marcadores relativos à formação, inovação e pesquisa avaliados por entrevista e questionário, na fase de diagnóstico. Além de possibilitarem informações sobre a Universidade de hoje e do próximo decénio, permitiram perspectivar a fase de intervenção e tirar algumas notas para trabalhos e atividades posteriores. Destacamos a elaboração de cenários prospectivos sobre a Universidade do futuro e designadamente, a Universidade dos próximos 20 anos, em que os radares de contrastes dos diferentes marcadores na formação, na inovação e na pesquisa nos possibilitarão a configuração de diferentes cenários. Estes cenários serão refletidos em grupos com a participação de um número determinado de professores, investigadores, bolsistas, estudantes e pesquisadores do projeto-marcadores com uma metodologia própria e original, em sessões, com uma dinâmica específica e materiais adequados e gravação em áudio e vídeo. Os dados daí decorrentes, após análise e estudo, poderão dar matéria para outros artigos e outras atividades a realizar. Estas sessões poderão ser realizadas em diferentes universidades com as devidas adaptações mas sem alterar o seu formato.

The problems in steam systems installations in Africa have three main causes: lack of proper preventive and predictive maintenance actions; lack of awareness of the importance of availability and efficiency of the systems during design stage; and lack of care and misuse of the steam systems during operation due to the scarcity of specialized workforce. The present study, carried out during a one year trainee program within the scope of Bosch Industrial Business Development in Africa, is focused on identifying po-tential improvement points and problems related to maintenance and efficiency in Steam Systems in Africa. Maintenance services offered by the company were studied and after thorough analysis, improvement points were identified using TRIZ methodol-ogy in order to propose enhanced solutions further adapted to the African necessities. A detailed analysis was performed regarding the problems of equipment availability in Africa, thus enlightening the importance of redundant solutions in a market where qual-ified workforce and spare parts for equipment are not easily and readily available. A remote predictive and preventive maintenance solution was identified using Contradiction Matrix. An improvement to a maintenance contract was proposed using Substance-Field analysis. A suggestion to increase the availability of a steam system, and a proposal to increase the efficiency of a steam system were both also developed using Substance-Field analysis. In this way, the study was focused on using innovative methodologies in the development of improvement proposals in Africa, without disregarding the importance of certain aspects that are often not taken into account as much as they should.

Este trabalho apresenta o procedimento, os resultados e a validacao da identificacao de um modelo ARX (autoregressive exogenous) para um conjunto experimental construido em laboratorio formado por uma Correia Rolante acionada por Motor CC. Para fins de comparacao foi tambem identificado um modelo de segunda ordem pelo ajuste dos parâmetros da resposta temporal a um degrau de entrada. Para validar o modelo ARX, controladores PI-D discretos para o conjunto foram projetados utilizando os dois modelos e os seus desempenhos foram comparados em ensaios de variacao de carga em degrau utilizando o indice NRMSE (normalized root mean square error ). Os resultados mostram que o controlador PI-D projetado com base no modelo ARX apresentou desempenho superior indicando sua melhor adequacao para este projeto.

Accurate transcriptional control of genes is fundamental for the correct functioning of organs and developmental processes. This control depends on the interplay between the promoter of genes and other noncoding sequences, whose interaction is mediated by 3D chromatin arrangements. Thus, the detailed description of transcriptional regulatory landscapes is essential to understand the mechanisms of transcriptional regulation. However, to achieve that, two important challenges have to be faced: (1) the identification of the noncoding sequences that contribute to gene transcription and (2) the association of these sequences to the respective genes they control. In this chapter, we describe two protocols that allow overcoming these important challenges: the assay for transposase-accessible chromatin using sequencing (ATAC-seq) and circularized chromosome conformation capture (4C-seq). ATAC-seq is a very efficient technique that, using a very low number of cells as starting material, allows the identification of active chromatin regions genome wide, whereas 4C-seq detects the subset of sequences that interact specifically with the promoter of a given gene. When combined, both techniques provide a comprehensive snapshot of the regulatory landscapes of developmental genes. The protocols we present here have been optimized for teleost fish samples, zebrafish and medaka, allowing the in-depth study of transcriptional regulation in these two emerging animal models. Given the amenability and easy genetic manipulation of these two experimental systems, we anticipate that they will be important in revealing general principles of the vertebrate regulatory genome., Work was funded by grants from Ministerio de Economía y Competitividad (BFU2013-41322-P and BFU2014-58449-JIN) and the Andalusian Government (BIO-396). JB was supported by an FCT Investigator grant (IF/00654/2013).

Unexpected child death investigation is a difficult area of forensic practice in view of the wide range of possible genetic, congenital, and acquired natural and nonnatural causes. Idiopathic infantile arterial calcification (IIAC) is a rare autosomic recessive disease usually diagnosed postmortem. Inactivating mutations of the ENPP1 gene were described in 80% of the cases with IIAC. We report a case of a 5-year-old girl submitted to a forensic autopsy due to sudden death and possible medical negligence/parents child abuse. Major alterations found (intimal proliferation and deposition of calcium hydroxyapatite around the internal elastic lamina and media of arteries; acute myocardial infarct, stenotic and calcified coronary artery; perivascular and interstitial myocardial fibrosis; and subendocardial fibroelastosis) were diagnostic of IIAC. We reviewed IIAC cases published in the English literature and highlight the importance of adequate autopsy evaluation in cases of sudden child death.

PMCID: PMC4434585.-- et al., The genomic regulatory programmes that underlie human organogenesis are poorly understood. Pancreas development, in particular, has pivotal implications for pancreatic regeneration, cancer and diabetes. We have now characterized the regulatory landscape of embryonic multipotent progenitor cells that give rise to all pancreatic epithelial lineages. Using human embryonic pancreas and embryonic-stem-cell-derived progenitors we identify stage-specific transcripts and associated enhancers, many of which are co-occupied by transcription factors that are essential for pancreas development. We further show that TEAD1, a Hippo signalling effector, is an integral component of the transcription factor combinatorial code of pancreatic progenitor enhancers. TEAD and its coactivator YAP activate key pancreatic signalling mediators and transcription factors, and regulate the expansion of pancreatic progenitors. This work therefore uncovers a central role for TEAD and YAP as signal-responsive regulators of multipotent pancreatic progenitors, and provides a resource for the study of embryonic development of the human pancreas., The research was supported by the National Institute for Health Research (NIHR) Imperial Biomedical Research Centre. Work was funded by grants from the Ministerio de Economía y Competitividad (CB07/08/0021, SAF2011-27086, PLE2009-0162 to J.F., BFU2013-41322-P to J.L.G-S.), the Andalusian Government (BIO-396 to J.L.G-S.), the Wellcome Trust (WT088566 and WT097820 to N.A.H., WT101033 to J.F.), the Manchester Biomedical Research Centre, ERC advanced starting grant IMDs (C.H-H.C. and L.V.) and the Cambridge Hospitals National Institute for Health Research Biomedical Research Centre (L.V.). R.E.J. is a Medical Research Council clinical training fellow. The authors are grateful to C. Wright (Vanderbilt University) for zebrafish Pdx1 antiserum, J. Postlethwait (Purdue University) for a Sox9b clone, H. Sasaki (Kumamoto University) for a TEAD–EnR clone, C. Vinod and L. Abi for research nurse assistance, and clinical colleagues at Central Manchester University Hospitals NHS Foundation Trust. The authors thank J. Garcia-Hurtado for technical assistance (IDIBAPS).

The pancreas hosts some of the most debilitating and deadly diseases, including pancreatic cancer and diabetes mellitus. In autoimmune diabetes, for example, there is a massive destruction of the insulin producing cells of the pancreas. Pancreatic developmental defects can also result in a deficit of this cell type. To revert these important pancreatic diseases, researchers are currently trying to artificially generate insulin producing beta-cells for implantation and, in this way, suppress insulin administration [1]. However, to derive cells that function properly it is necessary to understand in depth the instructions that our body uses to produce them during fetal development. On the other hand, regarding pancreatic cancer, it is known that tumor cells very often recover characteristics similar to those of embryonic cells [2]. These are clear examples of the relevance to understand the identity of pancreatic progenitor cells. The human genome contains the instructions to generate all the cell types that are formed during embryonic development. Building such complex systems is in part possible because the transcription of different sets of genes is sequentially activated or repressed generating cell-specific combinatorial profiles. Enhancers are one class of non-coding cis-regulatory sequences that control the transcription of genes. Also, the specific temporal and spatial regulatory activity of each enhancer is defined by clusters of transcription factors (TFs) that are bound to these sequences. Therefore, understanding the identity of pancreatic progenitor cells requires the identification of these regulatory sequences and the TFs that bind to them. Our recent work published in Nature Cell Biology characterized the identity of human pancreatic progenitor cells [3]. For this purpose, we used human fetal pancreas, which was dissected from embryos at an early stage of gestation (~ 6 weeks), as well as pancreatic progenitor cells derived in vitro from human embryonic stem cells (hESC), that matched the developmental stage of cells in the fetal pancreas. We took advantage of high throughput sequencing techniques to identify genes that are selectively expressed in both samples and to produce a map of the genomic sequences that act as enhancers in the human embryonic pancreas. The results showed that pancreatic progenitor cells derived in vitro effectively recapitulate the main properties of the transcriptional program of the fetal progenitor cells, validating the in vitro system. In addition, further analysis of the regulatory regions active in pancreatic progenitor cells uncovered how genes can be transcriptionally regulated at this specific cell stage. We report that these regulatory regions are recognized by several TFs that have been previously described to be mutated in patients with congenital defects in pancreas development. Additionally, we found that the majority of these enhancers are recognized by TEAD proteins and its co-activator factor YAP, two important effectors of the Hippo signaling pathway [4]. Two recent reports have shown that pancreas-specific disruption of the upstream Hippo kinases Mst1/2 leads to acinar differentiation defects and changes in pancreas architecture [5, 6]. These reports, however, do not address whether Hippo signaling or TEAD are important for pancreatic progenitors. The results reported in our recent work show that TEAD and YAP play a key role in the identity of pancreatic progenitor cells. This was confirmed by functional studies, performed in progenitors derived from hESC, pancreatic bud explants from mouse embryos, and zebrafish, which showed that TEAD and YAP not only play a key role in the activation of these regulatory regions, but also control cell proliferation in embryonic pancreatic progenitor cells. We end up with a model for early pancreas development in which specific non-coding regions of the genome are exquisitely defined by co-binding of several pancreas-specific TFs, which are timely co-expressed at the progenitor cell stage (green shapes in Figure ​Figure1).1). A subset of such regions is also bound by TEAD proteins (light blue in Figure ​Figure1),1), and this combination of factors somehow marks them as “poised” enhancers, awaiting timely enhancer activation. Proper activation is provided by YAP nuclear translocation and binding to TEAD-targeted enhancers (red circles in Figure ​Figure1),1), thus leading to increased expression of target genes. Given that the control of YAP intracellular localization is mainly driven by the Hippo kinase cascade (orange shapes in Figure ​Figure1),1), this would confer a means for pancreatic progenitors to integrate extracellular signaling cues to control a timely cell differentiation program. The identified role for TEAD and YAP in this process opens up new research avenues towards disease development in humans. YAP has been described as an oncogene involved in several types of cancer, including pancreatic ductal adenocarcinoma [4]. Our results suggest that the reactivation of the pancreatic embryonic program in adult pancreatic cells could contribute to dedifferentiation and uncontrolled growth during pancreatic carcinogenesis. In this context, YAP, TEAD, or their pancreatic-specific downstream genes could be used as targets of new anti-cancer drugs. On the other hand, the regulatory program described in our work could be potentially exploited to control the growth and differentiation of in vitro derived pancreatic beta cells, aiming for treatment of diabetic patients in which these cells have been destroyed. Diabetes can also result from the impairment of proper beta-cell embryonic development, which has been classically attributed as the result of coding mutations in key pancreatic developmental genes. Using a preliminary version of the regulatory regions that we recently reported, the groups of Prof. Ferrer and Prof. Hattersley have shown that, in addition to coding mutations, regulatory mutations might also cause severe developmental defects of the pancreas, such as pancreatic agenesis [7]. Therefore, the atlas of regulatory elements of pancreatic progenitor cells described in our work could be used to define genomic hotspots of mutations with the potential to impair proper pancreas development. Indeed, with current technologies allowing the sequencing of a patient's genome at an affordable cost, this will become a possibility in the near future. Figure 1 The role of YAP and TEAD in the activation of enhancers of pancreatic multipotent progenitor cells and their possible contribution to human pancreatic diseases In summary, we have now unveiled a novel role for YAP and TEAD proteins in the control of gene expression during pancreas development. These findings now turn the spotlight on YAP and the Hippo pathway, placing them at the crossroads of pancreas formation. Special attention should be directed from here on to check the expression or activity of YAP as well as kinases involved in Hippo signaling, since these might underlie defects in pancreas development resulting from non-coding genetic mutations or adverse environmental cues. A new path has been opened ahead and we now count with an epigenomic road map to evaluate causes of human pancreas disease that could be linked with its developmental process.

In Drosophila, the development of the compound eye depends on the movement of a morphogenetic furrow (MF) from the posterior (P) to the anterior (A) of the eye imaginal disc. We define several subdomains along the A–P axis of the eye disc that express distinct combinations of transcription factors. One subdomain, anterior to the MF, expresses two homeobox genes, eyeless (ey) andhomothorax (hth), and the zinc-finger geneteashirt (tsh). We provide evidence that this combination of transcription factors may function as a complex and that it plays at least two roles in eye development: it blocks the expression of later-acting transcription factors in the eye development cascade, and it promotes cell proliferation. A key step in the transition from an immature proliferative state to a committed state in eye development is the repression of hth by the BMP-4 homolog Decapentaplegic (Dpp).

Microbial cultures able to degrade xenobiotic compounds are the key element for biological treatment of waste effluents and are obtained from enrichment processes. In this study, two common enrichment methods, suspension batch and immobilized continuous, were compared. The main selection factor was the presence of 1,3-dichloro-2-propanol (1,3-DCP) as the single carbon source. Both methods have successfully enriched microbial consortia able to degrade 1,3-DCP. When tested in batch culture, the degradation rates of 1,3-DCP by the two consortia were different, with the consortia obtained by batch enrichment presenting slightly higher rates. A preliminary morphological and biochemical analysis of the predominant colonial types present in each degrading consortia revealed the presence of different constituting strains. Three bacterial isolates capable of degrading 1,3-DCP as single strains were obtained from the batch enrichments. These strains were classified by 16S rRNA analysis as belonging to the Rhizobiaceae group. Degradation rates of 1,3-DCP were lower when single species were used, reaching 45 mg 1(-1) d(-1), as compared to 74 mg 1(-1) d(-1) of the consortia enriched on the batch method. Mutualistic interactions may explain the better performance of the enriched consortia.