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Resumen Introduccion La tuberculosis (TB) continua siendo un reto en la edad pediatrica. Las dificultades en su diagnostico y la escasa experiencia en su tratamiento en la infancia y adolescencia son algunas de las razones para considerar la necesidad de centros especializados en tuberculosis pediatrica y priorizar a la poblacion pediatrica en los programas de control de tuberculosis, especialmente en paises de baja incidencia. Descripcion de casos de tuberculosis pediatrica tratados en un centro ambulatorio especializado en TB. Pacientes y metodos Analisis retrospectivo de datos epidemiologicos y clinicos de casos de TB en pacientes menores de 18 anos en el periodo de enero de 2007 a junio de 2017. Resultados Se identificaron 46 casos de TB, con una edad media de 5 anos (RIC: 1,75-13,25). Treinta casos (65,2%) se identificaron en contexto de cribado por exposicion conocida a la TB. En 36 pacientes (78%), la duracion mediana de los sintomas fue de 2 semanas, y los sintomas mas frecuentes fueron tos (54%) y fiebre (48%). La radiografia de torax fue anormal en el 73,9% de los pacientes, y se realizo TC en el 82,2%, cuyos hallazgos proporcionaron claves importantes para la decision de tratar en el 85,3%. A pesar de recogerse distintas muestras microbiologicas, solo se confirmo el diagnostico en 12 casos (26,1%). Las pruebas de cultivo y/o de amplificacion de acidos nucleicos fueron positivas en el 33,3% de las muestras de esputo, el 28,1% de las de lavado broncoalveolar y el 12,9% de las de aspirado gastrico. La TB pulmonar fue el diagnostico mas frecuente (n = 31), seguida de la TB pleural (n = 6), la linfadenitis tuberculosa (n = 3), la uveitis (n = 2), la TB osea, la TB diseminada, la TB cerebelar y el eritema nudoso (cada uno, n = 1). Conclusiones La TB en la edad pediatrica es un marcador epidemiologico de transmision reciente de Mycobacterium tuberculosis en la comunidad. Debe hacerse todo lo posible para recoger muestras microbiologicas antes de iniciar el tratamiento. Un equipo pediatrico especializado permite la realizacion de un diagnostico preciso incluso en ausencia de confirmacion microbiologica.

With the lack of effective treatments for low back pain, the use of extracellular matrix (ECM)-based biomaterials have emerged with undeniable promise for IVD regeneration. Decellularized scaffolds can recreate an ideal microenvironment inducing tissue remodeling and repair. In particular, fetal tissues have a superior regenerative capacity given their ECM composition. In line with this, we unraveled age-associated alterations of the nucleus pulposus (NP) matrisome. Thus, the aim of the present work was to evaluate the impact of ECM donor age on IVD de/regeneration. Accordingly, we optimized an SDS (0.1 %, 1 h)-based decellularization protocol that preserves ECM cues in bovine NPs from different ages. After repopulation with adult NP cells, younger matrices showed the highest repopulation efficiency. Most importantly, cells seeded on younger scaffolds produced healthy ECM proteins suggesting an increased capacity to restore a functional IVD microenvironment. In vivo, only fetal matrices decreased neovessel formation, showing an anti-angiogenic potential. Our findings demonstrate that ECM donor age has a strong influence on angiogenesis and ECM de novo synthesis, opening new avenues for novel therapeutic strategies for the IVD. Additionally, more appropriate 3D models to study age-associated IVD pathology were unveiled.

In the last decade, intervertebral disc (IVD) decellularization has gained significant attention for tissue regenerative purposes as a successful therapeutic alternative for low back pain (LBP). We discuss the recent advances in IVD decellularization, repopulation, and sterilization procedures, highlighting the major challenges that need to be addressed for clinical translation.

Assessing newborn infants at risk for early-onset sepsis (EOS) is a common clinical task conducted by pediatricians. A change in the internal protocol for managing neonates at risk was implemented in 2016. Unlike the previous protocol, which determined laboratory testing in asymptomatic newborns in the presence of one risk factor (RF) for sepsis; the new protocol advocates the screening in the presence of at least two RF. The purpose of this study was to characterize newborns at increased risk for EOS before and after the implementation of a diagnostic/treatment protocol.Retrospective analysis of asymptomatic newborns with RF to EOS who had laboratory testing performed at perinatology department in a central hospital in north of Portugal before and after the protocol was reviewed (2016), in the years 2015 and 2017, respectively. Patients were divided in two groups: preprotocol (2015) and postprotocol (2017), according to the date of admission.A total of 361 newborns were enrolled, 296 (82%) pre-protocol and 65 (18%) post-protocol. There was a significant raise in the number of preterm newborns (9.5 versus 30.8%, pre- and post-protocol, respectively;The modification of the approach protocol has led to a significant decline in the laboratory testing performed, minimizing newborn pain, unnecessary antibiotic exposure and costs, without increased risk of readmission for sepsis.

Intervertebral disc (IVD) degeneration has been linked to Low Back Pain (LBP) which affects over 80% of the population ranking first in terms of disability worldwide. Degeneration progresses with age and is often accompanied by annulus fibrosus (AF) tearing and nucleus pulposus (NP) herniation. Existing therapies fail to restore IVD function and may worsen AF defects, increasing the risk of reherniation in nearly 30% of patients. Current AF closure options are ineffective, presenting biological or mechanical limitations. Bioadhesives have potential use in this area, however methods to assess performance are limited. Herein, we propose a biomechanical testing method to assess bioadhesives’ capacity to seal AF tears. Two candidate bioadhesives to seal AF tears were evaluated; a tough hydrogel adhesive, and a cyanoacrylate-based glue. The adhesion energy at the interface between bovine discs and the tough hydrogel adhesive was quantified using a peel test (n=4). An experimental method to measure the burst pressure of IVDs was then developed. This method was used to quantify the burst pressure of intact (n=7), injured (AF punctured with a 21G needle; n=7), and sealed IVDs (after applying either the tough hydrogel adhesive patch as a sealant; n=5, or the cyanoacrylate-based glue over the AF tear; n=6). The tough adhesive yielded a strong adhesion energy of 239 ± 49 J/m2 during the peel tests. A maximum pressure of 13.2 ± 3.8 MPa was observed for intact discs in the burst pressure tests, which reduced by 61.4% to 5.1 ± 1.5 MPa in the injured IVDs (p

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Acute pancreatitis (AP) is a rare entity in pediatric age. Reports of AP in the context of diabetic ketoacidosis (DKA) have established the role of transient hyperlipemia as a consequent factor. Pharmacological etiology is responsible for 0.3−1.4% of AP cases, with no case reports of an association with the use of sertraline in the pediatric population to date.Herein is described the case of a 15-year-old girl with poorly controlled type 1 diabetes, taking sertraline for a depressive disorder, who developed AP associated with DKA. With this report, the authors intend to emphasize the importance of diagnosing AP in DKA setting. In addition, since the patient had normal serum triglyceride levels and no other risk factors for AP, it is hypothesized that sertraline may have been a triggering event in this context., A pancreatite aguda (PA) é uma entidade rara em idade pediátrica. Estão descritos casos clínicos que evidenciam uma associação entre PA e cetoacidose diabética (CAD), postulando o papel da hiperlipidemia transitória como um fator consequente.A etiologia farmacológica é responsável por 0.3−1.4% dos casos de PA, não existindo até o momento casos descritos na literatura que associem a condição ao uso de sertralina na população pediátrica. Os autores descrevem o caso de uma adolescente de 15 anos de idade com diabetes mellitus tipo 1 mal controlada, medicada com sertralina devido a um transtorno depressivo, que desenvolveu PA associada a CAD.Com este caso, os autores pretendem enfatizar a importância do diagnóstico de PA no contexto de CAD. Adicionalmente, dado que a adolescente apresentava níveis séricos de triglicerídeos normais e nenhum outro fator de risco para o desenvolvimento de PA, especula-se que o uso de sertralina pode ter atuado como um evento desencadeante neste contexto.

Intervertebral disc (IVD) degeneration is often the cause of low back pain. Degeneration occurs with age and is accompanied by extracellular matrix (ECM) depletion, culminating in nucleus pulpous (NP) extrusion and IVD destruction. The changes that occur in the disc with age have been under investigation. However, a thorough study of ECM profiling is needed, to better understand IVD development and age-associated degeneration. As so, iTRAQ LC-MS/MS analysis of foetus, young and old bovine NPs, was performed to define the NP matrisome. The enrichment of Collagen XII and XIV in foetus, Fibronectin and Prolargin in elder NPs and Collagen XI in young ones was independently validated. This study provides the first matrisome database of healthy discs during development and ageing, which is key to determine the pathways and processes that maintain disc homeostasis. The factors identified may help to explain age-associated IVD degeneration or constitute putative effectors for disc regeneration.

A importância do e-learning no seio das estruturas formativas existentes nos diferentes contextos profissionais tem vindo a tornar-se crescente. Elegendo a formação online como campo de estudo, este trabalho ambiciona contribuir para a modernização das práticas formativas implementadas ao nível da formação contínua dos magistrados através da adoção das tecnologias digitais e ambientes virtuais de aprendizagem. Para o efeito, estabeleceu-se o movimento prévio de análisar os modelos pedagógicos de formação a distância adotados pelos países pertencentes à Rede Europeia de Formação Judiciária. Esta investigação foi efetuada em 2014, tendo assumido os seguintes objetivos: i) identificar práticas de formação a distância e/ou em blended-learning; ii) efetuar um levantamento das principais orientações existentes ao nível da planificação, conpceção e avaliação de ações de formação em regime a distância e/ou em blended-learning; e iii) analisar os modelos pedagógicos adotados a este nível. Este artigo centra-se na análise dos resultados inerentes ao contexto internacional, mais concretamente dos modelos de suporte utilizados no âmbito da formação a distância, com vista ao desenvolvimento de um ‘state of the art’ em torno da temática. Os documentos recolhidos junto a 12 países europeus constituíram um corpus de dados conceitual que nos permitiu constatar que existe um esforço estabelecido no sentido da implementação de dispositivos de formação a distância em relação à atividade formativa proporcionada aos magistrados.

Being aware of the importance of e-learning within existing training structures and in different professional contexts, this chapter aims to contribute to the modernization of the training practices implemented in the continuous training of magistrates, through the design of a pedagogical model of distance training, using a hybrid regime, to be implemented in the continuous training of magistrates. The implementation of a model of this nature is of great value for the training carried out by the Centre for Judicial Studies (CEJ), an entity responsible for the initial and continuous training of Portuguese magistrates, as it can constitute a pedagogical innovation of the training practices. The pedagogical model of distance learning that has been developed for the ongoing training of Portuguese magistrates is described and understood as a set of pedagogical assumptions and guidelines for planning, designing, and evaluating the distance training action.

The extracellular matrix (ECM) constituents vary greatly from tissue to tissue, with age and disease. The process of ECM construction is highly regulated during embryonic development and organ homeostasis but is impaired or deregulated during disease and in the elderly. To design effective therapeutic strategies for tissue repair and regeneration, it is crucial to know the changes that occur and the mechanisms that lead to them. In this chapter, the constitution and function of the ECM in normal and diseased tissues will be covered, with an emphasis on bone, cartilage, skin, and nerve.

Estando conscientes da importância do e-learning no seio das atuais estruturas formativas e contextos profissionais, ambiciona-se com este estudo contribuir para a modernização das práticas formativas implementadas ao nível da formação contínua dos magistrados, através de uma análise dos modelos pedagógicos de formação a distância adotados pelos países pertencentes à Rede Ibero-Americana das Escolas Judiciais. Esta investigação assumiu os seguintes objetivos: i) identificar práticas de formação a distância e/ou em blendedlearning; ii) efetuar um levantamento dos principais guidelines existentes ao nível da planificação, conceção e avaliação de ações de formação em regime a distância e/ou em blended-learning; e iii) analisar os modelos pedagógicos adoptados a este nível. Este artigo centra-se na análise dos resultados inerentes ao contexto internacional, mais concretamente dos modelos de suporte utilizados no âmbito da formação a distância com vista ao desenvolvimento de um ‘state of the art’, tendo como propósito a implementação de um modelo pedagógico de formação a distância. Os documentos recolhidos constituíram um corpus de dados concetual que nos permitiu constatar que existe um esforço estabelecido para a implementação de dispositivos de formação a distância na atividade formativa proporcionada aos magistrados em serviço.

Intervertebral disc (IVD) degeneration often leads to low back pain, which is one of the major causes of disability worldwide, affecting more than 80% of the population. Although available treatments for degenerated IVD decrease symptoms' progression, they fail to address the underlying causes and to restore native IVD properties. Poly(γ-glutamic acid) (γ-PGA) has recently been shown to support the production of chondrogenic matrix by mesenchymal stem/stromal cells. γ-PGA/chitosan (Ch) nanocomplexes (NCs) have been proposed for several biomedical applications, showing advantages compared with either polymer alone. Hence, this study explores the potential of γ-PGA and γ-PGA/Ch NCs for IVD regeneration. Nucleotomised bovine IVDs were cultured ex vivo upon injection of γ-PGA (pH 7.4) and γ-PGA/Ch NCs (pH 5.0 and pH 7.4). Tissue metabolic activity and nucleus pulposus DNA content were significantly reduced when NCs were injected in acidic-buffered solution (pH 5.0). However, at pH 7.4, both γ-PGA and NCs promoted sulphated glycosaminoglycan production and significant type II collagen synthesis, as determined at the protein level. This study is a first proof of concept that γ-PGA and γ-PGA/Ch NCs promote recovery of IVD native matrix, opening new perspectives on the development of alternative therapeutic approaches for IVD degeneration.

International audience; A main challenge in cardiac tissue engineering is the limited data on microenvironmental cues that sustain survival, proliferation and functional proficiency of cardiac cells. The aim of our study was to evaluate the potential of fetal (E18) and adult myocardial extracellular matrix (ECM) to support cardiac cells. Acellular three-dimensional (3D) bioscaffolds were obtained by parallel decellularization of fetal- and adult-heart explants thereby ensuring reliable comparison. Acellular scaffolds retained main constituents of the cardiac ECM including distinctive biochemical and structural meshwork features of the native equivalents. In vitro, fetal and adult ECM-matrices supported 3D culture of heart-derived Sca-1(+) progenitors and of neonatal cardiomyocytes, which migrated toward the center of the scaffold and displayed elongated morphology and excellent viability. At the culture end-point, more Sca-1(+) cells and cardiomyocytes were found adhered and inside fetal bioscaffolds, compared to the adult. Higher repopulation yields of Sca-1(+) cells on fetal ECM relied on β1-integrin independent mitogenic signals. Sca-1(+) cells on fetal bioscaffolds showed a gene expression profile that anticipates the synthesis of a permissive microenvironment for cardiomyogenesis. Our findings demonstrate the superior potential of the 3D fetal microenvironment to support and instruct cardiac cells. This knowledge should be integrated in the design of next-generation biomimetic materials for heart repair.

Intervertebral disc (IVD) degeneration is characterized by significant biochemical and histomorphological alterations, such as loss of extracellular matrix (ECM) integrity, by abnormal synthesis of ECM main components, resultant from altered anabolic/catabolic cell activities and cell death. Mesenchymal Stem/Stromal Cell (MSC) migration towards degenerated IVD may represent a viable strategy to promote tissue repair/regeneration. Here, human MSCs (hMSCs) were seeded on top of cartilaginous endplates (CEP) of nucleotomized IVDs of bovine origin and cultured ex vivo up to 3 weeks. hMSCs migrated from CEP towards the lesion area and significantly increased expression of collagen type II and aggrecan in IVD, namely in the nucleus pulposus. Concomitantly, hMSCs stimulated the production of growth factors, promoters of ECM synthesis, such as fibroblast growth factor 6 (FGF-6) and 7 (FGF-7), platelet-derived growth factor receptor (PDGF-R), granulocyte-macrophage colony-stimulating factor (GM-CSF) and insulin-like growth factor 1 receptor (IGF-1sR). Overall, our results demonstrate that CEP can be an alternative route to MSC-based therapies for IVD regeneration through ECM remodeling, thus opening new perspectives on endogenous repair capacity through MSC recruitment.

9 páginas, 6 figuras. Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/136/5/761/DC1, Wnt molecules act as mitogenic signals during the development of multiple organs, and the aberrant activity of their pathway is often associated with cancer. Therefore, the production of Wnts and the activity of their signaling pathway must be tightly regulated. We have investigated the mechanisms of this regulation in the Drosophila hinge, a domain within the wing imaginal disc that depends on the fly Wnt1 ortholog wingless (wg) for its proliferation. Our results uncover a new feedback loop in the wg pathway in which the spatially restricted activation of the Sox gene SoxF (Sox15) by wg represses its own transcription, thus ensuring tight regulation of growth control. rotund, a wing proximodistal patterning gene, excludes SoxF from a thin rim of cells. These cells are thus allowed to express wg and act as the source of mitogenic signal. This novel mode of action of a Sox gene on the Wnt pathway - through transcriptional repression of a Wnt gene - might be relevant to human disease, as loss of human SoxF genes has been implicated in colon carcinoma., This work was funded through grants BMC2003-06248 and BFU2006-00349 to F.C. and the Consolider consortium `From Genes to Shape', of which F.C. is a participant researcher, all from the Spanish Ministry of Science and Innovation. M.L.-D.D. was funded by the FRM (Fondation pour la Recherche Medicale), the Juan de la Cierva Program (Spanish Ministry of Science and Education) and a Marie-Curie Intra-European Postdoctoral contract (EIF). J.C. is a predoctoral fellow of the Portuguese Fundação para a Ciência e a Tecnologia.

Epithelial-cadherin (Ecad) deregulation affects cell-cell adhesion and results in increased invasiveness of distinct human carcinomas. In gastric cancer, loss of Ecad expression is a common event and is associated with disease aggressiveness and poor prognosis. However, the molecular mechanisms underlying the invasive process associated to Ecad dysfunction are far from understood. We hypothesized that deregulation of cell-matrix interactions could play an important role during this process. Thus, we focussed on LM-332, which is a major matrix component, and in Ecad/LM-332 crosstalk in the process of Ecad-dependent invasion. To verify whether matrix deregulation was triggered by Ecad loss, we used the Drosophila model. To dissect the key molecules involved and unveil their functional significance, we used gastric cancer cell lines. The relevance of this relationship was then confirmed in human primary tumours. In vivo, Ecad knockdown induced apoptosis; nonetheless, at the invasive front, cells ectopically expressed Laminin A and βPS integrin. In vitro, we demonstrated that, in two different gastric cancer cell models, Ecad-defective cells overexpressed Laminin γ2 (LM-γ2), β1 and β4 integrin, when compared with Ecad-competent ones. We showed that LM-γ2 silencing impaired invasion and enhanced cell death, most likely via pSrc and pAkt reduction, and JNK activation. In human gastric carcinomas, we found a concomitant decrease in Ecad and increase in LM-γ2. This is the first evidence that ectopic Laminin expression depends on Ecad loss and allows Ecad-dysfunctional cells to survive and invade. This opens new avenues for using LM-γ2 signalling regulators as molecular targets to impair gastric cancer progression.

Intervertebral disc (IVD) degeneration is one of the major causes of low back pain, a problem with a heavy economic burden, which has been increasing in prevalence as populations age. Deeper knowledge of the complex spatial and temporal orchestration of cellular interactions and extracellular matrix remodelling is critical to improve current IVD therapies, which have so far proved unsatisfactory. Inflammation has been correlated with degenerative disc disease but its role in discogenic pain and hernia regression remains controversial. The inflammatory response may be involved in the onset of disease, but it is also crucial in maintaining tissue homeostasis. Furthermore, if properly balanced it may contribute to tissue repair/regeneration as has already been demonstrated in other tissues. In this review, we focus on how inflammation has been associated with IVD degeneration by describing observational andin vitrostudies as well asin vivoanimal models. Finally, we provide an overview of IVD regenerative therapies that target key inflammatory players.

E-cadherin (Ecad) is a well-known invasion suppressor and its loss of expression is common in invasive carcinomas. Germline Ecad mutations are the only known genetic cause of hereditary diffuse gastric cancer (HDGC), demonstrating the causative role of Ecad impairment in gastric cancer. HDGC-associated Ecad missense mutations can lead to folding defects and premature proteasome-dependent endoplasmic reticulum-associated degradation (ERAD), but the molecular determinants for this fate were unidentified. Using a Drosophila-based genetic screen, we found that Drosophila DnaJ-1 interacts with wild type (WT) and mutant human Ecad in vivo. DnaJ (Hsp40) homolog, subfamily B, member 4 (DNAJB4), the human homolog of DnaJ-1, influences Ecad localization and stability even in the absence of Ecad endogenous promoter, suggesting a post-transcriptional level of regulation. Increased expression of DNAJB4 leads to stabilization of WT Ecad in the plasma membrane, while it induces premature degradation of unfolded HDGC mutants in the proteasome. The interaction between DNAJB4 and Ecad is direct, and is increased in the context of the unfolded mutant E757K, especially when proteasome degradation is inhibited, suggesting that DNAJB4 is a molecular mediator of ERAD. Post-translational regulation of native Ecad by DNAJB4 molecular chaperone is sufficient to influence cell adhesion in vitro. Using a chick embryo chorioallantoic membrane assay with gastric cancer derived cells, we demonstrate that DNAJB4 stimulates the anti-invasive function of WT Ecad in vivo. Additionally, the expression of DNAJB4 and Ecad is concomitantly decreased in human gastric carcinomas. Altogether, we demonstrate that DNAJB4 is a sensor of Ecad structural features that might contribute to gastric cancer progression.

E-cadherin and P-cadherin are major contributors to cell-cell adhesion in epithelial tissues, playing pivotal roles in important morphogenetic and differentiation processes during development, and in maintaining integrity and homeostasis in adult tissues. It is now generally accepted that alterations in these two molecules are observed during tumour progression of most carcinomas. Genetic or epigenetic alterations in E- and P-cadherin-encoding genes (CDH1 and CDH3, respectively), or alterations in their proteins expression, often result in tissue disorder, cellular de-differentiation, increased invasiveness of tumour cells and ultimately in metastasis. In this review, we will discuss the major properties of E- and P-cadherin molecules, its regulation in normal tissue, and their alterations and role in cancer, with a specific focus on gastric and breast cancer models.

Mestrado em Engenharia do Ambiente Hoje em dia há uma necessidade crescente de encontrar uma solução viável, económica e sustentável para a substituição dos plásticos convencionais, que utilizam recursos não renováveis como matéria-prima para a sua produção. Além disso, quando depositados no ambiente tornam-se resíduos devido à sua alta resistência à biodegradação. O presente trabalho teve como objetivo principal o controlo biológico, através de análise microscópica, de doze reatores, sendo nove anaeróbios para acidificação, um MBBR e oito reatores descontínuos; sendo os restantes três reatores aeróbios, SBR. Efetuou-se a monitorização das variações morfológicas dentro dos reatores e correlacionaram-se com os parâmetros físico-químicos. A produção de AOVs nos reatores de acidificação para utilização como substrato dos microrganismos acumuladores de PHAs e a seleção de uma população dos microrganismos com elevada capacidade de acumulação desses polímeros nos reatores aeróbios foram também controladas através de análises microbiológicas. Ao longo do procedimento experimental dos reatores foram recolhidas amostras de lamas para a avaliação microbiológica qualitativa das mesmas ao microscópio e foram também determinados parâmetros físico-químicos e de operação dos reatores, assim como parâmetros biológicos. Os resultados obtidos permitem concluir que não é possível estabelecer correlações gerais entre parâmetros físico-químicos e biológicos, mas é possível estabelecê-las para casos de operação específicos, tal como a relação positiva entre a quantidade de flocos de pequenas dimensões e a intensidade de fluorescência emitida pelos grânulos de PHA nos reatores SBR1 e SBR2. Foi também possível obter um perfil de intensidades de fluorescência através da análise e tratamento de imagem em ImageJ. No entanto, não foi possível correlacionar o método de quantificação da fluorescência emitida pelos flocos corados com Azul do Nilo com as concentrações reais, pois não foi possível analisar quantitativamente o polímero acumulado no interior das células. Nowadays there is a growing need to find a viable, economic and sustainable solution for the replacement of conventional plastics, which use non-renewable resources as a raw material for their production. Additionally, when improperly disposed in the environment, they are considered waste because of their high resistance to biodegradation. Furthermore, when thrown away, they become waste because of their highly resistance to biodegradation. The present study was aimed to biologically control, through microscopic analysis, twelve reactors, nine of them being anaerobic acidification reactors, one MBBR and eight batch reactors, and the remainders were aerobic reactors, SBRs. Monitoring was carried out for the morphological variations within the reactors and they were correlated with the physicochemical parameters. The VFA production in the acidification reactors to use as substrate for PHA accumulating microorganisms and the selection of a high PHA accumulating microorganism population in the aerobic reactors were also monitored through microscopic analysis. Throughout the experimental procedure, sludge samples were taken for qualitative microbiologic evaluation under the microscope and physicochemical, operating and biological parameters were also determined The results indicate that it is not possible to establish a general correlation between physicochemical and biological parameters, but correlation can be found for specific reactor conditions, such as the one found for SBR1 and SBR2, where the quantity of small aggregates appeared to have a positive correlation with the fluorescence intensity emitted by the PHA granules. It was also possible to obtain a fluorescence intensity profile by image analysis and processing in ImageJ. However, it was not possible to correlate the method of emitted fluorescence intensity by the Nile Blue stained flocs with real concentration values, since it was not possible to determine the quantity of stored polymer inside the cells.

5 páginas, 2 figuras., Mutations in the CDH1 gene, which encodes the cell adhesion molecule E-cadherin, are associated with hereditary diffuse gastric cancer in humans. Although most of the CDH1 mutations found are truncating, leading to non-functional E-cadherin, some are missense. These missense E-cadherin mutants result in full-length proteins which, when assayed in cell culture, still retain some biological activity. In order to understand the molecular causes of the malfunction of the E-cadherin missense forms found in patients, we developed a Drosophila model, where the effects of expressing the mutant forms can be studied in vivo (Pereira et al., 2006). Here, we review the results obtained so far, and outline possible ways of exploiting the fly model system to screen for pathways affected by specific E-cadherin missense mutant forms and to identify mechanisms that contribute to tumourigenesis., Work reviewed here has been funded through grants POCTI/CBO/44770/2002 and POCI/SAU-OBS/57111/2004 from Fundação para a Ciência e a Tecnologia (Portugal), co-funded by FEDER.

Introduction Intervertebral disc (IVD) degeneration is often the cause of low back pain. With age, extracellular matrix (ECM) depletion occurs, leading to nucleus pulpous (NP) extrusion and IVD destruction. Concomitantly, there is a decline in viable cell populations, which in turn remain poorly characterized. As current clinical treatments have not provided adequate solutions, we propose an in-depth study in terms of NP cell subpopulation characterization and ECM changes with aging, in view of regeneration. Materials and Methods The first step was to characterize NP cell populations harvested from bovine IVDs, to better understand IVD's potential to endogenously regenerate. We started by optimizing cell isolation procedure, still not consensual among the literature. For that, distinct digestive enzymes (collagenase type-I, collagenase type-II, and collagenase type-XI) were used at three different concentrations (0.5, 1.0, and 2.0 mg/mL, respectively), for 4 and 19 hours. To understand the cellular effect of the digestion, we evaluated the cell yield (trypan blue exclusion); cell viability/apoptosis (AnnexinV/Propidium Iodide staining, Flow Cytometry), and cell morphology (actin (phalloidin-AlexaFluor488) and nuclei (DRAQ-5) staining, Imaging Flow Cytometry). The presence of specific cell subpopulations within NP and its phenotype was then investigated, by assessing the expression of CD29, CD44, CD45, CD146, CD34, Gly-A, and Brachyury. Due to microenvironments’ growing importance for regeneration, in parallel, we compared the NP ECM proteomic profile of bovine IVDs from fetus, young and old animals by quantitative iTRAQ LC-MS/MS. The candidates differentially expressed in the three age groups are being validated by Western blot. Results Cell yield and viability improved, in a concentration dependent manner, with increasing collagenase activity against collagen I and II—collagenase XI rendered the highest cell yield and greatest viability. In addition, cell yield was higher for shorter digestion periods (4 hours). Furthermore, NP cells did not reveal major morphological changes dependent on the enzymes used. Interestingly, three viable subpopulations, with different sizes and autofluorescence, were consistently identified by flow cytometry, immediately after isolation. Expression of cell markers differed among these subpopulations, proving the existence of an heterogeneous cell population within the NP. Concerning iTRAQ analysis, a few interesting candidates emerged that are expressed in different proportions in fetus, young and old animals. These quantitative differences are being validated. Conclusion This study shows that collagenase type XI is the most efficient enzyme to be used for isolation and that different cell populations can be identified within the bovine NP. It also demonstrates that distinct ECM proteins are expressed in the IVD at different ratios depending on bovine age groups. Acknowledgments This work was financed by FEDER funds through the Programa Operacional Factores de Competitividade—COMPETE, by Portuguese funds through FCT— Fundação para a Ciência e a Tecnologia. The authors would also like to acknowledge the AO SPINE Research Network (AO SRN) for funding. Disclosure of Interest None declared

E-cadherin plays a major role in cell–cell adhesion and inactivating germline mutations in its encoding gene predispose to hereditary diffuse gastric cancer. Evidence indicates that aside from its recognized role in early tumourigenesis, E-cadherin is also pivotal for tumour progression, including invasion and metastization. Herein, we discuss E-cadherin alterations found in a cancer context, associated cellular effects and signalling pathways, and we raise new key questions that will impact in the management of GC patients and families.