21,949 research outputs found

    Anu√°rio cient√≠fico da Escola Superior de Tecnologia da Sa√ļde de Lisboa - 2021

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    √Č com grande prazer que apresentamos a mais recente edi√ß√£o (a 11.¬™) do Anu√°rio Cient√≠fico da Escola Superior de Tecnologia da Sa√ļde de Lisboa. Como institui√ß√£o de ensino superior, temos o compromisso de promover e incentivar a pesquisa cient√≠fica em todas as √°reas do conhecimento que contemplam a nossa miss√£o. Esta publica√ß√£o tem como objetivo divulgar toda a produ√ß√£o cient√≠fica desenvolvida pelos Professores, Investigadores, Estudantes e Pessoal n√£o Docente da ESTeSL durante 2021. Este Anu√°rio √©, assim, o reflexo do trabalho √°rduo e dedicado da nossa comunidade, que se empenhou na produ√ß√£o de conte√ļdo cient√≠fico de elevada qualidade e partilhada com a Sociedade na forma de livros, cap√≠tulos de livros, artigos publicados em revistas nacionais e internacionais, resumos de comunica√ß√Ķes orais e p√≥steres, bem como resultado dos trabalhos de 1¬ļ e 2¬ļ ciclo. Com isto, o conte√ļdo desta publica√ß√£o abrange uma ampla variedade de t√≥picos, desde temas mais fundamentais at√© estudos de aplica√ß√£o pr√°tica em contextos espec√≠ficos de Sa√ļde, refletindo desta forma a pluralidade e diversidade de √°reas que definem, e tornam √ļnica, a ESTeSL. Acreditamos que a investiga√ß√£o e pesquisa cient√≠fica √© um eixo fundamental para o desenvolvimento da sociedade e √© por isso que incentivamos os nossos estudantes a envolverem-se em atividades de pesquisa e pr√°tica baseada na evid√™ncia desde o in√≠cio dos seus estudos na ESTeSL. Esta publica√ß√£o √© um exemplo do sucesso desses esfor√ßos, sendo a maior de sempre, o que faz com que estejamos muito orgulhosos em partilhar os resultados e descobertas dos nossos investigadores com a comunidade cient√≠fica e o p√ļblico em geral. Esperamos que este Anu√°rio inspire e motive outros estudantes, profissionais de sa√ļde, professores e outros colaboradores a continuarem a explorar novas ideias e contribuir para o avan√ßo da ci√™ncia e da tecnologia no corpo de conhecimento pr√≥prio das √°reas que comp√Ķe a ESTeSL. Agradecemos a todos os envolvidos na produ√ß√£o deste anu√°rio e desejamos uma leitura inspiradora e agrad√°vel.info:eu-repo/semantics/publishedVersio

    Formalin-free fixation and xylene-free tissue processing preserves cell-hydrogel interactions for histological evaluation of 3D calcium alginate tissue engineered constructs

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    Histological evaluation of tissue-engineered products, including hydrogels for cellular encapsulation, is a critical and invaluable tool for assessing the product across multiple stages of its lifecycle from manufacture to implantation. However, many tissue-engineered products are comprised of polymers and hydrogels which are not optimized for use with conventional methods of tissue fixation and histological processing. Routine histology utilizes a combination of chemical fixatives, such as formaldehyde, and solvents such as xylene which have been optimized for use with native biological tissues due to their high protein and lipid content. Previous work has highlighted the challenges associated with processing hydrogels for routine histology due to their high water content and lack of diverse chemical moieties amenable for tissue fixation with traditional fixatives. Thus, hydrogel-based tissue engineering products are prone to histological artifacts during their validation which can lead to challenges in correctly interpreting results. In addition, chemicals used in conventional histological approaches are associated with significant health and environmental concerns due to their toxicity and there is thus an urgent need to identify suitable replacements. Here we use a multifactorial design of experiments approach to identify processing parameters capable of preserving cell-biomaterial interactions in a prototypical hydrogel system: ionically crosslinked calcium alginate. We identify a formalin free fixative which better retains cell-biomaterial interactions and calcium alginate hydrogel integrity as compared to the state-of-the-art formalin-based approaches. In addition, we demonstrate that this approach is compatible with a diversity of manufacturing techniques used to fabricate calcium alginate-based scaffolds for tissue engineering and cell therapy, including histological evaluation of cellular encapsulation in 3D tubes and thin tissue engineering scaffolds (‚ąľ50¬†őľm). Furthermore, we show that formalin-free fixation can be used to retain cell-biomaterial interactions and hydrogel architecture in hybrid alginate-gelatin based scaffolds for use with histology and scanning electron microscopy. Taken together, these findings are a significant step forward towards improving histological evaluation of ionically crosslinked calcium alginate hydrogels and help make their validation less toxic, thus more environmentally friendly and sustainable

    A correlation between tellurite resistance and nitric oxide detoxification in Salmonella Typhimurium

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    Salmonella are important enteric pathogens that are responsible for causing various diseases from gastroenteritis to systemic typhoid fever. Salmonella are a major contributor to morbidity and mortality worldwide. Crucial to their pathogenesis is the survival in harmful conditions elicited by the host immune system, one of these being reactive oxygen and nitrogen species (ROS/RNS). These are produced by macrophages and neutrophils in an attempt to eliminate pathogens. Salmonella, have the unique ability to colonise macrophages and have dedicated nitric oxide (NO) detoxification systems. There are three prominent metalloenzymes (HmpA, NorVW and NrfA) heavily researched in the literature for NO detoxification. Previous work suggested that more proteins are responsible for the nitrosative stress response with these being regulated by the nitric oxide sensitive transcriptional repressor, NsrR. This study demonstrates a relationship between three putative tellurite resistance proteins regulated by NsrR (STM1808, YeaR and TehB) and NO detoxification. A Functional redundancy between these proteins was observed for anaerobic protection against NO and tellurite. Furthermore, this study identified that proteins responsible in NO protection such as HmpA and YtfE also provide resistance to tellurite during aerobic and anaerobic conditions, respectively. Tellurite resistant Salmonella strains were evolved by continued passage in this study that consequently had altered H2O2 resistance profiles and increased sensitivity to antibiotics. However, these strains were not significantly attenuated during macrophage survival or during the presence of NO in vitro. Additionally, the hypothetical protein YgbA, which has predicted roles in NO detoxification, was found to be important to Salmonella survival in macrophages. However, in vitro NO exposure with the NO donor deta NONOate only showed a role for anaerobic protection

    Characterization And Manipulation Of O-Glcnacylation In Granulosa Cells Of Bovine Ovarian Antral Follicles

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    Glucose is widely recognized as the preferred energy substrate for metabolism by granulosa cells (GCs). Yet in most cells, 2-5% of glucose is shunted through the hexosamine biosynthesis pathway (HBP) for O-linked N-acetylglucosaminylation (O-GlcNAcylation). O-GlcNAcylation is an evolutionarily-conserved, post-translational process that modifies serine and threonine residues on a variety of proteins. O-GlcNAcylation is also considered a nutrient sensor that can regulate cellular processes such as metabolism, signal transduction, and proliferation. In this respect, O-GlcNAcylation may be similar to, and possibly mediate, AMP-activated protein kinase (AMPK) signaling and its nutrient-sensing actions. However, the occurrence of O-GlcNAcylation and its relative importance to GC function has not been determined. Here, we characterized relative O-GlcNAcylation in bovine GCs from small and large antral follicles and determined its effects on GC proliferation. Bovine ovary pairs morphologically staged to the mid-to-late estrous period were used. Granulosa cells and follicular fluid were aspirated from small (3-5mm) and large (\u3e10mm) follicles. Freshly isolated GCs of small follicles exhibited greater immunodetectable expression of O-GlcNAcylation and the O-GlcNAcylation enzyme, O-GlcNAc transferase (OGT) expression than large follicles (P\u3c0.05, n=7 ovary pairs). Less glucose (0.4mM vs 2.2 mM, P\u3c0.05) and more lactate (33.3mM vs 9.6mM, P\u3c0.05) was present in the follicular fluid of small follicles compared to large follicles (P\u3c0.05, n=7). Steroid profiles revealed a progesterone to estradiol ratio \u3e10 in all small follicle pools, indicative of highly atretic follicles. Similarly, 5 of the 7 large follicles pools were highly atretic and 2 were intermediately atretic (\u3e1\u3c10). Culture of GCs in serum free conditions revealed that inhibition of the HBP via the glutamine fructose-6-phosphate aminotransferase (GFAT) inhibitor, DON (50¬ĶM), impaired O-GlcNAcylation for both follicle sizes (P\u3c0.05, n=5 independent expts.). The inhibitor DON also prevented GC proliferation regardless of follicle size (P\u3c0.05, n=4). Direct inhibition of O-GlcNAcylation via the OGT inhibitor, OSMI-1 (50¬ĶM), prevented proliferation of GCs from small follicles (P\u3c0.05, n=3). Augmentation of O-GlcNAcylation via the O-GlcNAcase (OGA) inhibitor, Thiamet-G (2.5¬ĶM), enhanced O-GlcNAcylation in GCs from both follicle sizes (P\u3c0.05, n=3) but had no effect (P\u3e0.05, n=3) on GC proliferation for either follicle size. Lastly, the use of the AMPK activator, Metformin (10mM), revealed that while AMPK activation inhibited GC proliferation from small follicles (P\u3c0.05, n=4) as anticipated, it had no effect on O-GlcNAcylation (P\u3e0.05 n=5). The results indicate that: 1) O-GlcNAcylation occurs in GCs of bovine antral follicles, 2) Relative expression of O-GlcNAcylation is associated with alterations of glucose and lactate within the follicle, 3) Disruption of O-GlcNAcylation impairs GC proliferation, and 4) AMPK activation does not affect O-GlcNAcylation. In conclusion, the HBP and O-GlcNAcylation in GCs constitute an alternative, potential nutrient-sensing pathway to influence GC function and folliculogenesis in the bovine ovary

    Investigating the mechanism of human beta defensin-2-mediated protection of skin barrier in vitro

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    The human skin barrier is a biological imperative. Chronic inflammatory skin diseases, such as Atopic Dermatitis (AD), are characterised by a reduction in skin barrier function and an increased number of secondary infections. Staphyloccocus aureus (S. aureus) has an increased presence on AD lesional skin and contributes significantly to AD pathology. It was previously demonstrated that the damage induced by a virulence factor of S. aureus, V8 protease, which causes further breakdown in skin barrier function, can be reduced by induction of human ő≤- defensin (HBD)2 (by IL-1ő≤) or exogenous HBD2 application. Induction of this defensin is impaired in AD skin. This thesis examines the mechanism of HBD2-mediated barrier protection in vitro; demonstrating that in this system, HBD2 was not providing protection through direct protease inhibition, nor was it altering keratinocyte proliferation or migration, or exhibiting specific localisation within the monolayer. Proteomics data demonstrated that HBD2 did not induce expression of known antiproteases but suggested that HBD2 stimulation may function by modulating expression of extracellular matrix proteins, specifically collagen- IVőĪ2 and Laminin-ő≤-1. Alternative pathways of protection initiated by IL-1ő≤ and TNFőĪ stimulation were also investigated, as well as their influence over generalised wound healing. Finally, novel 3D human skin epidermal models were used to better recapitulate the structure of human epidermis and examine alterations to skin barrier function in a more physiological system. These data validate the barrier-protective properties of HBD2 and extended our knowledge of the consequences of exposure to this peptide in this context

    Immune System, Gut Microbiota and Diet: An Interesting and Emerging Trialogue

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    The present chapter provides a comprehensive overview of the multifaceted links connecting the immune system, the intestinal microbiota, and the diet, covering also some recent, less explored, and emerging topics such as the ‚Äútrained immunity‚ÄĚ and the immune cell metabolic activity. The main characteristics of the innate and adaptive immune system are described, as well as the gut-associated lymphoid tissue (GALT). Gut microbiota structure and function are also presented. Particular emphasis is given to the diet as a modulator of the microbiota-immune system crosstalk, focusing on the impact of the three main dietary components (carbohydrates, proteins, and fats) and the different dietary profiles on the gut microbiota, by shaping its composition and the deriving microbial metabolites that influence host health, also through interaction with the immune system. Western and Mediterranean diets are described and chosen as representative models of detrimental and beneficial dietary patterns, respectively

    Caracterização do interactoma da proteina fosfatase 1 na polpa dentária humana

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    Tooth‚Äôs decay is the most common infectious disease worldwide. It is a pathological condition characterized by the (partial or total) breakdown of teeth‚Äôs mineralized structures, due to the activity of cariogenic bacteria, potentially developing into the affection of pulp and periapical tissues. After damage, the dental pulp has a regenerative and reparative potential that is activated in response to microrganisms. Serine/threonine phosphatase 1 (PP1) seems to play a role in the differentiation of odontoblasts (pulp cells) and in angiogenesis, since its expression is upregulated in these cellular processes. PP1 has regenerative activity in the cardiac and nervous systems and has been identified in the tongue and in the dental pulp. With this work, we aim to characterize the PP1 isoforms expressed in dental pulp cells and to identify PP1ő≥ interactors (RIPPOs), known to play a role in the modulation of the reparative/regenerative activity of dental pulp cells. We were able to show that PP1őĪ, PP1ő≤ and PP1ő≥ isoforms are expressed in dental pulp cells, while PP1ő≥2 expression was not observed. Also, the expression of RIPPOs involved in the signalling pathways that lead to regenerative and repair processes by dental pulp cells, like AKT, p38 MAPK and MAPK1 was confirmed. Our preliminary results suggest that PP1ő≥ is important in modulating the activation of signalling pathway involved in tooth repair/regeneration.A c√°rie dent√°ria √© a doen√ßa infecciosa mais comum em todo o mundo. √Č uma patologia caracterizada pela destrui√ß√£o (parcial ou total) das estruturas dent√°rias mineralizadas, devido √† atividade de bact√©rias cariog√©nicas, podendo evoluir para a afe√ß√£o dos tecidos pulpares e periapicais. Na sequ√™ncia do dano, a polpa dent√°ria apresenta um potencial regenerativo e reparador que √© ativado em resposta √† presen√ßa de microrganismos. A prote√≠na serina/treonina fosfatase 1 (PP1) parece desempenhar um papel importante na diferencia√ß√£o de odontoblastos (c√©lulas da polpa dent√°ria) e na angiog√©nese, uma vez que a sua express√£o √© regulada positivamente nesses processos celulares. A PP1 possui atividade regenerativa nos sistemas card√≠aco e nervoso e foi identificada na l√≠ngua e na polpa dent√°ria. Com este trabalho pretendemos caracterizar as isoformas da PP1 expressas nas c√©lulas da polpa dent√°ria e identificar interatores (RIPPOs) da PP1ő≥, conhecidos por modualrem a atividade reparadora/regenerativas das c√©lulas da polpa dent√°ria. Ap√≥s uma an√°lise por SDS-PAGE mostrou-se que as isoformas PP1őĪ, PP1ő≤ e PP1ő≥ s√£o expressas em c√©lulas da polpa dent√°ria, ao contr√°rio da isoforma PP1ő≥2. Al√©m disso, foi confirmada a express√£o de RIPPOs envolvidos nas vias de sinaliza√ß√£o que levam a processos regenerativos e de repara√ß√£o pelas c√©lulas da polpa dent√°ria, como a AKT, p38 MAPK e MAPK1. Estes resultados preliminares evidenciam que a PP1ő≥ √© importante na modula√ß√£o da ativa√ß√£o da via de sinaliza√ß√£o envolvida na repara√ß√£o/regenera√ß√£o dent√°ria.Mestrado em Biomedicina Molecula

    Cis-Regulation of Gremlin1 Expression during Mouse Limb Bud Development and its Diversification during Vertebrate Evolution

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    Embryonic development and organogenesis rely on tightly controlled gene expression, which is achieved by cis-regulatory modules (CRMs) interacting with distinct transcription factors (TFs) that control spatio-temporal and tissue-specific gene expression. During organogenesis, gene regulatory networks (GRNs) with selfregulatory feedback properties coordinately control growth and patterning and provide systemic robustness against genetic and/or environmental perturbations. During limb bud development, various interlinked GRNs control outgrowth and patterning along all three limb axes. A paradigm network is the epithelial-mesenchymal (e-m) SHH/GREM1/AER-FGF feedback signaling system which controls limb bud outgrowth and digit patterning. The BMP antagonist GREMLIN1 (GREM1) is central to this e-m interactions as its antagonism of BMP activity is essential to maintain both AER-Fgf and Shh expression. In turn, SHH signaling upregulates Grem1 expression, which results in establishment of a self-regulatory signaling network. One previous study provided evidence that several CRMs could regulate Grem1 expression during limb bud development. However, the cis-regulatory logics underlying the spatio-temporal regulation of the Grem1 expression dynamics remained obscure. From an evolutionary point of view, diversification of CRMs can result in diversification of gene regulation which can drive the establishment of morphological novelties and adaptions. This was evidenced by the observed differences in Grem1 expression in different species that correlates with the evolutionary plasticity of tetrapod digit patterning. Hence, a better understanding of spatio-temporal regulation of the Grem1 expression dynamics and underlying cis-regulatory logic is of interest from both adevelopmental and an evolutionary perspective. Recently, multiple candidate CRMs have been identified that might be functionally relevant for Grem1 expression during mouse limb bud development. For my PhD project, I genetically analyzed which of these CRMs are involved in the regulation of the spatial-temporal Grem1 expression dynamics in limb buds. Therefore, we generated various single and compound CRM mutant alleles using CRISPR/Cas9. Our CRMs allelic series revealed a complex Grem1 cis-regulation among a minimum of six CRMs, where a subset of CRMs regulates Grem1 transcript levels in an additive manner. Surprisingly, phenotypic robustness depends not on threshold transcript levels but the spatial integrity of the Grem1 expression domain. In particular, interactions among five CRMs control the characteristic asymmetrical and posteriorly biased Grem1 expression in mouse limb buds. Our results provide an example of how multiple seemingly redundant limb-specific CRMs provide phenotypical robustness by cooperative/synergistic regulation of the spatial Grem1 expression dynamics. Three CRMs are conserved along the phylogeny of extant vertebrates with paired appendages. Of those, the activities of two CRMs recapitulate the major spatiotemporal aspects of Grem1 expression in mouse limb buds. In order to study their functions in species-specific regulation of Grem1 expression and their functional diversification in tetrapods, I tested the orthologous of both CRMs from representative species using LacZ reporter assays in transgenic mice, in comparison to the endogenous Grem1 expression in limb buds of the species of origin. Surprisingly, the activities of CRM orthologues display high evolutionary plasticity, which correlates better with the Grem1 expression pattern in limb buds of the species of origin than its mouse orthologue. This differential responsiveness to the GRNs in mouse suggests that TF binding site alterations in CRMs could underlie the spatial diversification of Grem1 in limb buds during tetrapod evolution. While the fish fin and tetrapod limb share some homologies of proximal bones, the autopod is a neomorphic feature of tetrapods. The Grem1 requirement for digit patterning and conserved expression in fin buds prompted us to assess the enhancer activity of fish CRM orthologues in transgenic mice. Surprisingly, all tested fish CRMs are active in the mouse autopod primordia providing strong evidence that Grem1 CRMs are active in fin buds and that they predate the fin-to-limb transition. Our results corroborate increasing evidence that CRMs governing autopodial gene expression have been co-opted during the emergence of tetrapod autopod. Furthermore, as part of a collaboration with Dr. S. Jhanwar, I contributed to the study of shared and species-specific epigenomic and genomic variations during mouse and chicken limb bud development. In this analysis, Dr. S. Jhanwar identified putative enhancers that show higher chicken-specific sequence turnover rates in comparison to their mouse orthologues, which defines them as so-called chicken accelerated regions (CARs). Here, I analyzed the CAR activities in comparison to their mouse orthologues by transgenic LacZ reporter assays, which was complemented by analysis of the endogenous gene expression in limb buds of both species. This analysis indicates that diversified activity of CARs and their mouse orthologues could be linked to the differential gene expression patterns in limb buds of both species

    Watermelon-derived extracellular vesicles influence human ex vivo placental cell behavior by altering intestinal secretions

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    Scope During pregnancy, mother-to-fetus transfer of nutrients is mediated by the placenta; sub-optimal placental development and/or function results in fetal growth restriction (FGR), and the attendant risk of stillbirth, neurodevelopmental delay, and non-communicable diseases in adulthood. A maternal diet high in fruit and vegetables lowers the risk of FGR but the association cannot be explained fully by known macro- and micronutrients. Methods and results This study investigates if dietary-derived extracellular vesicles (EVs) can regulate placental function. The study characterizes the microRNA and protein cargo of EVs isolated from watermelon, show they are actively internalized by human intestinal epithelial cells in vitro, use mass spectrometry to demonstrate that they alter the intestinal secretome and bioinformatic analyses to predict the likely affected pathways in cells/tissues distal to gut. Application of the watermelon EV-modified intestinal secretome to human placental trophoblast cells and ex vivo tissue explants affects the trophoblast proteome and key aspects of trophoblast behavior, including migration and syncytialization. Conclusion Dietary-derived plant EVs can modify intestinal communication with distal tissues, including the placenta. Harnessing the beneficial properties of dietary-derived plant EVs and/or exploiting their potential as natural delivery agents may provide new ways to improve placental function and reduce rates of FGR
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