Elucidating the role of glycans in leucocyte function

RESUMO: O processo de glicosilação é a modificação pós-traducional de proteínas mais comum e está envolvido em vários processos fisiológicos e patológicos. Especificamente, certos perfis glicosídeos estão correlacionados a estados específicos de diferenciação celular, e podem modular vários eventos celulares, como sinalização celular, migração celular e interações hospedeiro-patogénio. Assim sendo, a glicosilação desempenha um papel crucial na modulação de vários processos imunológicos. No entanto, permanece por esclarecer como as estruturas glicosídicas influenciam a imunidade. Especificamente, algumas estruturas glicosídicas terminais que estão modificadas pela ligação de ácido siálico desempenham um papel importante em várias funções do sistema imune, nomeadamente migração leucocitária em contexto de inflamação e ativação de células imunes. Como tal, este trabalho teve como objectivo investigar como a expressão de certos glicanos influencia componentes importantes da resposta imune inata e adaptativa. Este trabalho está dividido em três componentes principais: 1) A imunidade está amplamente dependente da habilidade das células circulantes migrarem para os tecidos inflamados, sendo que a ligação de leucócitos à Eselectina endotelial é o primeiro passo. Assim, nós analisámos a estrutura e função dos ligandos de E-selectina que são expressos pelas células humanas mononucleares de sangue periférico (PBMCs), fornecendo novos conhecimentos para a compreensão dos intervenientes moleculares que mediam a ligação dos monócitos, células CD4+ e CD8+T e células B ao endotélio vascular. Surpreendentemente, os monócitos apresentaram maior capacidade de ligação à E-selectina comparativamente aos linfócitos. Esta observação pode ser explicada pelo facto de os monócitos humanos expressarem, uniformemente, um vasto reportório de glicoproteínas que exibem afinidade de ligação à E-selectina, nomeadamente: as glicoformas do CD43 (CD43E) e do CD44 (HCELL), em adição à já previamente reportada glicoforma da PSGL-1 (CLA). Consistentemente, a diferente capacidade que as diversas populações linfocitárias apresentam de se ligar à E-selectina, está integralmente relacionada com a sua expressão de glicoproteínas com afinidade de ligação à E-selectina. Enquanto que as células CD4+T apresentam uma elevada reatividade à E-selectina, as células CD8+T e B demonstram pouca ou nenhuma capacidade de ligação à E-selectina. Esta atividade de ligação à E-selectina das células CD4+T é conferida pela expressão de HCELL, em adição às já previamente reportadas CLA e CD43E. As células CD8+ T não expressam HCELL e apenas expressam pequenas quantidades de CLA e CD43E, enquanto que as células B não expressam ligandos de Eselectina. Mais, a exofucosilação da superfície destas células, levou ao dramático aumento da expressão dos ligandos de E-selectina em todos as populações leucocitárias, verificando-se que a criação de certos ligandos de E-selectina está dependente do tipo de célula, após fucosilação. Colectivamente, estes resultados redefinem o nosso conhecimento acerca dos mecanismos moleculares que governam o tráfico das células mononucleares de sangue periférico em contexto de inflamação. 2) A habilidade das células dendríticas (DCs) para extravasarem em locais de inflamação é crucial para o sucesso da terapia com DCs. Assim, analisámos a estrutura e função das moléculas de adesão que mediam a migração transendotelial (TEM) das DCs. Para isso, foram usadas DCs geradas a partir da diferenciação de monócitos (mo-DCS), obtidos quer pelo métodos de separação imuno-magnética de células CD14+ (CD14-S) ou por isolamento por aderência ao plástico (PA-S). Os resultados obtidos indicam que as glicoformas de ligação à Eselectina de PSGL-1, CD43 e CD44 são expressas pelas CD14-S mo-DCs, enquanto que as PA-S mo-DCs expressam apenas CLA. É importante notar que a ligação do CD44 nas mo-DCs, mas não nas PA-S mo-DCs, desencadeia a ativação e consequente adesão da VLA-4 ao endotélio na ausência de um gradiente de quimiocinas. Procedeu-se também à análise dos ligandos E-selectina expressos em mo-DCs geradas a partir de monócitos do sangue do cordão umbilical (UCB) e, inesperadamente, as UCB mo-DCs não expressam qualquer glicoproteína com reatividade à E-selectina. Além disso, a exofucosilação das mo- DCs humanas utilizando uma α(1,3)-fucosiltransferase aumenta significativamente a expressão de HCELL e, portanto, estas células apresentam uma capacidade aumentada para se ligarem à E-selectina em condições de fluxo hemodinâmico. Estes resultados destacam o papel do HCELL no desencadeamento do TEM das CD14-S mo-DCs e sugerem que estratégias para potenciar a expressão de HCELL poderão impulsionar o recrutamento de mo-DCs para locais de inflamação. 3) Outro obstáculo para alcançar o sucesso promissor de vacinas baseadas em DCs é o estabelecimento de abordagens eficientes que poderão melhorar o estado de maturação e apresentação antigénica das DCs. Por conseguinte, foram investigadas abordagens alternativas que podem superar este obstáculo. Através da remoção de ácido siálico de superfície celular das DCs, conseguiu-se induzir a maturação de DC humanas e de ratinhos. Notavelmente, tanto as DCs humanas como as de ratinho, ao serem desialiladas mostraram uma capacidade aumentada para induzir a proliferação de células T, para secretar citocinas Th1 e para induzir a morte específica de células tumorais. Em adição, as DCs desialiladas apresentam uma maior capacidade de apresentação cruzada de antigénios tumorais às células T citotóxicas. Colectivamente, o presente estudo oferece uma visão chave para optimizar a capacidade das DCs em induzir respostas imunitárias anti-tumorais, e indica que o tratamento com sialidase é uma nova tecnologia para melhorar a eficácia e aplicabilidade das vacinas baseadas em DCs. Coletivamente, os nossos resultados demostram como a glicosilação e a sua manipulação podem modular a imunidade. Concretamente, através de uma reação de exofucosilação conseguimos aumentar fortemente a capacidade de os leucócitos extravasarem para os tecidos afectados, enquanto que a remoção dos níveis de ácido siálico da superfície celular das DCs, induz potentes respostas anti-tumorais mediadas por células T citotóxicas. ------------------------------------ ABSTRACT: Glycosylation is the most widely form of protein post-translational modification and is involved in many physiological and pathological processes. Specifically, certain patterns of glycosylation are associated with determined stages of cell differentiation and can modulate processes like cell-signaling and migration and host-pathogen interactions. As such, glycosylation plays a crucial role in the modulation of several immune events. However, how glycans execute this immune-modulation and, therefore, influence immunity is still poorly unknown. Specifically, some terminal sialic acid-modified determinants are known to be involved in several physiological immune processes, including leukocyte trafficking into sites of inflammation and cell immune activation. Therefore, in this work, we sought to investigate more deeply how the expression of these glycosidic structures affects events form both innate and adaptive immune responses. To this end, we divided our work into three main parts: 1) Immunity critically depends on the ability of sentinel circulating cells to infiltrate injured sites, of which leukocyte binding to endothelial E-selectin is the critical first step. Thus, we first analyzed the structure and function of the E-selectin ligands expressed on native human peripheral blood mononuclear cells (PBMCs), providing novel insights into the molecular effectors governing adhesion of circulating monocytes, and of circulating CD4+T, CD8+T and B cells, to vascular endothelium under hemodynamic shear conditions. Strikingly, monocytes show a higher ability to tether and roll on endothelial cells than lymphocyte subsets. This is due to the fact that human circulating monocytes uniformly display a wide repertoire of E-selectin binding glycoproteins, namely the E-selectin-binding glycoforms of CD43 (CD43E) and CD44 (HCELL), in addition to the previously described E-selectin-binding glycoform of PSGL-1 (CLA). In addition, we also observed a differential ability of the different lymphocyte subsets to bind to Eselectin under hemodynamic shear stress conditions, and these differences were highly correlated with their individual expression of E-selectin binding glycoproteins. While CD4+T cells show a robust E-selectin binding ability, CD8+T and B cells show little to no E-selectin reactivity. CD4+T cell potent Eselectin rolling activity is conferred by HCELL expression, in addition to the previously reported E-selectin-binding glycoproteins CD43E and CLA. CD8+T cells display no HCELL and low amounts of CLA and CD43E, whereas B cells lack E-selectin ligand expression. Moreover, enforced exofucosylation of cell surface of these cells noticeably increases expression of functional E-selectin ligands among all leukocytes subsets, with cell type-dependent specificity in the protein scaffolds that are modified. Taken together, these findings redefine our understanding of the molecular mechanisms governing the trafficking patterns of PBMCs that are relevant in the context of acute or chronic inflammatory conditions. 2) The ability of circulating dendritic cells (DCs) to extravasate at inflammatory sites is critical to the success of DC-based therapies. Therefore, we assessed the structure and function of adhesion molecules mediating the transendothelial migration (TEM) of human monocyte derived-DCs (mo-DCs), obtained either by CD14 positive immune-magnetic selection (CD14-S) or by plastic adherence of blood monocytes (PA-S). We report for the first time that the E-selectin binding glycoforms of PSGL-1, CD43 and CD44 are all expressed on CD14-S mo-DCs, in contrast to PA-S mo-DCs that express only CLA. Importantly, CD44 engagement on CD14-S mo-DCs, but not on PA-S mo-DCs, triggers VLA-4-dependent adhesiveness and programs TEM in absence of chemokine gradient. We also analyzed the E-selectin ligands expressed on mo-DCs generated from umbilical cord blood (UCB) monocytes, and unexpectedly, UCB mo-DCs do not express any glycoprotein with E-selectin reactivity. Furthermore, exoglycosylation of human mo-DCs using an α(1,3)-fucosyltransferase significantly increases expression of HCELL, and therefore exofucosylated mo-DCs exhibit an augmented ability to bind to E-selectin under hemodynamic shear stress conditions. These findings highlight a role for HCELL engagement in priming TEM of CD14-S mo-DCs, and suggest that strategies to enforce HCELL expression could boost mo-DC recruitment to inflammatory sites. 3) Another obstacle to achieve the promising success of DC-based vaccines is the establishment of efficient approaches that could successfully enhance maturation and cross-presentation ability of DCs. Therefore, we investigated an alternative approach that can overcome this problem. Through removal of sialic acid content from DC cell surface we are able to elicit maturation of both human and mouse DCs. Notably, desialylated human and murine DCs showed enhanced ability to induce autologous T cell to proliferate, to secrete Th1 cytokines and to kill tumor cells. Moreover, desialylated DCs display enhanced cross-presentation of tumor antigens to cytotoxic CD8+ T cells. Collectively, this study offers key insight to optimize the ability of DCs to boost anti-tumor immune responses, and indicates that the treatment with an exogenous sialidase is a powerful new technology to improve the efficacy and applicability of DC-based vaccines. Overall, our findings show how glycosylation and its manipulation can modulate immunity. Concretely, through an exofucosylation reaction we are able to greatly augment the ability of leukocytes to extravasate into injured tissues, while removal of sialic acid moieties from cell surface of DCs, significantly potentiate their ability to induce anti-tumor cytotoxic T cell-mediate responses.

Elucidating the role of glycans in leucocyte function

RESUMO:O processo de glicosilação é a modificação pós-traducional de proteínas mais comum e está envolvido em vários processos fisiológicos e patológicos. Especificamente, certos perfis glicosídeos estão correlacionados a estados específicos de diferenciação celular, e podem modular vários eventos celulares, como sinalização celular, migração celular e interações hospedeiro-patogénio. Assim sendo, a glicosilação desempenha um papel crucial na modulação de vários processos imunológicos. No entanto, permanece por esclarecer como as estruturas glicosídicas influenciam a imunidade. Especificamente, algumas estruturas glicosídicas terminais que estão modificadas pela ligação de ácido siálico desempenham um papel importante em várias funções do sistema imune, nomeadamente migração leucocitária em contexto de inflamação e ativação de células imunes. Como tal, este trabalho teve como objectivo investigar como a expressão de certos glicanos influencia componentes importantes da resposta imune inata e adaptativa. Este trabalho está dividido em três componentes principais: 1) A imunidade está amplamente dependente da habilidade das células circulantes migrarem para os tecidos inflamados, sendo que a ligação de leucócitos à Eselectina endotelial é o primeiro passo. Assim, nós analisámos a estrutura e função dos ligandos de E-selectina que são expressos pelas células humanas mononucleares de sangue periférico (PBMCs), fornecendo novos conhecimentos para a compreensão dos intervenientes moleculares que mediam a ligação dos monócitos, células CD4+ e CD8+T e células B ao endotélio vascular. Surpreendentemente, os monócitos apresentaram maior capacidade de ligação à E-selectina comparativamente aos linfócitos. Esta observação pode ser explicada pelo facto de os monócitos humanos expressarem, uniformemente, um vasto reportório de glicoproteínas que exibem afinidade de ligação à E-selectina, nomeadamente: as glicoformas do CD43 (CD43E) e do CD44 (HCELL), em adição à já previamente reportada glicoforma da PSGL-1 (CLA). Consistentemente, a diferente capacidade que as diversas populações linfocitárias apresentam de se ligar à E-selectina, está integralmente relacionada com a sua expressão de glicoproteínas com afinidade de ligação à E-selectina. Enquanto que as células CD4+T apresentam uma elevada reatividade à E-selectina, as células CD8+T e B demonstram pouca ou nenhuma capacidade de ligação à E-selectina. Esta atividade de ligação à E-selectina das células CD4+T é conferida pela expressão de HCELL, em adição às já previamente reportadas CLA e CD43E. As células CD8+ T não expressam HCELL e apenas expressam pequenas quantidades de CLA e CD43E, enquanto que as células B não expressam ligandos de Eselectina. Mais, a exofucosilação da superfície destas células, levou ao dramático aumento da expressão dos ligandos de E-selectina em todos as populações leucocitárias, verificando-se que a criação de certos ligandos de E-selectina está dependente do tipo de célula, após fucosilação. Colectivamente, estes resultados redefinem o nosso conhecimento acerca dos mecanismos moleculares que governam o tráfico das células mononucleares de sangue periférico em contexto de inflamação. 2) A habilidade das células dendríticas (DCs) para extravasarem em locais de inflamação é crucial para o sucesso da terapia com DCs. Assim, analisámos a estrutura e função das moléculas de adesão que mediam a migração transendotelial (TEM) das DCs. Para isso, foram usadas DCs geradas a partir da diferenciação de monócitos (mo-DCS), obtidos quer pelo métodos de separação imuno-magnética de células CD14+ (CD14-S) ou por isolamento por aderência ao plástico (PA-S). Os resultados obtidos indicam que as glicoformas de ligação à Eselectina de PSGL-1, CD43 e CD44 são expressas pelas CD14-S mo-DCs, enquanto que as PA-S mo-DCs expressam apenas CLA. É importante notar que a ligação do CD44 nas mo-DCs, mas não nas PA-S mo-DCs, desencadeia a ativação e consequente adesão da VLA-4 ao endotélio na ausência de um gradiente de quimiocinas. Procedeu-se também à análise dos ligandos E-selectina expressos em mo-DCs geradas a partir de monócitos do sangue do cordão umbilical (UCB) e, inesperadamente, as UCB mo-DCs não expressam qualquer glicoproteína com reatividade à E-selectina. Além disso, a exofucosilação das mo- DCs humanas utilizando uma α(1,3)-fucosiltransferase aumenta significativamente a expressão de HCELL e, portanto, estas células apresentam uma capacidade aumentada para se ligarem à E-selectina em condições de fluxo hemodinâmico. Estes resultados destacam o papel do HCELL no desencadeamento do TEM das CD14-S mo-DCs e sugerem que estratégias para potenciar a expressão de HCELL poderão impulsionar o recrutamento de mo-DCs para locais de inflamação. 3) Outro obstáculo para alcançar o sucesso promissor de vacinas baseadas em DCs é o estabelecimento de abordagens eficientes que poderão melhorar o estado de maturação e apresentação antigénica das DCs. Por conseguinte, foram investigadas abordagens alternativas que podem superar este obstáculo. Através da remoção de ácido siálico de superfície celular das DCs, conseguiu-se induzir a maturação de DC humanas e de ratinhos. Notavelmente, tanto as DCs humanas como as de ratinho, ao serem desialiladas mostraram uma capacidade aumentada para induzir a proliferação de células T, para secretar citocinas Th1 e para induzir a morte específica de células tumorais. Em adição, as DCs desialiladas apresentam uma maior capacidade de apresentação cruzada de antigénios tumorais às células T citotóxicas. Colectivamente, o presente estudo oferece uma visão chave para optimizar a capacidade das DCs em induzir respostas imunitárias anti-tumorais, e indica que o tratamento com sialidase é uma nova tecnologia para melhorar a eficácia e aplicabilidade das vacinas baseadas em DCs. Coletivamente, os nossos resultados demostram como a glicosilação e a sua manipulação podem modular a imunidade. Concretamente, através de uma reação de exofucosilação conseguimos aumentar fortemente a capacidade de os leucócitos extravasarem para os tecidos afectados, enquanto que a remoção dos níveis de ácido siálico da superfície celular das DCs, induz potentes respostas anti-tumorais mediadas por células T citotóxicas. ---------------------------- ABSTRACT: Glycosylation is the most widely form of protein post-translational modification and is involved in many physiological and pathological processes. Specifically, certain patterns of glycosylation are associated with determined stages of cell differentiation and can modulate processes like cell-signaling and migration and host-pathogen interactions. As such, glycosylation plays a crucial role in the modulation of several immune events. However, how glycans execute this immune-modulation and, therefore, influence immunity is still poorly unknown. Specifically, some terminal sialic acid-modified determinants are known to be involved in several physiological immune processes, including leukocyte trafficking into sites of inflammation and cell immune activation. Therefore, in this work, we sought to investigate more deeply how the expression of these glycosidic structures affects events form both innate and adaptive immune responses. To this end, we divided our work into three main parts: 1) Immunity critically depends on the ability of sentinel circulating cells to infiltrate injured sites, of which leukocyte binding to endothelial E-selectin is the critical first step. Thus, we first analyzed the structure and function of the E-selectin ligands expressed on native human peripheral blood mononuclear cells (PBMCs), providing novel insights into the molecular effectors governing adhesion of circulating monocytes, and of circulating CD4+T, CD8+T and B cells, to vascular endothelium under hemodynamic shear conditions. Strikingly, monocytes show a higher ability to tether and roll on endothelial cells than lymphocyte subsets. This is due to the fact that human circulating monocytes uniformly display a wide repertoire of E-selectin binding glycoproteins, namely the E-selectin-binding glycoforms of CD43 (CD43E) and CD44 (HCELL), in addition to the previously described E-selectin-binding glycoform of PSGL-1 (CLA). In addition, we also observed a differential ability of the different lymphocyte subsets to bind to Eselectin under hemodynamic shear stress conditions, and these differences were highly correlated with their individual expression of E-selectin binding glycoproteins. While CD4+T cells show a robust E-selectin binding ability, CD8+T and B cells show little to no E-selectin reactivity. CD4+T cell potent Eselectin rolling activity is conferred by HCELL expression, in addition to the previously reported E-selectin-binding glycoproteins CD43E and CLA. CD8+T cells display no HCELL and low amounts of CLA and CD43E, whereas B cells lack E-selectin ligand expression. Moreover, enforced exofucosylation of cell surface of these cells noticeably increases expression of functional E-selectin ligands among all leukocytes subsets, with cell type-dependent specificity in the protein scaffolds that are modified. Taken together, these findings redefine our understanding of the molecular mechanisms governing the trafficking patterns of PBMCs that are relevant in the context of acute or chronic inflammatory conditions. 2) The ability of circulating dendritic cells (DCs) to extravasate at inflammatory sites is critical to the success of DC-based therapies. Therefore, we assessed the structure and function of adhesion molecules mediating the transendothelial migration (TEM) of human monocyte derived-DCs (mo-DCs), obtained either by CD14 positive immune-magnetic selection (CD14-S) or by plastic adherence of blood monocytes (PA-S). We report for the first time that the E-selectin binding glycoforms of PSGL-1, CD43 and CD44 are all expressed on CD14-S mo-DCs, in contrast to PA-S mo-DCs that express only CLA. Importantly, CD44 engagement on CD14-S mo-DCs, but not on PA-S mo-DCs, triggers VLA-4-dependent adhesiveness and programs TEM in absence of chemokine gradient. We also analyzed the E-selectin ligands expressed on mo-DCs generated from umbilical cord blood (UCB) monocytes, and unexpectedly, UCB mo-DCs do not express any glycoprotein with E-selectin reactivity. Furthermore, exoglycosylation of human mo-DCs using an α(1,3)-fucosyltransferase significantly increases expression of HCELL, and therefore exofucosylated mo-DCs exhibit an augmented ability to bind to E-selectin under hemodynamic shear stress conditions. These findings highlight a role for HCELL engagement in priming TEM of CD14-S mo-DCs, and suggest that strategies to enforce HCELL expression could boost mo-DC recruitment to inflammatory sites.3) Another obstacle to achieve the promising success of DC-based vaccines is the establishment of efficient approaches that could successfully enhance maturation and cross-presentation ability of DCs. Therefore, we investigated an alternative approach that can overcome this problem. Through removal of sialic acid content from DC cell surface we are able to elicit maturation of both human and mouse DCs. Notably, desialylated human and murine DCs showed enhanced ability to induce autologous T cell to proliferate, to secrete Th1 cytokines and to kill tumor cells. Moreover, desialylated DCs display enhanced cross-presentation of tumor antigens to cytotoxic CD8+ T cells. Collectively, this study offers key insight to optimize the ability of DCs to boost anti-tumor immune responses, and indicates that the treatment with an exogenous sialidase is a powerful new technology to improve the efficacy and applicability of DC-based vaccines. Overall, our findings show how glycosylation and its manipulation can modulate immunity. Concretely, through an exofucosylation reaction we are able to greatly augment the ability of leukocytes to extravasate into injured tissues, while removal of sialic acid moieties from cell surface of DCs, significantly potentiate their ability to induce anti-tumor cytotoxic T cell-mediate responses.

Açaí (Euterpe oleracea mart.) e camu-camu (Myrciaria dubia (H.B.K.) Mc Vaugh) possuem ação anti anêmica?

Avaliou-se o impacto da utilização do açaí e camu-camu em pré-escolares de uma Unidade Filantrópica de Manaus-AM. Foram selecionadas 85 crianças voluntárias, de dois a seis anos incompletos, de ambos os sexos, distribuídas aleatoriamente em 5 grupos, tendo como fonte de ferro e vitamina C: açaí e açaí + camu-camu, perfazendo um total de 2 mg de ferro e 40 mg de ácido ascórbico, assim como o ferro aminoácido quelato na concentração de 1 e 2 mg de ferro. O ferro foi distribuído diariamente na colação por um período de 120 dias. Na caracterização da anemia considerou-se o ponto de corte de hemoglobina <11g/dL. Os resultados demonstraram que de um universo de 85 crianças 6 (7%) apresentaram um quadro de desnutrição crônica, sendo ao final da intervenção reduzido para 4 (4,7%). O maior impacto da utilização do açaí foi como fonte energética, refletido no ganho de peso significativo das crianças (1,76 kg), mesmo quando adicionado de camu-camu (1,69 kg). Em relação a concentração de hemoglobina, não foi constatada diferença significativa entre as crianças dos diferentes grupos, independente da fonte de ferro: açaí (0,71 g/dL); açaí + camu-camu (0,60 g/dL), Ferro 2 mg (0,88 g/dL); água (0,85 g/ dL) e Ferro 1 mg (0,54 g/dL). Entretanto, a recuperação de crianças anêmicas foi maior no grupo que recebeu ferro aminoácido quelato na concentração de 2 mg de ferro. Conclui-se que o açaí tem um grande potencial como fonte energética e pouca expressividade como fonte de ferro, mesmo adicionado de camu-camu.

Effect of variables on the thickness of an edible coating applied on frozen fish establishment of the concept of safe dipping time

Glazing is a technique used to retard fish deterioration during storage. This work focuses on the study of distinct variables (fish temperature, coating temperature, dipping time) that affect the thickness of edible coatings (water glazing and 1.5% chitosan) applied on frozen fish. Samples of frozen Atlantic salmon (Salmo salar) at -15, -20, and -25 °C were either glazed with water at 0.5, 1.5 or 2.5 °C or coated with 1.5% chitosan solution at 2.5, 5 or 8 °C, by dipping during 10 to 60 s. For both water and chitosan coatings, lowering the salmon and coating solution temperatures resulted in an increase of coating thickness. At the same conditions, higher thickness values were obtained when using chitosan (max. thickness of 1.41±0.05 mm) compared to water (max. thickness of 0.84±0.03 mm). Freezing temperature and crystallization heat were found to be lower for 1.5% chitosan solution than for water, thus favoring phase change. Salmon temperature profiles allowed determining, for different dipping conditions, whether the salmon temperature was within food safety standards to prevent the growth of pathogenic microorganisms. The concept of safe dipping time is proposed to define how long a frozen product can be dipped into a solution without the temperature raising to a point where it can constitute a hazard.

Biofunctionalization of titanium surfaces for dental implants: osteogenic, anti-microbial and tribocorrosion resistant surfaces

PhD thesis in Biomedical Engineering

Assessment of a protease inhibitor peptide for anti-ageing

Ageing and skin exposure to UV radiation induces production and activation of matrix metalloproteinases (MMPs) and human neutrophil elastase (HNE). These enzymes are known to break down the extracellular matrix (ECM) which leads to wrinkle formation. Here, we demonstrated the potential of a solid-in-oil nanodispersion containing a competitive inhibitor peptide of HNE mixed with hyaluronic acid (HA), displaying 158 nm of mean diameter, to protect the skin against the ageing effects. Western blot analysis demonstrated that activation of MMP-1 in fibroblasts by HNE treatment is inhibited by the solid-in-oil nanodispersion containing the peptide and HA. The results clearly demonstrate that solid-in-oil nanodispersion containing the HNE inhibitor peptide is a promising strategy for anti-ageing effects. This effect can be seen particularly by ECM regulation by affecting fibroblasts. The formulation also enhances the formation of thicker bundles of actin filaments.

In vitro anti-Candida activity of Glycyrrhiza glabra L.

The severity and frequency of opportunistic fungal infections still growing, concomitantly to the increasing rates of antimicrobial drugs resistance. Natural matrices have been used over years due to its multitude of health benefits, including antifungal potential. Thus, the present work aims to evaluate the anti-Candida potential of the phenolic extract and individual phenolic compounds of Glycyrrhiza glabra L. (licorice), by disc diffusion assay, followed by determination of the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) for both planktonic cells and biofilms. Licorice extract evidenced inhibitory potential against the nineteen tested Candida strains, but no pronounced effect was observed by testing the most abundant individual phenolic compounds. Candida tropicalis strains were the most sensible, followed by Candida glabrata, Candida parapsilosis and, then, Candida albicans. Lower MIC and MFC values were achieved to C. glabrata and C. tropicalis, which confirms its susceptibility to licorice extract; however, for C. tropicalis strains a higher variability was observed. Anti-biofilm potential was also achieved, being most evident in some C. glabrata and C. tropicalis strains. In general, a twice concentration of the MIC was necessary for planktonic cells to obtain a similar potential to that one observed for biofilms. Thus, an upcoming approach for new antifungal agents, more effective and safer than the current ones, is stablished; notwithstanding, further studies are necessary in order to understand its mechanism of action, as also to assess kinetic parameters.

Impact of an educational hands-on project on the antimicrobial, antitumor and anti-inflammatory properties of plants on Portuguese students' awareness, knowledge, and competences

Promoting environmental and health education is crucial to allow students to make conscious decisions based on scientific criteria. The study is based on the outcomes of an Educational Project implemented with Portuguese students and consisted of several activities, exploring pre-existent Scientific Gardens at the School, aiming to investigate the antibacterial, antitumor and anti-inflammatory properties of plant extracts, with posterior incorporation in soaps and creams. A logo and a webpage were also created. The effectiveness of the project was assessed via the application of a questionnaire (pre- and post-test) and observations of the participants in terms of engagement and interaction with all individuals involved in the project. This project increased the knowledge about autochthonous plants and the potential medical properties of the corresponding plant extracts and increased the awareness about the correct design of scientific experiments and the importance of the use of experimental models of disease. The students regarded their experiences as exciting and valuable and believed that the project helped to improve their understanding and increase their interest in these subjects and in science in general. This study emphasizes the importance of raising students’ awareness on the valorization of autochthonous plants and exploitation of their medicinal properties.

Evaluation of antinociceptive and anti-inflammatory activities of the topical preparation of Cipura paludosa (Iridaceae)

Alho do mato (Cipura paludosa, Iridaceae) is a medicinal plant found in the Amazon rain forest, North of Brazil. It has been used to treat algic, inflammatory and infectious processes. The aim of this study was to evaluate the anti-inflammatory and antinociceptive action of the crude Cipura paludosa ethanolic extract at concentrations ranging between 2.0 and 4.0% in Oil and Water cream formulations for topical use. The physical-chemical stability of the formulations was monitored over a six-month period with the use of accelerated stability tests. In order to evaluate the anti-inflammatory and antinociceptive activities, we used a paw edema test induced by carrageenan and a formalin test, respectively. The paw edema test showed that there was a statistical difference in the control group in relation to the treatments. The formalin test did not confirm antinociceptive action of the treatments with the extract in the early phase of the test. However, statistical difference was confirmed for the treatments in relation to the control in the late phase. The antinociceptive and anti-inflammatory activities of Cipura paludosa preparations, as demonstrated in the results, at least partially support the ethno-medical uses of this plant.

In vivo antioxidant activity of phenolic compounds: facts and gaps

Background: Numerous diseases have been related with free radicals overproduction and oxidative stress. Botanical preparations possess a multitude of bioactive properties, including antioxidant potential, which has been mainly related with the presence of phenolic compounds. However, the mechanisms of action of these phytochemicals, in vivo effects, bioavailability and bio-efficacy still need research. Scope and Approach: The present report aims to provide a critical review on the aspects related with the in vivo antioxidant activity of phenolic extracts and compounds from plant origin. Key findings: Biological functions beyond the human metabolism were discussed, comparing in vivo vs. in vitro studies, as also focusing the conditioning factors for phenolic compounds bioavailability and bio-efficacy. Furthermore, an upcoming perspective about the use of phytochemicals as life expectancy promoters and anti-aging factors in human individuals was provided. Conclusions: Overall, and despite all of those advances, the study of the biological potential of numerous natural matrices still remains a hot topic among the scientific community. In fact, the available knowledge about the responsible phytochemicals for the biological potential, their mechanisms of action, the establishment of therapeutic and prophylactic doses, and even the occurrence of biochemical inter-relations, is considerable scarce.

The contribution of phenolic acids to the anti-inflammatory activity of mushrooms: Screening in phenolic extracts, individual parent molecules and synthesized glucuronated and methylated derivatives

In the present study, the ethanolic extracts of fourteen edible mushrooms were investigated for their anti-inflammatory potential in LPS (lipopolysaccharide) activated RAW 264.7 macrophages. Furthermore the extracts were chemically characterized in terms of phenolic acids and related compounds. The identified molecules (p-hydroxybenzoic, p-coumaric and cinnamic acids) and their glucuronated and methylated derivatives obtained by chemical synthesis were also evaluated for the same bioactivity, in order to establish structure-activity relationships and to comprehend the effects of in vivo metabolism reactions in the activity of the compounds. The extracts of Pleurotus ostreatus, Macrolepiota procera, Boletus impolitus and Agaricus bisporus revealed the strongest anti-inflammatory potential (EC50 values 96 ± 1 to 190 ± 6 µg/mL, and also the highest concentration of cinnamic acid (656 to 156 µg/g), which was also the individual compound with the highest anti-inflammatory activity. The derivatives of p-coumaric acid revealed the strongest properties, specially the derivative methylated in the carboxylic group (CoA-M1) that exhibited similar activity to the one showed by dexamethaxone used as anti-inflammatory standard; by contrast, the derivatives of p-hydroxybenzoic revealed the lowest inhibition of NO production. All in all, whereas the conjugation reactions change the chemical structure of phenolic acids and may increase or decrease their activity, the glucuronated and methylated derivatives of the studied compounds are still displaying anti-inflammatory activity.

Barramento delgado anti carbonatação

Dissertação de mestrado integrado em Engenharia Civil

MET is required for the recruitment of anti-tumoural neutrophils

Mutations or amplification of the MET proto-oncogene are involved in the pathogenesis of several tumours, which rely on the constitutive engagement of this pathway for their growth and survival. However, MET is expressed not only by cancer cells but also by tumour-associated stromal cells, although its precise role in this compartment is not well characterized. Here we show that MET is required for neutrophil chemoattraction and cytotoxicity in response to its ligand hepatocyte growth factor (HGF). Met deletion in mouse neutrophils enhances tumour growth and metastasis. This phenotype correlates with reduced neutrophil infiltration to both the primary tumour and metastatic sites. Similarly, Met is necessary for neutrophil transudation during colitis, skin rash or peritonitis. Mechanistically, Met is induced by tumour-derived tumour necrosis factor (TNF)-a or other inflammatory stimuli in both mouse and human neutrophils. This induction is instrumental for neutrophil transmigration across an activated endothelium and for inducible nitric oxide synthase production upon HGF stimulation. Consequently, HGF/MET-dependent nitric oxide release by neutrophils promotes cancer cell killing, which abates tumour growth and metastasis. After systemic administration of a MET kinase inhibitor, we prove that the therapeutic benefit of MET targeting in cancer cells is partly countered by the pro-tumoural effect arising from MET blockade in neutrophils. Our work identifies an unprecedented role of MET in neutrophils, suggests a potential 'Achilles' heel' of MET-targeted therapies in cancer, and supports the rationale for evaluating anti-MET drugs in certain inflammatory diseases.