Geopolymers as infill material for conservation of azulejos

The restoration materials currently used to fill gaps in architectural historical azulejos (e.g. lime or organic resin pastes) usually show serious drawbacks in terms of compatibility, effectiveness and durability. The existing solutions do not fully protect azulejos in outdoor conditions and frequently result in further deterioration. Geopolymers can be a potential solution for azulejo lacunae infill given the chemical-mineralogical similitude to the ceramic body, and also the durability and versatile range of physical properties that can be obtained through the manipulation of their formulation and curing conditions. This work presents and discusses the viability of the use of geopolymeric pastes to fill lacunae in azulejos or to act as “cold” cast ceramic tile surrogates reproducing missing azulejo fragments. The formulation of geopolymers, namely the type of activators, the aluminosilicate source, the amount of water (to meet adequate workability requirements) and curing conditions were studied. The need for post-curing desalination was also considered envisaging their application in the restoration of outdoor architectural historical azulejos frequently exposed to adverse environmental conditions. The possible advantages and disadvantages of the use of geopolymers in the conservation of azulejos are also discussed. Several techniques were used to study the chemical and physical behavior of geopolymers, namely FT-IR, XRD, MIP, SEM-EDS, WDXRF, electrical conductivity, open porosity, bending strength, adhesion strength, water vapour permeability, thermal expansion and hydric expansion. The results indicate that geopolymers are a promising material for restoration of azulejos, exhibiting some properties, such as adhesion to the ceramic substrate, higher than inorganic materials used nowadays, such as aerial lime based pastes.

Microfluidics: a new look at cell migration analysis

This thesis explores the development and employment of microfluidic devices as a tool for studying the effect of the surrounding environment on embryonic stem cells during the migration phenomena. Different single-cell microchips were designed and manufactured to study mouse embryonic fibroblasts (MEFs) migration towards an environmental variation (increase of serum concentration in the culture medium) that was expected to function as a motility stimuli. Considering the experimental, cells were injected into the microchips chambers and individually isolated by dedicated cell traps with view to a single-cell analysis. Once fribroblasts were attached to the surface, culture medium with an increased serum level was subsequently injected in an adjacent chamber to promote the formation of a serum concentration gradient. The gradient established between the chambers could be sensed by the fibroblasts and thus triggered the cells mobilization towards and in the direction of the richer serum medium. Additionally, the experiment allowed the observation of MEFs’ structural reorganization when migrating through micro-tunnels containing widths below the cell size, suggesting a cytoskeleton rearrangement on account of the nutritional stimulus introduced. Furthermore, results indicate that fibronectin promotes MEFs adhesion to the substrate and that MEFs migration is characterized as haptotactic.

Earthen plasters based on illitic soils from Barrocal region of Algarve: contributions for building performance and sustainability

Clayish earth-based mortars can be considered eco-efficient products for indoor plastering since they can contribute to improve important aspects of building performance and sustainability. Apart from being products with low embodied energy when compared to other types of mortars used for interior plastering, mainly due to the use raw clay as natural binder, earth-based plasters may give a significant contribution for health and comfort of inhabitants. Due to high hygroscopicity of clay minerals, earth-based mortars present a high adsorption and desorption capacity, particularly when compared to other type of mortars for interior plastering. This capacity allows earth-based plasters to act as a moisture buffer, balancing the relative humidity of the indoor environment and, simultaneously, acting as a passive removal material, improving air quality. Therefore, earth-based plasters may also passively promote the energy efficiency of buildings, since they may contribute to decreasing the needs of mechanical ventilation and air conditioning. This study is part of an ongoing research regarding earth-based plasters and focuses on mortars specifically formulated with soils extracted from Portuguese ‘Barrocal’ region, in Algarve sedimentary basin. This region presents high potential for interior plastering due to regional geomorphology, that promote the occurrence of illitic soils characterized by a high adsorption capacity and low expansibility. More specifically, this study aims to assess how clayish earth and sand ratio of mortars formulation can influence the physical and mechanical properties of plasters. For this assessment four mortars were formulated with different volumetric proportions of clayish earth and siliceous sand. The results from the physical and mechanical characterization confirmed the significantly low linear shrinkage of all the four mortars, as well as their extraordinary adsorption-desorption capacity. These results presented a positive correlation with mortars´ clayish earth content and are consistent with the mineralogical analysis, that confirmed illite as the prevalent clay mineral in the clayish earth used for this study. Regarding mechanical resistance, although the promising results of the adhesion test, the flexural and compressive strength results suggest that the mechanical resistance of these mortars should be slightly improved. Considering the present results the mortars mechanical resistance improvement may be achieved through the formulation of mortars with higher clayish earth content, or alternatively, through the addition of natural fibers to mortars formulation, very common in this type of mortars. Both those options will be investigated in future research.

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.

The perfect paint in modern art conservation: a comparative study of 21st century vinyl emulsions

Contemporary painting places, and will continue to place, several questions about its meaning, its chemical nature, its durability and the best way to preserve it. This research aims at putting together comprehensive data on vinyl based paints, including their components, their properties, their aging behavior and their response to selected cleaning products. In this project degradation mechanisms of vinyl binders and formulations used in the 20th and 21st century were studied. Stability over time of selected vinyl polymers was assessed through natural indoor and artificially aging. The objective was to enhance knowledge and understanding of vinyl emulsion formulations and their performance over time. Overall conservation state of pictorial layers namely, adhesion, cohesion and discoloration of selected case studies from the Portuguese artist Julião Sarmento (b.1948) was correlated with the observed molecular level changes studied in laboratory experiments. Sarmento’s paintings were chosen due to conservation concerns (discoloration) on some of his works from the 90’s. Besides, research was carried out to start increasing the knowledge of what can be expected of PVAc based paints in terms of response to conservation treatments namely, surface cleaning. Artificial aging showed that the most recent formulations which are based on a poly(vinyl acetate), poly(vinyl chloride) and polyethylene terpolymer are less stable when compared to some homopolymer formulations. From the four pigments studied, titanium dioxide rutile and a carbon based black proved to be stabilizers for both types of polymer. The mixture lithopone plus calcium carbonate has showed to have a photocatalytic effect on the binders. The studied paintings showed to be in an overall good state of conservation except for the paintings created in the 90’s with white glue and a mixture of white lithoponeand calcium carbonate. Discoloration of this white paint seems to be irreversible and ongoing and is still a major concern. The disapearance of the plasticizer was the only change detected. The current works created by Sarmento are expected to be more stable as they were painted using the rutile titanium dioxide. Immersion/cleaning tests showed that vinyl based paints can be susceptible to water and organic solvents like ethanol as some evidences point to the removal/diffusion of additives from the paint. The observations made point to the need to further proceed in this research field.

Arbitragem de consumo no direito brasileiro

This dissertation analyzes how the current Constitution and the Brazilian law establish consumer protection, arbitration and access to justice. Following we try to demonstrate why arbitration is a method rarely used in the resolution of consumer disputes in Brazil. It also examines the doctrinal and jurisprudential aspects of the conflict between the Brazilian Arbitration Law (Law nº. 9.307/96), which allows the arbitration clause in contracts of adhesion, and the Consumer Protection Code (Law nº 8.078/90) that in article 51, VII, considers as abusive the arbitration clause. Furthermore, analyzes new proposed bills under scrutiny by the National Congress on the issue and identifies the causes, in the Brazilian legal system, hampering the use of arbitration in consumer relations. Concludes that there are no principle obstacles preventing consumer litigations to be settled by arbitration. High costs, mistrust, oppression, misinformation of consumers and non-participation of the State, being a totally private institute, are factors that generate distrust, suspicion, and have prevented the development of arbitration in consumer relations in Brazil.

Towards a knowledge society

Economies are moving towards competitive stadiums based on knowledge and innovation. The changing environment and the level of globalization demand important efforts in order to sustain competitive advantages. Portugal has experienced a remarkable evolution since its adhesion to the European Union in several fields: economic development, Research & Development (R&D) consolidation, health parameters and social cohesion. As other developed economies, Portugal started its journey towards a knowledge-based economy and has been consolidating an innovation system during the last 35 years. The following report aims to analyze the evolution of a system since its creation to its last transformation within a globalized context. Challenges such as the lack of maturity of the system, the economic crisis, the European paradox, and closing the gap with other European countries are addressed in the next chapters. Likewise, recommendations on these points are provided by the end of the report as potential solutions.

Chondrogenic differentiation within magnetic-responsive multilayered liquified capsules containing collagen II/TFG-β3 coated microparticles

The use of stem cells is a promising therapeutic approach for the substantial challenge to regenerate cartilage. Considering the two prerequisites, namely the use of a 3D system to enable the chondrogenic differentiation and growth factors to avoid dedifferentiation, the diffusion efficiency of essential biomolecules is an intrinsic issue. We already proposed a liquified bioencapsulation system containing solid microparticles as cell adhesion sites1. Here, we intend to use the optimized system towards chondrogenic differentiation by encapsulating stem cells and collagenII-TGF-β3 PLLA microparticles. As a proof-of-concept, magnetite-nanoparticles were incorporated into the multilayered membrane. This can be a great advantage after implantation procedures to fixate the capsules in situ with the held of an external magnetic patch and for the follow-up through imaging. Results showed that the production of glycosaminoglycans and the expression of cartilage-relevant markers (collagen II, Sox9, aggrecan, and COMP) increased up to 28 days, while hypertrophic (collagen X) and fibrotic (collagen I) markers were downregulated. The presence of nanofibers in the newly deposited ECM was visualized by SEM, which resembles the collagen fibrils of native cartilage. The presence of the major constituent of cartilage, collagen II, was detected by immunocytochemistry and afranin-O and alcian blue stainings revealed a basophilic ECM deposition, which is characteristic of neocartilage. These findings suggest that the proposed system may provide a suitable environment for chondrogenic differentiation.

Fucoidan-based particles for diabetes mellitus treatment

Marine organisms are rich in a variety of materials with potential use in Tissue Engineering and Regenerative Medicine. One important example is fucoidan, a sulfated polysaccharide extracted from the cell wall of brown seaweeds.  Fucoidan is composed by L-fucose, sulfate groups and glucuronic acid. It has important bioactive properties such as anti-oxidative, anticoagulant, anticancer and reducing the blood glucose (1). In this work, the biomedical potential of fucoidan-based materials as drug delivery system was assessed by processing modified fucoidan (MFu) into particles by photocrosslinking using superamphiphobic surfaces and visible light. Fucoidan was modified by methacrylation reaction using different concentrations of methacrylate anhydride, namely 8% v/v (MFu1) and 12% v/v (MFu2). Further, MFu particles with and without insulin (5% w/v) were produced by pipetting a solution of 5% MFu with triethanolamine and eosin-y onto a superamphiphobic surface and then photocrosslinking using visible light (2). The developed particles were characterized to assess their chemistry, morphology, swelling behavior, drug release, insulin content and encapsulation efficiency. Moreover, the viability assays of fibroblast L929 cells in contact with MFu particles showed good adhesion and proliferation up to 14 days. Furthermore, the therapeutic potential of these particles using human beta cells is currently under investigation. Results obtained so far suggest that modified fucoidan particles could be a good candidate for diabetes mellitus therapeutic approaches.  

Organic-inorganic nanostructured multilayers for calcium phosphate biomineralization

The weak fixation of biomaterials within the bone structure is one of the major reasons of implants failures. Calcium phosphate (CaP) coatings are used in bone tissue engineering to improve implant osseointegration by enhancing cellular adhesion, proliferation and differentiation, leading to a tight and stable junction between implant and host bone. It has also been observed that materials compatible with bone tissue either have a CaP coating or develop such a calcified surface upon implantation. Thus, the development of bioactive coatings becomes essential for further improvement of integration with the surrounding tissue. However, most of current applied CaP coatings methods (e.g. physical vapor deposition), cannot be applied to complex shapes and porous implants, provide poor structural control over the coating and prevent incorporation of bioactive organic compounds (e.g. antibiotics, growth factors) because of the used harsh processing conditions. Layer-by-layer (LbL) is a versatile technology that permits the building-up of multilayered polyelectrolyte films in mild conditions based on the alternate adsorption of cationic and anionic elements that can integrate bioactive compounds. As it is recognized in natureâ s biomineralization process the presence of an organic template to induce mineral deposition, this work investigate a ion based biomimetic method where all the process is based on LbL methodology made of weak natural-origin polyelectrolytes. A nanostructured multilayer component, with 5 or 10 bilayers, was produced initially using chitosan and chondroitin sulphate polyelectrolyte biopolymers, which possess similarities with the extracellular matrix and good biocompatibility. The multilayers are then rinsed with a sequential passing of solutions containing Ca2+ and PO43- ions. The formation of CaP over the polyelectrolyte multilayers was confirmed by QCM-D, SEM and EDX. The outcomes show that 10 polyelectrolyte bilayer condition behaved as a  better site for initiating the formation of CaP as the precipitation occur at earlier stages than in 5 polyelectrolyte bilayers one. This denotes that higher number of bilayers could hold the CaP crystals more efficiently. This work achieved uniform coatings that can be applied to any surface with access to the liquid media in a low-temperature method, which potentiates the manufacture of effective bioactive biomaterials with great potential in orthopedic applications.

Saloplastic membranes as green devices for soft tissue regeneration

Implantable devices must exhibit mechanical properties similar to native tissues to promote appropriate cellular behavior and regeneration. Herein, we report a new membrane manufacture method based on the synthesis of polyelectrolyte complexes (PECs) that exhibit saloplasticity, i.e. variable physical-chemistry using salt as a plasticizer. This is a Green Chemistry approach, as PECs generate structures that are stabilized solely by reversible electrostatic interactions, avoiding the use of harmful crosslinkers completely. Furthermore, natural polyelectrolytes - chitosan and alginate - were used. Upon mixing them, membranes were obtained by drying the PECs at 37ºC, yielding compact PECs without resorting to organicsolvents. The plasticizing effect of salt after synthesis was shown by measuring tensile mechanical properties, which were lower when samples were immersed in high ionic strength solutions.Salt was also used during membrane synthesis in different quan- tities (0 M, 0.15 M and 0.5 M in NaCl) yielding structures with no significant differences in morphology and degradation (around 15% after 3 months in lysozyme). However, swelling was higher (about 10x) when synthesized in the presence of salt. In vitro cell studies using L929 fibroblasts showed that cells adhered and proliferated preferentially in membranes fabricated in the presence of salt (i.e. the membranes with lower tensile strength). Structures with physical-chemical properties controlled with precision open a path to tissue engineering strategies depending on fine tuning mechanical properties and cellular adhesion simply by changing ionic strength during membrane manufacture

Numerical simulation of galvanized rebars pullout

The usage of rebars in construction is the most common method for reinforcing plain concrete and thus bridging the tensile stresses along the concrete crack surfaces. Usually design codes for modelling the bond behaviour of rebars and concrete suggest a local bond stress – slip relationship that comprises distinct reinforcement mechanisms, such as adhesion, friction and mechanical anchorage. In this work, numerical simulations of pullout tests were performed using the finite element method framework. The interaction between rebar and concrete was modelled using cohesive elements. Distinct local bond laws were used and compared with ones proposed by the Model Code 2010. Finally an attempt was made to model the geometry of the rebar ribs in conjunction with a material damaged plasticity model for concrete.

Epidermis recreation in spongy-like hydrogels: New opportunities to explore epidermis-like analogues

[Excerpt] On the road to successfully achieving skin regeneration, 3D matrices/scaffolds that provide the adequate physico-chemical and biological cues to recreate the ideal healing environment are believed to be a key element [1], [2] and [3]. Numerous polymeric matrices derived from both natural [4] and [5] and synthetic [6], [7] and [8] sources have been used as cellular supports; nowadays, fewer matrices are simple carriers, and more and more are ECM analogues that can actively participate in the healing process. Therefore, the attractive characteristics of hydrogels, such as high water content, tunable elasticity and facilitated mass transportation, have made them excellent materials to mimic cells’ native environment [9]. Moreover, their hygroscopic nature [10] and possibility of attaining soft tissues-like mechanical properties mean they have potential for exploitation as wound healing promoters [11], [12], [13] and [14]. Nonetheless, hydrogels lack natural cell adhesion sites [15], which limits the maximization of their potential in the recreation of the cell niche. This issue has been tackled through the use of a range of sophisticated approaches to decorate the hydrogels with adhesion sequences such as arginine-glycine-aspartic acid (RGD) derived from fibronectin [16], [17] and [18], and tyrosine-isoleucine-glycine-serine-arginine (YIGSR) derived from laminin [18] and [19], which not only aim to modulate cell adhesion, but also influencing cell fate and survival [18]. Nonetheless, its widespread use is still limited by significant costs associated with the use of recombinant bioactive molecules.

Monitoring the early stiffness development in epoxy adhesives for structural strengthening

The present work aimed to assess the early-age evolution of E-modulus of epoxy adhesives used for Fibre-Reinforced Polymer (FRP) strengthening applications. The study involved adapting an existing technique devised for continuous monitoring of concrete stiffness since casting, called EMM-ARM (Elasticity Modulus Measurement through Ambient Response Method) for evaluation of epoxy stiffness. Furthermore, monotonic tensile tests according to ISO standards and cyclic tensile tests were carried out at several ages. A comparison between the obtained results was performed in order to better understand the performance of the several techniques in the assessment of stiffness of epoxy resins. When compared to the other methodologies, the method for calculation of E-modulus recommended by ISO standard led to lower values, since in the considered strain interval, the adhesive had a non-linear stress–strain relationship. The EMM-ARM technique revealed its capability in clearly identifying the hardening kinetics of epoxy adhesives, measuring the material stiffness growth during the entire curing period. At very early ages the values of Young׳s modulus obtained with quasi-static tests were lower than the values collected by EMM-ARM, due to the fact that epoxy resin exhibited a significant visco-elastic behaviour.