Study of multi-component systems in polybenzimidazole membrane formation and their impact on membrane performance

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

Comparative effects of sediments contaminated by carcinogenic and non-carcinogenic PAHs in Dicentrarchus labrax: a semi-quantitative histopathological approach

Dissertação para obtenção do Grau de Mestre em Engenharia do Ambiente

Multiresponsive supramolecular systems for information processing at the molecular level

Dissertação para obtenção do Grau de Doutor em Química Sustentável

On the way towards the understanding of suberin degradation by Aspergillus nidulans

Dissertation presented to obtain the Ph.D degree in Biochemistry.

Aqueous biphasic system based on cholinium ionic liquids: extraction of biologically active phenolic acids

Phenolic acids are aromatic secondary plant metabolites, widely spread throughout the plant kingdom. Due to their biological and pharmacological properties, they have been playing an important role in phytotherapy and consequently techniques for their separation and purification are in need. This thesis aims at exploring new sustainable separation processes based on ionic liquids (ILs) in the extraction of biologically active phenolic acids. For that purpose, three phenolic acids with similar chemical structures were selected: cinnamic acid, p-coumaric acid and caffeic acid. In the last years, it has been shown that ionic liquids-based aqueous biphasic systems (ABSs) are valid alternatives for the extraction, recovery and purification of biomolecules when compared to conventional ABS or extractions carried out with organic solvents. In particular, cholinium-based ILs represent a clear step towards a greener chemistry, while providing means for the implementation of efficient techniques for the separation and purification of biomolecules. In this work, ABSs were implemented using cholinium carboxylate ILs using either high charge density inorganic salt (K3PO4) or polyethylene glycol (PEG) to promote the phase separation of aqueous solutions containing three different phenolic acids. These systems allow for the evaluation of effect of chemical structure of the anion on the extraction efficiency. Only one imidazolium-based IL was used in order to establish the effect of the cation chemical structure. The selective extraction of one single acid was also researched. Overall, it was observed that phenolic acids display very complex behaviours in aqueous solutions, from dimerization to polymerization and also hetero-association are quite frequent phenomena, depending on the pH conditions. These phenomena greatly hinder the correct quantification of these acids in solution.

Development of an integrative approach for the characterization of ecological risk on marine transition ecosystems: the Sado Estuary as a case study

Estuaries and other transitional waters are complex ecosystems critically important as nursery and shelter areas for organisms. Also, humans depend on estuaries for multiple socio-economical activities such as urbanism, tourism, heavy industry, (taking advantage of shipping), fisheries and aquaculture, the development of which led to strong historical pressures, with emphasis on pollution. The degradation of estuarine environmental quality implies ecologic, economic and social prejudice, hence the importance of evaluating environmental quality through the identification of stressors and impacts. The Sado Estuary (SW Portugal) holds the characteristics of industrialized estuaries, which results in multiple adverse impacts. Still, it has recently been considered moderately contaminated. In fact, many studies were conducted in the past few years, albeit scattered due to the absence of true biomonitoring programmes. As such, there is a need to integrate the information, in order to obtain a holistic perspective of the area able to assist management and decision-making. As such, a geographical information system (GIS) was created based on sediment contamination and biomarker data collected from a decade-long time-series of publications. Four impacted and a reference areas were identified, characterized by distinct sediment contamination patterns related to different hot spots and diffuse sources of toxicants. The potential risk of sediment-bound toxicants was determined by contrasting the levels of pollutants with available sediment quality guidelines, followed by their integration through the Sediment Quality guideline Quotient (SQG-Q). The SQG-Q estimates per toxicant or class was then subjected to georreferencing and statistical analyses between the five distinct areas and seasons. Biomarker responses were integrated through the Biomarkers Consistency Indice and georreferenced as well through GIS. Overall, in spite of the multiple biological traits surveyed, the biomarker data (from several organisms) are accordant with sediment contamination. The most impacted areas were the shipyard area and adjacent industrial belt, followed by urban and agricultural grounds. It is evident that the estuary, although globally moderately impacted, is very heterogeneous and affected by a cocktail of contaminants, especially metals and polycyclic aromatic hydrocarbon. Although elements (like copper, zinc and even arsenic) may originate from the geology of the hydrographic basin of the Sado River, the majority of the remaining contaminants results from human activities. The present work revealed that the estuary should be divided into distinct biogeographic units, in order to implement effective measures to safeguard environmental quality.

Follow the red road of triheme cytochromes in Geobacter sulfurreducens

The bacterium Geobacter sulfurreducens (Gs) is capable of oxidizing a large variety of compounds relaying electrons out of the cytoplasm and across the membrane in a process designated as extracellular electron transfer. The Gs genome was fully sequenced and a family composed by five periplasmic triheme cytochromes c7 (designated PpcA-E) was identified. These cytochromes play an important role in the reduction of extracellular acceptors. They contain approximately 70 amino acids, three heme groups with bis-histidinyl axial coordination, and share between 57 and 77% sequence identity. The triheme cytochrome PpcA is highly abundant in Gs and is most likely the reservoir of electrons destined for outer surface. In addition to its role in electron transfer pathways this protein can perform e-/H+ energy transduction, a process that is disrupted when the strictly conserved aromatic residue phenylalanine 15 is replaced by a leucine (PpcAF15L). This Thesis focuses on the expression, purification and characterization of these proteins using Nuclear Magnetic Resonance and ultraviolet-visible spectroscopy. The orientations of the heme axial histidine ring planes and the orientation of the heme magnetic axis were determined for each Gs triheme cytochrome. The comparison of the orientations obtained in solution with the crystal structures available showed significant differences. The results obtained provide the paramagnetic constraints to include in the future refinement of the solution structure in the oxidized state. In this work was also determined the solution structure and the pH-dependent conformational changes of the PpcAF15L allowing infer the structural origin for e-/H+ energy transduction mechanism as shown by PpcA. Finally, the backbone and side chain NH signals of PpcA were used to map interactions between this protein and the putative redox partner 9,10-anthraquinone-2,6-disulfonate (AQDS). In this work a molecular interaction was identified for the first time between PpcA and AQDS, constituting the first step toward the rationalization of the Gs respiratory chain.

Photochemical control of rheology

The main objective of the research work developed in the framework of this PhD thesis was the preparation and development of novel photorheological fluids. This was pursued following two distinct strategies. The first one focused on the synthesis of tripodal compounds functionalized with photodimerizable moieties of cinnamic acid, coumarin and anthracene. Two sets of compounds were prepared, varying the central unit as well as spacers resulting in molecules with different solubilities and molecular weight. All compounds were characterized towards their photochemical properties and all exhibited photoreactivity upon irradiation with ultra-violet light. In particular, both coumarin derivatives exhibited the greatest photopolymerization reactivity, resulting in the formation of dendrimeric nanoparticles or in the increase of viscosity of organic solutions. The second strategy was focused on the careful design of photosensitive ionic liquids, based on the results of several quantitative structure-property relationship studies. Thus, photosensitive ionic liquids were synthesized bearing cinnamic acid or coumarin moieties in the organic cation. Upon irradiation, all compounds exhibited reactivity, which resulted in changes in their physical properties, such as melting point or viscosity. In addition, novel coumarin chromophores with different photophysical and photochemical properties were developed. It is expected that these compounds may find application in the preparation of new photosensitive ionic liquids.

Development of novel ionic liquids based on biological molecules

Ionic Liquids (ILs) belong to a class of compounds with unusual properties: very low vapour pressure; high chemical and thermal stability and the ability to dissolve a wide range of substances. A new field in research is evaluating the possibility to use natural chiral biomolecules for the preparation of chiral ionic liquids (CILs). This important challenge in synthetic chemistry can open new avenues of research in order to avoid some problems related with the intrinsic biodegradability and toxicity associated to conventional ILs. The research work developed aimed for the synthesis of CILs, their characterization and possible applications, based on biological moieties used either as chiral cations or anions, depending on the synthetic manipulation of the derivatives. Overall, a total of 28 organic salts, including CILs were synthesized: 9 based on L-cysteine derivatives, 12 based on L-proline, 3 based on nucleosides and 4 based on nucleotides. All these new CILs were completely characterized and their chemical and physical properties were evaluated. Some CILs based on L-cysteine have been applied for discrimination processes, including resolution of racemates and as a chiral catalyst for asymmetric Aldol condensation. L-proline derived CILs were also studied as chiral catalysts for Michael reaction. In parallel, the interactions of macrocyclic oligosugars called cyclodextrins (CDs) with several ILs were studied. It was possible to improve the solubility of CDs in water and serum. Additionally, fatty acids and steroids showed an increase in water solubility when ILs-CDs systems were used. The development of efficient and selective ILs-CDs systems is indispensable to expand the range of their applications in host-guest interactions, drug delivery systems or catalytic reactions. Novel salts derived from nucleobases were used in order to enhance the fluorescence in aqueous solution. Additionally, preliminary studies regarding ethyl lactate as an alternative solvent for asymmetric organocatalysis were performed.

Fishing the key biological components of the bacterium Geobacter metallireducens for optimal biotechnological applications

Os resultados apresentados no capítulo 2 foram incluídos no artigo Dantas JM, Campelo LM, Duke NEC, Salgueiro CA, Pokkuluri PR (2015) "The structure of PccH from Geobacter sulfurreducens – a novel low reduction potential monoheme cytochrome essential for accepting electrons from an electrode", FEBS Journal, 282, 2215-2231.

Nanocomposite polymer beads for cell detection

Circulating tumor cells (CTCs) may induce metastases when detached from the primary tumor. The numbers of these cells in blood offers a valuable prognostic indication. Magnetoresistive sensing is an attractive option for CTC counting. In this technique, cells are labeled with nancomposite polymer beads that provide the magnetic signal. Bead properties such as size and magnetic content must be optimized in order to be used as a detection tool in a magnetoresistive platform. Another important component of the platform is the magnet required for proper sensing. Both components are addressed in this work. Nanocomposite polymer beads were produced by nano-emulsion and membrane emulsification. Formulations of the oil phase comprising a mixture of aromatic monomers and iron oxide were employed. The effect of emulsifier (surfactant) concentration on bead size was studied. Formulations of polydimethilsiloxane (PDMS) with different viscosities were also prepared with nano-emulsion method resulting in colloidal beads. Polycaprolactone (PCL) beads were also synthetized by the membrane emulsification method. The beads were characterized by different techiques such as dynamic light scattering (DLS), thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). Additionally, the magnet dimensions of the platform designed to detect CTCs were optimized through a COMSOL multiphysics simulation.

Monoterpene isolated from the essential oil of Trachyspermum ammi is cytotoxic to multidrug-resistant Pseudomonas aeruginosa and Staphylococcus aureus strains

Abstract INTRODUCTION: The aim of this study was to determine whether an herbal extract containing monoterpene exhibited activity against multidrug-resistant Staphylococcus aureus and Pseudomonas aeruginosa isolated from clinical infection samples. METHODS: The essential oil of Trachyspermum ammi (L.) Sprague ex Turrill (Apiaceae) fruit was extracted by hydrodistillation. Fruit residues were treated with hydrochloric acid and re-hydrodistilled to obtain volatile compounds. Compounds in the distilled oil were identified using gas-chromatography (GC) and GC-mass spectrometry (MS). The antibiotic susceptibility of all bacterial isolates was analyzed using both the disc diffusion method and determination of the minimum inhibitory concentration (MIC). The sensitivity of antibiotic-resistant isolates to essential oil was also determined by using the disc diffusion method and MIC determination. RESULTS: Of 26 clinical isolates, 92% were multidrug-resistant (MDR). Aromatic monoterpenes (thymol, paracymene, and gamma-terpinene) were the major (90%) components of the oil. Growth of S. aureus strains was successfully inhibited by the oil, with an inhibitory zone diameter (IZD) between 30-60mm and MIC <0.02μL/mL. The oil had no antimicrobial activity against clinical isolates of P. aeruginosa; rather, it prevented pigment production in these isolates. CONCLUSIONS: This study revealed that the essential oil of Trachyspermum ammi, which contains monoterpene, has good antibacterial potency. Monoterpenes could thus be incorporated into antimicrobial ointment formulas in order to treat highly drug-resistant S. aureus infections. Our findings also underscore the utility of research on natural products in order to combat bacterial multidrug resistance.

Gastric and colorectal cancer mortality in an urban and industrialized area of Brazil

PURPOSE: To study the gastric and colorectal cancer mortalities and their relation to the urban-industrialization in Baixada Santista, located in the southeastern region of Brazil. METHODS: Selected from the registries of the State System of Data Analysis Foundation (SEADE) were 1105 deaths due to gastric cancer (ICD 153--154) and 690 due to colorectal cancer (ICD 151) that occurred from 1980 to 1993 in males, above 10 years of age, residing in Baixada Santista. For each of these types of cancer, the standardized mortality rates, age-adjusted by world population in the 1960s, for 4 industrialized and 4 non-industrialized urban communities in that region were calculated. The ratios among those rates were calculated in order to compare the mortality in the periods 1980--93, 1980--1986, and 1987--1993. RESULTS: Standardized mortality rates for colorectal cancer were significantly higher in industrialized area, with ratios of 1.6 [95% CI 1.22 -- 2.29], 1.6 [95% CI 1.2 -- 2.0], and 1.6 [95% CI 1.3 -- 2.0] in the periods 1980--86, 1987--1993 and 1980--93, respectively. Gastric cancer did not show any statistical difference between the industrialized and non-industrialized areas, but there was a significant decrease in BS from the period 1980--1986 to 1987--1993. CONCLUSIONS: The significant elevation of colorectal cancer mortality in the industrialized area could be related to exposure to numerous carcinogens such as aromatic hydrocarbon, organic-chloride, metals, and industrial-port dust present in the region. Alternatively, the non-significant difference in gastric cancer between industrialized and non-industrialized areas and significant decrease in the last few years could be predominately reflecting the advances in the quality of life in urban areas. These results require further case-control studies that could help with the analysis of the associations among cancer and environmental factors (occupational, urban-industrial, habit, and life condition) and genetic susceptibility.

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.