Weathering resistance of thin plasma polymer films on pre-coated steel

O trabalho apresentado teve origem no projecto de investigação “Tailored Thin Plasma Polymers Films for Surface Engineering of Coil Coated Steel”, financiado pelo Programa Europeu ECSC Steel Research. Sistemas de aço galvanizado pré-pintado em banda à base de poliéster e poliuretano foram submetidos a um processo de polimerização por plasma onde um filme fino foi depositado de modo a modificar as propriedades de superfície. Foram usados reactores de cátodo oco, microondas e rádio frequência para a deposição do polímero fino. Os sistemas preparados foram analisados de modo a verificar a influência do processo de polimerização por plasma na alteração das propriedades barreira dos sistemas pré-pintados em banda. Foi estudado o efeito dos diferentes passos do processo de polimerização por plasma, bem como o efeito de diferentes variáveis operatórias. A mistura precursora foi variada de modo a modificar as propriedades da superfície de modo a poder vir a obter maior hidrofobicidade, maior resistência a marcas digitais, bem como maior facilidade de limpeza. Os testes foram conduzidos em solução de NaCl 0,5 M. Para o trabalho foram usadas técnicas de análise da morfologia da superfície como Microscopia de Força Atómica e Microscopia Electrónica de Varrimento. As propriedades electroquímicas dos sistemas foram estudadas por Espectroscopia de Impedância Electroquímica. A estrutura dos filmes gerados no processo de polimerização por plasma foi caracterizada por Microscopia de Transmissão Electrónica. A modificação das propriedades ópticas devido ao processo de polimerização por plasma foi também obtida.

Oil hydrocarbon-bacteria interactions in coastal environments

The ability of microorganisms to use oil hydrocarbons as a source of carbon and energy is crucial for environmental oil detoxification. However, there is still a lack of knowledge on fundamental aspects of this process on specific habitats and under different climate scenarios. In the first phase of this work, the culturable fraction of the oil hydrocarbon (OH) degrading bacteria from the sea surface microlayer (SML) of the estuarine system Ria de Aveiro was characterized. In the second phase, the impact of oil contamination on the active bacterial community was studied under climate change scenarios. Pseudomonas emerged as the prevailing genera among OH degrading bacteria in the SML. Moreover, culture-independent methods revealed that the relative abundance and diversity of Gammaproteobacteria, in which Pseudomonas is included, varies along an estuarine gradient of contamination. In order to access the impact of oil contamination on microbial communities under climate change scenarios, an experimental life support system for microcosm experiments (ELLS) was developed and validated for simulation of climate change effects on microbial communities. With the ELSS it is possible to simulate, in controlled conditions, fundamental parameters of the dynamics of coastal and estuarine systems while maintaining community structure in terms of the abundance of the most relevant members of the indigenous bacterial community. A microcosm experiment in which the independent and combined impact of ultraviolet radiation, ocean acidification and oil contamination on microbial communities was conducted. The impact on bacterial communities was accessed with a 16S RNA (cDNA) based barcode pyrosequencing approach. There was a drastic decrease of Desulfobacterales relative abundance after oil contamination under the reduced pH value estimated for 2100, when compared to present values. Since members of this order are known OH degraders, such a significant decrease may have consequences on OH detoxification of contaminated environments under the pH levels of the ocean expected for the future. Metagenome predictions based on the 16S RNA database indicated that several degradation pathways of OH could be affected under oil contamination and reduced water pH. Taken together, the results from this work bring new information on the dynamics of OH degrading bacteria in coastal and estuarine environments under present and future climate scenarios.

Surface reactivity enhancement of silica-based glass-ceramic scaffolds

A paradigm shift is taking place from using transplanting tissue and synthetic implants to a tissue engineering approach that aims to regenerate damaged tissues by combining cells from the body with highly porous scaffold biomaterials, which act as templates, guiding the growth of new tissue. The central focus of this thesis was to produce porous glass and glass-ceramic scaffolds that exhibits a bioactive and biocompatible behaviour with specific surface reactivity in synthetic physiological fluids and cell-scaffold interactions, enhanced by composition and thermal treatments applied. Understanding the sintering behaviour and the interaction between the densification and crystallization processes of glass powders was essential for assessing the ideal sintering conditions for obtaining a glass scaffolds for tissue engineering applications. Our main goal was to carry out a comprehensive study of the bioactive glass sintering, identifying the powder size and sintering variables effect, for future design of sintered glass scaffolds with competent microstructures. The developed scaffolds prepared by the salt sintering method using a 3CaO.P2O5 - SiO2 - MgO glass system, with additions of Na2O with a salt, NaCl, exhibit high porosity, interconnectivity, pore size distribution and mechanical strength suitable for bone repair applications. The replacement of 6 % MgO by Na2O in the glass network allowed to tailor the dissolution rate and bioactivity of the glass scaffolds. Regarding the biological assessment, the incorporation of sodium to the composition resulted in an inibition cell response for small periods. Nevertheless it was demonstrated that for 21 days the cells response recovered and are similar for both glass compositions. The in vitro behaviour of the glass scaffolds was tested by introducing scaffolds to simulated body fluid for 21 days. Energy-dispersive Xray spectroscopy and SEM analyses proved the existence of CaP crystals for both compositions. Crystallization forming whitlockite was observed to affect the dissolution behaviour in simulated body fluid. By performing different heat treatments, it was possible to control the bioactivity and biocompatability of the glass scaffolds by means of a controlled crystallization. To recover and tune the bioactivity of the glass-ceramic with 82 % crystalline phase, different methods have been applied including functionalization using 3- aminopropyl-triethoxysilane (APTES). The glass ceramic modified surface exhibited an accelerated crystalline hydroxyapatite layer formation upon immersion in SBF after 21 days while the as prepared glass-ceramic had no detected formation of calcium phosphate up to 5 months. A sufficient mechanical support for bone tissue regeneration that biodegrade later at a tailorable rate was achievable with the glass–ceramic scaffold. Considering the biological assessment, scaffolds demonstrated an inductive effect on the proliferation of cells. The cells showed a normal morphology and high growth rate when compared to standard culture plates. This study opens up new possibilities for using 3CaO.P2O5–SiO2–MgO glass to manufacture various structures, while tailoring their bioactivity by controlling the content of the crystalline phase. Additionally, the in vitro behaviour of these structures suggests the high potential of these materials to be used in the field of tissue regeneration.

Structural analysis of glycans and development of microarrays from Helcobacter pylori cell surface glycome

Helicobacter pylori is a bacterial pathogen that affects more than half of the world’s population with gastro-intestinal diseases and is associated with gastric cancer. The cell surface of H. pylori is decorated with lipopolysaccharides (LPSs) composed of three distinct regions: a variable polysaccharide moiety (O-chain), a structurally conserved core oligosaccharide, and a lipid A region that anchors the LPS to the cell membrane. The O-chain of H. pylori LPS, exhibits unique oligosaccharide structures, such as Lewis (Le) antigens, similar to those present in the gastric mucosa and are involved in interactions with the host. Glucan, heptoglycan, and riban domains are present in the outer core region of some H. pylori LPSs. Amylose-like glycans and mannans are also constituents of some H. pylori strains, possibly co-expressed with LPSs. The complexity of H. pylori LPSs has hampered the establishment of accurate structure-function relationships in interactions with the host, and the design of carbohydrate-based therapeutics, such as vaccines. Carbohydrate microarrays are recent powerful and sensitive tools for studying carbohydrate antigens and, since their emergence, are providing insights into the function of carbohydrates and their involvement in pathogen-host interactions. The major goals of this thesis were the structural analysis of LPSs from H. pylori strains isolated from gastric biopsies of symptomatic Portuguese patients and the construction of a novel pathogen carbohydrate microarray of these LPSs (H. pylori LPS microarray) for interaction studies with proteins. LPSs were extracted from the cell surface of five H. pylori clinical isolates and one NCTC strain (26695) by phenol/water method, fractionated by size exclusion chromatography and analysed by gas chromatography coupled to mass spectrometry. The oligosaccharides released after mild acid treatment of the LPS were analysed by electrospray mass spectrometry. In addition to the conserved core oligosaccharide moieties, structural analyses revealed the presence of type-2 Lex and Ley antigens and N-acetyllactosamine (LacNAc) sequences, typically found in H. pylori strains. Also, the presence of O-6 linked glucose residues, particularly in LPSs from strains 2191 and NCTC 26695, pointed out to the expression of a 6-glucan. Other structural domains, namely ribans, composed of O-2 linked ribofuranose residues were observed in the LPS of most of H. pylori clinical isolates. For the LPS from strain 14382, large amounts of O-3 linked galactose units, pointing to the occurrence of a galactan, a domain recently identified in the LPS of another H. pylori strain. A particular feature to the LPSs from strains 2191 and CI-117 was the detection of large amounts of O-4 linked N-acetylglucosamine (GlcNAc) residues, suggesting the presence of chitin-like glycans, which to our knowledge have not been described for H. pylori strains. For the construction of the H. pylori LPS microarray, the structurally analysed LPSs, as well as LPS-derived oligosaccharide fractions, prepared as neoglycolipid (NGL) probes were noncovalently immobilized onto nitrocellulosecoated glass slides. These were printed together with NGLs of selected sequence defined oligosaccharides, bacterial LPSs and polysaccharides. The H. pylori LPS microarray was probed for recognition with carbohydratebinding proteins (CBPs) of known specificity. These included Le and blood group-related monoclonal antibodies (mAbs), plant lectins, a carbohydratebinding module (CBM) and the mammalian immune receptors DC-SIGN and Dectin-1. The analysis of these CBPs provided new information that complemented the structural analyses and was valuable in the quality control of the constructed microarray. Microarray analysis revealed the occurrence of type-2 Lex and Ley, but not type-1 Lea or Leb antigens, supporting the results obtained in the structural analysis. Furthermore, the H. pylori LPSs were recognised by DC-SIGN, a mammalian lectin known to interact with this bacterium through fucosylated Le epitopes expressed in its LPSs. The -fucose-specific lectin UEA-I, showed restricted binding to probes containing type-2 blood group H sequence and to the LPSs from strains CI-117 and 14382. The presence of H-type-2, as well Htype- 1 in the LPSs from these strains, was confirmed using specific mAbs. Although H-type-1 determinant has been reported for H. pylori LPSs, this is the first report of the presence of H-type-2 determinant. Microarray analysis also revealed that plant lectins known to bind 4-linked GlcNAc chitin oligosaccharide sequences bound H. pylori LPSs. STL, which exhibited restricted and strong binding to 4GlcNAc tri- and pentasaccharides, differentially recognised the LPS from the strain CI-117. The chitin sequences recognised in the LPS could be internal, as no binding was detected to this LPS with WGA, known to be specific for nonreducing terminal of 4GlcNAc sequence. Analyses of the H. pylori LPSs by SDS-PAGE and Western blot with STL provided further evidence for the presence of these novel domains in the O-chain region of this LPS. H. pylori LPS microarray was also applied to analysis of two human sera. The first was from a case infected with H. pylori (H. pylori+ CI-5) and the second was from a non-infected control.The analysis revealed a higher IgG-reactivity towards H. pylori LPSs in the H. pylori+ serum, than the control serum. A specific IgG response was observed to the LPS isolated from the CI-5 strain, which caused the infection. The present thesis has contributed to extension of current knowledge on chemical structures of LPS from H. pylori clinical isolates. Furthermore, the H. pylori LPS microarray constructed enabled the study of interactions with host proteins and showed promise as a tool in serological studies of H. pyloriinfected individuals. Thus, it is anticipated that the use of these complementary approaches may contribute to a better understanding of the molecular complexity of the LPSs and their role in pathogenesis.