Browsing by Author "Ferreira, Susana Margarida Paraíso"
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- Produção de celulases e hidrólise enzimática de materiais lenhocelulósicos para a produção de biocombustíveisPublication . Ferreira, Susana Margarida Paraíso; Domingues, Fernanda da ConceiçãoUm dos maiores desafios para a sociedade de século XXI é acompanhar a grande procura de energia para transportes, aquecimento e processos industriais. Durante as últimas décadas, o consumo excessivo de combustíveis fósseis, tem levado à procura de novos combustíveis, principalmente de origem renovável. Assim, a produção de energias renováveis (biocombustíveis) tais como etanol, a partir da biomassa proveniente dos resíduos florestais, poderá apresentar uma fonte alternativa aos combustíveis fósseis. A área florestal de Portugal pode ser vista como uma fonte de grandes quantidades de resíduos florestais, nomeadamente giesta (Cytisus striatus) e esteva (Cistus ladanifer), arbustos nativos do país e considerados em muitas regiões como plantas invasivas. Para além, de a aplicação destes resíduos em bioprocessos ser favorável, pois apresentam-se como um substrato alternativo, também facilita a resolução do problema da sua eliminação. Assim é importante a optimização dos diferentes passos de produção de etanol a partir destes materiais lenhocelulósicos, designadamente do processo de hidrólise da celulose, que neste trabalho foi desenvolvido como um passo de hidrólise enzimática. A produção de celulases é um assunto chave na hidrólise enzimática de materiais lenhocelulósicos, pelo que é de grande importância conhecer o comportamento do fungo durante a cultura. Em muitos casos, a morfologia do fungo influencia directa ou indirectamente a produtividade das fermentações com fungos, sendo, nosso objectivo avaliar os efeitos da suplementação do meio de cultura com tampões na morfologia e produção de celulases pelo Trichoderma reesei Rut C30. Neste trabalho, foi estudada a morfologia e produção de celulases em culturas em balão pela suplementação do meio com diferentes sistemas tamponantes a duas concentrações e pH diferentes, usando-se o meio sem tampão como controlo. Os resultados sugerem que a morfologia do fungo é significativamente dependente da adição de diferentes tampões ao meio de crescimento. A morfologia micelial mostra uma clara transição, em culturas com variação do sistema tamponante e concentração dos tampões, de uma forma de agregados a “pellets”. A mais elevada actividade sobre o papel de filtro foi obtida usando o tampão succinato a uma concentração de 100mM e pH 4.8, correspondendo a uma morfologia micelial dispersa. Neste trabalho, a hidrólise enzimática da esteva e da giesta foi estudada empregando numa primeira fase ensaios preliminares e de seguida estratégias estatísticas para resolução de problemas, como o método de Plackett- Burman e o “Central Composite Design”. O primeiro método foi utilizado com o objectivo de proceder a uma selecção das variáveis a estudar mais importantes a partir de um estudo simultâneo da influência de diversas condições operacionais e reaccionais, e o segundo de optimizar o processo de hidrólise enzimática, por variação de pH, temperatura, concentração de celulases, concentração do polímero adicionado e tempo de reacção. O estudo do processo de hidrólise enzimática segundo uma metodologia de resposta de superfície não permitiu o ajuste de um modelo estatístico para optimização do processo, mas possibilitou um estudo do processo quanto à influência das variáveis estudadas, verificando-se uma variabilidade devida ao tipo de substrato usado.
- Study of molecular and cellular pathogenicity mechanisms of Arcobacter speciesPublication . Ferreira, Susana Margarida Paraíso; Domingues, Fernanda da Conceição; Queiroz, João António de Sampaio Rodrigues; Oleastro, Mónica Alexandra de SousaArcobacter is a genus of Gram-negative, spiral-shaped bacteria in the Epsilonproteobacteria class. It was first proposed in 1991 and was included in the Campylobacteraceae family together with the genera Campylobacter and Sulfurospirillum. There are currently 18 species described, among which A. butzleri, A. cryaerophilus and A. skirrowii are known to be human and animal pathogens. Arcobacter genus shows an unusually wide range of habitats, having been isolated from food, water, food processing and handling facilities, diverse environmental, animal and human samples. In fact, consumption of Arcobacter-contaminated food or water is regarded as the most probable cause of infection by this bacterium. Therefore it is important to evaluate its distribution in food or food processing environment, together with the assessment of its genetic variability and other phenotypic and genotypic features associated with virulence potential, to be able to understand the persistence mechanism of Arcobacter in the food chain. Thus, this study intend to evaluate the presence, genetic diversity, and virulence characteristics (namely antibiotic resistance, biofilm-forming capacity and detection of putative virulence genes) of A. butzleri strains isolated from poultry and from the environment of a Portuguese slaughterhouse. A. butzleri isolates revealed a high genetic diversity, with all isolates showing to be susceptible to gentamicin, in contrast to 55.8% that were resistant to ciprofloxacin. The latter resistance was associated with the presence of a cytosine to thymine transition in the quinolone resistance determining region of the gyrA gene. Among selected isolates, 72.2 % presented biofilm-forming ability and in all strains putative virulence genes were detected. These results highlight the relevance of A. butzleri relevance as food-borne pathogen. Taking into account that Arcobacter is potentially transmitted through contaminated food and is resistant to common antimicrobials, it is important to develop alternative control strategies that could be both effective and safe for human consumption. Therefore, the antimicrobial properties of resveratrol against A. butzleri and A. cryaerophilus were studied. Resveratrol exhibited a bacteriostatic or bactericidal activity dependent on cellular growth phase and resveratrol concentration, leading to both DNA content and metabolic activity reduction on Arcobacter cells. Resveratrol also showed the ability to act as an efflux pump inhibitor, and to induce cellular damage. Thus, resveratrol showed anti-Arcobacter activity, with the results obtained suggesting that this compound inhibits this microorganism through different pathways, which together with resveratrol beneficial properties described for human health may encourage its use as a food preservative. Some of the species of Arcobacter genus have been associated with gastrointestinal disease in humans, however there were a lack of studies evaluating its prevalence in Portugal, with the same happening for non-Campylobacter jejuni/coli species. Therefore, the frequency of Arcobacter and Campylobacter species in faeces from patients with diarrhoea in Portugal was assessed using a molecular approach. Concerning, Arcobacter and Campylobacter prevalence and distribution, 298 diarrhoeal samples from Portuguese patients were analysed, 1.3% of the samples were positive for A. butzleri and 0.3 % for A. cryaerophilus. Campylobacter species were found in 31.9 % of diarrhoeic faeces samples, with C. jejuni and C. concisus being the most prevalent species of this genus (13.7 % and 8.0 %, respectively). In this cohort of samples, A. butzleri was the fourth most frequent species. These results evidence the importance of Arcobacter and Campylobacter species as aetiological agents of acute gastroenteritis among Portuguese patients, affecting particularly the paediatric age group. Although A. butzleri has been implicated in human diseases, much of its pathogenesis and virulence factors remain unclear. Thus, A. butzleri virulence potential was also investigated, through the characterization of genotypic and pathogenic properties of human and non- human isolates. The isolates showed to be susceptible to tetracyclines and aminoglycosides, however displaying high resistance to quinolones. A. butzleri demonstrated a weak haemolytic activity and the ability to form biofilms in polystyrene surfaces. Adhesion levels similar to Salmonella Typhimurium were found for A. butzleri on Caco-2 cells, with pre- existing inflammation showing no significant effect on its adherence ability, yet invasion ability showed to vary among the isolates. A. butzleri was able of intracellular survival in Caco-2 cells and to induce a significant up-regulation of interleukin-8 secretion, as well as to promote structural cell disturbance. These data brings new insights to A. butzleri virulence and highlights its pathogenic potential. Overall in this work, the prevalence of Arcobacter species both in human and food-related samples was evaluated, contributing to understanding the epidemiology of Arcobacter in Portugal. The survival and persistence of this organism in the environment was highlighted due to its ability to form biofilms. Its relevance as a human pathogen was underlined by the resistance to antimicrobials, the presence of several putative virulence genes, along with its adherence, invasion, intracellular survival abilities and induction of proinflammatory cytokine secretion in intestinal epithelial cells. Finally, resveratrol was tested as an alternative to control the growth of Arcobacter. This work provided new insights on the epidemiology and pathogenicity of Arcobacter, and also identified a natural compound with anti-Arcobacter activity, which may contribute for future developments of new control approaches.