Electrochemical immunosensor for multiplexed detection of food-borne pathogens using nanocrystal bioconjugates and MWCNT screen-printed electrode

Bacterial food poisoning is an ever-present threat that can be prevented with proper care and handling of food products. A disposable electrochemical immunosensor for the simultaneous measurements of common food pathogenic bacteria namely Escherichia coli O157:H7 (E. coli), campylobacter and salmonella were developed. The immunosensor was fabricated by immobilizing the mixture of anti-E. coli, anticampylobacter and anti-salmonella antibodies with a ratio of 1:1:1 on the surface of the multiwall carbon nanotube-polyallylamine modified screen printed electrode (MWCNT-PAH/SPE). Bacteria suspension became attached to the immobilized antibodies when the immunosensor was incubated in liquid samples. The sandwich immunoassay was performed with three antibodies conjugated with specific nanocrystal ( -E. coli-CdS, -campylobacter-PbS and -salmonella-CuS) which has releasable metal ions for electrochemical measurements. The square wave anodic stripping voltammetry (SWASV) was employed to measure released metal ions from bound antibody nanocrystal conjugates. The calibration curves for three selected bacteria were found in the range of 1 × 103 – 5 × 105 cells mL−1 with the limit of detection (LOD) 400 cells mL−1 for salmonella, 400 cells mL−1 for campylobacter and 800 cells mL−1 for E. coli. The precision and sensitivity of this method show the feasibility of multiplexed determination of bacteria in milk samples.

Diversity and characterisation of coagulasenegative staphylococci isolated from healthy individuals in Portugal

In the past few years the interest in coagulase-negative staphylococci (CoNS) has significantly increased in human medicine. CoNS are common commensal colonisers of the human skin, although now also recognised as major nosocomial pathogens. Over the last decades, several studies have been carried out in order to understand the pathogenicity mechanisms of CoNS. The well known determinants in the pathogenesis of CoNS infections are their ability to form biofilms and an exceptional resistance to several antibiotics. Nevertheless, there is a lack of studies regarding the commensal lifestyle of these microorganisms. Additionally, it is now hypothesised that commensal bacteria might be a reservoir of pathogenic determinants. Therefore, the work described throughout this thesis was aimed to perform a phenotypic and genotypic characterisation of different CoNS species isolated from healthy Portuguese individuals. A total of 61 CoNS isolates, comprising 7 different species, were obtained and characterised at the level of biofilm formation and antibiotic susceptibility profiles. According to the results, biofilm formation ability and presence of biofilm-associated genes were commonly found features, highlighting their pivotal role in the colonising lifestyle of CoNS. This study also addressed the correlation between phenotypic and genotypic characteristics of biofilm formation, corroborating and raising questions about the importance of some genes in this process. Moreover, it was observed a great proportion of isolates with decreased susceptibility and multiple resistances to some important antibiotics. A significant association between antibiotic resistance and biofilm formation was also demonstrated, and some hypotheses about the nature of such association were provided. Lastly, the expression patterns of two biofilm-associated genes at two distinct biofilm developmental stages were determined, confirming their importance in the accumulative stage of biofilm formation. Overall, the results presented in this thesis indicate that staphylococcal skin flora might be an important reservoir of potentially pathogenic bacteria and, simultaneously, bring to light new perceptions about the molecular basis of staphylococcal biofilm formation, and the nature of the association between antibiotic resistance and biofilm formation.

Diagnóstico e avaliação da segurança de estruturas de madeira existentes

A utilização da madeira enquanto material estrutural é um aspeto abordado com algum receio ou até desconhecimento por parte dos elementos intervenientes no processo de construção civil, sendo ainda uma área pouco lecionada e muitas vezes excluída dos planos curriculares em Engenharia Civil. Assim sendo é importante o surgimento de trabalhos relacionados com esta matéria, relembrando ao sector da construção civil e ao ramo de estudo relacionado, que a madeira já teve e possivelmente voltará a ter um papel importante a desempenhar na área da construção. Um dos aspetos evidenciados nos estudos realizados consiste na importância da reabilitação de edifícios e do seu papel cada vez mais determinante na sociedade. Esta metodologia de intervenção apresenta-se muitas vezes como uma vantagem económica assim como contribui para a preservação do património arquitetónico e cultural, cujo valor histórico é incalculável. A importância da reabilitação de edifícios habitacionais nas zonas históricas, bem como de monumentos de grande importância cultural apresenta-se cada vez mais como um desafio devido à falta de conservação dos mesmos ao longo da sua vida útil, e da necessidade de preservar a identidade do local, beneficiando assim a qualidade do tecido urbano. Assim sendo, este trabalho apresenta um levantamento dos danos possíveis de ocorrer em elementos de madeira, dando especial destaque aos agentes patológicos e às consequências da sua atividade na madeira assim como às metodologias de inspeção e diagnóstico que poderão ser aplicadas em estruturas de madeira existentes, favorecendo assim a vertente da reabilitação ao invés da demolição. Estas metodologias recorrem a ensaios não destrutivos, como é exemplo o Pilodyn, o Resistógrafo, o Ultra-sons, etc., referindo também o recurso a ensaios destrutivos, cuja utilização terá de ser uma questão muito bem ponderada devido às consequências que implica. Por fim, apresenta-se um caso real de estudo em que se analisa um pavimento em madeira através da aplicação de metodologias de ensaio não destrutivas e destrutivas a amostras recolhidas. O objetivo será determinar o estado de conservação da madeira e algumas características físicas tais como a sua densidade e os módulos de elasticidade, sendo posteriormente efetuada uma análise estrutural. Os resultados obtidos permitem efetuar uma avaliação qualitativa do estado geral do pavimento sendo as principais conclusões que o pavimento se encontra atacado por parte de insetos xilófagos e apresenta problemas a nível de verificação a estados limite de utilização, deformação e vibração, fator que condiciona a possível utilização do pavimento.

Brazilian fruit pulps as functional foods and additives: Evaluation of bioactive compounds

Eight tropical fruit pulps from Brazil were simultaneously characterised in terms of their antioxidant and antimicrobial properties. Antioxidant activity was screened by DPPH radical scavenging activity (126–3987 mg TE/100 g DW) and ferric reduction activity power (368–20819 mg AAE/100 g DW), and complemented with total phenolic content (329–12466 mg GAE/100 g DW) and total flavonoid content measurements (46–672 mg EE /100 g DW), whereas antimicrobial activity was tested against the most frequently found food pathogens. Acerola and açaí presented the highest values for the antioxidant-related measurements. Direct correlations between these measurements could be observed for some of the fruits. Tamarind exhibited the broadest antimicrobial potential, having revealed growth inhibition of Pseudomonas aeruginosa. Escherichia coli, Listeria monocytogenes, Salmonella sp. and Staphylococcus aureus. Açaí and tamarind extracts presented an inverse relationship between antibacterial and antioxidant activities, and therefore, the antibacterial activity cannot be attributed (only) to phenolic compounds.