Escola Superior de Biotecnologia
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- Design of paper-based analytical devices for chemical and biochemical assays of biomarkers in biological fluids of non-invasive collectionPublication . Ferreira, Francisca Teixeira Soares da Mota; Mesquita, Raquel Beatriz Ribeiro de; Rangel, Antonio Osmaro Santos SilvaEnsuring basic healthcare access around the world remains a challenge, particularly in lowincome regions. In response, international initiatives such as the Sustainable Development Goals (SDGs) and the World Health Organization (WHO) guidelines emphasize the need to develop innovative, accessible, and cost-effective diagnostic tools. Point-of-care testing has emerged as an ideal solution, enabling rapid and decentralized analysis. Among these type of devices, microfluidic paper-based analytical devices (µPADs) have gained attention due to their affordability, portability, and ease of use, making them a particularly valuable tool in resource-limited locations. The purpose of the work presented throughout this thesis was to design and develop innovative point-of-care methodologies based on the µPAD approach for the determination of several healthrelated parameters, that could serve as an adding tool in the diagnosis and monitoring of several health conditions. The use of biological samples of non-invasive collection, such as saliva and urine, enhances diagnostic accessibility, particularly in point-of-care settings where traditional sample collection may be impractical or even impossible. Additionally, this thesis also explores the use of colorimetric detection methods, a straightforward approach to quantifying the targeted analytes by producing visible colour changes. To further improve the specificity and accuracy of these diagnostic tools, enzymatic reactions were incorporated into some of the developed µPADs. The first developed device was dedicated to the quantification of total iron in urine samples by using the colorimetric reaction of bathophenanthroline with iron (II) coupled with hydroxylamine, a well-known reducing agent capable of converting iron (III) in iron (II). To handle the potential colour of the urine samples, a sample blank was included in the device. This feature was vital to ensure the applicability of the developed μPAD, as urine may present a wide variability of colour range, from light yellow to brownish. The determination of nitrate was also accomplished in urine samples with a newly developed µPAD. This device included the enzymatic reaction of nitrate reductase to perform the conversion of nitrate to nitrite and the Griess reagent which provided the colorimetric detection of the resulting nitrite. In order to delay the vertical flow and increase the extent of the enzymatic reaction, a hydrophilic membrane layer was also incorporated into the device. The small porosity of this membrane also led to the retention of the compound responsible for the colour of urine, enabling a direct analysis of the samples. The next devices developed performed the quantification of NHX and urea in saliva samples but with a more complex approach. Not only were the devices composed of four layers, but the detection relies on the diffusion of NH3 (g) through a gas-diffusion membrane to produce a colorimetric change of a pH indicator. The hydrophobicity of this membrane also helped eliminate possible interferences of the saliva sample in the colorimetric reaction, since it did not allow the sample to pass through the membrane and reach the colour reagent. The urea determination was accomplished by including urease in the device, since it selectively catalyzes the dissociation of urea in ammonia and carbon dioxide. The urease activity determination was accomplished using a similar structure and reactions to the one used for the urea µPAD. However, the determination itself was achieved using the kinetic capabilities of the enzyme by correlating to the urease activity with the variation of the signal obtained between two different enzymatic reaction times. This strategy not only allowed a more accurate quantification but also suppressed the influence of NHX already present in the samples. The last device developed using the µPAD structure approach was to quantify glucose in saliva samples. With only two layers in its composition, this device uses a combination of two enzymatic reactions. First, glucose oxidase converts glucose into gluconic acid and hydrogen peroxide, then followed by the release of oxygen from hydrogen peroxide performed by peroxidase. The colorimetric detection is accomplished with the oxidation of o-dianisidine. In this work, a correlation between the concentration of glucose in saliva and blood was also successfully established, using #5 saliva samples of diabetic patients and the correspondent glucometer measurements provided by the patients. A rather different approach was used to perform the semi-quantitative analysis of E. coli DNA. Instead of developing a µPAD, an option was made to design a barcode-style lateral flow strip that would allow the semi-quantitative detection of E. coli. The amplification of the DNA in the sample was performed using the Loop-Mediated Isothermal Amplification (LAMP) technique since it can be performed at a constant temperature and provides high sensitivity and specificity results. The developed methodology, although requiring further optimization, showed reliable performance and offers a rapid, cost-effective method for E. coli detection. Overall, this thesis demonstrates the potential of microfluidic paper-based analytical devices and lateral flow assays as innovative, cost-effective diagnostic tools for point-of-care applications. By addressing critical challenges such as sample complexity, reagent stability, and measurement accuracy, the developed devices enhance accessibility to reliable testing, particularly in resourcelimited settings. Their affordability and ease of use further emphasize their role in expanding diagnostic capabilities outside traditional laboratory environment.
- Ecology and antimicrobial resistance of Ralstonia spp. in drinking waterPublication . Mayhua, Felix Pompeyo Ferro; Rodrigues, Célia Maria Manaia; Moreira, Ivone Cristina VazAs Betaproteobactérias são ubíquas no ciclo urbano da água, com múltiplas oportunidades de interacção com os seres humanos. A combinação das propriedades fisiológicas e ecológicas de algumas Betaproteobactérias explica sua capacidade de sobreviver no meio ambiente, persistir após desinfecção da água potável e, por vezes, colonizar animais, incluindo humanos. Versatilidade metabólica, capacidade de formar biofilme e tolerância a agentes antimicrobianos e metais, são exemplos de características subjacentes à ubiquidade acima mencionada. Porém, esta informação não se encontra claramente disponível na literatura científica, pelo que um dos objectivos deste trabalho, foi o de investigar e sistematizar informação sobre a ocorrência de Betaproteobactérias em água potável. Três géneros bacterianos de Betaproteobacteria, cuja presença em água exemplifica a referida ubiquidade são Achromobacter, Burkholderia e Ralstonia, por vezes relacionados a surtos de infecção associados a água potável. Ralstonia spp. despertou particular atenção visto ter sido recentemente isolada no grupo de fontes tão diversas como água mineral ou água de esgoto hospitalar. Ralstonia spp., principalmente as espécies Ralstonia pickettii e R. mannitolilytica, têm sido recentemente associadas casos de infecção e esta ubiquidade suscitou interesse. Membros destas espécies podem apresentar tolerância a metais, antibióticos e desinfetantes, o que pode contribuir para a sua ubiquidade e eventual severidade como agentes infecciosos. Neste estudo procurou compreender-se se existia uma inter-relação entre resistência a metais e antibióticos e como é que tal poderia influenciar o comportamento das estirpes. O trabalho experimental envolveu a caracterização de estirpes das espécies R. pickettii e R. mannitolilytica, isoladas de água de esgoto hospitalar, água da torneira e de água mineral engarrafada, e teve como objectivo identificar possíveis associações de fenótipos de resistência a antibióticos e metais e outros tipos de stresse, bem como as suas bases genéticas. Especificamente, num primeiro ensaio estudaram-se isolados de água de Ralstonia pickettii e R. mannitolilytica de efluente hospitalar (n = 2), água da torneira (n = 2), água mineral (n = 1), e avaliaram-se os fenótipos de resistência a antibióticos e a metais bem como resposta a desinfecção e alguns parâmetros de cinética de crescimento. Para atingir esses objetivos, utilizou-se o método de difusão em disco e de microdiluição para determinar concentrações inibitórias mínimas (MIC), fizeram-se curvas de crescimento em meio Mueller-Hinton suplementado com os agentes antimicrobianos de interesse, de modo a estudar os parâmetros de cinética de crescimento, avaliou-se a capacidade de formação de biofilme e testou-se o comportamento em presença de radiação ultravioleta, hipoclorito de sódio e peróxido de hidrogénio. Este estudo sugeriu existir uma associação entre resistência a gentamicina (MIC > 256 mg/L) e tolerância aumentada a arsenito (MIC = 1.4 mmol/L) A hipótese de se tratar de resistência cruzada foi descartada visto, concentrações sub-inibitórias de gentamicina ou de arsenito diminuíram significativamente a taxa de crescimento e produção de Ralstonia spp. embora apenas o arsenito causasse um aumento significativo na fase de lag. Além disso, em estirpes resistentes a gentamicina, a formação de biofilme foi estimulada na presença de aminoglicosídeos ou de arsenito. A desinfecção com radiação ultravioleta ou hipoclorito apresentou taxas de inativação semelhantes nas linhagens resistentes e suscetíveis a gentamicina. Em contraste, a mesma dose de peróxido de hidrogénio causou inativação mais rápida em estirpes sensíveis a gentamicina do que em resistentes. A associação entre resistência a gentamicina e arsenito foi explorada num grupo mais alargado de R. pickettii, pelo que se incluíram 37 estirpes de, 14 de água mineral, 17 de água de torneira e 6 de esgoto hospitalar. Analisaram-se os perfis de resistência a antibióticos e a arsenito, conforme se descreve acima e fez-se o rastreio de determinantes genéticos de resistência selecionados, incluindo os relacionados com Elementos Conjugativos Integrativos (ICE), plasmídeos, e genes associados com uma bomba de efluxo e resistência ao arsénio. A maioria dos isolados (32/37) era resistente a gentamicina, beta-lactâmicos e colistina. Porém, nem todos os isolados eram resistentes a gentamicina e a associação deste fenótipo com o aumento da tolerância ao arsenito foi confirmada. Esta divisão de estirpes resistentes a gentamicina e arsenito vs. suscetíveis concidia com a separação de dois grupos filogenéticos, com base na análise do gene rRNA 16S. Os isolados resistentes a gentamicina e arsenito apresentaram ICEs e os genes arsH e acr3, relacionados à resistência ao arsenito. A maioria dos isolados de R. pickettii (36) apresentava um ou dois plasmídeos com tamanhos entre 77 e 260 kpb. As sequências de aminoácidos da bomba de efluxo, cujo gene amplificou em todas as estirpes, diferiam em isolados resistentes e suscetíveis. Embora os genótipos de estirpes resistentes e suscetíveis a gentamicina diferissem de forma consistente com os fenótipos e com a distinção filogenética de ambos os grupos, não foi possível encontrar uma explicação genética para o fenótipo observado, nem para a associação de resistência arsenito e gentamicina. Porém, para todos os elementos genéticos analisados verificou-se uma divisão clara entre ambos os grupos, sugerindo que abordagens de genómica comparativa poderiam contribuir para elucidar as bases genéticas dos fenótipos em análise – resistência vs. suscetibilidade a gentamicina e arsenito. Neste sentido, compararam-se os genomas de uma estirpe resistente e de uma estirpe suscetível e com especial enfoque nas categorias funcionais “Transporte Membranar”, “Stress e Defesa” e “Virulência” analisaram-se os genes presentes apenas na estirpe resistente ou as mutações detetadas entre ambos. Esta análise mostrou haver algumas funções que podem explicar quer a capacidade para adquirir ou desenvolver novos mecanismos de resistência. Este tipo de estudo permite elucidar os processos de desenvolvimento ou aquisição de resistência em espécies ubíquas caracterizadas por mecanismos de resistência assumidamente intrínseca, visto serem comuns à maioria de membros de uma espécie.
- Functional proteins and peptides obtained from fish by-productsPublication . Ghalamara, Soudabeh; Pintado, Maria Manuela Estevez; Silva, Sara Nunes da Costa e; Ferreira, Carla Maria Carvalho Gil Brazinha de BarrosMarine processing industries face significant challenges due to excessive by-products, which contribute to both environmental and economic issues. By-products from fish processing, which can constitute between 30% and 70% of the fish's total weight, exacerbate these challenges. Activities like washing, thawing, cooking, and fishmeal production generate more wastewater, intensifying the industry's environmental impact. This research explored using fish by-products like sardine cooking effluent and codfish bloodwater (CFBW) in a sustainable, zero-waste approach. The functional proteins and peptides extracted from fish by-products enhanced their value. The research evaluated their properties and potential food uses, aligning with circular bioeconomy principles. Fish by-products were fractionated using membrane technology at laboratory and pilot scales, aligning with zero-waste and biorefinery approaches. Ultrafiltration (UF) membranes were used to extract bioactive peptide-enriched fractions from sardine cooking effluent and CFBW at the laboratory scale. The process involved analyzing the selective permeation of small peptides (<1 kDa) using different membranes. The membranes effectively rejected (>10 kDa) of proteins and peptides but had relatively low rejection of <1 kDa peptides, with UP010 from CFBW achieving a 2% rejection rate and GH from sardine cooking effluent operating at minimum pressure (1 bar) achieving a 23% rejection rate. Peptides from CFBW using MW and UP010 membranes demonstrated potent antioxidant activity (high ABTS+ and ORAC values). However, the peptide fractions from sardine cooking effluent using the GH membrane did not enhance antioxidant activity. Nevertheless, the peptide fractions from CFBW (UP010 membrane) and sardine cooking effluent (GH membrane, 1 bar pressure) inhibited E. coli growth. Peptide-enriched fractions from CFBW were successfully obtained using a pilot-scale process involving microfiltration (MF), UF, and reverse osmosis (RO). The process aimed to fractionate CFBW into protein- and peptide-rich fractions. Chemical and biological characterization revealed that CFBW and pretreated CFBW consisted primarily of proteins and peptides. UF membrane fractions had lower protein content but higher ash levels. The UF retentate with a MWCO of 50 kDa, followed by RO, displayed the highest antioxidant values, indicating the presence of potent antioxidants. Additionally, the UF permeate obtained with a MWCO of 50 kDa from the MF-PFG showed antimicrobial activity against E. coli. A pilot-scale integrated membrane process was developed to obtain protein- and peptiderich fractions from sardine cooking effluent. The process used UF, nanofiltration (NF), and RO combined with enzymatic hydrolysis. Specifically, the RO retentate fractions of NF retentate were derived from diluted sardine cooking effluent, diluted hydrolyzed UF retentate, and diluted non-hydrolyzed UF retentate. The process involved UF, nanofiltration (NF), and RO combined with enzymatic hydrolysis. Specifically, the RO retentate fractions of NF retentate fractions were derived from water-diluted sardine cooking effluent (sardine cooking effluent-NF-RO), water-diluted UF retentate hydrolyzed (UF-H-NF-RO), and water-diluted UF retentate non-hydrolyzed (UF-NH-NF-RO). UF-H-NF-RO showed enhanced antioxidant and antimicrobial activities. The UF-NH-NF-RO peptide fraction displayed significantly enhanced functional properties in terms of WHC, FBC, emulsifying properties, and foaming properties at pH 4. In vitro digestion analysis showed this fraction also had the highest antioxidant activity, and none of the fractions exhibited cytotoxicity. Finally, foamy fish sauces (FFSs) were developed and evaluated using protein/peptide and lipofish fractions obtained from centrifugation of sardine cooking effluent. The control sample (CS) used a butter-based sauce emulsified with lecithin. An alternative lipofish sauce (LS) substituted unsalted butter with fish oil and included lecithin as the emulsifier. Three other formulas, namely lipofish-sardine cooking effluent-NF-RO-sauce (LSS), lipofish-UFH-NF-RO-sauce (LHS), and lipofish-UF-NH-NF-RO-sauce (LNHS), replaced unsalted butter with fish oil and incorporated a combination of protein/peptide fractions and lecithin. Despite minor physicochemical differences, the sauce formulas showed improvements compared to the control, including enhanced fatty acid (FA) content and profile, slightly reduced viscosity, improved foaming capacity, and enhanced foam stability. In vitro digestion analysis demonstrated high recovery of FAs, with the formula LNHS exhibiting the highest efficacy in scavenging ABTS radicals, indicating strong antioxidant properties. Furthermore, the FFS received remarkable acceptance from the trained panelists, who highly praised its texture, aroma, color, and flavor. This study's findings on fish by-products have significant implications for sustainable development in the fish processing industry. The research showcased the potential to obtain functional ingredients through eco-friendly strategies, preserving their bioactivity. These outcomes promote responsible and sustainable fish processing, reducing waste and maximizing by-product utilization.
- Hortas públicas, biológicas e urbanas : que efeito nos comportamentos de saúde, na qualidade de vida e nas práticas ambientais?Publication . Nova, Paulo Ricardo Santos Pontes da; Silva, Margarida Carvalho e; Jane, Elisabete PintoIntrodução: Num mundo em crescente urbanização, caracterizado por uma forte poluição e ausência de espaços verdes, as hortas urbanas desempenham um papel importante contribuindo para a sustentabilidade ambiental, económica e alimentar das cidades. Estes espaços permitem também melhorar a paisagem, criando espaços para a socialização e a prática de atividades lúdicas ou desportivas e desempenham um papel pedagógico ao permitir o contacto da população urbana com a base da sua sobrevivência. O recente aumento deste tipo de estruturas, e a clara adesão das populações, tem despertado o interesse da comunidade científica. Neste sentido, vários estudos têm evidenciado um conjunto de benefícios nos indivíduos expostos à horticultura relativamente à saúde física e mental, à alimentação, ao bem-estar e à qualidade de vida. O presente trabalho torna-se pertinente dada a expansão recente destes espaços em Portugal e a ausência de estudos que tenham medido o seu impacto nos comportamentos de saúde e qualidade de vida dos utilizadores. Objetivos: Foram objetivos da presente investigação i) caracterizar os horticultores de uma horta urbana em termos do seu estado de saúde, comportamentos de saúde e práticas ambientais, bem como ii) avaliar o efeito da prática da horticultura nos comportamentos de saúde, qualidade de vida e práticas ambientais. Metodologia: O estudo foi realizado na horta do parque José Avides de Moreira, localizado no Centro Hospitalar Conde Ferreira, com uma amostra de 115 hortelões que estavam a iniciar a horticultura ou a tinham iniciado recentemente. A recolha de dados foi realizada por entrevista, através da aplicação de questionários semiestruturados elaborados para o efeito. Todos os participantes foram entrevistados no momento em que foram convidados para integrar o estudo e num segundo momento (n=102), que ocorreu sensivelmente seis meses após a avaliação inicial e cujo objetivo era avaliar eventuais alterações, devido à horticultura, nos comportamentos dos indivíduos. Resultados: Foram encontradas alterações relevantes e estatisticamente significativas da primeira para a segunda avaliação no que concerne aos parâmetros antropométricos, hábitos alimentares, prática de atividade física, hábitos tabágicos, qualidade de vida e práticas de sustentabilidade ambiental, tendo-se observado uma melhoria clara na segunda avaliação. Para o efeito da intensidade da exposição (até 3 horas vs. mais de 3 horas semanais) não foram, de uma forma geral, encontradas diferenças estatisticamente significativas entre os dois grupos. Contudo, foi possível constatar valores mais elevados no grupo associado a um maior tempo de exposição. Conclusões: A horticultura apresentou um efeito benéfico nos comportamentos de saúde, na qualidade de vida e nas práticas ambientais dos participantes. No futuro justifica-se a realização de estudos com períodos mais longos de seguimento de modo a identificar i) o impacto positivo máximo que a horticultura acarreta nas variáveis aqui estudadas e ii) outros eventuais tipos de benefício em áreas não abordadas na presente investigação.
- Isolation and characterisation of plastic polyethylene terephthalate (PET) degrading and polyhydroxyalkanoates (PHAs) producing bacteria from soil and waterPublication . Saidu, Muhammad Bashir; Gonçalves, David Manuel Flores; Castro, Paula Maria Lima e; Moreira, Irina Susana SousaThe demand for plastic has led to enormous plastic waste in the environment, which persist and negatively impact the ecosystems. Polyethylene terephthalate (PET) is one of the most common thermoplastic polymers available on the market. The concerns about plastic waste generated an interest in strategies to enhance its biodegradation and finding alternative polymers. In this work was investigated the possibility of using bacteria to degrade PET and to produce bioplastics (Polyhydroxyalkanoates, PHAs). Finally, the integration of the two processes was tested. Overall, the work aimed to investigate the potential to recycle PET into bioplastic using bacteria. The potential of bacterial consortia from various environmental samples to degrade PET granules in liquid matrix was investigated. The results revealed maximum PET granules degradation of 1.1 % by one of the tested consortia. PET degradation intermediate terephthalic acid (TPA) was not detected at the end of 55 days. Fourier-transform infrared spectroscopy (FTIR) results showed major spectral peak shifts and bends on PET chemical structure compared to non-inoculated control. The biodegradation of PET films buried in the soil (A), with mangrove plants (B), and bioaugmented with a bacterial consortium (C) was also investigated. The experiments were conducted for 270 days at ambient conditions. The results revealed no difference between treatments in the degradation, with a maximum weight loss of 0.118 % in the bioaugmented treatment. Nevertheless, Scanning Electron Microscope (SEM) and FTIR results indicated significant surface changes, spectral peak shifts, and stretches in PET chemical structures. Bacterial consortia isolated from the soil of the experimental treatments were assessed for degradation of PET monomers, TPA and monoethylene glycol (MEG), and intermediate Bis(2-hydroxyethyl) terephthalate (BHET). The consortia were inoculated in flasks V containing minimal media with 1000 mg/L TPA or BHET or1113 mg/L MEG as the sole carbon source. Results showed complete degradation of TPA and significant degradation of BHET (96.09%), and MEG (83.65%) by the consortia. In the second part of the study, bacteria were isolated from various environmental samples and screened for PHA production using Sudan Black B staining on colonies and smeared glass slides. Transmission Electron Microscope images were captured to confirm the intracellular PHA inclusions. A total of 35 isolates were screened for PHA, and 22 showed positive staining. The isolate showing higher levels of PHA synthesis (EC2-30-3) was identified based on 16S rRNA gene sequence as Bacillus sp. and selected for PET monomers degradation and fermentation cultures for PHA production. It was cultured in minimal (Moreira et al., 2013) media with 1000 mg/L TPA and 1113 mg/L MEG as the carbon source for eight days. The isolate grew better in media containing MEG, which was selected as a substrate model for PHA fermentation. To integrate PET monomers biodegradation and production of PHA, the isolate was cultured in 0.2 % MEG. A control with 0.2 % of glucose was prepared, and the cultures were incubated for 96 hours. Bacillus sp. EC2-30-3 showed higher PHA accumulation in media supplied with MEG (40.31%) than glucose (25.53%). This is the first report showing that Bacillus sp. uses PET monomer as carbon source to produce a biopolymer. FTIR results of the extracted PHA identified its functional units as C–H, CH3, C=O, and C–O groups. The absorption bands obtained are closely related to the structure of PHB. The study thus confirmed the ability of the isolated bacteria to degrade PET monomers and produce biopolymers. The results of this work open the possibility for upscaling the use of bacteria to mitigate the impact of PET on the environment while producing environmentally friendly bioplastics.
- Relatório de actividade profissionalPublication . Oliveira, Gonçalo Nuno Santos Mercier e; Castro, Paula Maria Lima e