ESB - Dissertações de Mestrado / Master Dissertations
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- Metagenomic analyses of pristine, Arctic environments for the risk assessment : evaluation of antibiotic resistance genes presencePublication . Diniz, Vitor Gaspar; Dziewit, Lukasz Jacek; Decewicz, PrzemysławAntimicrobial resistance is a major global public health challenge, and it is essential to understand its origin and spread in different ecosystems. This study investigated microbial diversity and antibiotic resistance genes in soils from the Arctic, an extreme and underexplored environment. Classical microbiological and metagenomic approaches were used to characterize the microbiota and assess the presence of antibiotic resistance genes. The results revealed significant microbial diversity and the presence of resistance genes, even in areas with no apparent anthropogenic impact. The metagenomic analysis indicated that resistance may be associated with natural mechanisms of microbial adaptation, reinforcing the need for environmental monitoring to better understand the risks of antimicrobial resistance spread. These findings contribute to the assessment of the ecological risk of antimicrobial resistance.
- Characterization of codY regulon in staphylococcus epidermidis culturesPublication . Pinheiro, Ana Paula de Souza Cristal; França, Ângela Maria Oliveira de SousaStaphylococcus epidermidis, a commensal bacterium found on human skin and mucous membranes, has emerged as an opportunistic pathogen responsible for nosocomial infections. Given its increasing clinical relevance, the importance of studying and understanding the genetic mechanisms behind its infectious capabilities has become more evident. CodY, a global regulator present in many Gram-positive bacteria, has been previously identified as a potential controlling factor of mechanisms that contribute to infections caused by S. epidermidis. Considering this, a comparative analysis of the absence (strain with codY gene deletion) and presence (wild type strain) of this key regulator becomes essential to understand how differential gene expression coordinates the microorganism's metabolism, nutrition, and virulence expression. Using high-throughput Illumina RNA-seq technology, we identified 19 differentially expressed genes: 18 repressed and 1 activated by CodY, when comparing the mutant to the wild-type strain. This reduced number of differentially expressed genes, combined with the predominance of repression, contradicted our initial expectations and prompet us to explore possible explanations. Given that CodY activity is modulated by intracellular levels of branched-chain amino acids (BCAAs) and GTP, metabolites known to fluctuate with growth, we hypothesized that the time point chosen for RNA-seq (16 hours) might have coincided with a phase of nutrient depletion, leadind to reduced CodY activation. To test this codY expression was evaluated throughout the bacterium growth phase, revealing stable transcript levels that were not influenced by nutritional supplementation. We also detected transcriptional variability among samples cultivated under identical conditions, suggesting heterogeneity in gene expression within the bacterial population. Finally, in silico analysis of CodY-binding motifs suggested that regulation may involve non-canonical sequences, pointing to an alternative control mechanism. These results contribute to an initial understanding of CodY’s role in S. epidermidis.
- Detergent-free supercritical carbon dioxide : assisted protocol for the production of sustainable and highly preserved decellularized porcine meniscus for orthopedic applicationsPublication . Ho, Chou I; Costa, João Pedro Bebiano e; Oliveira, Ana Leite de Almeida Monteiro deThe development of biologically functional scaffolds for meniscal applications using the extracellular matrix (ECM) as a source has been shown to be a promising alternative to tissue engineered strategies. Therefore, there is a need to achieve efficient decellularization strategies that can effectively eliminate cellular and genetic materials to minimize immunogenic responses, while maximally preserving the ECM to support subsequent cellular activities essential for tissue regeneration. Conventional decellularization methods generally rely on harsh chemicals that are associated with ECM damage, cytotoxic residues, and prolonged processing times, compromising tissue integrity and biocompatibility. These limitations have driven the exploration of supercritical carbon dioxide (scCO2)4a green and minimally invasive technology4as an alternative decellularization agent to develop faster, more cost-effective, and environmentally sustainable approaches. This study evaluated a series of scCO2-assisted decellularization protocols, where operational parameters including number of cycles, depressurization speed, applied pressures, exposure time, co-solvent application, as well as washing strategies with distinct solvents, washing modes, and physical interventions were tested. Decellularization efficiency was assessed via quantification of residual dsDNA. Moreover, ECM preservation was assessed using biochemical assays for glycosaminoglycans (GAGs), soluble collagen, and insoluble collagen, alongside Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR) and the corresponding chemometric analysis. These assessments for ECM preservation were further conducted after sterilization applying scCO2 with the additive NovaKillTM, a mixture comprising peracetic acid (PAA) and hydrogen peroxide (H2O2). The scCO2-assisted protocol4employing nine cycles at 160 bars and integrating freeze-thaw, direct sonication with an ultrasonic processor, and lyophilization4 achieved the highest decellularization efficiency, with residual dsDNA of 88.74 ± 10.25 ng/mg of dry sample. Additionally, biochemical assay results demonstrated great preservation of ECM, with only minimal degradation suggested by spectroscopic analysis. Following sterilization, no significant differences were observed in the ECM quantification results and FTIR spectral analysis, indicating an optimal ECM preservation. These findings highlight the potential of generating decellularized ECM (dECM) by scCO2-assisted approaches as a promising candidate for advanced meniscal regenerative applications.
- Development of fish analogues with algae : innovation and characterizationPublication . Maia, Daniela Dias; Vieira, Dalila da Assunção MaiaThe growing need for alternatives to marine-derived products has driven the development of two fish analogues: one inspired by sardines and the other by salmon pâté. This choice was made based on their popularity in Portugal, where sardines are culturally iconic and salmon pâté is widely consumed. The primary aim was to create products that closely resemble the nutritional and sensory attributes of their conventional counterparts, with a focus on the use of algae and plant-based protein, particularly pea protein. Ingredients—such as algae and hydrocolloids—were selected based on functionality tests, and the formulations were optimised in terms of texture, flavour, and appearance. The prototypes were characterised for pH, water activity, texture, and lipid profile. The sardine analogue (SA) exhibited a firm and cohesive texture (hardness: 348 g; cohesiveness: 27%), while the salmon spread analogue (SSA) demonstrated good spreadability (adhesion work: −192 g·s). Sensory acceptance of the SA was assessed by 21 participants using a Just-About-Right (JAR) and a hedonic scale, yielding average ratings close to the ideal (3) for flavour (3.1), freshness (3.0), marine aroma (3.05), and texture (2.62). The overall appreciation scored 6.5 out of 9 points. From a nutritional standpoint, both products are eligible for nutritional claims. The SA provides 18 g of protein per 100 g and the SSA 12 g/100 g, supporting the claim "source of protein". Both are also sources of dietary fibre and iodine, and their omega-3 fatty acid contents meet the criteria for the "rich in omega-3" claim, with SA containing 591 mg and SSA 1114 mg, both well above the regulatory threshold. Life Cycle Assessment (LCA) revealed that environmental impacts varied depending on the category analysed. Both analogues showed advantages over their conventional counterparts in terms of CO₂ emissions and land use, although they exhibited higher impacts in other categories of environmental impact. It is concluded that it is feasible to develop seafood analogues that are both sensorially acceptable and nutritionally balanced. While their overall environmental footprint may not be lower than that of conventional products, these analogues contribute to more diverse diets and help avoid the direct degradation of marine ecosystems.
- Análise comparativa da vida útil da borra de café em diferentes embalagensPublication . Pires, Gabriela Maria Cunha; Teixeira, AntónioA borra de café, é um dos subprodutos produzidos pela indústria do café, que pela sua composição em diversos compostos bioativos, revela-se um potencial substrato para o desenvolvimento de produtos de valor acrescentado. No entanto, os desafios logísticos associados à sua recolha, assim como a rápida degradação microbiológica, representam uma barreira à conservação e subsequente reutilização. Neste contexto, o presente trabalho teve como objetivo realizar uma análise comparativa da estabilidade e vida útil da borra de café armazenada em cinco embalagens diferentes ao longo de 15 dias. As embalagens selecionadas para o estudo apresentam um conjunto de propriedades promissoras à redução da proliferação de microrganismos e durabilidade da borra, e foram as seguintes: amido de milho (E1), kraft (E2), kraft com 50g de sílica gel (E3), kraft com 25g de cloreto de cálcio (E4) e polietileno a vácuo (E5). Para cumprir com o objetivo proposto, foram colocadas 100g de borra de café, proveniente da recolha após 24h de atividade de produção de uma cafetaria, em cada embalagem, e monitorizaram-se alguns parâmetros físico-químicos (cor, pH, humidade, atividade da água) e microbiológicos (microrganismos a 30 ºC e bolores e leveduras) da borra armazenada aos dias 0, 2, 5, 7 e 15. Paralelamente, foi realizada uma análise ao ciclo de vida, com base na compilação de dados disponíveis na literatura, e com o propósito de avaliar o impacto ambiental associado a cada material das embalagens em estudo. Os resultados deste projeto, evidenciaram que a E5, foi estatisticamente mais eficaz na preservação dos parâmetros da cor e do pH, principalmente devido à ausência de oxigénio. A E1 e a E4, permitiram uma maior redução da humidade e atividade da água, justificada pela elevada taxa de transferência de vapor de água e capacidade dessecante do cloreto de cálcio, respetivamente. Do ponto de vista microbiológico, comprovou-se que a borra tem uma elevada carga inicial de microrganismos a 30 ºC, e no caso dos bolores e leveduras, a E5 foi a mais eficaz no controlo da proliferação dos mesmos ao longo do tempo. A análise do ciclo de vida, permitiu percecionar que a E5 apresenta uma menor pegada ambiental para as categorias de impacto consideradas, sobretudo pelo facto da produção do papel kraft e do amido de milho apresentarem uma elevada variabilidade e maior potencial de poluição em certas etapas. Em conclusão, este estudo demonstrou uma clara correlação entre o tipo de embalagem utilizada e a estabilidade da borra de café ao longo do tempo, sendo que a E5 parece ser a mais promissora para solucionar os desafios interligados com a reutilização desta matéria-prima. Apesar de tudo, é de extrema importância destacar que a borra presente na E5, apenas deverá ser utilizada na formulação de subprodutos que apresentem limites microbiológicos mais flexíveis que os definidos no guia do INSA.
- Cutting-edge hybrid dressing materials : combining medical textiles and decellularized extracellular matrices for burn wound applicationsPublication . Vale, Inês Carvalho do; Ribeiro, Viviana Pinto; Oliveira, Ana Leite de Almeida Monteiro deThe World Health Organization (WHO) estimates that each year, some 11 million people suffer burn injuries, and that these incidences are responsible for about 180,000 deaths. Regardless of the underlying causes, burn injuries result in complicated wounds that are difficult to cure and have a higher death rate. The present thesis describes the development of hybrid dressing materials, combining silk fibroin (SF)-based medical textiles and highly-preserved decellularized porcine small intestine (HP-dPSI) as a possible advanced solution for burn wounds treatment. The thesis discusses the capability of creating a multilayer dressing with stable integrity, superior biological performance and mechanical properties. To do so, a silk sericin (SS) hydrogel-based interface layer was proposed due to the biocompatibility of the protein and well-established glue-like properties. The decellularization process of the PSI is critical as it ensures the removal of cellular material preventing adverse immune reactions while preserving the extracellular matrix (ECM). The decellularization process significantly reduced the DNA content in the PSI, indicating effective removal of cellular material. The goal was to reduce DNA content to levels below 50 ng/mg of dry tissue, which was achieved, demonstrating the process's success. This result was also confirmed by histological analysis, in which there was a marked reduction in cellular components in all the sections observed. Regarding the hybrid biomaterial, the SS-based hydrogel was crucial for integrating the serous and submucous layers of the dSI into the SF textile structure, which maintained its structural integrity and binding properties after rehydration. SEM microscopy was used to evaluate the morphology of the HP-dPSI, the SF-based textile and ultimate hybrid material. The preservation of decellularized ECM was confirmed for the distinct surfaces (serosa and submucosa) and cross-section of the HP-dPSI, maintained after conjugation with the remaining layers of the multilayer hybrid materials. This is critical for the material's functionality as a wound dressing. The results obtained from mechanical properties showed that the tested materials presented consistent strength and flexibility, essential for adapting to the wound environment and supporting healing. The HP-dPSI, particularly its fracture point and maximum elongation, are crucial to its performance as a burn dressing. In comparison, SF-based textiles showed consistent strength and flexibility. All combined, the mechanical performance of the hybrid materials showed promising results, especially in adapting to the wound environment and supporting healing. These findings highlight the potential of the proposed hybrid dressing materials to provide an effective treatment for burn wounds by combining the structural benefits of SF-based textiles and biologically active ECM from HP-dPSI.
- Validação de sistemas de inspeção por visão artificial e implementação de processos de monitorização em linhas de engarrafamentos de vinhosPublication . Almeida, João Miguel Pereira Dias Vasques de; Santos, Cláudia Maria da SilvaNo âmbito da componente curricular de Tese do programa de Mestrado em Engenharia Alimentar na Escola Superior de Biotecnologia da Universidade Católica Portuguesa do Porto, foi selecionada a opção de realizar em formato de estágio curricular, tendo sido escolhido o departamento de Qualidade de Materiais e Produto Acabado (QMPA) da empresa Symington Family Estates, Vinhos, S.A. (SFE). Durante o estágio curricular foi desempenhada a função de Assistente de Controlo da Qualidade na Quinta de Santo António, Vila Nova de Gaia, no Porto. O presente relatório tem como objetivo descrever as atividades desenvolvidas ao longo dos cinco meses de estágio, de fevereiro de 2024 até julho de 2024. Este período permitiu adquirir uma visão prática do ambiente fabril e aperfeiçoar conhecimentos apreendidos ao longo do percurso académico, contribuindo adicionalmente para aquisição de competências técnicas na área de controlo de materiais de embalagem, controlo do processo de engarrafamento e acabamento, bem como uma grande melhoria de soft skills, como a comunicação, gestão de tempo e resolução de problemas. O relatório de estágio é composto pela apresentação da empresa de acolhimento e do departamento QMPA e descrição das tarefas realizadas. Estas tarefas incluíam a receção de materiais e controlo do processo de engarrafamento e foram realizadas diariamente, de acordo com as atividades da empresa. Adicionalmente, foi efetuada a validação de equipamentos de inteligência artificial acoplados às linhas de produção, nomeadamente de um inspetor de defeitos no vidro de garrafa vazia, inspetor de nível de enchimento e de progressão capilar de vinho na rolha. Por fim, retiraram-se conclusões e foram feitas reflexões sobre o trabalho desenvolvido e propostas futuras. Em suma, foi uma experiência extremamente enriquecedora, que proporcionou um contato direto com o mercado de trabalho e permitindo o crescimento em diversos aspetos, tanto no âmbito profissional quanto no pessoal.
- Integration of soft tissues with intraosseous protheses : a tissue engineering approachPublication . Lima, José Miguel Sousa; Scalera, Francesca; Piccirillo, ClaraEnhancing the quality of life for individuals with lower limb amputation is a fundamental goal in prosthetic development and rehabilitation. Biomaterials that replicate the properties of natural tissue can play a pivotal role in improving the integration and functionality of prostheses. This thesis focuses on the synthesis and characterization of cryogels based on Gelatin Methacrylate (GelMA), designed specifically to enhance the integration of soft tissue with osseointegrated transcutaneous lower limb prostheses. Methacrylation reactions were employed to create GelMA and CsMA derivatives, with methacrylation levels reaching 55% and 16%, respectively, as confirmed through Fourier-transform infrared (FTIR) spectroscopy and ninhydrin assays. Further optimization included incorporating Polyethylene Glycol Diacrylate (PEGDA) and Pectin with GelMA. Stability assessments highlighted the superior stabilizing effect of PEGDA, with GelMA_PEGDA cryogels exhibiting only 20% weight loss over 14 days compared to 40% for GelMA alone. By contrast, GelMA_Pectin formulations degraded quickly. Swelling tests indicated that the increased crosslinking conferred by PEGDA reduced water uptake, resulting in controlled swelling behavior suitable for moisture-sensitive applications. Scanning Electron Microscopy (SEM) analysis showed a porous network in all cryogels, with GelMA_PEGDA presenting smaller, more uniform pores4an ideal structure for promoting cell infiltration. Mechanical testing further supported the potential of GelMA_PEGDA cryogels, revealing enhanced viscoelastic properties and strong compressive strength, both of which are crucial for load-bearing tissue applications. Following refinements in preparation protocols, the GelMA_PEGDA cryogels exhibited high biocompatibility, as evidenced by substantial cell viability and proliferation rates. Overall, GelMA_PEGDA cryogels exhibit a promising combination of stability, mechanical strength, and biocompatibility, making them well-suited for further investigation as scaffolds to support soft tissue integration with the abutment in transcutaneous lower limb prostheses.
- Evaluation of activated sludge and treated water at wastewater treatment plant Gaia coastline : performance indicators using protozoa and filamentous bacteriaPublication . Madrigal, Luisa Fernanda Real; Barreiro, Ermelinda de Lurdes LopesThis study analyzed the operational dynamics of the Wastewater treatment plant (WWTP) SIMDOURO S.A. in Águas de Gaia, focusing on microbial communities in the activated sludge system. Design aids odor control, but efficiency depends on the microbial population. Initially, the activated sludge process was described using the Sludge Biotic Index (SBI) to evaluate how various factors influence microfauna. The goal was to find reliable indicators for daily monitoring and assess seasonal variations in microbial communities. Sludge, Affluent, and effluent samples were collected in fall, winter, and spring, while sensors monitored dissolved oxygen (O2) levels, temperature (Temp), and other parameters. Microscopic analysis quantified protozoan and metazoan populations, and laboratory analyses provided data on Total Suspended Solids (TSS), pH, Biochemical Oxygen Demand (COB), Chemical Oxygen Demand (COD), and nutrients like Ammonium (NH₄⁺) and Nitrate (NO₃⁻). Statistical analyses included t-tests, Wilcoxon tests, and Principal Component Analysis (PCA). Biological reactors 1 and 2 (T1 and T2) showed no significant differences, justifying their grouping. Seasonal shifts in microbial composition were noted: mobile ciliates dominated in autumn, while sessile ciliates prevailed in winter and spring. Key taxa such as Epistylis and Microthrix were identified, with SBI consistently at an ideal score of 10. PCA showed distinct seasonal clusters, and Modelo Linear Geral (GLM) indicated significant influences from seasonality and protozoan abundance on key parameters. Dominant filamentous bacteria included Nocardia spp. and Microthrix parvicella, correlated with specific Affluent conditions. Reliable ciliate indicators of system efficiency were identified, while flagellates and other organisms appeared sporadically. Future research should focus on advanced bacterial quantification methods, explore Artificial Intelligence (AI) and machine learning models, and extend the study into summer for a comprehensive understanding of microbial ecology.
- Analysis of sound-induced angiogenesis in the development of a 3D tumor modelPublication . Barbosa, Margarida Alice Leal Coelho; Raghunath, MichaelThe tumor environment arises the appearance of cells with altered mechanisms and uncontrolled growth. The use of mechanisms that result in their expansion is what allows them to grow indefinitely and evolve. Tumor angiogenesis is one of the mechanisms used by cancer cells to survive, expand and establish their own environment. Through tumor cells, several factors are secreted in response to hypoxia and the need for nutrients, resulting from the rapid growth and multiplication typical of these cells. Angiogenesis, with the formation of new blood vessels, arises not only to meet these needs, but also to create a microenvironment that facilitates the invasion and spread of cancer cells to other tissues, playing a crucial role in the growth and metastasis of tumors. By creating a 3D model where the tumor environment is present, this work aims to observe this mechanism. To do so, it uses bioacoustics, through sound waves and vibrations at a certain frequency and acceleration, to determine cellular rearrangement. Thus, through a 3D model consisting of two distinct layers, one formed by endothelial cells in combination with mesenchymal cells, and a top layer, consisting of breast cancer cells, this work aims to analyze the influence of the presence of cancer cells, namely at the level of the promotion of sprouting by endothelial cells, and thus the initiation of the angiogenic mechanism.