ESB - Dissertações de Mestrado / Master Dissertations
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- Exploring the discriminatory potential of intestinal microbiome for Alzheimer's disease detectionPublication . Apolinário, Eduardo Guarniero; Rodrigues, Pedro Miguel de Luís; Batista, Patrícia Sofia PinhançosAlzheimer’s Disease (AD) is a progressive neurodegenerative disorder marked by cognitive decline, commonly linked to the accumulation of amyloid plaques and tau tangles in the brain. Recent studies suggest that gut microbiota plays a role in AD pathogenesis, with gut dysbiosis potentially contributing to neuroinflammation through the gut-brain axis. This thesis explores the discriminatory potential of gut microbiota composition between AD patients and healthy controls (HC), aiming to identify microbiome-based biomarkers for early AD diagnosis. The clinical and demographic analysis included age, sex, apolipoprotein-E (ApoE4) allele presence, and body mass index (BMI), with significant age differences and ApoE4 allele frequency observed between AD and HC groups. Microbiome analysis examined alpha diversity indices (Shannon and Simpson) and taxonomic composition, uncovering specific microbial shifts, particularly within Escherichia-Shigella and Adlercreutzia, that may be associated with AD pathology. Machine learning models were used to assess the predictive power of microbiome features for AD, incorporating clinical variables. The models were optimized for accuracy and robustness through feature selection and dimensionality reduction (data fusion). The best performing model, evaluated through Leave-One-Out Cross-Validation (LOOCV), achieved an AUC of 0.88 and an accuracy of 0.89, reflecting an improvement over the model in the referenced study, which attained a maximum AUC of 0.83. These scores underscore the model's enhanced discriminatory capacity. These findings underscore the gut microbiota’s potential in AD biomarker discovery, with identified microbial alterations offering insights for future diagnostic and therapeutic research. Further studies are recommended to refine these predictive models and explore the microbiome’s influence on AD progression, advancing towards a reliable and accessible microbiome-based diagnostic tool.
- Pseudomonadota on human skin : effects of antimicrobial agentsPublication . Murugesan, Saravanan; Moreira, Ivone Cristina Vaz; Ribeiro, Inês Maria Melo Vaz PintoPseudomonadota are recognized as important harbours of antibiotic resistance, and some of them are also ubiquitous bacteria able to survive in multiple environments. The aim of this study was to understand if bacteria of the phylum Pseudomonadota that are common colonizers of a healthy human skin may be carriers of antibiotic resistance, and if their survival may be affected by the use of cosmetics or antimicrobial agents and if carbapenems resistance among Acinetobacter species is affected by phylogenomics. To answer this question, it was first done an analysis of the human healthy skin bacterial diversity based on metagenomic data available in public databases, and identified the most common Pseudomonadota genera and species. From this analysis, was identified the genus Acinetobacter as one of the most frequent in human skin, namely the species Acinetobacter johnsonii, Acinetobacter lwoffii and Acinetobacter junii. The genomes of those three species were collected from public databases (n=232) and compared in terms of antibiotic resistance and virulence profiles, aiming to understand if the resistance/virulence profiles were determined by the species or if other factors, such as the phylogeny or the strains origin (clinical, clinical associated, and environmental) may interfere. Based on this genome’s comparison it was possible to conclude that they are important carriers of antibiotic resistance genes. The strains from environmental origin presented a higher diversity of resistance genes, while the strains from clinical origin have poorer profiles of resistance but higher abundance. The blaNDM positive genomes (n=23) were processed to understand the genomic environment of the carbapenemase gene and if it varies according to the species or the strain origin. It was observed that the blaNDM gene was mainly associated to mobile genetic elements, namely the IS66 family, and associated with other genes of resistance (e.g. aminoglycosides, macrolides). Some Acinetobacter isolates in the laboratory culture collection, all from environmental origin, were tested for their tolerance to antiseptics (hydrogen peroxide, iodine, bleach, water) and cosmetics, using the disk diffusion method. For these isolates it was not possible to infer any correlation between the antibiotic resistance profile and the tolerance to cosmetic compounds, but it was observed some variance between strains in the tolerance to hydrogen peroxide. In the future, it would be interesting to extend these tests to strains from different origins (e.g. skin isolates) and test higher concentration of the cosmetics.
- Insect-derived bioactive peptides with protective potential upon brain disordersPublication . Mesias, Ana Sofia Belino; Silva, Sara Isabel Macedo Baptista da; Pintado, Maria Manuela EstevezIn an increasingly ageing world where neurodegenerative diseases (NDs) are exponentially rising, research into more effective and innovative treatments is paramount. Bioactive peptides (BPs) emerge as promising compounds with revolutionary potential in treating NDs, particularly in prevalent conditions like Alzheimer's disease (AD). The biological potential of these compounds is primarily attributed to their enhanced cellular penetration, low toxicity, and antioxidant properties bio-linked to neuroprotective effects. BPs can be sourced from common dietary origins or emerging sources like edible insects. However, to isolate active BPs with beneficial biological effects they must first be released from their parent protein, followed by a purification process. While in silico approaches can predict a BPs’ potential bioactivity and structural characteristics, in vitro, cell-based, and in vivo assays should be conducted to validate these properties. Therefore, this thesis presents the extraction of Tenebrio molitor (T. molitor) BPs through enzymatic hydrolysis using alcalase, obtaining a degree of hydrolysis of 20% and a yield of 22.5%. This was followed by an ultrafiltration process to isolate BPs with a molecular weight (MW) below 3 kDa, confirmed by high-performance size-exclusion chromatography (HPSEC). These BPs were submitted to gastrointestinal tract (GIT) digestion using the INFOGEST protocol. The peptide MW profile, antioxidant activity, and amino acids composition were evaluated in the main phases of the GIT digestion and intestinal absorption. T. molitor BPs showed high antioxidant activity, especially permeate (the fraction that reaches the systemic circulation) with 3.83 µmol of Trolox equivalents/mg of protein for ABTS assay. T. molitor protein hydrolysate and permeate could inhibit ROS production in the HMC3 cell line. Moreover, T. molitor protein hydrolysate peptidome was identified and based on their predicted bioactive properties using in silico tools, four peptide sequences were synthesized. Their antioxidant activity was evaluated and AM3 (AAPVCYLF) peptide sequence showed the highest antioxidant activity for ABTS and ORAC methods. Further studies using AD C. elegans models were carried out to assess the protective effects of hydrolysate, permeate, and the four peptide sequences. Together, these results highlight the therapeutic potential of the BPs from T. molitor, presenting them as a sustainable and innovative approach for the treatment of Alzheimer's disease (AD), which could significantly contribute to future effective therapeutic alternatives.
- A interdependência entre engenharia alimentar e nutrição no contexto do retalho alimentarPublication . Lage, Raquel Carvalho; Delgado, Mayumi Thaís Fernandes deO presente relatório de estágio é o resultado do estágio curricular realizado no âmbito do Mestrado em Engenharia Alimentar. O estágio decorreu na Sonae MC, no complexo de escritórios da Maia, onde foi integrada a Direção de Qualidade e Investigação, mais concretamente a Equipa de Desenvolvimento de Produto. A estrutura do relatório adapta-se ao trabalho desenvolvido e está organizada em três partes: Parte I – Alimentação, Nutrição e Consumo; Parte II – A Empresa, a Direção, a Equipa e as Atividades Realizadas no Estágio; Parte III – O Meu Contributo no Estágio. A Equipa de Desenvolvimento de Produto está organizada em três áreas. A estagiária participou ativamente na área de Alimentar & Frescos, na Unidade de Negócio de Mercearia Salgada, na categoria de suplementação alimentar e nutrição desportiva. Paralelamente, prestou apoio transversal a outras unidades de negócio. As funções desempenhadas no âmbito do estágio foram inicialmente delineadas em três áreas de atuação: (1) Avaliações Nutricionais de Gama, (2) Validação de Produtos de Marca Própria (MP) e (3) Projetos de Investigação e Desenvolvimento (I&D). À data de término do estágio, foram concluídas 2 avaliações nutricionais, 2 processos de validação de produtos de MP e 4 projetos de I&D. Estes serão descritos em detalhe na Parte II, nos pontos 3.1, 3.2 e 3.3, respetivamente. No âmbito destas funções, foram avaliados 613 produtos alimentares, realizadas 17 visitas a unidades comerciais e analisados 107 artigos científicos que apoiaram tecnicamente as atividades e a redação do relatório. Após a conclusão do estágio e do relatório, evidencia-se a relevância da interdependência entre a Engenharia Alimentar e a Nutrição, duas áreas cuja complementaridade é essencial para responder aos desafios do setor alimentar, especialmente no contexto dinâmico e exigente do retalho.
- Valorização da funcionalidade da biomassa de chlorellaPublication . Aldana, Paula Andrea Martínez; Barros, Ana Isabel Faustino; Pereira, Camilly FratelliAs microalgas têm ganho crescente relevância devido ao seu elevado valor nutricional e ao potencial para aplicação em produtos alimentares sustentáveis. A Chlorella branca (Chlorella sp. “white variant”), obtida por cultivo heterotrófico e caracterizada pelo baixo teor de clorofila, apresenta vantagens sensoriais face às microalgas verdes convencionais, tornando-se um ingrediente promissor para formulações de base vegetal. Este trabalho foi desenvolvido no âmbito do estágio curricular realizado na Allmicroalgae – Natural Products S.A., e teve como objetivo avaliar o potencial tecnológico e alimentar da biomassa de Chlorella branca. O estudo foi dividido em três partes: (i) análise do efeito da variação do rácio C/N na composição da biomassa, (ii) caracterização das propriedades tecno-funcionais da biomassa húmida e em pó e (iii) incorporação da biomassa numa bebida vegetal. Na primeira parte verificaram-se alterações no teor de amido e proteína em função do rácio C/N, embora esses efeitos não tenham sido reproduzidos à escala de fermentador. A segunda parte mostrou que a biomassa apresenta propriedades tecnológicas relevantes, nomeadamente solubilidade dependente da temperatura e elevada capacidade emulsificante, com a biomassa seca a revelar maior estabilidade em emulsão. A análise colorimétrica indicou ainda boa estabilidade cromática em condições térmicas moderadas. Na terceira parte, a formulação de uma bebida vegetal contendo 3% de biomassa em pó demonstrou um perfil nutricional favorável, permitindo alegações nutricionais de: “fonte de fibra”, “fonte de proteína” e “alto teor de vitamina B12”. Contudo, a avaliação sensorial revelou uma diminuição da aceitação global com a adição de Chlorella branca, especialmente ao nível do sabor, textura e apreciação global, quando comparada com a bebida base e a referência comercial. Em conclusão, a Chlorella branca apresenta potencial tecnológico para aplicação alimentar, sobretudo como agente funcional e emulsificante natural. No entanto, o impacto sensorial observado realça a necessidade de otimização futura das formulações para melhorar a aceitação do consumidor.
- Beyond bandages : a smart, glucose-responsive polyurethane for diabetic chronic wound treatmentPublication . Magalhães, Adriana Isabel Gomes; Laurano, RossellaThe treatment of diabetic chronic wounds represents a significant clinical challenge due to impaired healing capacity and complex local microenvironments, contributing to the high occupancy of acute hospital beds in Europe (up to 50 %). In this context, glucose-responsive materials offer a promising strategy for intelligent drug delivery systems adaptable to hyperglycaemic conditions. This work describes the synthesis and characterisation of a poly (ether)urethane-based hydrogel functionalised with phenylboronic acid groups, with potential for payload release in response to glucose. The synthesis of the multi-stimuli responsive polymer followed a three-step procedure: chain extension of Pluronic® P407 via diisocyanate mediated coupling with N-Boc diethanolamine, acid-mediated Boc deprotection, and functionalisation with 3-carboxyphenylboronic acid (3-CPBA). Characterisation by Attenuated Total Reflectance Fourier Transform Infrared spectroscopy and Size Exclusion Chromatography confirmed urethane bond formation and polymer integrity upon Boc cleavage, while the colourimetric Kaiser test revealed a 93 % increase in accessible amines after Boc removal. Furthermore, Proton Nuclear Magnetic Resonance spectroscopy indicated feed-dependent incorporation, with levels ranging from 6.0 × 1018 to 1.7 × 1019 molecules⁄gram by increasing the COOH:NH ratio from 2:1 to 10:1; Swelling and dissolution assays demonstrated that the functionalised hydrogel (GlucoDHP; 15 % (w/v) polymeric concentration) maintained structural stability over 72 h and exhibited lower mass loss compared to the non-functionalised control—i.e., D-DHP407 hydrogels) (p<0.05). Indirect Cytocompatibility assays using NIH-3T3 fibroblasts confirmed all formulations were non-toxic, with GlucoDHP5 promoting a 23.5 ± 3.2 % increase in cell viability compared to D-DHP407 (p < 0.01). In conclusion, the developed hydrogel showed improved stability, biocompatibility, and chemical responsiveness, supporting its potential for application in diabetic wound treatment. Future work will involve therapeutic agent encapsulation (e.g., insulin) and in vitro release assays to assess responsiveness under hyperglycaemic conditions.
- Desenvolvimento do plano HARPC da Prozis.NowPublication . Sousa, Beatriz Isabel Silva e; Hogg, Timothy AlunA segurança alimentar constitui um desafio central num mercado cada vez mais globalizado, caracterizado por cadeias de abastecimento cada vez mais exigentes e complexas. No âmbito do estágio realizado na Prozis, desenvolvido na unidade Prozis.Now, o projeto teve como objetivo responder aos requisitos do Food Safety Modernization Act (FSMA), com especoal foco na metodologia Hazard Analysis and Risk-Based Preventive Controls (HARPC). Este trabalho articula a componente analítica e prática, resultando na elaboração de um plano que abrange a análise de perigos, a definição de controlos de risco, a rastreabilidade, a higienização, a gestão da cadeia de fornecimento e os planos de recall. Para além de assegurar a conformidade legal, o estágio contribuiu para o desenvolvimento de competências técnicas e operacionais na área da segurança alimentar, reforçando a compreensão da importância dos sistemas preventivos em contextos organizacionais complexos.
- Validation of an automatic projective mapping acquisition system used in the sensory description of food by consumersPublication . Laverde Martinez, Diego Alejandro; Rocha, Célia Ferreira da; Lima, Rui Jorge Moreira da CostaProjective mapping is a sensory analysis technique widely used for the evaluation of consumer perceptions based on the application of a similarity criteria and represented spatially on a two-dimensional map. Traditionally, this method is conducted on a sheet of paper, in which the participants place and describe product samples, using additional methods like Ultra-flash Profile (UFP) for attribute generation. This methodology has been successfully applied with panels of consumers as well as trained assessors, providing similar insights as the descriptive analysis, although the traditional paper-based approach presents efficiency limitations as well. This study introduces a new and alternative projective mapping approach, focusing on the digitalization of the positioning data acquisition by implementing an automatic system that effectively identifies and captures the coordinates of the samples placed over the designated mapping area, with the objective of streamlining the development of projective mapping sessions and considerably reducing the time required. The method was established using robust detection hardware paired with an enhanced sample marker identification system and integrated to the projective mapping software developed by Sense Test. To validate the method, seven Verdinho canned fish samples were evaluated using three approaches: the traditional paper-based method, the proposed digital projective mapping and the screen-based projective mapping method developed by Sense Test. Additionally, the panellists evaluated the overall liking of the product samples through a nine point hedonic scale. Results showed that the digital projective mapping method strongly correlated with the traditional paper-based approach (R² = 0.9922), confirming its efficiency improving and error reducing capabilities. Moreover, the screen-based projective mapping showed a moderate correlation with the paper-based method (R² = 0,7028) as well as with the digital projective mapping (R² = 0,7000), proving to be a resourceful alternative.
- Design of thermoreversible peptide loaded liposomes for atopic dermatitisPublication . Carvalho, Laura Judite Benge Dias de; Costa, Eduardo Manuel Aguiar da; Pintado, Maria Manuela EstevezAtopic dermatitis (AD) is a chronic inflammatory skin disorder associated with impaired barrier function, immune dysregulation and intense pruritus, severely affecting patients’ quality of life. Despite its high prevalence, current therapies remain centred on symptomatic control and often show limited efficacy, highlighting the need for safer and more sustainable alternatives. This study explored bioactive marine peptides (<3 kDa) derived from fishery by-products as therapeutic agents, incorporated into liposomal nanosystems designed for topical delivery. The formulations defined as optimal, which included two systems composed of different amounts of lecithin and one containing both lecithin and cholesterol, exhibited encapsulation efficiencies between 89.7% and 91.3% and polydispersity index values ranging from 0.188 to 0.577 at laboratory scale. After scale-up, these same formulations maintained average particle sizes below 20 nm and low PDI (<0.4), with encapsulation efficiencies between 50.8% and 80.9%. The cholesterol-containing system demonstrated the greatest stability and resilience throughout the scale-up process, confirming the importance of moderate cholesterol content in maximising peptide retention and preserving colloidal stability. FTIR and DSC analyses revealed constituent compatibility and bilayer reorganisation without achieving thermoreversibility, as transition temperatures remained above the physiological range. Release studies fitted the Korsmeyer–Peppas model, indicating diffusion-controlled release, with cholesterol promoting a more gradual and sustained peptide delivery. No antimicrobial effects were observed against Staphylococcus aureus. Cytotoxicity was concentration-dependent, with all formulations biocompatible at 0.3 mg/mL, while the free peptide showed no cytotoxicity. Under these conditions, the phospholipid-rich formulation and cholesterol containing formulation induced metabolic stimulation. Overall, this work demonstrates the feasibility of integrating marine peptides into stable liposomal carriers for topical administration. The cholesterol based formulation stands out as the most balanced system in terms of encapsulation, stability and release performance, providing a solid foundation for future nanotechnology driven strategies in AD therapy that also promote sustainable valorisation of marine resources.
- Electrospinning a mechano-biocompatible external support for the Ross procedurePublication . Reis, Mariana Sofia Gomes; Mignon, Arn; Ribeiro, Viviana PintoThe Ross procedure is a surgical technique used to treat aortic valve diseases, in which the diseased aortic valve is replaced with the patient9s own pulmonary valve, offering unique advantages. However, progressive autograft dilatation remains a major limitation. To mitigate this problem some external supports currently exist, such as Dacron wraps, PEARS meshes or semi-resorbable grafts, which provide reinforcement but restrict root9s valve motion, or have biocompatibility challenges. This thesis aimed to develop a temporary, biodegradable and bioactive electrospun support capable of preventing early dilatation while allowing long-term tissue remodeling. The electrospun support was composed of polycaprolactone (PCL), elastin and decellularized extracellular matrix (dECM). dECM was optimized to achieve 97.4% deoxyribonucleic acid (DNA) reduction while preserving key extracellular matrix (ECM) components, including 61% elastin and 92% insoluble collagen. It was processed into fine powder (~24 µm) and incorporated into PCL:elastin electrospun blends to fabricate tubular scaffolds. Morphological analysis of the optimized structure (PCL:elastin:dECM, 19:0.5:0.5 w/v%) exhibited a well distributed fiber network, with partial circumferential alignment on the outer surface and randomly oriented fibers on the inner surface. Mechanical tests demonstrated a low-strain modulus of ~2 MPa and compliance values of ~2.2 %/100 mmHg. Accelerated degradation showed a more controlled mass loss profile than pure PCL. In conclusion, the PCL:elastin:dECM scaffold demonstrates potential as a resorbable external support for the Ross procedure, balancing compliance and benefiting from the presence of elastin and dECM, which may contribute to improved adaptation. Future studies should combine computational modelling and in vivo validation to confirm long-term performance.