ESS - DM - Bioquímica em Saúde
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- Deciphering the influence of CD276 glycode in colorectal cancer immunomodulationPublication . Magalhães, Mariana Moura; Peixoto, Andreia; Ferreira, José Alexandre; Santos, Lúcio Lara; Ferraz, RicardoColorectal cancer (CRC) remains a major contributor to cancer-related illness and death worlwide, with aggressive subtypes often resistant to conventional therapies. The imune checkpoint protein CD276 (B7-H3) is highly expressed in CRC and is associated with poor prognosis, tumor progression, and imune evasion. CD276 undergoes extensive glycosylation, a modification that influences protein stability, localisation, and imune interactions, yet the specific roles of its glycosylation in CRC are not well understood. This study examines how immature 0-glycosylation of CD276 affects CRC aggressiveness. To model the altered glycan landscape typical of cancer cells, C1GALT1-knockout CRC cells, which lack the enzyme necessary for 0-glycan elongation, were used. The effects of CD276 knockdown using small interfering RNA (siRNA) on cell proliferation and inasion were assessed by BrdU ELISA and Matrigel invasion assays, respectivily. Additionally, proteomic and phisphoproteomic analyses were conducted to explore how CD276 influences oncogenic signalling pathways. Our fundings reveal that aberrant, immature 0-glycosylation of CD276 promotes CRC cell proliferation and invasion by sustaining oncogenic biosynthetic and phosphorylation networks. Conversely, CD276 depletion activates stress responses involving RNA processing. DNA repair, and mitochondrial reorganisation, highlighting CD276 as a glycosylation-dependent driver of CRC aggressiveness and a potential therapuetic target.
- Avaliação in vivo do efeito de um produto cosmético na queda de cabeloPublication . Costa, Mariana Cristina Ferreira da; Ferreira, Marta de Oliveira; Ferraz, RicardoO tratamento da alopecia androgenética e do eflúvio telogénico crónico envolve principalmente intervenções farmacológicas, como o minoxidil e a finasterida. No entanto, ambos estão associados a efitos colaterais frequentes, fazendo crescer a procura por produtos cosméticos naturais para reduzir a queda de cabelo. Apesar disso, muitos produtos ainda não possuem evidências científicas apoiadas por estudos clínicos, sendo estes necessários para comprovar a sua eficácia. Este estudo clínico teve compo objetivo investigar o efeito de um sérum capilar composto por óleos essenciais n queda de cabelo em mulheres caucasianas entre os 25 e os 55 anos e com alopecia androgenética e/ou eflúvio telogénico durante 84 dias. Os resultados demonstraram um aumento estatisticamente significativo na espessura acumulada do cabelo e no rácio anagénese/telogénese, medidos com Trichoscan, após 84 dias. Além disso, o produto aumentou a ancoragem do cabelo, avaliado através do teste de tração capilar, e o brilho do cabelo após 42 e 84 dias de aplicação. No geral, o sérum capilar foi bem tolerado pelas participantes, não tendo sido registados quaisquer eventos adversos graves. A dermatologista que supervisionou o estudo classificou a eficácia do produto como muito boa, considerando que existiram efeitos significativos após um curto período de uso.
- Umbilical cord banking: exploring cryopreservationPublication . Monteiro, João Miguel de Barbosa; Gomes, Andreia; Oliveira, Melissa; Vieira, MónicaStem cells play a crucial role in tissue renewal, serving as reservoirs for the formation of new differentiated cells. The umbilical cord is a source of stem cells, which have a higher proliferative capacity compared to cells isolated from adult tissues and, therefore, greater clinical application. Cryopreservation is a widely used technique that keeps stem cells, including those from the umbilical cord, viable for several years for later clinical applications. Cryopreservation allows cell and tissue banks, which can be public or private, to store and preserve biological material. The main objectives of this work were the observation and involvement in the routine laboratory processes of BebéVida, specifically the processing of umbilical cord tissue and the validation of an umbilical cord tissue freezing curve. The validation is based on comparing the test curve with the routine curve, assessing cell viability. The results confirmed that the test curve was successful, showing cell viability comparable to the routine curve, reinforcing its clinical applicability. By validating different freezing protocols and evaluating long-term viability, this work addresses the need for standardized and reproducible methods suitable for clinical use. In this way, it contributes to the advancement of stem cell-based therapies. Additionally, the ERASMUS+ chapter focused on genes involved in the ageing process, using the C. elegans model.
- Valorization of the nutraceutical potential by Salicornia ramisissima by-product - Impact of gastrointestinal digestion on in vitro bioactivityPublication . Santos, Inês Maria Manso; Rodrigues, Francisca; Pinto, Diana; Vieira, MónicaThis work focused on the valorization of the halophyte Salicornia ramosíssima, already known for its bioactive compounds, by evaluating the Effect of in vitro gastrointetinal digestion on its phenolic contente, bioacessibility, and bioactivity. The main objective of this study was to assess the impact of in vitro gastrointestinal digestion on the phytochemical profile of this halophyte by-product, as well as to evaluate their antioxidante and antiradical properties and intestinal premeability of the bioactive compounds. For that, various assays were performed to evaluate the total phenolic and flavonoid contentes, antioxidant/antiradical activities by FRAP, ABTS, and DPPH assays, the scavening capacity of reactive oxygen species (ROS), neuroprotective properties by acetylcholinesterase (AChE) inhibition, intestinal cell viability, and the extracts Effect and their digested fractions in the antioxidante enzymes activities and lipid peroxidation. The intestinal permeability was assessed in a Caco-2/HT29-MTX co-culture model by identifying and quantifying the bioactive compounds that permeated the intestinal barrier by liquid chromatography coupled to mass spectometry. The results revealed the richness of this by-product in phenolic compounds and flavonoids, confirming the effects of the in vitro digestion on its bioactivity. The antioxidante and antiradical properties increased throughout the different stagies of digestion, particularly after the intestinal phase, with the S. ramosissima by-product extract (SE) showing higher activity when compared to the by-product (SP). Additionally, the enzymatic assays – AchE (Acetylcholinesterase), SOD (Superoxide dismutase), CAT (Catalase), GSH-Px (Glutathione peroxidase), and LPO (Lipid peroxidation) demosntrated significant health benefits, highlighting the neuroprotective properties, prevention of lipid peroxidation, and capacity to stimulate the activity of the various antioxidant enzymes studied. Cell viability assays indicated that SE extracts were safe at the highest concentrations studied (1000µg/mL), demonstrating greater cell viability in the HT29-MTX cell line. The intestinal permeability assays demonstrated that 4-caffeoylquinic acid exhibited the highest permiability (34,84%). This study highlights the potential health benefits of S. ramosíssima phenolic compounds after the digestion process. The results obtained provide a solid Foundation for future research on the development of nutraceuticals and functional foods using the bioactive componentes of this plant. From a future perspective, subsequente studies will focus on in vivo assays to confirm the bioactivity of the digested compounds and invetigate their long-term effects on health. Furthermore, exploring other bioactive compounds available in S. ramosíssima could enhance its applications in therapeutic contexts.
- Biophysical cellular cues as predictive biomarkers of bladder cancerPublication . Azevedo, Ana Catarina Correia; Calejo, Isabel; Canadas, Raphael; Ferraz, RicardoBladder cancer (BC) is a common neoplasm characterized by high recurrence and rapid progression. Current diagnostic methods have limitations: cytoscopy is invasive and expensive, and urinary cytology is not very sensitive for low-grade tumors. Therefor, there is increasing interest in non-invasive biophysical biomarkers. In this dissertation, label-free methodologies were used, such as imaging flow cytometry (IFC), eletrophoretic light scattering (ELS) and acoustofluidics, which were essential for the deepening of the morphological and biophysical characteristics of BC cells, which were relevant for their label-fre separation from other cells commonly found in urine. IFC allowed the quantification of several morphological parameters in five urinary populations, such as cancerous epithelial cells, non-cancerous epithelial cells, squamous cells, erythrocytes, and lymphocytes. The data obtained with this technique also allowed us to infer critical biophysical properties for acoustic migration, particularly cell density. The increased diameter and reduced contrast and gradient RMS of cancer epitelial cells indictaed less internal organization and compaction, indicating lower density than, erythrocytes, and lymphocytes, that promotes different accoustic mobility between cell types. ELS outlined distinct zeta potential profiles, revealing that there could be a correlation between epithelial-mesenchymal transition (EMT) and tumor cell load, with a tendency to be more, negative values in cells with a more mesenchymal profile. EMT unscored cell lines presented intermediate values. Clinical isolates from bladder tumours exhibited variation consistente with that was observed in cell lines, suggesting zeta potential as a functional biomarker of tumor progression. Finally, aoustofluidics allowed the separation of epitelial cancer cells from the other cell populations presente in clinical urine samples. The results of this technique were compared with histological (reference control) and cytological (technique for direct comparison) results. Cytology obtaines high specificity, but limited sensivity; while acoustofluidics received superior sensivity, although with lower specificity. However, considering that almost 90% of the studied clinical cases were histologically positive, the high sensivity of the acoustic seperation technique is clinically more relevant.
- Study of the relevance of antioxidant enzymes in thyroid cancerPublication . Freitas, Sílvia Daniela da Silva; Soares, Paula; Peixoto, Joana; Máximo, Valdemar; Vieira, MónicaThyroid cancer, the most common endocrine malignancy, has exhibited a marked rise in incidence worldwide in recent decades, with significant impact on women and a broad clinical and histological spectrum. The thyroid gland intrinsic physiology is closely intertwined with oxidative processes, as the biosynthesis of thyroid hormones depends on the generation of reactive oxygen species (ROS). Oxidative stress and consequent redox imbalance are increasingly recognized as crucial drivers in the pathogenesis and progression of thyroid neoplasms. This dissertation aim to elucidate the role of the antioxidant enzymes SOD1, SOD2, and GPX1 in thyroid carcinogenesis by assessing their gene and protein expression in a cohort of 90 human thyroid tissue samples, comprising adjacent non-tumor tissue, benign, and malignant neoplasms. Methodologies included validated protocols for quantitative RT-PCR and immunohistochemistry, ensuring robust analysis across all tissue types. The results showed that SOD1 tends to be more highly expressed in malignant lesions, particularly in classic papillary carcinomas, and GPX1 showed increased mRNA expression in malignant lesions with statistically significant differences compared to adjacent non-tumor tissue. SOD2, however, did not prove to be a robust marker for malignancy. The study supports the involvement of redox regulation in thyroid tumor biology and suggests that SOD1 and GPX1 could be interesting biomarkers of thyroid disease. Overall, the work contributes to the understanding of how oxidative imbalance influences thyroid cancer and highlights the potential for redox-modulatory therapeutic strategies in the management of this disease.
- Levedura de Cerveja como agente encapsulante de compostos bioativos de macroalgas: preparação, incorporação num iogurte e caracterizaçãoPublication . Santos, Rúben Manuel Barbosa dos; Vieira, Elsa F.Neste trabalho foi avaliada a capacidade da biomassa excedente de levedura do processo cervejeiro (BLC) em ser explorada como material biossorvente de compostos fenólicos e antioxidantes presentes nos extratos das macroalgas: Laminaria digitata (LD), Fucus vesiculosus (FV), Himanthalia elongata (HE) e Eisenia bicyclis (EB). Numa primeira fase foi avaliado o efeito da modificação da parede celular da BLC (alcalinização) na capacidade de biossorção dos compostos de interesse. A biossorção dos compostos bioativos pela BLC foi avaliada por estudos cinéticos e a retenção de compostos fenólicos totais e antioxidantes na BLC foi avaliado por métodos espetrofotométricos. Após 3 horas de ensaio cinético, os resultados sugerem que a BLC alcalinizada apresentou melhor capacidade de biossorção de compostos fenólicos e antioxidantes da macroalga FV e LD, pelo que estes extratos foram os selecionados para a formulação das cápsulas de alginato. Numa segunda fase, foram preparadas diferentes cápsulas de alginato incorporando: (i) BLC (ensaio controlo); (ii) BLC enriquecida nos extratos de macroalga FV e LD – “BLCLD e BLCFV”; (iii) BLC e extratos de macroalga FV e LD - “BLC+LD e BLC+FV”; (iv) BLC enriquecida nos extratos de macroalga FV e LD e BLC e extratos de macroalga FV e LD - “BLCLD+LD e BLCFV+FV”, bem como (v) extratos de macroalga FV e LD – “LD e FV”. Para efeitos comparativos, todas as imobilizações foram realizadas usando a mesma proporção de 1:20 (p/v) de suspensão: 2% (p/v) de alginato de sódio. As cápsulas desenvolvidas foram caracterizadas e incorporadas (1% p/p) na formulação de iogurtes. Os resultados indicaram que as formulações de iogurte contendo as cápsulas enriquecidas (BLCLD e BLCFV) apresentaram um aumento significativo no conteúdo de fenólicos totais (TPC) e na atividade antioxidante (avaliada pelos métodos DPPH, ABTS e FRAP) em comparação com o iogurte controlo e a formulação BLC. A caracterização físico-química das formulações demonstrou que a adição das cápsulas manteve as propriedades essenciais do iogurte (como pH, acidez titulável e sinérese) dentro dos limites aceitáveis para este tipo de produtos, evidenciando a estabilidade da matriz láctea perante o enriquecimento. Desta forma, o estudo valida a eficácia da BLC como um veículo seguro e sustentável para a fortificação funcional de alimentos, permitindo a transferência bem-sucedida dos compostos bioativos das macroalgas para um produto lácteo, e alinhando-se com os princípios da economia circular e do desenvolvimento de nutracêuticos de elevado valor.
- Relação entre o estado de saúde física e mental com o microbioma oral e da pele em jovens no Ensino SuperiorPublication . Costa, Gabriel Galete; Vieira, Mónica; Pereira, Cassilda; Rodrigues, João MiguelO microbioma humano é cada vez mais reconhecido como um regulador central da saúde sistémica e mental, interagindo com dieta, atividade física e stress por vias metabólicas, imunitárias e neurocomportamentais. Estudantes universitários constituem um grupo particularmente vulnerável, frequentemente exposto a stress crónico, rotinas irregulares e hábitos de vida subótimos, o que pode impactar simultaneamente a composição corporal, a saúde mental e os microbiomas oral e cutâneo. Este projeto teve como objetivo caracterizar o perfil de saúde física e mental de estudantes universitários e explorar a sua relação com padrões microbiológicos orais e cutâneos, em vez de se centrar exclusivamente na descrição do microbioma. No total, 55 estudantes preencheram questionários validados para depressão (PHQ-9), ansiedade (GAD-7), atividade física (IPAQ) e adesão à dieta mediterrânea (MEDAS-7), realizaram avaliação de composição corporal por bioimpedância e forneceram amostras orais e cutâneas por zaragatoa para análise microbiológica dependente de cultura. A amostra apresentou, em média, índice de massa corporal na faixa superior da normalidade e uma carga substancial de sintomas depressivos e ansiosos, enquanto a maioria relatou níveis moderados a elevados de atividade física e adesão baixa a moderada à dieta mediterrânea. Estatisticamente, as associações mais consistentes foram observadas entre massa muscular esquelética, ansiedade e atividade física: maior massa muscular correlacionou-se com menores pontuações de ansiedade (r = –0,368; p = 0,027) e com níveis mais elevados de atividade física (r = 0,398; p = 0,016), sustentando o papel da aptidão muscular como correlato relevante de saúde mental em adultos jovens. Em contraste, a adesão à dieta mediterrânea não apresentou correlações significativas com saúde mental ou indicadores antropométricos nesta amostra. Do ponto de vista microbiológico, as amostras orais exibiram cargas bacterianas marcadamente superiores às cutâneas, enquanto os locais de pele apresentaram maior heterogeneidade de morfotipos de colónias; leveduras e fungos filamentosos foram detetados em ambos os nichos. Apenas possíveis géneros bacterianos e fúngicos foram sugeridos com base em morfologia e coloração, a serem confirmados em futuros estudos com sequenciação utilizando o mesmo material biobancado. De forma global, os achados evidenciam pontos de melhoria relacionados a saúde dos estudantes universitários e fornecem evidência preliminar para investigações futuras mais aprofundadas sobre a relação complexa entre o microbioma e o corpo humano.
- Polyphenol encapsulation and skin models: a comparative in vitro and ex vivo approach to cosmetic safety testingPublication . Marques, Mariana Isabel Gonçalves; Rodrigues, Francisca; Vieira, MónicaPolyphenols are bioactive compounds with skin antioxidant and anti-aging potential, yet their cosmetic application remains limited due to instability and poor solubility, so encapsulation offers a promising strategy to improve their stability and controlled release (1). This thesis aimed to develop a novel cosmetic ingredient by enhancing the stability and safety of polyphenols through encapsulation, while employing physiologically relevant 3D in vitro and ex vivo skin models for toxicity and permeation testing. Catechin, epicatechin, chlorogenic acid (CGA), neochlorogenic acid (NCGA), and their mixture were microencapsulated in sodium alginate by spray-drying, achieving encapsulation efficiencies between 19 and 27% and product yields from 37 to 53%. The microparticles displayed enhanced thermal stability and physical entrapment of the phenolic compounds. Moreover, microencapsulation enhanced the antioxidant and antiradical activities, and synergistic effects were detected in the phenolic mix. In vitro biocompatibility assays revealed viabilities above 60% in keratinocytes (HaCaT) and over 80% in fibroblasts (HDF), whereas pure polyphenols displayed dose-dependent reductions in viability. A 3D in vitro co-culture model composed by dermal (HDF) and epidermal (HaCaT) cell lines at air-liquid interface conditions was constructed. The permeability assays revealed compound-dependent permeation. Encapsulation reduced the overall permeation but provided a gradual and controlled release, particularly for catechin and epicatechin. MTT assays demonstrated that catechin and epicatechin-loaded microparticles increased the cell viability in the 3D co-culture and the commercial SkinEthicTM Reconstructed Human Epidermis (RHE) model, whereas NCGA reduced the viability only in the 3D co-culture model (82.55%). Histological analysis of SkinEthicTM RHE model confirmed the absence of phenotypical alterations in keratinocytes with exposure to alginate microparticles. Discrepancies were noted between the monocultures and the 3D co-culture skin model, highlighting the critical role of epidermal-dermal communication in safety evaluations. Permeation studies with Franz diffusion cells in ex vivo human explants revealed negligible permeation of catechin and epicatechin, while CGA, NCGA, and the mix permeated up to 50% and displayed more than 60% of skin retention. Overall, this work demonstrates that alginate-based encapsulation enhances the stability, antioxidant activity, and safety profile of polyphenols, while enabling their controlled release and modulating their skin permeation. The integration of advanced 3D in vitro and ex vivo skin models provided valuable mechanistic insights into skin interactions, underscoring their potential as effective and safe cosmetic ingredients. These findings contribute to the development of alternative testing strategies and highlight the relevance of encapsulation for translating bioactive compounds into cosmetically applicable formulations.
- Bioadhesive hydrogels based on collagen for bone regenerationPublication . Fernandes, Maria Margarida Nogueira; Alves, Natália; Peixoto, Daniela; Vieira, MónicaBone exhibits a unique characteristic of self-regeneration in response to a fracture; however, when the fracture is extensive, bone grafts may be necessary. Since this option presents several limitations and risks for the patient, tissue engineering emerges as a promising alternative, offering solutions for treating these fractures by creating devices or materials for bone regeneration. Hydrogels represent a promising alternative for bone regeneration, as they possess a structure similar to the extracellular matrix (ECM), are biocompatible, and enable the incorporation of molecules that stimulate osteogenesis. Collagen is a primary component of the extracellular matrix (ECM) and the primary component of bone, making it an ideal material for developing new scaffolds for bone applications. When modified with catechol groups, the hydrogels exhibit enhanced adhesive capacity, a desirable characteristic for regenerative applications. Gallium-doped bioglass nanoparticles are promising for bone regeneration because they stimulate osteogenesis and have antibacterial properties. In this dissertation, composite hydrogels of collagen modified with catechol groups and gallium-doped bioglass nanoparticles were developed. It was found that these hydrogels possess high adhesive capacity, bioactivity, antibacterial properties, and cytocompatibility, thereby having potential applications in bone regeneration.