ESS - TBIO/Rise Health - Posters apresentados em eventos científicos
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- Impact of simulated digestion on microplastics: Surface changes and intestinal in vitro exposurePublication . Lamas, Mariana; Fernandes, Virgínia Cruz; Oliveira, Marta; Rodrigues, Francisca; Fernandes, VirgíniaMicroplastics (MP) have been widely detected in a variety of food products1, and ingestion is one of the main pathways for human exposure to these contam-inants. Once ingested, MP might interact with the gastrointestinal (GI) environment, yet it remains un-clear whether digestive processes alter their chemical structure or mainly influence their surface properties, and possibly their interaction capacity3. This study in-vestigates digested-induced modifications in MP and establishes suitable conditions for intestinal in vitroassays. Simulated digestion of six polymers (PTFE; PMMA, PA6, uPVC, PET, and LDPE) followed the IN-FOGEST model2. Following digestion, particles were analysed using Fourier-transform infrared spectros-copy (FTIR) to assess structural and surface changes. Cell viability was evaluated, by MTT, in Caco-2 and HT29-MTX intestinal cell lines exposed to different dilutions (1:10, 1:20, and 1:40) of digestion fluids, with respective MP. FTIR results indicated that all polymers maintained their fundamental chemical structure after digestion. Instead, consistent surface-related alter-ations were observed, including reduced intensity of characteristic polymer bands and enhanced signals in regions associated with proteins and hydroxyl groups, suggesting biomolecules adsorption and increased hy-dration. Cell studies demonstrated that 1:20 dilution of digestive fluids with MP maintained the cell viability above 70%, representing the most suitable condition tested. Simulated GI digestion appears to preserve the intrinsic chemical identity of MP, while promoting surface modifications that may affect their biological interactions. The identification of 1:20 digestion fluid dilution supports its application in future studies using advanced intestinal models, including three-dimen-sional systems and assessment of cellular oxidative stress responses.
- Profiling airway microbiome composition through volatilomicsPublication . Neto, Filipa; Ferraz, Ricardo; Vieira, Mónica; Prudêncio, Cristina; Rufo, João; Almeida Vieira, Mónica Andreia; Cavaleiro Rufo, JoãoThe airway microbiome is known to mediate respi-ratory health. However, current available methods for microbiome analysis are time-consuming and/or rep-resent significant costs for a generalized application in clinical practice. Volatilomics has been suggested as a rapid and low-cost approach to screen microbial pro-files in human samples. Therefore, we aimed to study the efficacy of volatilomics in discriminating microbi-al isolates collected from human breath condensate samples. Bacterial strains showing significant growth under conditions simulating the respiratory environ-ment were isolated. Each strain was standardised to an inoculum of 10⁸ CFU/mL and analysed using an elec-tronic nose equipped with a six-sensor matrix. Data were explored through principal component analysis, cluster analysis, pattern analysis, random forests and recursive partitioning regression. One sensor was re-moved from the analysis due to high collinearity. Prin-cipal component analysis was able to separate strains and the control mainly through the second principal component (p = 0.024), characterized by high MQ3 and MQ8 sensor responses. Sensor profile maps showed distinct volatile patterns across strains (Fig-ure 1), suggesting the presence of distinct microbial signatures. However, reproducibility was low between replicas and time since culture. Recursive partitioning for separating sterile controls from inoculated sam-ples showed the highest accuracy (AUC = 0.73). These results show the potential of separation of microbial strains based on volatilomics. Nonetheless, relative robustness was only achieved for the discrimination of sterile vs inoculated samples.
- Enhancing antifungal activity against Candida albicans using novel organic compoundsPublication . Alves, Beatriz; Silva, Rui; Vieira, Mónica; Ferraz, Ricardo; Fernandes, Silvia; Teixeira, Vitor; Pereira, Clara; Almeida Vieira, Mónica Andreia; Fernandes, SílviaCandida albicans is the predominant cause of inva-sive candidiasis, a major global health threat [1 The increasing emergence of resistance to commonly used antifungals (ex.:iazoles and polyenes) demonstrates an urgent need for alternative therapeutic strategies. The objective of this study was to evaluate the antifungal activity of novel organic derivatives from bioactive natural compound, including Naphthalene, Betulinic Acid (BA) [2]; Protocatechuic Acid (PCA) and Gallic Acid (GA), alone, or in combination with conventional antifungals to investigate synergistic effects that en-hance their efficacy. Antifungal susceptibility assays were performed in C. albicans ATCC 10231 using the broth microdilution method, according to CLSI M27 guidelines, to determine Minimum Inhibitory Concen-trations (MICs) and Minimum Fungicidal Concentra-tions (MFC). The interaction between BA derivatives and amphotericin B was assessed by checkerboard assay and fractional inhibitory concentration index (FICI). Naphthalene, PCA and GA derivatives showed low antifungal activity, falling within the moderate to weak bioactivity range (MIC ≥ 100 μg mL−1; [3]). Among BA derivatives, BA-4 and BA-5 exhibited the best antifungal activity, with MIC values of 0,95 and 1,91 μg mL−1, respectively, achieving 99% fungicidal activity at the same concentration. Furthermore, in combination assays, both compounds enhanced Am-photericin B activity, showing synergistic effects (FICI = 0.50), resulting in a decrease in MIC. Although most newly synthesized compounds exhibited limited anti-fungal potential, BA-4 and BA-5 demonstrated very strong bioactivity and fungicidal effects against C. albicans. The observed synergy with amphotericin B highlights their potential role as antifungal adjuvants and supports the further exploration of betulinic acid derivatives as viable approaches to overcoming anti-fungal resistance.
- Optimization of SPE-HPLC-FLD methodologies for the extraction and detection of steroid hormones in plasmaPublication . Bracchi, Isabella; Paíga, Paula; Delerue-Matos, Cristina; Pestana, Diogo; Negrão, Rita; Keating, Elisa; Fernandes, VirgíniaThe quantification of steroid hormones, such as es-tradiol and ethinylestradiol, in plasma requires sensi-tive and reliable analytical methodologies1. This study presents the optimization of a bioanalytical method based on Solid-Phase Extraction (SPE) coupled with High-Performance Liquid Chromatography with Flu-orescence Detection (HPLC-FLD). For sample ex-traction, Strata-X (polymeric reversed-phase) cartridg-es were used, where critical SPE parameters, including cartridge conditioning, matrix washing, and analyte elution, were evaluated. Chromatographic separation was achieved on a C18 stationary phase using a mobile phase of acetonitrile and 0.1% formic acid in ultrapure water. Both isocratic and gradient elution programs were tested to maximize resolution and peak shape. The method was validated for linearity, recovery, and precision (intra- and inter-day), meeting all established criteria for bioanalytical applications. This combined optimization resulted in improved selectivity and sen-sitivity for the determination of estradiol and ethinyl-estradiol in plasma (Figure 1), demonstrating a robust bioanalytical tool for monitoring these compounds in complex biological matrices.
- Betulinic acid modulates adipocyte differentiation and secretome activity: Downstream effects on Glioblastoma cell migrationPublication . Gouveia, Afonso; Ferraz, Ricardo; Prudêncio, Cristina; Fernandes, Silvia; Fernandes, SílviaBetulinic acid (BA) has been recognized for its po-tential to modulate tumor progression and adipocyte function, yet its effects on adipocyte secretome re-modeling and the downstream consequences for glio-blastoma behavior remain incompletely understood1-4. In this study, we investigated the influence of BA on adipocyte secretory activity and its functional impact on glioblastoma cell migration. Direct exposure of U251 glioblastoma cells to BA significantly reduced cell migration, confirming its intrinsic anti-migratory potential. To further explore microenvironmental in-teractions, adipocyte-conditioned media (ACM) de-rived from BA-treated adipocytes was evaluated for its effects on glioblastoma behavior. Secretome anal-ysis demonstrated that BA altered matrix metallopro-teinase (MMP) expression and reduced gelatinolytic activity, indicating significant modulation of extracel-lular matrix–related proteolytic pathways. Functional assessment using wound healing assays revealed that ACM from BA-treated adipocytes did not consistent-ly suppress glioblastoma migration. Intermediate BA concentrations were associated with increased migratory behavior relative to basal conditions, although differences compared with control ACM were not statistically significant. Additionally, transwell migra-tion assays did not demonstrate significant differ-ences between treatment conditions, suggesting that BA-induced alterations in adipocyte secretome com-position do not uniformly translate into measurable changes in glioblastoma migratory capacity across dif-ferent experimental models. These findings indicate that modulation of gelatinolytic pathways alone is insufficient to predict glioblastoma cell migration and support the contribution of additional adipocyte-de-rived signalling mechanisms in regulating tumor be-havior. Overall, BA induces substantial remodelling of adipocyte secretory function, producing complex and non-linear downstream effects on glioblastoma cells. This study highlights the importance of tumor micro-environment crosstalk and underscores the need to evaluate multiple regulatory pathways when consid-ering the therapeutic potential of BA and related bio-active compounds.
- Evaluation of the functional properties of kombucha-derived Brettanomyces yeastsPublication . Areal-Hermida, Lara; Coelho, Pedro; Pichardo-Gallardo, Ángeles; Prudêncio, Cristina; Sieiro, Carmen; Prudêncio, Cristina; Coelho, PedroKombucha is a fermented beverage traditionally produced from tea and widely associated with benefi-cial health effects. These properties have been mainly attributed to the fermentable substrate and, to a less-er extent, to the microorganisms involved in the fer-mentation process. Therefore, the characterization of kombucha-associated microorganisms is essential to better understand and validate their potential health benefits. In this study, four probiotic yeast strains isolated from kombucha tea were identified, by the PCR-RFLP analysis of the ribosomal ITS region and the sequence of the D1/D2 domain of the 26S rDNA, as Brettanomyces bruxellensis (UVI55 and UVI56) and B. anomalus (UVI57 and UVI58). The evaluated strains exhibited remarkable antioxidant capacity, the abil-ity to hydrolyze bile salts, and antimicrobial activity against several key pathogens. Additionally, they displayed significant cytotoxic effects across various tumor cell lines. Notably, in the Caenorhabditis ele-gans in vivo model, strain UVI56 significantly extend-ed the nematode’s lifespan and conferred protection against S. enterica infection. Although additional in vivo studies are necessary to further elucidate the un-derlying biological mechanisms and validate the ther-apeutic potential of these yeasts, the present findings highlight the relevance of Brettanomyces species as key members of the kombucha microbial consortium. These strains exhibit multiple biological activities of potential biomedical interest and may contribute syn-ergistically to the health-promoting properties tradi-tionally associated with kombucha beverages.
- Sustainable development: an educational laboratory exercise using bacteriophagesPublication . Teixeira, Dulce; Vieira, Mónica; Prudêncio, Cristina; Almeida Vieira, Mónica Andreia; Prudêncio, CristinaSustainable development is a model that seeks to balance growth, social inclusion, and environmental protection, ensuring present and future well-being. The United Nations 17 Sustainable Development Goals (SDGs) guide this goal until 2030. Sustainable biotechnology is a branch that refers to the set of tech-niques and processes that use living organisms, their parts, or derivatives to develop products and solutions that provide economic, social, and environmental ben-efits. The growing development of the techniques that use living organisms, alongside the multidisciplinary nature of a career in medicinal biotechnology, under-scores the need for students in this sector to acquire a robust understanding of sustainable biotechnology as a fundamental component of their education. To this end, we propose a class focused on bacteriophages and their added value, covering five laboratory ses-sions: (i) design of a strategy for the recovery, enrich-ment, and isolation of a bacteriophage; (ii) extraction and recovery of bacteriophages from an environ-mental water sample; (iii) bacteriophage enrichment (Staphylococcus aureus ATCC 6538P, Staphylococcus epidermidis ATCC 1457, Escherichia coli ATCC 25922, and E. coli ATCC 8739); (iv) isolation and propagation of bacteriophages; and (v) research on the applicabili-ty of bacteriophages in the field of sustainable devel-opment. The major goals of the present work were (i) to provide students with the necessary tools to adapt protocols in the context of a laboratory class; (ii) to assess the students’ skills to carry out the assays in accordance with the expected results, and (iii) to fos-ter awareness for sustainable development. All assays were performed by students of the B.Sc in Medicinal Biotechnology. The results showed that Escherichia coli ATCC 8739 was the only strain that allowed the isolation of bacteriophages. Overall, these laboratory sessions demonstrated the practical implementation of sustainability concepts while also expanding the knowledge of biotechnological laboratory techniques.
- Glioma behavior under the influence of Cinnamic Acid: a cellular approachPublication . Moreira, Ana; Almeida, Joana; Ferraz, Ricardo; Ferraz, RicardoCancer is one of the biggest health problems worldwide, characterized by uncontrolled proliferation, rapid growth and the ability to invade other tissues (1). Gliomas are highly challenging tu-mours from a therapeutic perspective, so new and more effective therapeutic strategies have been in-vestigated, with particular focus on the therapeu-tic potential of plant-derived compounds, such as Cinnamic Acid (2,3). Cinnamic Acid, extracted from Cinnamomum cassia, has proved to be a promising therapeutic agent, as it exhibits antitumor properties and low cytotoxicity toward healthy cells (4–6). This phenolic compound has the ability to cross the blood-brain barrier and affect tumour cell survival by reduc-ing their proliferation rate, inducing apoptosis and inhibiting key signalling pathways involved in tumour growth (7–9). This study aims to evaluate the glioma behaviour under the influence of cinnamic acid, using a GL261 cell model. To this end, cells were cultured and treated with different concentrations of cinnamic acid (1x10-3 mol/L to 4x10-3 mol/L), followed by in vi-tro assays to assess its impact on cell viability and cell migration. The MTT assay performed on the GL261 cell line demonstrated that Cinnamic Acid is capable of reducing cell viability at all tested concentrations, with a cytotoxic effect that tends to be dose-dependent. At lower concentrations (1x10-3 mol/L and 2x10-3 mol/L), cell viability was approximately 83% (SD=13,3% and 13,9%, respectively) and the most pronounced reduc-tion in cell viability was detected at the highest tested concentration (4x10-3 mol/L), with a viability of 76% ± 13,9%, corresponding to an approximately 24% re-duction relative to the control (viability of 100%).
- Insights into the structure and activity of LONP2, a peroxisomal AAA+ ATPasePublication . Silva, Ana Rita; Francisco, Tânia; Rodrigues, Tony; Pedrosa, Ana G.; Fransen, Marc; Azevedo, Jorge E.Peroxisomes are single membrane-bound organelles that participate in a wide range of metabolic pathways, often involving multiple H2O2-producing oxidases. The large amounts of H2O2 generated inside these organelles, combined with the absence of protein re-pair systems renders peroxisomal proteins particular-ly susceptible to oxidative damage. Notably, distinct peroxisomal matrix proteins were shown to display heterogeneous half-lives, pointing to the existence of Protein Quality Control mechanisms (PQC) that de-grade damaged proteins. In mammalian peroxisomes, two proteases were identified: TYSND1 (Peroxisomal leader peptide processing protease) and LONP2 (Lon protease homolog 2). TYSND1 cleaves flexible loops within some peroxisomal proteins and the type 2 per-oxisomal targeting signals; however its restricted sub-strate specificity makes a role in PQC system unlike-ly. In contrast, LONP2 belongs to the AAA+ family of ATP- dependent proteases, a family typically involved in PQC. Structurally, LONP2 comprises an N-terminal domain (NTD) of ~200 amino acid residues, an ATPase AAA+ domain, a Lon proteolytic domain, and a per-oxisomal targeting signal type 1. Mammalian LONP2 is most related to mitochondrial LONP1 (38% identi-ty), the second LONP member expressed in mammals. LONP1 degrades misfolded, oxidized, or unassembled proteins using its NTD to both recruit substrates and regulate allosterically the proteolytic domain. Unfor-tunately, the NTDs of LONP1 and LONP2 are poorly conserved, and so data on LONP1 cannot be easily extrapolated to LONP2. Thus, the nature of the sub-strates targeted by LONP2 and the mechanisms reg-ulating its activity remain largely unknown. In this work, we aim to comprehensively investigate the role of LONP2 in peroxisomes. To this end, LONP2 was heterologously expressed in E. coli and purified us-ing affinity and size-exclusion chromatography. The hexameric, catalytically active fraction of LONP2 was subsequently used in proteolytic assays with β-casein as a substrate. Structural analysis of LONP2 reveals distinct features compared to mitochondrial LONP1. Further studies, including the characterization of mu-tant variants, are currently underway.
- Reduction of gallic acid by in situ BHPublication . Silva, Rui Pedro; Prudêncio, Cristina; Vieira, Mónica; Almeida Vieira, Mónica AndreiaLithium aluminum hydride (LiAlH₄, LAH) is one of the most widely used reducing agents in organic synthesis due to its high reactivity and broad applicability. It effi-ciently reduces carbonyl-containing compounds such as esters, carboxylic acids, amides, and nitriles to the corresponding alcohols or amines, whereas sodium borohydride (NaBH₄) is generally limited to reducing aldehydes and ketones1 under mild conditions. The superior reactivity of LAH arises from the highly po-lar Al-H bond, which enables hydride transfer to less reactive carbonyl groups.2 However, LAH presents important drawbacks: it is highly moisture-sensitive, reacts violently with water or protic solvents, and re-quires strictly anhydrous conditions, aprotic solvents such as THF or diethyl ether, and an inert atmosphere. On the other hand, some studies are shown a new al-ternative to use NaBH4 in the presence of an electro-phile, as iodine, generating new alternatives to the use of LAH.3 This work presents a safer and more conve-nient alternative for the reduction of carboxylic acids using gallic acid as a model substrate. The methodolo-gy employs NaBH₄ in the presence of boron trifluoride diethyl etherate (BF₃·Et₂O), which generates borane (BH₃) in situ, a much stronger reducing species capable of efficiently converting carboxylic acids into primary alcohols.4 Compared with LAH, this approach offers milder reaction conditions, improved operational safe-ty, and easier handling, since in situ generation of BH₃ avoids direct manipulation of highly reactive reducing agents while maintaining high reduction efficiency. The reduction of gallic acid is particularly significant due to its multiple hydroxyl groups and the sensitivity of the aromatic structure, making selectivity essential. Controlled addition of gallic acid to the BH₃ solution proved effective for conversion into 3,4,5-trihydroxy-benzyl alcohol while minimizing possible interactions between free BF₃ and phenolic groups. Overall, this methodology represents a practical and valuable al-ternative for safer and milder organic reductions with good functional-group tolerance.