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- 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.
- 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.
- Use of natural compounds in obesity: A study on knowledge, attitudes, and practices regarding supplementsPublication . Castro, Bruno; Vieira, Mónica; Prudêncio, Cristina; Fernandes, Sílvia; Fernandes, Sílvia; Almeida Vieira, Mónica Andreia; Prudêncio, CristinaObesity represents a major public health challenge, being associated with high morbidity, mortality, and significant socioeconomic impact [1]. Consequently, there has been growing interest in the use of natural compounds with potential anti-obesity effects, driven by the perception that these compounds may repre-sent a safer and more accessible alternative to conven-tional therapies. Considering that scientific evidence regarding their efficacy and safety remains limited [2], the present study aims to analyze the knowledge, at-titudes, and practices of the Portuguese population regarding the use of natural compounds in obesity management. Following institutional ethical approval, data collection was carried out through an anonymous questionnaire developed on the Microsoft Forms plat-form and disseminated online through the academ-ic community of E2S|IPP. The questionnaire includes questions designed to obtain information on sociode-mographic variables, patterns of use, perceptions of efficacy and safety, level of knowledge about natural compounds, and the occurrence of adverse effects. Data were analyzed using descriptive and inferential statistics in SPSS v31 to explore possible associations between variables. To date, 683 individuals have par-ticipated in the study, the majority being female (79%), with the 45–65 age group (40%) being the most rep-resented. The results show that 52% of participants reported having heard about natural compounds used for weight loss, while approximately 10% reported us-ing them, with the most common being Conjugated Linoleic Acid, Berberine, and Caffeine. Among individ-uals who reported use, 7% indicated the occurrence of adverse effects associated with consumption. The data obtained contribute to a better understanding of population behaviors and perceptions regarding nat-ural compounds, fostering the development of new educational strategies and promoting more informed, safe, and evidence-based use.
- Psychostimulants and neuroinflammation: finding critical players in the crostalk between glial cells and neuronsPublication . Bravo, Joana; Andrade, Elva Bonifácio; Vieira, Renato; Lorga, Inês; Azevedo, M.; Rodrigues, J.; Magalhães, Ana; Relvas, João B.; Summavielle, Teresa; Summavielle, Teresa; Bonifácio Andrade, Elva; Bravo, JoanaExposure to psychostimulants has been classically associated with damage to neuronal terminals. However, it is now accepted that interaction between neuronal and glial cells also contributes to the addictive behavior. We have recently shown that acute methamphetamine (Meth), a powerful psychostimulant, causes microgliosis and increases microglia activation through astrocytic-TNF release. We are now interested in clarifying the progression of neuroinflammation under chronic drug exposure and how different brain and immune cells contribute to this inflammatory process.To explore this, firstly, we performed a proteomic analysis, in different phases of the addictive process, in mice exposed to an escalating dosing of Meth for ten days (Meth10d). To validate the conditioning power of our model,mice were tested in a condition place preference (CPP) at 10d of Meth, and 2 or 10 days of withdrawal (WD). At all these time points, mice were seen to be strongly conditioned by Meth. Next, we conducted a proteomic analysis to compare the different time points (using the hippocampus, where we previously found robust microgliosis underMeth). We found a proteome profile that varied substantially with exposure (Meth10d) and after a short- (WD2d)and long-term withdrawal (WD10d) periods. Interestingly, the most altered pathways were neuro transmitter-related. However, we also identified significant differences in Wnt signaling, which was previously linked to regulation of microglia reactivity. As such, we evaluated the microglia profile after chronic Meth exposure and at withdrawal. In the hippocampus, the number of microglia cells was significantly increased at Meth10d and remained also increased at WD2d. Microglia presented a more ameboid-like shape at Meth10d, but its ramified morphology was recovered atWD2d. Importantly, our proteomic data also revealed that during Meth withdrawal, several microglial receptors were down regulated, suggesting that microglia was in a “primed” state. In addition, as the crosstalk between neurons and microglia seems to be relevant for the behavioral expression of Meth, we are dissecting the modulation of microglia by neurons under Meth exposure, to evaluate neuroimmune regulatory ligand-receptor pairs that seem to impact onthe neuron-microglia interaction. Of note, some these ligand-receptor pairs seem to be down regulated by chronic Meth and during abstinence, which may be associated with reduced neuronal ability to down regulate microglia reactivity, and lead to increased neuronal damage.We fore see that these receptors may prove to be interesting therapeutic targets for the treatment of addiction, and therefore we will manipulate them to confirm their value in reducing relapse rates and improve addiction treatments.
- Blockingmethamphetamine-induced microglia reactivity by targeting glutamate receptorsPublication . Summavielle, Teresa; Canedo, Teresa; Silva, Ana Isabel; Andrade, Elva Bonifácio; Almeida, Tiago O.; Bravo, Joana; Terceiro, Ana Filipa; Canedo, Teresa; Silva, Ana Isabel; Magalhães, Ana; Relvas, João B.; Bonifácio Andrade, Elva; Bravo, JoanaExposure to psychostimulants has been classically associated with damage to neuronal terminals. However, it is now accepted that interaction between neuronal and glial cells also contributes to the addictive behavior. We have recently shown that acute methamphetamine (Meth), a powerful psychostimulant, causes microgliosis and increases microglia activation through astrocytic-TNF release1. We are now interested in clarifying the progression of neuroinflammation under chronic drug exposure and how different brain and immune cells contribute to this inflammatory process.To explore this, firstly, we performed a proteomic analysis, in different phases of the addictive process, in mice exposed to an escalating dosing of Meth for ten days (Meth10d). To validate the conditioning power of our model, mice were tested in a condition place preference (CPP) at 10d of Meth, and 2 or 10 days of withdrawal (WD). At all these time points, mice were seen to be strongly conditioned by Meth. Next, we conducted a proteomic analysis to compare the different time points (using the hippocampus, where we previously found robust microgliosis underMeth1). We found a proteome profile that varied substantially with exposure (Meth10d) and after a short- (WD2d)and long-term withdrawal (WD10d) periods. Interestingly, the most altered pathways were neuro transmitter-related.However, we also identified significant differences in Wnt signaling, which was previously linked to regulation of microglia reactivity. As such, we evaluated the microglia profile after chronic Meth exposure and at withdrawal. In the hippocampus, the number of microglia cells was significantly increased at Meth10d and remained also increased at WD2d. Microglia presented a more ameboid-like shape at Meth10d, but its ramified morphology was recovered at WD2d. Importantly, our proteomic data also revealed that during Meth withdrawal, several microglial receptors were down regulated, suggesting that microglia was in a “primed” state. In addition, as the crosstalk between neurons and microglia seems to be relevant for the behavioral expression of Meth, we are dissecting the modulation of microgliaby neurons under Meth exposure, to evaluate neuroimmune regulatory ligand-receptor pairs that seem to impact on the neuron-microglia interaction. Of note, some these ligand-receptor pairs seem to be down regulated by chronic Meth and during abstinence, which may be associated with reduced neuronal ability to down regulate microglia reactivity, and lead to increased neuronal damage. We fore see that these receptors may prove to be interesting therapeutic targets for the treatment of addiction, and therefore we will manipulate them to confirm their value in reducing relapse rates and improve addiction treatments.
- Uso de filtros no processamento de Citologia de base líquida: Importante discriminar?Publication . Monteiro, Tiago; Fernandes, Sílvia; Silva, Regina; Silva, ReginaRegularmente em todo o mundo, sendo a filtração com recurso a filtros descartáveis a metodologia mais utilizada. Apesar de existirem filtros específicos para diversos tipos de processamento, pouco se sabe acerca das suas características, da forma como são empregues e do impacto da sua utilização no processamento de citologia de base líquida e consequente diagnóstico citológico. O estudo teve como principal objetivo avaliar e registar as características dos filtros usados em citologia de base líquida, e avaliar o impacto do seu uso indiscriminado, em Portugal. Para tal, estudou-se microscopicamente os poros de uma área de 7500µm2 de membrana de policarbonato de filtros de citologia de base líquida, indicados para processamento de amostras ginecológicas (Gyn) e não ginecológicas (Não Gyn), de três marcas diferentes. Procedeu-se à sua caracterização quanto ao número, diâmetro e distribuição dos poros. Foi ainda administrado um questionário a laboratórios de citologia em Portugal, e os dados recolhidos foram analisados com o intuito de caracterizar o uso das metodologias, os resultados obtidos permitem concluir que existem diferenças entre os filtros destinados aos vários tipos de processamento e entre filtros de diferentes marcas. Os filtros destinados ao processamento de amostras Gyn, para além de poros de maiores dimensões (7.2-7.5 µm), apresentam um menor número de poros (92-98) e respetiva sobreposição em 55 a 58% da área em análise. Por sua vez, os filtros recomendados para amostras Não Gyn apresentam menor diâmetro (5.7-6.1 µm), maior número de poros (107-122) e respetiva sobreposição numa maior área (60 a 83%).Considerados globalmente, os resultados revelam que as diferenças verificadas entre os filtros podem resultar na alteração ao nível da representação de estruturas celulares e de microrganismos, e o seu uso indiscriminado a nível laboratorial pode comprometer a avaliação citopatológica.
- Computacional pathology: What’s newPublication . Coelho, Daniel; Assunção, Teresa; Borrecho, Gonçalo; Geraldes, Mariana; Vinagre, Tiago; Ferreira, Inês; Ferreira, Ana; Fernandes, Ana Isabel; França, Amélia; Vale, João; Curado, Mónica; Mendes, Fernando; Martins, DianaThe term computacional pathology (CPath) has become a buzz-word among the digital pathology community. Adances in scanning systems, imaging technologies and storage devices are generating an ever-increasing volume of whole-slide images (WSI) acquired in clinical settings, which can be computacionally analyzed using artificial intelligence (AI), such as deep learning technologies, in a new área of development called CPath. The purpose of the review is to disseminate the latest news and futures perspectives by CPath. Deep learning in the context of CPath has methodological contributions that can be distinguished into approaches based on the final purpose of the analysis: predicting clinical endpoints such as cancer subtype, patient survival or genetic mutations from WSI and AI-based assistive tools, such as segmentation methods for WSI or virtual staining. The emergence of multipex imaging, spatially resolver genomic assays and 3D pathology, among other methodologies, will accelerate this trend, providing new opportunities for multimodal integration and discovering new biomarkers. Additionally, these developments will help automating labor-intensive manual work and reducing inter-observer variability diagnosis between pathologists, contributing to a better patient care. CPath will underpin the development of the next generation of cancer therapies and diagnostics, changing the clinical research and ultimately leading towards new cures or improved patient outcomes.
- Caraterização de tumores sólidos numa amostra pediátrica do Instituto Português de Oncologia do Porto Francisco Gentil, EPEPublication . Santos, Ana; Serra, Carla; Mota, Marlene; Sousa, Manuela; Mendes, CarlosCaracterizar uma amostra pediátrica com TS quanto à sua frequência, óbito, sexo e idade ao diagnóstico. Estudar o possível envolvimento da MO ao diagnóstico, recorrendo a dados qualitativos de mielogramas. Estudo observacional descritivo transversal, dos registos dos dados de pacientes pediátricos com TS (n=148), diagnosticados entre 2000-2008. O TS mais frequente foi o Neuroblastoma (NB=37,2%), e os com maior registo de óbitos foram Rabdomiossarcoma (RMS=30,4%) e Família Tumores Ewing (FTE=28,0%). Nas raparigas os Tumor Wilms (TW=68,0%) e Osteossarcoma (58,3%) foram mais frequentes, enquanto nos rapazes foram os Retinoblastoma (75,0%), RMS (65,2%) e NB (61,8%). O NB manifestou-se, em 30,9% dos casos, até aos 12 meses de idade, o TW, em 32,0% dos casos, até aos 2 anos, e o RMS, em 69,6% dos casos, entre os 1-10 anos. A maioria dos pacientes com FTE (80,0%), NB (74,6%) e RMS (73,9%) realizaram mielograma ao diagnóstico. Observando os resultados qualitativos do mielograma, os tumores onde poderá ter ocorrido invasão da MO foram o NB e RMS. Na frequência dos TS verificou-se que seguem o descrito na literatura, excepto a FTE que surge em segundo lugar conjuntamente com o TW, sendo o NB mais frequente. Nos óbitos, o tumor com maior registo, RMS, não corresponde ao com maior gravidade (NB) descrito na investigação. Para estes resultados pode ter contribuído o número reduzido da amostra. Relativamente ao sexo, o NB e o RMS são mais frequentes nos rapazes, enquanto o TW é mais frequente nas raparigas, como descrito na literatura. Doentes com NB, TW e RMS apresentaram idades ao diagnóstico que indicam um melhor prognóstico. O NB, RMS e FTE, em alguns casos, disseminam-se para a MO, podendo ser esta a explicação para os resultados obtidos quanto à realização ou não de mielograma. Dos resultados obtidos nos mielogramas, verifica-se que poderá ter ocorrido invasão medular nos NB e RMS. Propõe-se continuar este estudo com amostra, por TS, mais representativa e com dados dos hemogramas e biópsias ósseas.
- Molecular profiling of prognostic biomarkers in MelanomaPublication . Jesus, Marta; Correia, Beatriz; Costa, Catarina; João, Alexandre; Lopes, José Manuel; Barata, Catarina; Soares, Paula; Pópulo, HelenaCutaneous melanoma (CM) is the most aggressive and deadliest form of skin cancer (1). Its development is influenced by the genetic background, genetic mutations and environmental factors. The three most frequent mutations of CM appear in BRAF gene, which leads to uncontrolled cell growth, in NRAS gene, that disrupts normal cell signaling, and in TERT promoter, that increases telomerase activity and play a crucial role in tumor progression, influencing melanoma aggressiveness and patient outcome (2, 3). Despite advances in treatment, the prognosis of patients with advanced-stage remains poor (4). This work aims to improve prognosis determination by correlating identified molecular alterations with clinicopathological data. The analysis is being conducted on primary tumors collected at Hospital dos Capuchos. The study currently includes 29 samples collected between 2023 and 2024, 18 from male subjects and 11 from female subjects, with an average age of 69. Most of the melanomas were located on the trunk, and the most common diagnoses were nodular melanoma and superficial spreading melanoma. The mean Breslow thickness of the series is 3.78 mm, and 46% of samples have a mitotic index greater than 1. The samples were subjected to histopathological evaluation, followed by PCR and sequencing techniques, to identify the three mutations. Subsequent statistical analysis will be performed to assess the correlation between these genetic changes and clinicopathological features, with a focus on determining their impact on patient outcomes. The work is still ongoing, but the results will provide further information about the three mutations and their corresponding aggressiveness, as well as how the coexistence of different mutations influences patient prognosis. This study underlines the significance of molecular profiling in the prognosis assessment of melanoma patients, and it will provide a foundation for further research on molecular-based prognosis models.
- CytoPath®Easy: screening of cervical cancerPublication . Fernandes, Sílvia; Vilarinho, Ana Sofia; Silva, ReginaRecently, CytoPath®Easy kit was created by DiaPath S.p.A. and started to be commercially available for the screening of cervical cancer. Using this methodology, epithelial cells are immersed in a preservative liquid, and a thin-layer of cells in the slide is obtained through gravity sedimentation and filtration. The main objectives of this study are to evaluate the efficacy for the processing of cervical samples, for the detection of pre-neoplastic lesions and for the nucleic preservation and extraction by the kit. For this purpose, 215 cervical samples obtained by self-sampling were used: 174 were collected and processed by CytoPath® Easy and, as a control, 41 were collected and processed by the Thinprep® method. The samples were processed, stained by the Papanicolaou method, and independently evaluated microscopically for various morphological parameters; nucleic acids were isolated and evaluated for purity and integrity by spectrophotometry. Results obtained showed that both methods have a good performance, allowing the morphological evaluation of the cervical epithelium. However, the statistical analysis reveals that the methods are different from each other, with overall lower results being obtained in the method under study (p<0.001). In turn, both methods allow the extraction of good quality and quantity of DNA. Although some differences were found regarding morphology of the cells fixed and processed by the CytoPath®Easy method, this new methodology reveals efficient for the preservation of nucleic acids. Thus, its use in cervical cancer screening is recommended.
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