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- Synthesis of ionic liquids based on Betulinic Acid-based for Glioblastoma therapyPublication . Nunes, Ana; Fernandes, Sílvia; Prudêncio, Cristina; Gomes, Paula; Ferraz, Ricardo; Fernandes, Sílvia; Prudêncio, Cristina; Ferraz, RicardoIonic liquids (ILs) are a novel category of organic salts that exhibit diverse physicochemical properties and a broad spectrum of potential applications. Driven by the increasing relevance of Ils in various scientific fields, recent studies have shown that it is feasible to synthesize ILs involving betulinic acid (BA) and a range of cations with distinct characteristics, using straightforward and cost -effective procedures. Betulinic acid (BA) is recognized for its selective cytotoxicity against tumour cells, primarily through its modulation of apoptotic and angiogenic pathways. These properties highlight its promise as an alternative therapeutic candidate for the treatment of glioblastoma. Our work seeks to leverage the antitumor capabilities of BA in conjunction with the unique features of ILs to establish innovative and effective strategies to counteract the aggressive nature of glioblastoma. To synthesize the desired ILs, we employed a neutralization approach involving acid-base reactions between BA and equimolar quantities of selected ammonium and phosphonium cations. This method enabled the efficient production of the target compounds, with high yields and confirmed structural integrity. Buiding upon our previous findings-where BA-derived lLs demonstrated promising activity against lung and liver cancers-we are confident that the newly developed lLs will also exhibit significant therapeutic potential in glioblastoma treatment.
- Glioblastoma: therapeutic effect of betulinic acid under an adipocytic environmentPublication . Azevedo, Lara; Ferraz, Ricardo; Vieira, Mónica; Fernandes, Silvia; Almeida Vieira, Mónica Andreia; Fernandes, SílviaGlioblastoma is the most common malignant tumor of the central nervous system, characterized by high incidence and poor survival rates, Current treatment strategies face significant challenges due to therapy resistance and tumor recurrence, highlighting the need for novel therapeutic approaches. Moreover, obesity has emerged as a potential risk factor in glioblastoma progression, being linked to chronic inflammation, metabolic dysregulation, and elevated levels of growth factors, all of which may promote tumor growth and invasiveness, Betulinic acid (BA) has shown anticancer activity in several tumor models, which along with anti-adipogenic properties, makes it a promising candidate for addressing these conditions, This study aims at: 1) assessing the BA effect on tumoral cell's apoptosis; 2) assessing the BA effect on adiposity and adipocytes secretome pattern, Thus, U251 and A172 human glioblastoma cells, and normal 3T3-Ll cell line were treated with 1-20 μM BA and cell's viability was quantified through MTT assays, Adiposity pattern and adipocytes differentiation with or without BA treatment were assessed and quantified. Results show that BA treatment significantly decreases tumoral cells viability without affecting non-tumoral cells, including for higher doses of the compound. These in vitro studies were essential to define BA doses of 1μ M, 2.5μ M, and 10μM as suitable for the adipocytes' differentiation and production of adipocytes-conditioned medium using 3T3-Ll cells. These preliminary results will promote the assessment of the secretome composition under BA treatment and will allow to explore the anti-tumoral potential of BA under an adipocytic environment. By exploring the potential therapeutic effects of BA in the glioblastoma-obesity relationship, this study seeks to contribute to the development of novel treatment approaches for glioblastoma and obesity.
- 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.
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