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- Betulinic acid and obesity-related disordersPublication . Azevedo, Lara; Ferraz, Ricardo; Vieira, Mónica; Prudêncio, Cristina; Fernandes, Sílvia; Ferraz, Ricardo; Almeida Vieira, Mónica Andreia; Prudêncio, Cristina; Fernandes, SílviaThe obesity epidemic is not just a health issue, it is increasingly driving a shift in the prevalence of chronic diseases, affecting 890 million adults and straining healthcare systems worldwide. Conditions such as type 2 diabetes mellitus, cardiovascular diseases, non-alcoholic fatty liver disease, and various cancer types are closely tied to this growing crisis. Betulinic acid has anti-inflammatory, antioxidant and anti-cancer properties and modulates key metabolic pathways such as NF-κB and AMPK signaling. This compound improves insulin sensitivity, reduces hepatic steatosis, mitigates the progression of atherosclerosis and fibrosis, and suppresses inflammatory responses, which are important in treating those obesity-related disorders. Additionally, betulinic acid use in cancer treatment has been explored due to its potential in angiogenesis and metastasis inhibition and promotion of apoptosis. This review spotlights the therapeutic potential of the natural compound betulinic acid in processes such as insulin sensitivity, glucose and lipid metabolism, adiposity, inflammation, oxidative stress, intestinal microbiota, and other mechanisms underlying different obesity-related disorders. Overall, besides strong therapeutic potential of betulinic acid, described limitations such as poor aqueous solubility, limited bioavailability, production and extraction have resulted in scarce clinical data making it premature to draw definitive conclusions regarding its application in clinical practice.
- Combination therapy with CD147‐targeted nanoparticles carrying phenformin decreases lung cancer growthPublication . Pereira-Nunes, Andreia; Ferreira, Helena; Abreu, Sara; Guedes, Marta; Neves, Nuno M.; Baltazar, Fátima; Granja, Sara; Granja, SaraLung cancer is one of the most fatal cancers worldwide. Resistance to conventional therapies remains a hindrance to patient treatment. Therefore, the development of more effective anti-cancer therapeutic strategies is imperative. Solid tumors exhibit a hyperglycolytic phenotype, leading to enhanced lactate production; and, consequently, its extrusion to the tumor microenvironment. Previous data reveals that inhibition of CD147, the chaperone of lactate transporters (MCTs), decreases lactate export in lung cancer cells and sensitizes them to phenformin, leading to a drastic decrease in cell growth. In this study, the development of anti-CD147 targeted liposomes (LUVs) carrying phenformin is envisioned, and their efficacy is evaluated to eliminate lung cancer cells. Herein, the therapeutic effect of free phenformin and anti-CD147 antibody, as well as the efficacy of anti-CD147 LUVs carrying phenformin on A549, H292, and PC-9 cell growth, metabolism, and invasion, are evaluated. Data reveals that phenformin decreases 2D and 3D-cancer cell growth and that the anti-CD147 antibody reduces cell invasion. Importantly, anti-CD147 LUVs carrying phenformin are internalized by cancer cells and impaired lung cancer cell growth in vitro and in vivo. Overall, these results provide evidence for the effectiveness of anti-CD147 LUVs carrying phenformin in compromising lung cancer cell aggressiveness.
- Prevenção do Cancro Cervical - O caso angolanoPublication . Andrade, P.; Silva, Regina Augusta; Rocha, Helder; Silva, ReginaO cancro cervical é a principal causa de mortalidade por cancro em Angola, colocando sérios desafios ao sistema de saúde do país. Esta situação resulta de deficiências nas infraestruturas de saúde pública, sensibilização da população e acesso a cuidados de saúde preventivos, apesar da disponibilidade de intervenções com boa relação custo-eficácia, como a vacinação contra o HPV e o rastreio do colo do útero. A implementação de programas de rastreio organizado é desafiante em contextos com poucos recursos, mesmo com a aplicação de métodos de inspeção visual, estes enfrentam limitações na precisão e cobertura diagnóstica. Similarmente, campanhas de vacinação contra o HPV, embora promissoras, são limitadas por barreiras sociogeográficas e logísticas. Esta revisão explora a situação atual da prevenção do cancro cervical em Angola, identificando os principais desafios, como a falta de estudos epidemiológicos, infraestruturas de rastreio e as barreiras socioeconómicas no acesso aos cuidados de saúde. Há ainda um trabalho considerável pela frente para cumprir a estratégia da Organização Mundial de Saúde para 2030 -90% de cobertura vacinal, 70% de rastreio e 90% de tratamento. No entanto, podem ser feitos progressos com a adoção de métodos de rastreio inovadoras que incluam a auto-amostragem e unidades de saúde móveis, expandindo esforços de vacinação e reduzindo obstáculos culturais e logísticos.
- ZYG-12/Hook's dual role as a dynein adaptor for early endosomes and nuclei is regulated by alternative splicing of its cargo binding domainPublication . Carvalho, Cátia; Moreira, Matilde; Barbosa, Daniel J.; Chan, Fung-Yi; Koehnen, Carlota Boal; Teixeira, Vanessa; Rocha, Helder; Green, Mattie; Carvalho, Ana Xavier; Cheerambathur, Dhanya K.; Gassmann, RetoThe microtubule motor cytoplasmic dynein-1 transports and positions variousorganelles, but the molecular basis of this functional diversity is not fully understood. Cargoadaptors of the Hook protein family recruit dynein to early endosomes (EE) in fungi andhuman cells by forming the FTS–Hook–FHIP (FHF) complex. By contrast, the Caenorhabdi-tis elegans Hook homologue ZYG-12 recruits dynein to the nuclear envelope (NE) in themeiotic gonad and mitotic early embryo by forming a Linker of Nucleoskeleton and Cy-toskeleton (LINC) complex. Here, we demonstrate that ZYG-12 recruits dynein to EE inepithelia. We identify and functionally characterize the homologues of FTS (UBC-19) andFHIP (FHIP-1) that constitute the C. elegans FHF complex, validate the predicted FHIP-1–RAB-5 binding interface in vivo, and show that ZYG-12 forms FHF via a conserved segmentthat precedes, and is distinct from, its C-terminal NE targeting domain. Finally, we showthat C-terminal ZYG-12 splice isoforms differ in their ability to target to the NE and EE.We conclude that the C. elegans Hook adaptor evolved to recruit dynein to two distinctorganelles, and that cargo specificity of ZYG-12 is regulated by alternative splicing.
- Application of CytoPath®easy vials in Cervical Cancer screening: Self‑sampling approachPublication . Fernandes, Sílvia P. M.; Vilarinho, Ana Sofia; Frutuoso, Amaro; Teixeira, Cidália; Silva, Regina Augusta A. P."CytoPath®Easy kit (DiaPath S.p.A.) offers a major advantage compared to other commercially available kits available for the screening of cervical cancer, as it does not require additional equipment for sample processing. Using this methodology, collected epithelial cells are immersed in a preservative liquid before setting as a thin layer on a slide via gravity sedimentation. Aims: To evaluate the suitability of the CytoPath®Easy kit for the processing of cervicalsamples, detection of pre‑neoplastic lesions, and nucleic preservation and extraction for HR‑HPV diagnosis. A total of 242 self‑sampled cervicalspecimens were utilized, with 192 collected in CytoPath®Easy vials and 50 collected and processed using the ThinPrepTM for comparative analysis. The samples underwent processing, Papanicolaou staining, and microscopic evaluation for morphological parameters. The extracted nucleic acids were assessed for purity and integrity, and the detection of high‑risk human papillomavirus (HR‑HPV) was carried out using the Alinitym HR HPV system kit (Abbott Laboratórios Lda). Both methods demonstrated effective performance, enabling the morphological assessment of the cervical epithelium. Statistical analysis indicated that ThinPrepTM yielded significantly better results in terms of cellularity. Conversely, CytoPath®Easy exhibited superior performance in terms of the quantity of extracted DNA and its degree of purification. Concerning the time consumed during processing, both methods were comparable, with the CytoPath®Easy methodology standing out for its cost‑effectiveness, as it does not necessitate additional instruments and consumables. The novel CytoPath®Easy methodology proves effective in preserving both nucleic acids and cell morphology characteristics, two crucial features for cervical cancer screening."
- An evolutionary perspective on the relationship between kinetochore size and CENP-E dependence for chromosome alignmentPublication . Almeida, Ana C.; Almeida, Ana C.; Rocha, Hélder; Rocha, Hélder; Raas, Maximilian W. D.; Raas, Maximilian W. D.; Witte, Hanh; Witte, Hanh; Sommer, Ralf J.; Sommer, Ralf J.; Snel, Berend; Snel, Berend; Kops, Geert J. P. L.; Kops, Geert J. P. L.; Gassmann, Reto; Gassmann, Reto; Maiato, Helder; Maiato, HelderChromosome alignment during mitosis can occur as a consequence of bi-orientation or is assisted by the CENP-E (kinesin-7) motor at kinetochores. We previously found that Indian muntjac chromosomes with larger kinetochores bi-orient more efficiently and are biased to align in a CENP-E-independent manner, suggesting that CENP-E dependence for chromosome alignment negatively correlates with kinetochore size. Here, we used targeted phylogenetic profiling of CENP-E in monocentric (localized centromeres) and holocentric (centromeres spanning the entire chromosome length) clades to test this hypothesis at an evolutionary scale. We found that, despite being present in common ancestors, CENP-E was lost more frequently in taxa with holocentric chromosomes, such as Hemiptera and Nematoda. Functional experiments in two nematodes with holocentric chromosomes in which a CENP-E ortholog is absent (Caenorhabditis elegans) or present (Pristionchus pacificus) revealed that targeted expression of human CENP-E to C. elegans kinetochores partially rescued chromosome alignment defects associated with attenuated polar-ejection forces, whereas CENP-E inactivation in P. pacificus had no detrimental effects on mitosis and viability. These data showcase the dispensability of CENP-E for mitotic chromosome alignment in species with larger kinetochores.
- Glucose metabolism as a potential therapeutic target in Cytarabine-resistant acute Myeloid leukemiaPublication . Pereira-Vieira, Joana; Weber, Daniela D.; Silva, Sâmia; Barbosa-Matos, Catarina; Granja, Sara; Reis, Rui Manuel; Queirós, Odília; Ko, Young H.; Kofler, Barbara; Casal, Margarida; Baltazar, Fátima; Granja, SaraAltered glycolytic metabolism has been associated with chemoresistance in acute myeloid leukemia (AML). However, there are still aspects that need clarification, as well as how to explore these metabolic alterations in therapy. In the present study, we aimed to elucidate the role of glucose metabolism in the acquired resistance of AML cells to cytarabine (Ara-C) and to explore it as a therapeutic target. Resistance was induced by stepwise exposure of AML cells to increasing concentrations of Ara-C. Ara-C-resistant cells were characterized for their growth capacity, genetic alterations, metabolic profile, and sensitivity to different metabolic inhibitors. Ara-C-resistant AML cell lines, KG-1 Ara-R, and MOLM13 Ara-R presented different metabolic profiles. KG-1 Ara-R cells exhibited a more pronounced glycolytic phenotype than parental cells, with a weaker acute response to 3-bromopyruvate (3-BP) but higher sensitivity after 48 h. KG-1 Ara-R cells also display increased respiration rates and are more sensitive to phenformin than parental cells. On the other hand, MOLM13 Ara-R cells display a glucose metabolism profile similar to parental cells, as well as sensitivity to glycolytic inhibitors. These results indicate that acquired resistance to Ara-C in AML may involve metabolic adaptations, which can be explored therapeutically in the AML patient setting who developed resistance to therapy.
- In vitro and in vivo evaluation of novel chromeno[2,3-d]pyrimidinones as therapeutic agents for triple negative breast cancerPublication . Carvalho, Luísa; Lima, Fábio Pedroso de; Cerqueira, Mónica; Silva, Ana; Pontes, Olívia; Oliveira-Pinto, Sofia; Guerreiro, Sara; Costa, Marta D.; Granja, Sara; Maciel, Patrícia; Longatto-Filho, Adhemar; Baltazar, Fátima; Proença, Fernanda; Costa, Marta; Granja, SaraTriple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and the limited therapeutic options show poor efficacy in patients, associated to severe side effects and development of resistance. Considering that chromene-based scaffolds proved to be attractive candidates for cancer therapy, herein we prepared new chromeno[2,3-d]pyrimidinone derivatives by a simple two step procedure, starting from the reaction of cyanoacetamide and a salicylaldehyde. A cell viability screening in several breast cancer cell lines allowed to identify two promising compounds with IC50 values in the low micromolar range for TNBC cells. These chromenes inhibited cell proliferation, induced cell cycle arrest and triggered cell death through apoptosis. In vivo studies revealed a safe profile in invertebrate and vertebrate animal models and confirmed their capacity to inhibit tumor growth in the CAM model, inducing significant tumor regression after 4 days of treatment. The two compounds identified in this study are promising drug candidates for TNBC treatment and valuable hits for future optimization, using the versatile synthetic platform that was developed.
- Development of bioluminescent Group B streptococcal strains for longitudinal infection studiesPublication . Lorga, Inês; Geraldo, Rafaela; Soares, Joana; Oliveira, Liliana; Firon, Arnaud; Bonifácio Andrade, ElvaGroup B Streptococcus (GBS) remains the leading bacterial cause of invasive neonatal disease, resulting in substantial morbidity and mortality. New therapeutic approaches beyond antibacterial treatment to prevent neonatal disease outcomes are urgent. One significant limitation in studying GBS disease and progression is the lack of non-invasive technologies for longitudinal studies. Here, we develop and compare three bioluminescent GBS strains for in vivo pathogenic analysis. Bioluminescence is based on the luxABCDE operon on a replicative vector (luxGBS-CC17), and the red-shifted firefly luciferase on a replicative vector (fflucGBS-CC17) or integrated in the genome (glucGBS-CC17). We show that luxGBS-CC17 is suitable for in vitro analysis but does not produce a significant bioluminescent signal in infected pups. In contrast, the fflucGBS-CC17 results in a strong bioluminescent signal proportional to the organ colonisation level. However, the stability of the replicative vector depends on the route of infection, especially when pups acquire the bacteria from infected vaginal mucosa. Stable chromosomal integration of luciferase in glucGBS-CC17 leads to significant bioluminescence in both haematological and vertical infection models associated with high systemic colonisation. These strains will allow the preclinical evaluation of treatment efficacy against GBS invasive disease using wholemouse bioluminescence imaging.
- The project Trauma-Informed Practice for workers in public service settings: New strategies for the same old objectivePublication . Dores, Artemisa Rocha; Letica-Crepulja, Marina; Silva, ReginaMany professionals are confronted in their practice with clients who show post-traumatic symptoms (PTS). “Trauma-informed practice” helps professionals recognize, understand, and appropriately respond to the effects of trauma. This work presents the “Trauma-Informed Practice for Workers in Public Service Settings” – TIPS (Erasmus+). The project sought to: (1) increase awareness among professionals about the occurrence of PTS; (2) equip them with skills to identify PTS and reduce barriers related to discrimination; (3) improve the care and support provided; and (4) reflect on innovative pedagogical strategies. This was a distance learning experience aimed at professionals from various sectors, namely education. The project is based on training through new digital spaces and learning tools, combined with strategies of storytelling and teaching practices based on narrativization. The project includes the following final outputs (1) guidelines; (2) a catalog; (3) a resource pack; (4) an interactive e-platform; and (5) a mobile application. The diverse learning opportunities and formats aims to promote new and improved learning strategies, respecting personal preferences and diversity of learning styles, thus ensuring new paths to the “old” objective of effective learning