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- 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
- Polymorphisms and haplotypes of TOLLIP and MUC5B are associated with susceptibility and survival in patients with fibrotic hypersensitivity pneumonitisPublication . Mota, P.C.; Soares, M.L.; Ferreira, A.C.; F. Santos, Rita; Cavaleiro Rufo, J; Vasconcelos, D.; Carvalho, A.; Guimarães, S.; Vasques-Novo, F.; Cardoso, C.; Melo, N.; Alexandre, A.T.; Coelho, D.; Novais-Bastos, H.; Morais, A.Hypersensitivity pneumonitis (HP) is an interstitial lung disease with diverse clinical features that can present a fibrotic phenotype similar to idiopathic pulmonary fibrosis (IPF) in genetically predisposed individuals. While several single nucleotide polymorphisms (SNPs) have been associated with IPF, the genetic factors contributing to fibrotic HP (fHP) remain poorly understood. This study investigated the association of MUC5B and TOLLIP variants with susceptibility, clinical presentation and survival in Portuguese patients with fH. A case-control study was undertaken with 97 fHP patients and 112 controls. Six SNPs residing in the MUC5B and TOLLIP genes and their haplotypes were analyzed. Associations with risk, survival, and clinical, radiographic, and pathological features of fHP were probed through comparisons among patients and controls. MUC5B rs35705950 and three neighboring TOLLIP variants (rs3750920, rs111521887, and rs5743894) were associated with increased susceptibility to fHP. Minor allele frequencies were greater among fHP patients than in controls (40.7% vs 12.1%, P<0.0001; 52.6% vs 40.2%, P = 0.011; 22.7% vs 13.4%, P = 0.013; and 23.2% vs 12.9%, P = 0.006, respectively). Haplotypes formed by these variants were also linked to fHP susceptibility. Moreover, carriers of a specific haplotype (G-T-G-C) had a significant decrease in survival (adjusted hazard ratio 6.92, 95% CI 1.73–27.64, P = 0.006). Additional associations were found between TOLLIP rs111521887 and rs5743894 variants and decreased lung function at baseline, and the MUC5B SNP and radiographic features, further highlighting the influence of genetic factors in fHP. These findings suggest that TOLLIP and MUC5B variants and haplotypes may serve as valuable tools for risk assessment and prognosis in fibrotic hypersensitivity pneumonitis, potentially contributing to its patient stratification, and offer insights into the genetic factors influencing the clinical course of the condition.
- Pesquisa de mutação T790M sequencial e alteração da abordagem terapêutica após 2 linhas de quimioterapia – caso clínicoPublication . Estevinho, Fernanda; Silva, Ana Catarina; Cirnes, Luís; Amaro, TeresinaA mutação do EGFR é identificada em 10 -50% dos doentes com cancro do pulmão de não pequenas células (CPNPC). Após terapêutica de primeira linha com inibidores da tirosinacinase (TKI) de 1.ª ou 2.ª linha, em doentes com CPNPC e mutação sensibilizadora do EGFR, o mecanismo de resistência mais frequente é a aquisição de mutação T790M, em 50 -70%. Apresenta -se o caso clínico de uma mulher com CPNPC, cT2bN1M1b, estadio IV, com metastização cerebral e suprarrenal ao diagnóstico e mutação do EGFR. Realizou radioterapia holocraniana e tratamento com erlotinib. Deteção de mutação T790M após 2 esquemas de quimioterapia, com pesquisas prévias em biópsia líquida e histológica negativas para a pesquisa de mutação T790M. Oito anos após o diagnóstico, está funcionalmente autónoma, assintomática, sob terapêutica com osimertinib. Este caso ilustra a importância do conhecimento do mecanismo de resistência nos doentes com CPNPC e mutações do EGFR, e o papel de biopsias sequenciais
- Combined germline and tumor mutation signature testing identifies new families with NTHL1 tumor syndromePublication . Pinto, Carla; Guerra, Joana; Pinheiro, Manuela; Escudeiro, Carla; Santos, Catarina; Pinto, Pedro; Porto, Miguel; Bartosch, Carla; Silva, João; Peixoto, Ana; Teixeira, Manuel R.NTHL1 tumor syndrome is an autosomal recessive rare disease caused by biallelic inactivating variants in the NTHL1 gene and which presents a broad tumor spectrum. To contribute to the characterization of the phenotype of this syndrome, we studied 467 index patients by KASP assay or next-generation sequencing, including 228 patients with colorectal polyposis and 239 patients with familial/personal history of multiple tumors (excluding multiple breast/ovarian/polyposis). Three NTHL1 tumor syndrome families were identified in the group of patients with polyposis and none in patients with familial/personal history of multiple tumors. Altogether, we identified nine affected patients with polyposis (two of them diagnosed after initiating colorectal cancer surveillance) with biallelic pathogenic or likely pathogenic NTHL1 variants, as well as two index patients with one pathogenic or likely pathogenic NTHL1 variant in concomitance with a missense variant of uncertain significance. Here we identified a novel inframe deletion classified as likely pathogenic using the ACMG criteria, supported also by tumor mutational signature analysis. Our findings indicate that the NTHL1 tumor syndrome is a multi-tumor syndrome strongly associated with polyposis and not with multiple tumors without polyposis.
- Adenocarcinoma numa mulher histerectomizada – Estudo de casoPublication . Nunes, Catarina; Maia, C.; Leite, P.; Dominguez, R.A presença de células de tipo glandular na vagina em mulheres histerectomizadas pode ter várias origens. Estas células podem sofrer alterações malignas e originar adenocarcinoma primário da vagina. Esta neoplasia é rara, sendo o fator de risco mais frequente para o seu desenvolvimento a exposição intrauterina ao dietilestilbestrol. Relatamos o caso de uma paciente de 70 anos, com história clínica de histerectomia realizada aos 36 anos de idade para controlar hemorragia pós-aborto. No exame ecográfico foi detetada uma massa na parede anterior da vagina. Na citologia vaginal foram observadas células de tipo glandular atípicas, com características de adenocarcinoma, sem outras especificações. A biópsia realizada posteriormente mostrou um adenocarcinoma da vagina. Não há informação clínica acerca de uma possível exposição intrauterina ao composto dietilestilbestrol.
- Metaplasia transicional em citologia cérvico-vaginal: um estudo de casoPublication . Ferreira, Filipa; Ferreira, M.; Fialho, C.; Amaro, T.A metaplasia transicional cérvico-vaginal é uma condição benigna que está associada com atrofia e ocorre em mulheres na peri e pós-menopausa. O caso em análise reporta uma citologia cérvico-vaginal convencional corada pelo método de Papanicolaou de uma mulher de 57 anos de idade, cujo diagnóstico foi Células Pavimentosas Atípicas não excluindo Lesão Intraepitelial de Alto Grau (ASC-H). Posteriormente, o exame histológico revelou metaplasia transicional. A metaplasia transicional apresenta características citológicas que podem ser confundidas com atipia de células pavimentosas, dificultando a sua interpretação citológica. As caraterísticas mais determinantes para a sua identificação e distinção de situações de lesão são a ausência de atipia citológica e a presença de fendas longitudinais, apesar de estas não serem específicas desta condição.