Departamento de Genética Humana
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- Acute venous thromboembolism plasma and red blood cell metabolomic profiling reveals potential new early diagnostic biomarkers: observational clinical studyPublication . Febra, Claúdia; Saraiva, Joana; Vaz, Fátima; Soares, Nelson; Penque, PenqueBackground: Venous thromboembolism (VTE) is a leading cause of cardiovascular mortality. The diagnosis of acute VTE is based on complex imaging exams due to the lack of biomarkers. Recent multi-omics based research has contributed to the development of novel biomarkers in cardiovascular diseases. Our aim was to determine whether patients with acute VTE have differences in the metabolomic profile compared to non-acute VTE. Methods: This observational trial included 62 patients with clinical suspicion of acute deep vein thrombosis or pulmonary embolism, admitted to the emergency room. There were 50 patients diagnosed with acute VTE and 12 with non-acute VTE conditions and no significant differences were found between the two groups for clinical and demographic characteristics. Metabolomics assays identified and quantified a final number of 91 metabolites in plasma and 55 metabolites in red blood cells (RBCs). Plasma from acute VTE patients expressed tendency to a specific metabolomic signature, with univariate analyses revealing 23 significantly different molecules between acute VTE patients and controls (p < 0.05). The most relevant metabolic pathway with the strongest impact on the acute VTE phenotype was D-glutamine and D-glutamate (p = 0.001, false discovery rate = 0.06). RBCs revealed a specific metabolomic signature in patients with a confirmed diagnosis of DVT or PE that distinguished them from other acutely diseased patients, represented by 20 significantly higher metabolites and four lower metabolites. Three of those metabolites revealed high performant ROC curves, including adenosine 3',5'-diphosphate (AUC 0.983), glutathione (AUC 0.923), and adenine (AUC 0.91). Overall, the metabolic pathway most impacting to the differences observed in the RBCs was the purine metabolism (p = 0.000354, false discovery rate = 0.68). Conclusions: Our findings show that metabolite differences exist between acute VTE and nonacute VTE patients admitted to the ER in the early phases. Three potential biomarkers obtained from RBCs showed high performance for acute VTE diagnosis. Further studies should investigate accessible laboratory methods for the future daily practice usefulness of these metabolites for the early diagnosis of acute VTE in the ER.
- Alternative Splicing at the Crossroad of Inflammatory Bowel Diseases and Colitis-Associated Colon CancerPublication . Matos, Paulo; Jordan, PeterSimple Summary: Patients with ulcerative colitis (UC) face a higher risk of developing colorectal cancer (CRC) due to chronic inflammation, a known promoter of tumour growth. Here, we review the molecular differences between colitis-associated cancer (CAC) and sporadic CRC, with a focus on “alternative splicing”, a mechanism by which the same gene can produce various protein forms. We explore how inflammation triggers changes in this process, increasing cancer risk for UC patients. The revised data emphasize that additional research into these molecular changes could help identify new biomarkers (molecules that indicate disease progression) and pave the way for innovative treatments targeting these alterations. Such advances would improve outcomes and quality of life for patients while contributing to cancer prevention and care.
- Analysis and evaluation of genotoxicity and carcinogenicity assessment in EU legislation to improve regulatory implementation of NAMs: A focus on in silico approachesPublication . Bossa, Cecília; Raitano, Giuseppa; Benfenati, Emilio; Alivernini, Silvia; Andreoli, Cristina; Aquilina, G.; Attias, L.; Dusinska, Maria; El Yamani, N.; Louro, Henriqueta; Marcon, Francesca; Rundèn-Pran, E.; Russo, Maria Teresa; Silva, Maria João; Battistelli, Chiara LauraGenotoxicity and carcinogenicity are key endpoints for the risk assessment of all types of substances. Research on alternatives to animal testing for these endpoints has been active for decades, leading to the development of short-term in vitro tests that are integrated into current testing strategies. Nevertheless, high relevance is still devoted to data from in vivo studies. In parallel, progress in the comprehension of mechanisms underpinning genotoxicity and genotoxic carcinogenicity processes, together with the analysis of the great wealth of experimental data produced, allowed the discovery of structural determinants utilized in quantitative and qualitative structure-activity relationships and enabling in silico predictions of these endpoints. Presented here is a case study part of the collective effort carried out within the European Partnership for the Assessment of Risks from Chemicals (PARC) to address the challenges associated with innovation in chemical risk assessment, including the phasing out of animal testing through the introduction of New Approach Methodologies (NAMs) [1,2]. The case study aims to analyze current practices of the regulatory evaluation of genotoxicity and carcinogenicity hazard in several EU frameworks, in order to highlight needs and challenges in the actual or potential use of NAMs as well as short-and long- term goals towards the overcoming of animal testing. Among other NAMs, we are focusing on the role of in silico approaches highlighting strategies to increase the regulatory application and acceptance of QSAR based approaches. To this aim, the OECD QSAR Assessment Framework [3,4] has been identified as a suitable tool for evaluating the models and their predictions and will be applied to selected case studies. Moreover, a list of human relevant carcinogens has been developed as reference chemicals to evaluate and possibly refine in silico methodologies supporting a human-centric paradigm shift in toxicology.
- Assessing exposure and early biological effects in waste management workers using a harmonized occupational biomonitoring studyPublication . Tavares, Ana; Rosário, Rita; Aimonen, K.; Louro, Henriqueta; Martins, Carla; Viegas, Susana; Santonen, Tiina; Silva, Maria JoãoObjective: Human biomonitoring provides measurements of internal exposure to all chemicals by all routes of exposures and also addresses associated early biological effects. An occupational biomonitoring study requires a comprehensive and harmonised planning, including participants’ recruitment, sampling strategy, biomarkers definition and analysis, data collection, management, and reporting, in order to generate comparable and replicable data. In the framework of the European Partnership for the Assessment of Risks from Chemicals (PARC), a multi-centric biomonitoring campaign was planned to assess occupational exposure to hazardous chemicals from electronic and plastic waste management industries. We here present its design, comprising the assessment of external and internal exposure to several substances and characterization of early biological effects. Methods: Target participants are workers involved in e-waste and plastic recycling activities, and non-exposed individuals as controls. Besides industrial hygiene samples (e.g., air, settle dust) also biological samples (hair, blood, epithelial buccal cells and urine) are collected after informed consent, and a questionnaire is applied to all participants. Exposure biomarkers comprise measurements of heavy metals, flame retardants, per- and polyfluoroalkyl substances (PFAS), polycyclic aromatic hydrocarbons (PAHs), bisphenols, polychlorobiphenyls (PCBs), and other plasticizers in hair, blood, or urine samples by qualified laboratories. Effect biomarkers are assessed in blood (genotoxicity – micronucleus and comet assay - inflammation, epigenetic and oxidative stress markers), buccal cells (micronucleus) and urine samples (metabolic changes). Occupational hygiene measurements and contextual information will facilitate the interpretation of biomarkers data and the identification of potential confounding variables. Expected Results: The findings are expected to provide valuable information on exposure and associated early (preclinical) effects, and also to uncover groups at increased risk, supporting the promotion of good work practices and/or the implementation of efficient risk management strategies further improving worker’s health protection.
- Assessment of genotoxicity biomarkers in the scope of a human biomonitoring study in workers from E-waste management industriesPublication . Lopes Rosário, Rita Isabel; Silva, Maria João; Pina Martins, FranciscoElectrical and electronic waste (e-waste) represents a pressing global challenge with its rapid growth and hazardous composition. This recycling sector often involves inadequate worker safety, exposing e-workers to harmful substances like heavy metals and flame retardants via several routes, causing significant short- and long-term health risks. Human Biomonitoring (HBM) is a useful tool in assessing exposure and associated health outcomes through biomarkers like micronucleus (MN) in blood or epithelial cells, enabling the identification of early biological changes and linking exposure to disease. This HBM study used two assays,- the Buccal Micronucleus Cytome (BMNCyt) assay and the Cytokinesis-Block Micronucleus Cytome (CBMNCyt) assay,- to assess potential genotoxic effects from occupational e-waste exposure. The BMNCyt assay, conducted under the HBM4EU initiative, targets buccal mucosa epithelial cells, a primary barrier against hazardous agents, thus assessing local genotoxic effects. The CBMNCyt assay in peripheral blood lymphocytes, conducted under the PARC Project, reliably measures structural and numerical chromosomal changes, reflecting a systemic effect. This research aimed to assess early biological effects from exposure to pollutants from e-waste in in PBL and buccal epithelial cells of European e-waste workers, comparatively with control groups. The BMNCyt assay showed no significant differences in MN frequency between the exposed and control groups, while the CBMNCyt assay detected significantly increased frequencies of MN in exposed compared with non-exposed groups. Factors like small sample sizes, interindividual differences, and the use of protective equipment might have influenced results. Demographic/lifestyle variables showed differing impacts on MN formation between assays, but also potential influence, thus the importance of their consideration. Concluding, expanding e-waste occupational health research to include more workers/activities within the waste management industry and broader biomonitoring efforts is paramount. Boosting the understanding of health risks associated with those activities will help developing protective measures and mitigation strategies to safeguard the exposed workers’ health.
- CCL2 expression predicts clinical outcomes and regulates E-cadherin and angiogenesis in pituitary tumoursPublication . Silva, Ana Luísa; Barry, Sayka; Lopes-Pinto, Mariana; Joaquim, Rita; Miranda, Catarina; Reis, Fábio; Miranda, Micaella; Matos, Paulo; Suleyman, Oniz; Oliveira, Tiago; López-Presa, Dolores; Borrecho, Gonçalo; Tortosa, Francisco; Faria, Claúdia C.; Korbonits, Márta; Marques, PedroThe crosstalk between tumour cells and microenvironment components in pituitary neuroendocrine tumours (PitNETs), including chemokines, may impact tumour behaviour and clinical outcomes. CCL2 was previously identified as a key chemokine in PitNETs, but its role remains unknown. We aimed to study the role of CCL2 in defining the phenotype and clinical outcomes of PitNETs and in regulating macrophage chemotaxis, epithelial-to-mesenchymal transition (EMT) and angiogenesis. We studied CCL2 and E-cadherin expression, macrophages (CD68 and CD163) and vessels (CD31) in samples from 86 PitNET patients. Higher CCL2 mRNA expression was found in patients who required multimodal and multiple treatments and had active disease at the last follow-up. Higher CCL2 immunoreactivity was observed in patients with larger PitNETs. Among somatotroph tumours, CCL2 mRNA expression correlated with serum IGF-1 at the last follow-up. CCL2 mRNA expression levels correlated negatively with CDH1 expression and with E-cadherin complete membranous staining. In vitro, CCL2 downregulated E-cadherin expression in GH3 cells but did not affect cell morphology or migration. CCL2 expression correlated with the number of vessels, vessel perimeter and vessel area in PitNETs but not with PitNET-infiltrating macrophages. Our data suggest that CCL2 may lead to (or is at least a predictive marker of) poorer clinical outcomes and more difficult-to-treat PitNETs, potentially through its regulatory effects on different tumour-related mechanisms beyond immune cell chemotaxis, including in the activation of the EMT pathway and modulation of angiogenesis in PitNETs. Further studies are needed to corroborate our findings and to validate CCL2 as a potential predictive marker and therapeutic target in PitNETs.
- Chemicals in food: from toxicological research to regulatory challengesPublication . Silva, Maria JoãoCommunication on toxicological research and regulatory challenges related to chemicals in food.
- Dysregulated gene expression in colorectal cancer upon exposure to bisphenol A alternatives - a new approachPublication . Lacerda, Rafaela; Ventura, Célia; Louro, Henriqueta; Silva, Maria João; Romão, LuísaBisphenol A (BPA) has been widely used in plastics and resins since the 1950s, making it a common part of everyday products like food containers and bottle linings. Alternative substances are increasingly replacing BPA, but they are raising health and environmental concerns. Some mimic BPA’s endocrine-disrupting effects, while others affect different biological pathways. Substitutions in bisphenols can alter their biological properties, including nuclear receptor activation. Some BPA alternatives, like BPS, BPF and BPZ, may also pose cancer risks by activating oestrogen receptors, potentially even more than BPA itself. They may also contribute to colorectal cancer (CRC). Research suggests that BPA and its substitutes can influence cancer progression by altering cellular pathways, promoting metastasis and affecting gene expression. One of the key steps in gene expression regulation is translation initiation, whose canonical pathway is globally impaired under stress conditions, like exposure to BPA alternatives. Thus, we will subject NCM460 (normal intestinal mucosa) and HCT116 (colorectal carcinoma) cells to BPS, BPF and BPZ exposure and identify the transcripts actively being translated in such conditions, using ribosome profiling. We will analyse data with the R package anota2seq and evaluate the positively identified targets (compared to total RNA sequencing) for the existence of alternative mechanisms of translation initiation regulating their expression. The accurate characterisation of such mechanisms will be crucial for designing antisense RNA oligomers (ASOs) for potential therapeutic approaches. We will evaluate the cytotoxic effects of BPS, BPF and BPZ in the presence or absence of the selected alternatively translated transcripts (functional or targeted with the designed ASOs). Cytotoxic effects will be assessed through in vitro assays, analysing metabolic activity, membrane integrity, and cell proliferation. Thus, our research explores protein synthesis dysregulation to reduce CRC risks from BPS, BPF and BPZ exposure — an emerging public health issue.
- Economia circular e reciclagem de resíduos eletrónicos: o papel da biomonitorização humana na identificação de potenciais riscos ocupacionaisPublication . Silva, Maria João; Tavares, Ana; Rosário, Rita Lopes; Ventura, Célia; Namorado, Sónia; Martins, Carla; Louro, Henriqueta; Viegas, SusanaA reciclagem dos resíduos produzidos pela sociedade moderna, incluindo os resíduos elétricos e eletrónicos (REEE), é fundamental para a economia circular, um dos pilares do Pacto Ecológico Europeu. Porém, pode também representar um problema de saúde ocupacional, devido à exposição dos trabalhadores a metais (p.ex. chumbo e cádmio) e a compostos orgânicos (p.ex. ftalatos, PCBs) tóxicos. Como consequência, poderão surgir, a longo prazo, patologias respiratórias, renais, neurológicas ou oncológicas. Apresenta-se um estudo multicêntrico harmonizado de biomonitorização humana envolvendo indústrias de gestão de REEE localizadas em diversos países europeus, cujo objetivo consistiu em avaliar a exposição dos trabalhadores a substâncias químicas nocivas para a saúde e os seus efeitos precoces. Caracterizou-se a exposição através da medição dos compostos ou seus metabolitos em amostras de sangue e urina dos trabalhadores, comparativamente a um grupo de controlo, e os seus potenciais efeitos através da análise de biomarcadores de genotoxicidade em células bucais e sanguíneas. Os resultados evidenciaram a exposição de alguns grupos de trabalhadores a metais, com destaque para o chumbo, e a compostos orgânicos, incluindo PCBs e ftalatos. Relativamente aos biomarcadores de efeito, observou-se uma tendência para um acréscimo de instabilidade cromossómica local e sistémica nos trabalhadores expostos vs. controlos. Este estudo, iniciado no âmbito da Iniciativa Europeia em Biomonitorização Humana (HBM4EU) e continuado na Parceria para Avaliação do Risco de Químicos (PARC), permitiu identificar potenciais riscos para a saúde de trabalhadores envolvidos na gestão de REEE. Esses riscos deverão ser mitigados através da revisão/implementação de regulamentação, promoção do uso de novas tecnologias e/ou sistemas de proteção mais seguros e capacitação dos trabalhadores para a sua correta utilização.
- Evaluating the Impact of Newborn Screening for Cystic Fibrosis in Portugal: A Decade of Insights and OutcomesPublication . Camacho, Bernardo; Pereira, Luísa; Bragança, Raquel; Castanhinha, Susana; Penteado, Raquel; Silva, Teresa Reis; Miragaia, Pedro; Silva, Sónia; Cardoso, Ana L.; Barbosa, Telma; Freitas, Cristina; Gonçalves, Juan; Marcão, Ana; Vilarinho, Laura; Barreto, Celeste; Constant, CarolinaThe implementation of newborn screening (NBS) has revolutionized the diagnostic landscape of cystic fibrosis (CF). In Portugal, NBS was initiated in October 2013 through a pilot study and was subsequently fully integrated into a nationwide program by December 2018. Infants with positive screening results are referred to a specialized CF reference center for diagnostic confirmation, employing Sweat Chloride Testing (SCT) and genetic testing for CFTR variants. We aimed to analyze infants with a positive CF screening and determine the false positive and false negative rates, as well as to calculate the positive predictive value and sensitivity of our NBS program. A retrospective nationwide analysis was conducted on infants with a positive NBS for CF between October 2013 and February 2023. Two hundred and forty infants were referred from the NBS program; 74 (30.8%) were confirmed to have CF through SCT and genetic testing. Sensitivity was 93.2%, and the positive predictive value (PPV) was 30.8%. In addition, 48.5% were homozygous for F508del variants, and 87.8% had at least one F508del variant. Guidelines set forth by the European Cystic Fibrosis Society advise NBS programs to achieve a minimum PPV of 30% and a minimum sensitivity of 95%. Our report demonstrated good compliance with these recommendations.