INSA - Dissertações de mestrado
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- Assessing the pro inflammatory effects of bisphenol compounds using exposure relevant in vitro co culture modelsPublication . Pereira, Gonçalo Alexandre Candeia; Jordan, Peter; Rodrigues, CecíliaInflammation has reached epidemic proportions in industrialized countries, mainly due to unhealthy habits, poor diet, environmental pollution and other factors not yet understood. If uncontrolled or prolonged, inflammation can become chronic and contribute to the development of a number of human diseases, including autoimmune diseases, intestinal diseases and, in the worst cases, tumorigenesis and tumor progression. Exposure to endocrine disrupting chemicals (EDCs) is one environmental factor contributing to inflammation, and recent studies have brought the bisphenol (BP) group of EDCs into the scientific spotlight. They have been strongly linked to various pathologies, including chronic inflammation, and their effect on human gut health is a hot topic in the scientific community. With this in mind, the aim of this work was proposed to analyze the effects of four bisphenols, BPA, BPS-MAE, BPAP and BPP, on intestinal barrier stress and associated pro-inflammatory effects. To achieve this, a co-culture system was optimized and established, consisting of an improved protocol of polarized Caco-2 epithelial cells seeded on PET insert filters in an apical compartment, together with THP-1 derived macrophages in a basolateral compartment. Subsequently, the effects of BPs exposure on barrier integrity, cellular stress and pro-inflammatory cytokine were tested in a wide range of concentrations (from 100 μM to 0.1 μM). Experimentally, we found that the model was capable of delivering BP-specific data on potential health effects. In terms of transepithelial resistance and epithelial stress, we were able to identify some clear trends that need to be consolidated with more independent experimental replicates. In particular, BPA was the least potent inducer of cellular stress responses and changes in epithelial polarization, whereas the BP analogues tested proved to be more disruptive than BPA, with BPP appearing to be the most potentially hazardous, followed by BPAP and then BPS-MAE. To access the inflammation-modulating effects of these compounds, we tested macrophages, either directly or as co-cultured cells, for expression of the pro-inflammatory marker IL-1β using a semiquantitative RT-PCR approach. An important optimization was their priming with IFN-γ to increase the sensitivity of the model and allow for more physiological relevance. Our observations showed that, once again, the BP analogues induced greater effects compared to BPA. BPP appeared to be the more potent inducer of inflammation, followed by BPS-MAE. Both showed elevated levels of the IL-1β marker at all concentrations tested. BPAP and BPA produced more attenuated effects, although significant at higher concentrations. In conclusion, this work has provided us with landmark results on these BPA analogues and their effects on gut health, adding new insights into the 'new generation' of emerging BPs and their potential adverse health effects.
- 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.
- Hazard characterisation of bisphenol A alternatives to improve risk assessment for human health: genotoxic and carcinogenic effects in mammalian cellsPublication . Pereira, Maria João Rodrigues; Silva, Maria João; Farinha, CarlosBisphenol A (BPA) is a compound used in the production of epoxy resins, polycarbonate plastics and as an additive (e.g. in thermal paper). It is present in several different products and has been detected both in the environment and in human matrices. BPA is a known endocrine disruptor, with several studies linking it to multiple harmful health effects. Therefore, there is an effort to phase out its use, but also to replace it with alternative substances. Various alternatives are already being used and have been detected in the environment and human matrices. Furthermore, some harmful health effects have also been reported for those substances (albeit not as many as BPA), justifying the need for more research on these alternatives. This thesis is part of the Partnership for the Assessment of Risks from Chemicals (PARC), that prioritized alternatives based on the lack of data on their effects on human health and due to human exposure to them occurring or being likely to occur. This thesis focused on BPS-MAE and BPAP, aiming to contribute to their hazard assessment, through the characterisation of their in vitro genotoxic and carcinogenic potential. The former was assessed for BPS-MAE and BPAP with the Cytokinesis-Block Micronucleus assay, in human peripheral blood lymphocytes. The results suggest that BPS-MAE and BPAP do not have a genotoxic effect at the concentrations and exposure times tested. Genotoxic and non-genotoxic carcinogenicity of BPAP and BPA was ascertained with the Cell Transformation assay, in Bhas 42 cells. BPA was tested to allow inter-laboratory comparison of results and assay optimisation. The results indicate that neither BPAP nor BPA have potential carcinogenic activity at the concentrations and treatment durations tested. The data obtained will help eliminating existing data gaps, aid to improve these alternatives’ risk assessment and contribute to the formulation of legislation, if necessary.
- Impact of BCL-6 downregulation in the oncogenic properties of breast cancer cellsPublication . Jorge, João Miguel Dyson de Lima; Barros, Patrícia; Jordan, PeterBreast cancer (BC) incidence has risen over the past two decades, now being the second most prevalent cancer worldwide and the fourth leading cause of cancer-related deaths. Despite advancements in BC treatment, challenges like acquired resistance, recurrence, and metastasis persist. BCL-6, a transcriptional repressor, plays a controversial role in BC development. It is overexpressed in approximately half of primary tumors across all subtypes, correlating with poorer patient prognosis. Conversely, its downregulation is linked to disease progression and metastasis, highlighting the critical need for a deeper understanding of BCL-6's dual role in BC pathogenesis. This study used RNA interference to explore the impact of BCL-6 depletion on the oncogenic progression of MCF-7 cells, a low-tumorigenic estrogen receptor-alpha-positive (ERα+) cell line. While BCL-6 is known to regulate mammary cell proliferation and differentiation, its depletion did not affect MCF-7 cell proliferation or viability but significantly reduced their individual and collective migratory properties. An RNA microarray analysis identified a set of genes upregulated following BCL-6 depletion, including S100A7, previously reported to inhibit MCF-7 cell migration and invasion in ERα+ BC cells. However, our findings showed that S100A7 downregulation alone did not affect MCF-7 migration. Moreover, simultaneous depletion of BCL-6 and S100A7 failed to restore MCF-7 cell migratory behavior. Our results suggest that increased expression of BCL-6 is linked to increased cell migration but is independent of S100A7 upregulation. Further studies are required to clarify the role of BCL-6 in BC, including disease progression.
- The mechanism of nonsense-mediated mRNA decay and its playersPublication . Subtil, Catarina; Loison, Luísa; Santos, Rafaela LacerdaNonsense-mediated mRNA decay (NMD) is a post-transcriptional surveillance mechanism harbouring two functions: identification and degradation of transcripts containing premature translation-termination codons (PTC), preventing deleterious effects in the cell; and downregulation of mRNAs in response to cellular needs, maintaining the quality of gene expression. One-third of gene mutations in human genetic diseases, including cancer, are due to nonsense mutations or frameshift that result in transcripts harbouring nonsense codons and can be eliminated by NMD. The several factors involved in this mechanism may act in diverse ways depending on the set of transcripts to be regulated, contributing to the branching of this pathway. Cytoplasmic DIS3 exosome independent 3′-5′ exoribonuclease 2 (DIS3L2) has been reported as one of the factors to induce NMD targets decay. Therefore, this study aims to enlighten how DIS3L2 functions in NMD: i) analyse the correlation between the distinct branches of NMD and cervical and uterus cancer; ii) investigate which branch guides DIS3L2-mediated degradation; and iii) test which region of the NMD/DIS3L2-targets mediate DIS3L2 degradation through a system of hybrid-genes. For our first aim, we detected no correlation between any of the branches of NMD and uterus and cervical cancer. Each factor acts independently. In our second objective, we analysed the mRNA expression of five transcripts, but none displayed a significant alteration in their expression to infer a correlation between DIS3L2 and a particular NMD branch. Relatively to the last aim, we successfully cloned three out of the four constructs but due to time constrictions we could not continue. Nonetheless, further testing is needed to better understand this mechanism and how transcript degradation is mediated, including the diverse factors needed for its activation, which might be the key to future advanced therapeutic strategies.
- Pesquisa de Deleções/Duplicações em Genes Associados a Cancro Hereditário por MLPA DigitalPublication . Alves, Beatriz Correia; Gonçalves, João; Melo, Maria Joana Lima BarbosaA presença, na linha germinativa, de Variações do Número de Cópias (CNV) em genes de predisposição para cancro hereditário pode aumentar a suscetibilidade a esta doença. A identificação de uma CNV patogénica ou provavelmente patogénica num doente oncológico tem um impacto significativo na gestão clínica do indivíduo afetado e dos seus familiares. Tradicionalmente, a pesquisa de CNV no diagnóstico molecular de cancro hereditário é realizada apenas para alguns genes através do MLPA (Multiplex Ligation-dependent Probe Amplification) convencional. Nos últimos anos, o desenvolvimento de softwares de análise in silico de CNV com base em dados de NGS (Next-Generation Sequencing) representou um avanço significativo, ao possibilitar a pesquisa de deleções e duplicações em múltiplos genes em simultâneo. No entanto, estas ferramentas apresentam ainda limitações. Dada a relevância de uma análise abrangente que integre o maior número possível de genes relevantes no âmbito da patologia em causa, este trabalho teve como principal objetivo implementar uma nova metodologia de pesquisa de CNV em genes associados a cancro hereditário, que combina os princípios do MLPA convencional com a capacidade da NGS de analisar vários genes em simultâneo: o MLPA digital. Neste estudo, foi realizada a pesquisa de CNV por MLPA digital em amostras de doentes com história clínica e familiar de cancro, seguida de validação dos resultados utilizando outras metodologias de genética molecular e classificação das variantes identificadas segundo as recomendações da CanVIG-UK. O MLPA digital demonstrou ser eficaz na deteção de deleções e duplicações em genes associados a cancro hereditário, apresentando um desempenho adequado para a utilização em laboratórios clínicos, com sensibilidade de 100% e especificidade de 98%. A eficácia dos softwares de pesquisa in silico panelcn.MOPS e DRAGEN Enrichment foi confirmada através da concordância entre os resultados destas ferramentas e do MLPA digital. Foram identificadas cinco variantes patogénicas ou provavelmente patogénicas nos genes APC, BRCA1, BRCA2 e CHEK2, que justificam os fenótipos dos doentes. Este estudo demonstra que o MLPA digital é uma alternativa ao MLPA convencional na primeira fase de pesquisa molecular de CNV germinativas em genes associados a cancro hereditário, permitindo a análise de múltiplos genes em várias amostras em simultâneo.
- Regulation of PERK gene expression by its upstream open reading framesPublication . Ponte, João Gonçalves da; Zilhão, Rita; Romão, LuísaUpstream open reading frames (uORFs) are cis-acting elements located within the 5’ untranslated region (5’UTR) of transcripts, which can regulate the translation of the corresponding main open reading frame (mORF). In normal conditions, uORFs are typically repressors of downstream translation, as they can block ribosomal access to the mORF or even induce mRNA degradation through the nonsense-mediated mRNA decay (NMD) pathway. However, in stress conditions, phosphorylation of the eukaryotic initiation factor 2 (eIF2) allows the expression of several stress-responsive proteins via uORF-mediated mechanisms, while global mRNA translation is inhibited. During endoplasmic reticulum (ER) stress, the accumulation of unfolded proteins leads to activation of the ER-resident PKR-like ER kinase (PERK), which will phosphorylate the α-subunit of eIF2 as part of the stress-protective mechanisms of the unfolded protein response (UPR) and integrated stress response (ISR). This results in selective uORF-mediated translation of downstream effectors, which will drive stress resolution or cell death in case of prolonged stress. The dual role of PERK in regulating cell fate is implicated in a growing list of pathophysiological conditions, including neurological and cardiovascular diseases, ophthalmological disorders, viral infections, cancer, and diabetes. Moreover, mutations in the EIF2AK3 gene encoding PERK are implicated in a rare autosomal recessive disorder, the Wolcott-Rallison Syndrome (WRS). Data from ribosome-profiling (Ribo-seq) studies indicate the existence of uORFs within PERK 5’UTR which could be involved in regulating PERK expression. This work aims to study the translational regulatory role of the uORFs identified in PERK’s 5’UTR and estimate its impact on cell homeostasis and human disease. We wish to highlight the importance of including 5’UTRs in the screening of disease-related mutations, as well as the necessity of functional studies to assess their relevance in the pathogenesis of human diseases, as it may provide vital information for developing new therapeutic strategies.