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Percorrer FCB1-Teses por Objetivos de Desenvolvimento Sustentável (ODS) "03:Saúde de Qualidade"
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- Epigenetic regulation of ZNF687 in bone cells: elucidation of its role in the progression of Paget’s disease of bonePublication . Domingos Varela, Débora Cristina; Cancela, Leonor; Conceição, NatérciaPaget’s disease of bone (PDB) is characterized by focal areas of intense bone resorption by hyperactive osteoclasts followed by excessive bone formation by osteoblasts. Mutations and increased expression of ZNF687 have been associated with PDB. However, the role of ZNF687 in bone metabolism is poorly understood, and the molecular mechanisms that regulate its expression remain unknow. Therefore, the main objective of this study was to investigate the regulation of ZNF687 in bone cells, focusing on epigenetic mechanisms, in order to elucidate its involvement in the pathophysiology of PDB. In addition, we performed a genetic analysis of ZNF687, along with other candidate genes, in a cohort from southern Portugal. First, we characterized the human ZNF687 promoter, evaluated the functionality of predicted binding sites for bone-related transcription factors, and assessed the impact of CpG methylation on its regulatory activity. Our results indicate that NFκB, PU.1, DLX5, and SOX9 act as transcriptional regulators of ZNF687, and that DNA methylation inhibits their regulatory activity. Next, we analyzed mice Zfp687 expression and epigenetic regulation in MC3T3-E1 osteoblast differentiation and in hindlimb bones throughout mice life stages. Our results suggest that miR-142a-3p targets Zfp687 3′UTR, contributing to its downregulation during osteoblastogenesis, while DNA methylation does not appear to regulate Zfp687. In the PDB genetic study of Portuguese population, we identified the ZNF687 c.2810C>A variant that was predicted in silico to be pathogenic and shown in vitro to enhance nuclear import. In addition, OPTN rs2234968 variant was significantly associated with PDB. Finally, we examined ZNF687 expression and CpG methylation during in vitro osteoclast differentiation. ZNF687 was upregulated during murine osteoclastogenesis and overexpressed in osteoclasts from PDB patients in comparison to those of healthy controls. Moreover, methylation levels in -506/-396 promoter region were significantly higher in osteoclasts from PDB patients compared to their undifferentiated precursors and healthy osteoclasts. In summary, this work evidences the involvement of epigenetic mechanisms in bone cells’ differentiation, particularly in the regulation of the ZNF687 gene during osteoclast and osteoblast differentiation. Furthermore, it suggests that DNA methylation may contribute to the upregulation of ZNF687 in PDB.
- Exploring angiogenesis and cardiomyocyte differentiation in left ventricular noncompaction using patient derived hiPSCPublication . Carmo, Sara Martins do; Bragança, JoséLeft ventricular noncompaction (LVNC), is a rare cardiomyopathy characterized by a spongy myocardial structure, hyper-trabeculation and intra-trabecular recesses, resulting from failure of normal embryonic compaction of the myocardium. The prevalence of LVNC varies globally, between 9.5% in paediatric age and 0.05-0.25% in the general population. Multiple genetic mutations are associated with LVNC, affecting sarcomeric, cytoskeletal and mitochondrial functions. This study investigates the genetic mechanisms underlaying LVNC, focusing on mutations involved in heart diseases and their impact in cardiomyocyte differentiation and angiogenesis, with a special focus on ZSCAN10, SCN10A and VE-PTP, found mutated in the patient’s cells used in this study. Cardiomyocytes were differentiated from induced pluripotent stem cells (iPSCs) derived from a LVNC patient and a 1st degree healthy relative. Gene relative expression analysis of samples collected during cardiomyocyte differentiation, highlighted significant differences on in key genes, such as GATA4, ISL1, SOX17, KDR, VE-PTP, VEGFA, TNNT2 and NKX2.5 in patients derived cells compared to control cells. Among those, GATA4, ISL1 and NKX2.5, which were previously showed to cooperate for the differentiation and proliferation of cardiomyocytes, presented a significantly lower expression. SOX17, KDR and VEGFA have functions in cardiac vascularization, and the expression of SOX17 was higher in patient’s cells, while in the same conditions KDR and VEGFA were decreased at critical time points for endothelial cells differentiation. Thus, the expression of these genes, crucial for cardiomyocyte development and angiogenesis, were markedly altered in LVNC-derived cells compared to control cells. A Tube formation assay using endothelial cells from both LVNC-derived and control cells, to assess their ability to form capillary like structures. Remarkable differences were observed, with patient cells showing a delayed and an impaired tube formation, and a reduced vascular network complexity. Overall, our results argue that novel genetic and cellular mechanisms might be altered in the LVNC patient cells analysed.
- Genetic diversity of rotavirus A causing diarrhea in patients admitted to the Clinic University Hospital in Valencia, Spain (2022-2024)Publication . Conjo, Carolina da Glória Dinis; Ferreira, Bibiana I.; Gomez, Javier Buesa; Deus, Nilsa deA diarreia é uma das principais causas de mortalidade infantil em todo o mundo, e o rotavírus destaca-se como o principal agente etiológico associado. Neste contexto, muitos países introduziram a vacina contra o rotavírus no seu calendário de vacinação infantil, incluindo a Espanha. No entanto, a carga das doenças diarreicas continua elevada. Existe uma lacuna de informação em relação a infecção por rotavírus em pacientes que não tenham idade pediátrica e muitos fatores podem estar implicados na suscetibilidade a infeção por rotavírus entre eles os fatores genéticos do hospedeiro denominados Histo Blood Group Antigens (HBGA´s), que podem reconhecer agentes entéricos que modulam doenças entéricas infecciosas, conferindo risco ou suscetibilidade à população. Foi realizada uma análise transversal de dados de base hospitalar, de abril de 2022 a fevereiro de 2024, em 136 pacientes atendidos com diarreia no Hospital Clínico Universitário de Valência. A triagem inicial das amostras foi feita por Real-Time PCR no Hospital Clínico Universitário de Valencia, 136 amostras foram positivas para Rotavírus A (RVA) e testadas por RT-PCR para a identificação do genótipo no laboratório de Microbiologia da Universidade de Valência. A maior diversidade de estirpes de rotavírus foi encontrada em crianças menores de 2 anos e os genótipos mais comuns nesta faixa etária foram G4P[8] e G12P[8]. Cerca de 26,5% das amostras eram não tipificáveis, 16,9% correspondiam a G4P[8], 16,2% eram NTP[8] e 11,8% eram G12P[8]. A sazonalidade foi associada à distribuição das estirpes de rotavírus (p-valor<0,001), com maior pico de infecção em maio, julho e abril de 2023. O status secretor do gene FUT2 foi determinado em 7,7% (2/26) das amostras testadas. A presente análise mostrou uma alta proporção de infecção e diversidade genotípica em crianças com menos de 24 meses de idade. No futuro, será necessário investigar a diversidade genética e a dinâmica evolutiva das estirpes de rotavírus. Embora o estudo tenha encontrado dificuldades na determinação do status secretor para FUT2 a partir das amostras fecais, apresentou informações inovadoras sobre o potencial e as limitações desta abordagem.
- Molecular tools to study SCA2: from new advanced disease models to CRISPR-mediated editing approachesPublication . Gonçalves, Rebekah Cavaco Koppenol; Nóbrega, Clévio; Matos, Carlos A.; Almeida, Luís Pereira deSpinocerebellar ataxia type 2 (SCA2) is a rare neurodegenerative disease caused by an abnormal expansion of the trinucleotide CAG in the coding region of ATXN2. This overexpanded CAG region is translated into an abnormally long tract of glutamines within the ATXN2 protein, which above 32 repetitions drives pathology. SCA2 comprehends a complex network of pathological mechanisms, progressively leading to neuronal dysfunction and cell death. As a result of the expanded ATXN2-mediated neurodegeneration, especially affecting the cerebellum and the brainstem, SCA2 patients suffer from several motor and non-motor signs and symptoms, with ataxia as the most frequent. Currently, there is no therapy capable of delaying or stopping disease progression, leading to the premature death of patients. Disease models have proven to be a valuable tool for the study of the pathological mechanisms underlying SCA2. In this work we develop a new transgenic mouse model for SCA2 with early motor and neuropathologic phenotype to study the role of the ATXN2 expanded protein in the pathogenesis of the disease. Additionally, we generated a SCA2 patient-derived iPSC line to serve as a platform to test new advanced therapeutic strategies. Taking advantage of the CRISPR toolbox to manipulate gene expression, we designed three CRISPRbased strategies targeting the ATXN2 gene: a CRISPR-Cas9 indel directing the nuclease activity of Cas9 to an early site of the ATXN2 gene; a CRISPRi using the dCas9-KRAB complex to hinder transcription; and a CRISPR-Cas9 excision directing Cas9 to two sites of the ATXN2 to excise the CAG region. We tested these strategies in the newly generated SCA2 patient-derived iPSC line, inducing its differentiation into mature neurons. The CRISPR strategies resulted in a decrease of the ATXN2 protein levels or the complete ablation of ATXN2 expression, preventing several pathological traits of SCA2. The tools developed in this project support the development of CRISPR-based disease-modifying strategies for SCA2, enlightening the action of ATXN2-mediated pathogenesis.