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Percorrer FCB1-Teses por Objetivos de Desenvolvimento Sustentável (ODS) "09:Indústria, Inovação e Infraestruturas"
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- Elucidação do papel de Brachyury no desenvolvimento do sistema nervoso centralPublication . Militão, Inês Torradinhas; Andrade, Raquel; Araújo, InêsBrachyury é um fator de transcrição responsável pelos movimentos morfogénicos durante o processo embriológico da gastrulação para formar as camadas germinativas mesoderme e endoderme. Participa ainda na manutenção de características estaminais das células no botão caudal, garantindo a elongação do eixo anteroposterior do embrião. No adulto, a expressão de Brachyury é diminuta na maioria dos tecidos provenientes da mesoderme, endoderme e ectoderme não-neural. No tecido cerebral adulto, no entanto, é possível detetar expressão de Brachyury. Experiências prévias no laboratório mostraram que Brachyury participa da diferenciação neural, e a diminuição da sua expressão está associada a Gliomas, o tumor mais agressivo do cérebro. Neste trabalho procurou-se compreender quando e onde é expresso Brachyury ao longo do desenvolvimento do sistema nervoso central e qual é a sua função. Através da técnica de hibridação in situ aplicada a embriões de galinha em diversos estadios de desenvolvimento, observou-se que Brachyury é primeiramente expresso nas células da crista neural cranial no estadio HH13 e que, mais tarde, é expresso nas regiões dorsais do telencéfalo e diencéfalo, assim como na região do istmo. Brachyury co-localiza com os genes Pax6 e Wnt8b nestas estruturas. Com recurso à técnica de eletroporação in ovo, foi feita a sobre-expressão de Brachyury e observou-se que não alterou a expressão do gene Wnt8b no cérebro em desenvolvimento. Neste trabalho, foi também iniciada a otimização da técnica de imunohistoquímica in toto, para localização da proteína Brachyury, bem como da técnica de RT-qPCR de forma a genotipar embriões de murganho provenientes do cruzamento de mutantes T+/-, para estudos futuros. É necessário continuar o processo de otimização, focando no desenho de novos primers, os Intron-spanning primers.
- 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.
- Gene therapy for Cockayne syndrome: in vivo studiesPublication . Vaz, Adriana Afonso; Nóbrega, ClévioCockayne Syndrome (CS) is a rare, severe, multi-systemic disorder inherited in an autosomal recessive pattern, with an incidence of 2.77 cases per million births. First documented by Dr. Edward Cockayne in 1936, this syndrome presents a variety of clinical features, primarily impacting the vision, hearing, growth, and motor and cognitive functions. Neuropathologically, it involves white matter loss, microcephaly, and brain calcifications. CS can be categorized into three severity groups: CS type II (most severe), CS type I (moderate), and CS type III (least severe). It can also be classified according to the underlying genetic mutation: ERCC8 mutation causes CS type A (CS-A) and ERCC6 mutation leads to CS type B (CS-B), with 65% of cases being CS-B. This study focuses on CS-B, due to its therapeutic relevance. The ERCC6 gene, which translates the CSB protein, is crucial in several cellular mechanisms, such as DNA damage repair (induced by ultraviolet radiation or oxidative stress), transcription regulation, and mitochondrial function. Mutations in ERCC6 lead to DNA damage accumulation, transcriptional arrest, and mitochondrial dysfunction. Currently, treatments are limited to symptom management, highlighting the need for gene-based therapies. Gene therapy aims to treat genetic disorders by delivering genetic material to human cells, through vectors. There are several gene therapy strategies and more than a dozen have been approved for clinical use. As a monogenic disorder with recessive inheritance, CS-B poses a strong candidate for a gene therapy-based treatment. Therefore, the aim of this work is to determine the therapeutic potential of a gene therapy for CS-B in vivo. This strategy is based on delivering a functional ERCC6 gene through an AAV9 vector. The first step in this study was to test eight therapeutic strategies in vitro, with the objective of narrowing it down to one (Cure1) for further in vivo testing in a CS-B mouse model, CSB m/m. Following, we injected a CS-B mouse model with Cure1 and these preliminary results showed promising CSB expression in the injected brain hemisphere. Lastly, given the success of preliminary tests, this strategy was injected CSB m/m mice for further behavioural assessment. However, these tests showed no significant improvements, suggesting Cure1’s limited effectiveness. Additionally, histological analysis of these brains showed no expression of CSB in mice injected with Cure1, further supporting the inference that Cure1 has limited therapeutic potential. In conclusion, the findings indicate that Cure1 gene therapy does not significantly enhance CSB expression or improve the phenotype in CS-B mice. Further studies are required to confirm the reliability of these results and assess Cure1's therapeutic potential comprehensively.
- 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.
- Operacionalização da ferramenta do Norwegian General Practice-Nursing Home à realidade portuguesaPublication . Tomé, Daniela Katrine Gudmand; Leitão, Helena; Nascimento, TâniaA prescrição de Medicamentos Potencialmente Inapropriados (MPI) é uma preocupação crescente entre a população idosa institucionalizada, devido às suas características específicas e à elevada prevalência de uso inadequado de medicamentos. Em Portugal, não existe uma lista adaptada de MPI para esta população, o que motivou a adaptação dos critérios Norwegian General Practice Nursing Home criteria (NORGEP-NH) para a realidade nacional. O objetivo principal desta dissertação foi operacionalizar os critérios NORGEP-NH para idosos institucionalizados em Portugal, utilizando o Método Delphi com um painel de peritos composto por médicos, farmacêuticos e enfermeiros com experiência na gestão de cuidados a idosos. Os critérios foram avaliados quanto à sua relevância clínica numa escala de Likert de 0 (sem relevância clínica) a 10 (alta relevência clínica). O processo de consenso necessitou de apenas uma ronda online. A lista final, resultante do consenso, incluiu 35 critérios explícitos para o uso potencialmente inapropriado de medicamentos em lares de idosos, divididos em três secções: substâncias únicas, combinações e desprescrição. A adaptação dos critérios NORGEP-NH para Portugal mostrou-se relevante e ajustada ao contexto clínico dos idosos institucionalizados, o que facilitará a prática clínica diária dos profissionais de saúde em lares de idosos. Conclui-se que a operacionalização dos critérios NORGEP-NH para a realidade portuguesa é uma ferramenta valiosa para melhorar a segurança medicamentosa e otimizar os cuidados aos idosos institucionalizados, contribuindo para uma prática clínica mais informada e orientada para as necessidades específicas dos utentes.
- PLCy1: Tumour suppressor or oncogene in triple negative breast cancerPublication . Cordeiro, Andreia Sofia de Sá Barreira; Martins, Marta Sofia AlvesIn 2020, breast cancer represented the cancer with highest incidence among women worldwide, with 2.26 million new cases estimated. Triple-negative breast cancer (TNBC), which lack HER2 expression and hormonal receptor markers, is a particular type of extremely aggressive breast cancer with a faster growth rate, higher risk of metastasis and recurrence. Interestingly, we found that phospholipase C gamma 1 (PLC𝛾1) expression is a good prognostic marker in TNBC. However, PLC𝛾1 is overall considered an oncogene involved in cancer development and progression. To unravel the effective role of PLCy1 in TNBC prognosis, MDA-MB-231, MDAMB- 468 and Hs578T cell lines were used. With a PLC𝛾1 overexpression model we evaluated the capacity of this cells to form colonies, calculate doubling time and confirm cell viability levels. Through BrdU assay we compared cell proliferation rates. We used western blot to detect proteins that play a role in tumorigenesis. By analysing mRNA, we identified that there is only one gene commonly upregulated in the PLC𝛾1 KO cells, ESM1. In the meaning of trying to understand its regulation in these cell lines, we performed Immunofluorescence assays, ELISA, and qRT-PCR. Further, we sent dry samples to Aveiro ́s University to do a nuclear magnetic resonance assay. In this study MDA-MB-231 PLC𝛾1 OE cells were found to be less capable of colony formation and proliferation and have a higher doubling time, although PLC𝛾1 OE cells presented a higher expression of tumour promoters. PLC𝛾1 KO cells had an increased proliferation rate as well as a higher expression of known tumour promoters, when compared to WT cells. PLC𝛾1 was found to alter metabolism in TNBC cell lines. ESM1/Endocan was found to be increased in MDA- MB-231 PLC𝛾1 KO cells. These results reveal that PLC𝛾1 may have a protective potential and be a determinator of good prognosis in TNBC. Eventually, a dual effect of the expression of this phospholipase is also a possibility.
- Proliferação celular na ausência de função do gene Mob3Publication . Oliveira, Bruna Isabel dos Santos; Tavares, ÁlvaroO desenvolvimento e a manutenção dos organismos dependem do equilíbrio entre a proliferação celular e a apoptose, essenciais à morfogénese e ao crescimento dos tecidos. Este equilíbrio é controlado por várias vias de sinalização, incluindo a via Hippo, cuja atividade é modulada por proteínas da família Mob. Além de regular negativamente a atividade da via Hippo, dados preliminares sugerem que, em células humanas, o MOB4 tem um papel essencial na progressão da mitose. Adicionalmente, estudos mostraram uma expressão elevada dos genes MOB3 e MOB4 em tumores humanos. Desta forma, o principal objetivo deste projeto foi estudar a função molecular dos genes Mob3 e Mob4 na proliferação celular, utilizando a Drosophila melanogaster. Começou-se por seguir a localização subcelular da proteína GFP-Mob3 durante a mitose, em neuroblastos larvares. Além disso, foram analisados os efeitos mitóticos, provocados pela ausência do Mob3, em cérebros larvares de indivíduos nulos Mob3. Observou-se que a localização intracelular da proteína Mob3 é constante nos vários estadios de desenvolvimento da mosca, sugerindo uma função conservada desta proteína. Os indivíduos nulos Mob3 revelaram ainda problemas na proliferação celular, evidenciados pela redução do tamanho dos cérebros larvares e do número de mitoses. Estes indivíduos apresentaram também problemas na segregação do DNA. No Mob4, a abordagem adotada consistiu na observação dos fenótipos no indivíduo adulto e na análise dos efeitos mitóticos, provocados pela depleção específica do Mob4 em três tecidos larvares diferentes, utilizando o sistema UAS-Gal4. Analisou-se ainda a atividade desta proteína como um potencial componente do complexo STRIPAK. A depleção do Mob4 resultou numa diminuição do tamanho dos tecidos selecionados. Observou-se ainda uma diminuição do número de células em mitose e um aumento da apoptose. Em conjunto, os nossos dados sugerem uma função conservada do Mob4, enquanto elemento do complexo STRIPAK, na manutenção do equilíbrio entre a proliferação e a apoptose. Além disso, o Mob4 parece ser necessário ao correto estabelecimento da polaridade nos tecidos, observando-se uma desorganização no posicionamento das células nos tecidos com depleção do Mob4. Em suma, os nossos dados sugerem que os genes Mob3 e Mob4 são essenciais ao controlo da proliferação celular.
- Study of BUB1 functions in the neurodevelopmentPublication . Silva, Anita Ferreira Triguinho da; Carvalhal, Sara; Calado, SofiaBudding uninhibited by benzimidazole 1 (BUB1) is a kinase protein essential for proper chromosome segregation. Recently, we identified two biallelic BUB1 mutations linked to primary microcephaly, a rare condition causing reduced head size. The transmission pattern of BUB1’s individuals was consistent with the Autosomal Recessive Primary Microcephaly 30 (MCPH30). The present thesis aimed to understand how the BUB1 protein functions during human neurodevelopment, and how, when affected, it leads to microcephaly. We successfully generated a new induced pluripotent stem cells (iPSCs) line from patient 2’s fibroblasts. Two clones - clone 4 (C4) and clone 7 (C7) - were characterised by standard norms. Both showed pluripotency and the ability to differentiate in cells from all germ layers. However, while C4 had a diploid karyotype, C7 held trisomy on chromosome 8. To evaluate BUB1’s role in neurodevelopment, wild-type iPSCs were differentiated in neuro progenitor cells (NPCs) and then submitted to differentiation and maturation steps. Our findings reveal that when NPCs were treated with the BUB1 inhibitor drug, BAY1816032, they could not eliminate the signal for the main substrate of the BUB1 kinase, the phosphorylation on H2A-T120 (pH2A-T120), as demonstrated in somatic cells. However, pH2A-T120 levels on forebrain cells were sensible to BAY1816032. These cells hold several morphological modifications. In addition, diploid C4 iPSCs were differentiated into NPCs. However, C4-NPCs exhibited poor stability under multipotent conditions. Additionally, C4-forebrain cells also displayed morphological changes. Previous lab results showed that BAY1816032-treatment induced microcephaly in chicken embryos. However, our preliminary study revealed that microcephalic size depends on the developmental stage. This project successfully developed a new cell line for studying BUB1, made available to the community (Ferreira et al., 2024). And, despite preliminary, our overall results support BUB1's crucial role in NPCs maintenance and proper forebrain neurodevelopment.