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- Isolamento, cultura e caracterização de células musculares lisas derivadas da artéria cerebral média de ratoPublication . Quelhas, Patrícia Alexandra Silveira; Baltazar, Graça Maria Fernandes; Oliveira, Maria Elisa Cairrão RodriguesEmbora o cérebro represente apenas 2% da massa corporal do ser humano consome cerca de 20% da energia do organismo. Este apresenta um armazenamento de energia limitado, tornando o correto funcionamento do suprimento sanguíneo de grande importância, pois é este que fornece todo o oxigénio e glucose necessário ao desempenho das suas funções. A irrigação sanguínea cerebral é feita a partir das duas artérias carótidas internas e das artérias vertebrais. Uma das artérias mais importantes é a artéria cerebral média, que deriva da carótida interna e recebe cerca de 60 a 80% do fluxo sanguíneo proveniente da mesma. Os principais componentes das artérias são as células musculares lisas, consideradas as principais reguladoras do tónus vascular, do diâmetro dos vasos sanguíneos, pressão sanguínea e do fluxo sanguíneo. A sua principal função é a contração e o seu mediador principal o cálcio. Tendo em consideração o enorme impacto social e económico que as doenças cerebrovasculares têm na sociedade ocidental é da maior importância desenvolver modelos experimentais que facilitem o estudo destas patologias. A informação atualmente disponível sobre o funcionamento das artérias cerebrais, e sobre as células que as constituem, é bastante escassa. Assim, o principal objetivo deste trabalho foi desenvolver um modelo in vitro de células musculares lisas derivadas da artéria cerebral média de rato. Foram recolhidos explantes da artéria cerebral média de rato os quais foram aderidos a placas de cultura revestidas com colagénio. A partir destes explante foram obtidas células com morfologia típica de células musculares. Estas células expressavam a proteína a-actina, característica do fenótipo contrátil. Para além disso as células presentes na cultura não expressaram marcadores endoteliais. Para avaliar a funcionalidade destas células foi analisada a resposta aos agentes contráteis, serotonina e noradrenalina e ao agente relaxante, nitroprussiato de sódio, através da técnica de Plannar Cell Surface Area. Os resultados obtidos indicaram que as células se mantinham funcionais in vitro, contraindo em resposta à serotonina e noradrenalina e relaxando em resposta ao nitroprussiato de sódio. Assim, em conjunto os dados obtidos indicam que a cultura implementada pode ser utilizada como modelo para caracterização e estudo do comportamento funcional da artéria cerebral média, bem como estudos da interação entre o sistema vascular e neuronal.
- Adenosine inhibits human astrocyte proliferation independently of adenosine receptor activationPublication . Marcelino, Helena; Nogueira, Vanda Cristina Simões; Santos, Cecilia; Quelhas, Patricia; Carvalho, Tiago; Gomes, João Fonseca; Tomás, Joana; Diógenes, Maria José; Sebastião, Ana M; Cascalheira, JoséBrain adenosine concentrations can reach micromolar concentrations in stressful situations such as stroke, neurodegenerative diseases or hypoxic regions of brain tumours. Adenosine can act by receptor-independent mechanism by reversing the reaction catalysed by S-adenosylhomocysteine (SAH) hydrolase, leading to SAH accumulation and inhibition of S-adenosylmethionine (SAM)-dependent methyltransferases. Astrocytes are essential in maintaining brain homeostasis but their pathological activation and uncontrolled proliferation plays a role in neurodegeneration and glioma. Adenosine can affect cell proliferation, but the effect of increased adenosine concentration on proliferation of astrocytes is not clarified and was addressed in present work. Human astrocytes (HA) were treated for 3 days with test drugs. Cell proliferation/viability was assessed by the MTT assay and by cell counting. Cell death was evaluated by assessing lactate dehydrogenase (LDH) release and by western blot analysis of αII-Spectrin cleavage. 30µM-Adenosine caused a 40%±3% (p < .05, n = 5) reduction in cell proliferation/viability, an effect reversed by 2U/ml-adenosine deaminase, but unchanged in the presence of antagonists of any of the adenosine receptors. Adenosine alone did not induce cell death. 100µM-Homocysteine alone caused 16%±3% (p < .05) decrease in HA proliferation. Combined action of adenosine and homocysteine decreased HA proliferation by 76%±4%, an effect higher (p < .05) than the sum of the effect of adenosine and homocysteine alone (56%±5%). The inhibitory effect of adenosine on HA proliferation/viability was mimicked by two adenosine kinase inhibitors and attenuated in the presence of folate (100µM) or SAM (50-100µM). The results suggest that adenosine reduces HA proliferation by a receptor-independent mechanism probably involving reversal of SAH hydrolase-catalysed reaction.
- Ciliopathy in cholangiocytes of biliary atresia patients – association with ischemic cholangiopathy, patterns of ductular reaction and disease severityPublication . Quelhas, Patrícia Alexandra Silveira ; Santos, Jorge Luiz dos; Vieira, Sandra Maria Gonçalves; Lusquinos, José Ignacio VerdeBiliary atresia (BA) is a rare neonatal cholangiopathy characterized by the progressive obstruction of the extrahepatic bile ducts, leading to cholestasis, liver fibrosis, and often the need for liver transplantation. Although affected infants appear clinically healthy at birth, laboratory evidence of cholestasis is already detectable. Despite advances in understanding the underlying pathophysiological mechanisms, the etiology of BA remains unclear, with proposed causes ranging from viral infections and immune dysregulation to genetic predispositions and vascular anomalies. Recent evidence suggests that hypoxia and primary cilia dysfunction in cholangiocytes may play a central role in BA pathogenesis, influencing cholangiopathy progression and clinical outcomes. This thesis investigates the interplay between hypoxia—specifically, the activation of the hypoxia-inducible factor 1-alpha (HIF-1α) pathway in cholangiocyte nuclei (ischemic cholangiopathy)—and the morphological and functional alterations of primary cilia in cholangiocytes of BA patients. The methodological approach included immunohistochemical, immunofluorescence, and molecular analyses of liver samples obtained from patients undergoing portoenterostomy, detailed characterization of ciliary morphology using advanced digital imaging techniques, and correlation of these findings with clinical and laboratory parameters and post-operative outcomes, including native liver survival. The results demonstrated increased nuclear HIF-1α expression in cholangiocytes of BA patients, particularly in areas near the peribiliary vascular plexus and progenitor cell niches. This activation was absent in liver samples from control patients with other neonatal cholestases (non-BA), suggesting a specific role of hypoxia in BA pathogenesis. Furthermore, gene expression analysis revealed the upregulation of molecular pathways related to ductular reaction, oxidative stress, and angiogenesis, reinforcing the hypothesis that hypoxia actively contributes to bile duct injury and tissue remodeling. These findings justify further studies focused on the mechanisms involved in HIF-1α activation and the clinical effects of hypoxia and/or oxidative stress on cholangiocytes. Additionally, the characterization of primary cilia in cholangiocytes revealed significant structural alterations in BA patient samples. A reduction in ciliary length and a possible disruption in the transport of acetylated tubulin 4α—a key marker of ciliary stability— from the cytoplasm to the cilium was observed. These alterations were associated with worse clinical outcomes, including a significant reduction in native liver survival. Regarding the association between hypoxia and ciliopathy, the co-localization of HIF- 1α and TUBA4A in BA samples suggests an inverse relationship between HIF-1α positivity and the presence of primary cilia on the luminal membrane of cholangiocytes, both in the portal tract and other microanatomical regions of the liver. Quantitative analysis of ciliary characteristics showed that biliary cells without hypoxic features maintained primary cilia integrity, suggesting that hypoxia may negatively impact ciliary formation or maintenance. The correlation between HIF-1α activation and ciliary dysfunction implies that hypoxia not only promotes inflammation and fibrosis but also compromises the structural and functional integrity of primary cilia, exacerbating disease progression. These findings offer new insights into the pathophysiological mechanisms underlying BA, suggesting that HIF-1α activation and ciliary dysfunction may serve as relevant biomarkers for prognosis and potential therapeutic targets. Identifying these molecular alterations not only facilitates the prediction of clinical outcomes but also opens avenues for innovative interventions aimed at modulating the hypoxic response and restoring ciliary function to improve liver survival and patient quality of life. Overall, this work advances the understanding of the relationship between hypoxia, ciliary dysfunction, and cholangiopathy in BA, paving the way for new research directions to develop more effective diagnostic and therapeutic strategies that mitigate the effects of hypoxia and preserve liver function in pediatric patients with this condition.