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  • Ability of the MSC secretome to repair synaptic structure and function after hypoxia-induced injury
    Publication . Marceta, Bragança Lourenço Moisés; Baltazar, Graça Maria Fernandes
    Ischemic stroke (IS) remains one of the leading causes of death and disability worldwide, representing a major clinical and economic challenge. It is caused by the temporary or permanent interruption of a blood vessel, leading to disruption of the central nervous system and consequent neuronal loss. In the acute phase, therapeutic strategies include thrombolysis and thrombectomy to restore blood flow, whereas in the chronic phase, approaches aimed at promoting neural plasticity, such as cell therapy, are employed. However, these techniques face several limitations, as their efficacy is directly influenced by multiple variables, making their response unpredictable. Recent studies have attributed the effects of mesenchymal stem cells (MSCs) mainly to their paracrine action, thus positioning the use of their secretome as a promising alternative. In this work, we evaluated the impact of secretome derived from umbilical cord MSCs (UC-MSCs), obtained under normoxic and hypoxic conditions, on neuronal viability and on the recovery of synaptic structure after oxygen and glucose deprivation (OGD) in vitro. The results suggest that the secretome can modulate cell survival, preserve the length of neuronal arborization processes, and modulate synaptic markers. Although some results are promising, further studies are required to explore alternative approaches in order to better understand the action of MSC-derived secretome in the treatment of ischemic stroke and other neurological disorders associated with hypoxicischemic injury.
    2025-11-19Dissertação de mestrado Acesso embargado Ver mais
  • Benefícios terapêuticos do humor
    Publication . Vidal, Carlos Alexandre Machado de Lemos; Borges, Luís Filipe; Sousa, Miguel Castelo Branco Craveiro de
    Introdução: A palavra “humor” surgiu no passado, derivada da “Teoria Humoral” de Hipócrates, pai da Medicina (1,2). O humor e o riso são, desde a antiguidade, temas fracturantes. Devido à sua importância, complexidade e relevância histórica, o riso foi abordado em diferentes áreas por diversos pensadores e escritores, como Umberto Eco (3). O grande inspirador e pioneiro nos benefícios terapêuticos do humor, foi Norman Cousins (4). Esta dissertação centra-se nestes benefícios provocados pelo humor e pelo riso, no sistema endócrino, tegumentar, reprodutor e cardiovascular. Os estudos foram realizados em indivíduos saudáveis e com patologia: eczema atópico, diabetes mellitus tipo 2 e infertilidade. Pretende-se uma revisão sistemática para avaliar cientificamente, os benefícios terapêuticos do humor. Métodos: Foram pesquisadas publicações indexadas nas bases electrónicas científicas, Pubmed, B-On, Elsevier, ScienceDirect. Das 218 publicações identificadas, foram seleccionadas 10 para análise. Resultados: Doentes com eczema atópico mostraram significativa diminuição da produção de IgE pelas células B seminais cultivadas com espermatozóides e a expressão de galectina-3 nos espermatozóides foi reduzida. Os níveis de peptídeo derivado da dermicidina aumentaram sem afectar os níveis de proteína total no suor. Em mães saudáveis e com EA, os níveis de melatonina no leite materno aumentaram e as reacções alérgicas ao latéx e a ácaros diminuíram nas crianças alimentadas com leite materno após ambas as mães se rirem antes da amamentação. Doentes com diabetes mellitus tipo 2 tiveram uma diminuição do nível de pró-renina no sangue e uma regulação crescente do gene receptor da pró-renina, assim como uma supressão do aumento da glucose sanguínea pós-prandial 2 horas após a refeição. Em doentes inférteis, verificou-se que a taxa de gravidez aumentou. Em indivíduos saudáveis, houve um maior gasto de energia e um aumento da frequência cardíaca existindo uma correlação entre eles e deles com a duração do riso. Verificou-se também um aumento dos valores da capacidade de eliminação de radicais livres na saliva. Há um aumento da pressão arterial, da vasodilatação da artéria braquial mediada por fluxo induzido por isquémia, da complacência da artéria carótida apesar de voltar ao estado basal após 24 horas. Observou-se em idosos que o fluxo salivar aumentou e em jovens os níveis de CgA diminuiram. Conclusão: Este estudo leva-nos a supor que o humor e o riso apresentam benefícios terapêuticos em indivíduos saudáveis e com patologia, nomeadamente eczema atópico, diabetes mellitus tipo 2 e infertilidade, demonstrando a abrangência de efeitos benéficos nos sistemas endócrino, tegumentar, reprodutor e cardiovascular.
    2014-7-7Dissertação de mestrado Acesso aberto Ver mais
  • Burnout e Vinculação em Oncologia e Fim de Vida
    Publication . Gonçalves, Florbela dos Santos; Viana, Joaquim Manuel da Silva; Sousa, Miguel Castelo Branco Craveiro de
    Introduction The current shortage of human resources in the healthcare sector is increasingly recognized as a critical public health issue, with projections indicating that this challenge will escalate in the coming years. While poor working conditions have traditionally been regarded as the primary cause of burnout, emerging evidence underscores the pivotal role of personality traits, including attachment styles, in the development of this syndrome. The concept of burnout was first introduced by Herbert Freudenberger in the 1970s, defining it as a state characterized by despersonalization, emotional exhaustion and demotivation linked to work. Over time, tools to assess and measure burnout have been developed and refined. One of the most widely used instruments is the Maslach Burnout Inventory (MBI), created by Christina Maslach. According to this framework, burnout syndrome is defined by three core dimensions: emotional exhaustion, depersonalization, and a diminished sense of personal accomplishment. Despite its widespread application across various professional domains, some critics have questioned the validity of the MBI, particularly the interpretation of depersonalization, which may function as an adaptive coping mechanism rather than an unequivocal symptom of burnout. Burnout typically arises from prolonged exposure to stress, especially when an individual’s expectations are incongruent with their work environment. Although unfavorable working conditions are strongly associated with the syndrome, not all individuals respond to such conditions in the same way. This has led to increasing interest in the role of individual personality traits and their contribution to the susceptibility to burnout. The Copenhagen Burnout Inventory (CBI), developed by Kristensen et al, offers an alternative, open-access instrument designed to evaluate burnout. It assesses three distinct dimensions: personal burnout, work-related burnout, and client/patient-related burnout. The presence of burnout significantly jeopardizes healthcare professionals' performance and well-being, as it has been associated with adverse outcomes such as addictive behaviors, sleep disorders, and depression. Burnout is a prevalent issue among healthcare professionals, particularly those caring for patients experiencing profound suffering, such as individuals with cancer or other advanced chronic diseases. Recognizing that not all individuals exposed to similar work environments develop burnout, increasing attention has been directed toward understanding the role of individual characteristics in the syndrome's onset. Among these factors, the study of attachment traits revealed useful as a promising root to understand variability in burnout production and development of preventive strategies. Attachment theory asserts that early childhood experiences critically shape emotional bonds and interpersonal relationships, which, in turn, influence workplace behavior. John Bowlby’s pioneering research on emotional attachment demonstrated its relevance to various organizational and professional contexts. Individuals with a secure attachment style, often linked to a positive outlook, are more resilient to workplace stressors. Conversely, an insecure attachment style is generally associated with greater susceptibility to burnout and diminished professional performance. This insight may help explain why some healthcare professionals develop burnout under similarly stressful conditions, while others remain unaffected. Objectives The primary objective of this study was to determine whether there is an association between attachment style and the risk of burnout in a population of healthcare professionals working in an Oncology Hospital. The secondary objectives of this study are as follows: - To assess the risk of burnout in individuals working with oncology and palliative care patients; - To identify the various attachment styles exhibited by healthcare professionals in oncology and palliative care; - To determine potential predictors of burnout in the population of healthcare professionals working in oncology and palliative care; - To assess the professional quality of life in the population of professionals working at the Oncology hospital; - To explore the potential association between burnout and the quality of professional life in individuals working with cancer patients and in palliative care. Materials and Methods This was a cross-sectional, descriptive, and correlational study conducted between January and December 2018. The study was carried out at the Portuguese Institute of Oncology of Coimbra Francisco Gentil, EPE, involving 1003 healthcare professionals from the institution who were invited to participate The inclusion criteria encompassed healthcare professionals aged ≥18 years, currently employed at the institution, willing to participate, and able to provide written informed consent, with an adequate understanding of the study objectives. Healthcare professionals who declined participation and those with diagnosed psychopathologies were excluded. Of the 1003 professionals invited, 337 participated, 626 declined to participate, and 40 were excluded due to psychiatric conditions. Thus, a convenience sample of 337 healthcare professionals was obtained, yielding a response rate of 36%. The assessment protocol included a sociodemographic questionnaire, burnout assessments via the Maslach Burnout Inventory (MBI) and the Copenhagen Burnout Inventory (CBI), the Adult Attachment Scale, the Professional Quality of Life-5 Scale (ProQOL-5), and a single question: "Is it common to work with patients in palliative care?" This question allowed for the categorization of the sample into two groups: professionals who had exclusively worked with non-palliative oncology patients and those who had worked with palliative oncology patients. Statistical analyses were performed using IBM SPSS Statistics V.25 software, with significance tests conducted at the 5% level. Results It was observed that 76.8% of the healthcare professionals in the sample were involved in the care of non-palliative oncology patients. Upon comparing the two groups of professionals using CBI, it was found that more than 50% of the participants reported high levels of personal burnout, with no statistically significant differences between the groups (53.5% in one group and 56.8% in the other, p=0.619). Similar findings were noted for the work-related (p=0.626) and patient-related (p=0.672) dimensions of burnout. The analysis of the correlation between burnout dimensions and attachment style demonstrated that higher scores in emotional exhaustion, depersonalization, work-related burnout, and patient-related burnout were significantly associated with increased levels of anxiety (p<0.001). These findings were consistent across both groups, including professionals working with patients in the advanced stages of oncological diseases. The exploration of the correlation between burnout dimensions and the dimensions of the Adult Attachment Scale revealed that elevated scores in emotional exhaustion, depersonalization, work-related burnout, and patient-related burnout were significantly associated with higher levels of anxiety (p<0.001). These findings were consistent in the sample of professionals working with patients in the advanced stages of oncological diseases. Further exploration of the correlation between burnout dimensions and the dimensions of Professional Quality of Life (ProQOL-5) did not reveal any statistically significant differences between the two groups within the sample. Discussion and Conclusions Working in oncology and palliative care requires effective communication and a resilient personality from healthcare professionals. Without these two key factors, the likelihood of burnout increases. The etiology of burnout is multifactorial, involving both occupational factors and the personality traits of healthcare professionals, with attachment style being particularly significant. In the studied sample, no statistically significant differences were found between the two groups of healthcare professionals. Higher levels of anxiety were correlated with increased levels of both patient-related and work-related burnout, suggesting that an insecure attachment pattern may predispose individuals to burnout. Both groups experienced a similar quality of working life. The most significant contributor to burnout in this sample was the number of hours worked per week, leading to prolonged exposure to human suffering. Preventive measures, including the pursuit of personal well-being, regular physical activity, mindfulness practices, and the maintenance of proper sleep hygiene, can help reduce the risk of burnout. This study was enhanced by the use of two established burnout scales, including the Copenhagen Burnout Inventory (CBI), and by examining the correlation between burnout levels, attachment style, and the professional quality of life among healthcare professionals caring for cancer patients, some of whom were in end-of-life stages.
    2025-12-17Tese de doutoramento Acesso aberto Ver mais
  • Casuística da Esclerose Múltipla no Centro Hospitalar Cova da Beira entre os anos de 2005 e 2015
    Publication . Alcantara, Hilário dos Ramos Quaresma; Rosado, Maria Luiza Constante
    Introdução: A Esclerose Múltipla é um dos distúrbios neurológicos mais comuns. Em muitos países é a principal causa de incapacidade não traumática em adultos jovens. Trata-se de uma doença autoimune do Sistema Nervoso Central, caraterizada por inflamação crónica, desmielinização, gliose e perda neuronal. É aproximadamente três vezes mais comum em indivíduos do sexo feminino em relação aos do sexo masculino. A idade no início da doença tipicamente situa-se entre os 20 e os 40 anos. No entanto, em cerca de 10 % dos casos, esta ocorre antes dos 18 anos e numa pequena percentagem antes dos 10 anos. Quando a esclerose múltipla surge depois dos 50 anos, é denominada esclerose múltipla de início tardio, ocorrendo em cerca de 5% da população Metodologia: foi feito um estudo observacional transversal usando a base de dados iMed® do Centro Hospitalar Cova da Beira. Resultados: a amostra é composta de 108 doentes, sendo que 77 são do sexo feminino e 31 do sexo masculino, com idade compreendida entre os 19 e os 82 anos, e com média situada nos 48,35 anos. Encontrou-se uma maior prevalência da doença no sexo feminino com uma relação mulher/homem de 2,48:1. A média da idade aquando do início da doença é ligeiramente superior no sexo masculino (36,18) em relação ao feminino (33,97). Estas não são significativamente diferentes (p=0,354). Os sintomas inaugurais mais prevalentes nesta população são os distúrbios motores (40,7%) e visuais (33,3%). Quanto ao curso da doença 70,4% dos nossos doentes possuem EMSR, 14,8% EMSP, 6,5% EMPP e 1,9% EMPR. A Expanded Disability Status Scale nesta população tem valores compreendidos entre 0 e 9 com média de 2,091. Doentes que tiveram um curso progressivo inaugural têm a média da idade no início da doença significativamente superior (p=0,00215) aos que não tiveram. Embora 9 dos nossos doentes tiveram esclerose múltipla de inicio tardio, apenas 2 tiveram progressão de início. Conclusão: conclui-se que as características da nossa população e as variáveis em estudo não são muito diferentes daquelas observadas em estudos internacionais. A exceção a essa generalização é a maior frequência de esclerose múltipla de início tardio em comparação com outros estudos internacionais.
    2016-7-11Dissertação de mestrado Acesso aberto Ver mais
  • Chemically crosslinked Injectable hydrogels laden with graphene-based nanostructures for chemo-photothermal anticancer applications
    Publication . Cunha, João Pedro Rolo da; Diogo, Duarte Miguel de Melo; Correia, Ilídio Joaquim Sobreira; Pouso, Manuel António do Rosário
    Breast cancer is among the foremost causes of mortality on a global scale, exemplifying the disease's resilience and adaptability. The conventional clinical approaches, notably encompassing surgery, radiotherapy, chemotherapy, and immunotherapy, present significant limitations: subpar efficacy and adverse events/side effects. In view of these challenges, it is crucial to develop innovative strategies that can contribute for reducing the mortality rates related to breast cancer. In recent years, strategies based on nanomaterials for chemo-photothermal therapy have started to light the path, showcasing promising results in cancer treatment. The unique physical and chemical properties of these nanomaterials enable the encapsulation of chemotherapeutic drugs as well as their journey into the tumor site. In turn, the optical features enable these nanomaterials to absorb light in the near-infrared (NIR) region, deploying a localized heat. The combined action of these two modalities (drug delivery and photoinduced heat) amplifies the destruction of cancer cells. However, the limited capacity of nanomaterials to reach the tumors, following systemic administration, continues to represent a significant impediment to its full therapeutic capacity. The pursuit for overcoming the systemic administration problems has led to the development of injectable in situ forming hydrogels. These injectable hydrogels have the capacity to encapsulate anticancer agents (including drugs and nanomaterials), and to deliver them locally into the tumors. Among these, injectable hydrogels formed in situ by chemical crosslinking have gained popularity due to their stability and greater control over structural and functional properties (e.g., degradation rate, swelling capacity). In particular, injectable in situ forming hydrogels crosslinked by Thiol-Maleimide reaction are emerging due to the advantageous features of this chemistry: chemo-selectivity and rapid kinetics. In this MSc dissertation, a chemically crosslinked injectable in situ forming hydrogel was developed based on the Thiol-Maleimide reaction between thiolated poly(acrylic acid) and 4-arm poly(ethylene glycol)-maleimide. This hydrogel was incorporated with dopamine-reduced graphene oxide (DOPA-rGO; nanosized photothermal agent) and Doxorubicin (DOX; chemotherapeutic drug), being exploited for the chemo-photothermal therapy of breast cancer. The assembled formulations exhibited injectability and capacity to gelate in situ through the Thiol-Maleimide reaction. The degradation studies highlighted the excellent integrity of the hydrogels. Furthermore, the incorporation of DOPA-rGO into the hydrogel enabled the attainment of a minimal and well-controlled swelling. As importantly, the inclusion of DOPA-rGO into the hydrogel allowed a high photothermal effect under NIR irradiation, that also enhanced the release of DOX by up to 1.5-times. In vitro tests displayed the hydrogels’ favorable cytocompatibility. Besides this, the hydrogels incorporating both DOPA-rGO and DOX generated an effect under NIR irradiation that reduced the breast cancer cells’ viability to just ≈25%, hence confirming their chemo-photothermal capacity. In summary, the injectable hydrogels developed in this dissertation, based on the Thiol-Maleimide reaction between thiolated poly(acrylic acid) and 4-arm poly(ethylene glycol)-maleimide, that incorporated DOPA-rGO and DOX, have showcased auspicious results for application in the chemo-phototherapy of breast cancer cells.
    2025-11-20Dissertação de mestrado Acesso embargado Ver mais
  • Chronotherapy of Brain Diseases: Assessment of the Circadian Rhythms of Efflux Transporters at the Blood-cerebrospinal Fluid Barrier
    Publication . Furtado, André Filipe Lino ; Paixão, Telma Alexandra Quintela; Santos, Cecília Reis Alves; Gallardo Alba, Maria Eugénia
    The choroid plexus (CP) is an integral part of the blood cerebrospinal-fluid barrier (BCSFB). The CP is formed by a monolayer of cuboidal epithelial cells united by tight junctions. On the apical side, these cells present microvilli and are in contact with the cerebrospinal fluid (CSF). On the basal membrane, these cells are surrounded by a vast network of capillary blood vessels. The CP is responsible for several functions that are vital to the homeostasis of the central nervous system (CNS) where we include the production of the CSF, synthesis of several proteins, CNS protection against foreign elements, CSF detoxification from noxious compounds that result from normal cell metabolism and the transport of multiple molecules across the BCSFB. The CP has an essential role on the transport across the BCSFB of therapeutic molecules targeting the CNS. For that, it expresses multiple membrane transporters that have been described in the literature as essential for the transport of therapeutic compounds across CNS biological barriers. Recently, a functional molecular clock was described in the CP. This means that the biological functions of this structure might have a circadian rhythmicity associated. There's the possibility that this circadian clock influences membrane transporters' expression and activity at the CP which would result in circadian changes of the bioavailability of therapeutic compounds in the CNS depending on the time of administration. As such, the main goal of this doctoral thesis was to analyse the influence of circadian rhythms on the expression of multiple membrane transporters on the CP. Additionally, we used therapeutic compounds, namely methotrexate (MTX) and donepezil (DNPZ) to assess the relation between the CP's membrane transporters circadian expression and their drug transport function across the BCSFB. One of the objectives of this project, as mentioned earlier, was to assess the circadian expression of multiple CP’s membrane transporters. For that, CP primary cell cultures of neonate rats were used. We concluded that rSlc9a1 and rSlc1a5 expression was rhythmic during a 24-hour period while rSlc47a1 did not reveal a circadian pattern. This work also aimed at disclosing the influence of sex on the daily expression oscillations of several ABC and SLC membrane transporters expressed by the CP. For this we used CPs from male, female, ovariectomized and sham-operated female rats. The results showed that the membrane transporter rAbcc1 is expressed in a circadian manner in the CP of male rats, while rAbcg2 presented circadian rhythmic expression in the CP of female rats. Both rAbcc4 and rOat3 were rhythmically expressed in the CP of male and female rats. Next, we used an in vitro model of the CP in order to evaluate the relevance of Abcc4’s circadian expression in the transport of MTX across the BCSFB. We demonstrated that MTX transport across the BCSFB was rhythmic. Besides, we also concluded that Abcc4 circadian expression might influence the MTX circadian transport across the BCSFB. Finally, this project also aimed to describe the impact of circadian rhythms on CP Abcg2 expression and also on the circadian transport profile of DNPZ across the BCSFB. Using CP primary cell cultures of neonate rats, we demonstrated the presence of rAbcg2 circadian expression. Next, using primary cell cultures, an in vitro model of the BCSFB was established and we discovered that DNPZ transport across the BCSFB presents circadian rhythmicity. Furthermore, it was also proposed that besides rABCG2, SLC22A4 could also be involved in the DNPZ circadian transport across the BCSFB. The results obtained in this project demonstrate that membrane transporters present circadian expression in the BCSFB. Moreover, the transport of therapeutic compounds, such as MTX and DNPZ, across the BCSFB is also influenced by the circadian rhythm of CP membrane transporters. In the future, it is essential to further exploit the role of circadian rhythms on the expression of membrane transporters at the CP and its influence on the transport of therapeutic compounds across the BCSFB. This information might prove vital in the treatment of CNS diseases. By timing drug administration with the period when they are more prone to reach the target tissue at the CNS, we are ensuring their maximum target tissue concentration, and a reduction in side effects.
    2025-04-01Tese de doutoramento Acesso aberto Ver mais
  • Ciências Farmacêuticas: Aprendizagem em ambiente profissional
    Publication . Gaiolas, Carla Sofia Cardona Jorge; Granadeiro, Luiza Augusta Tereza Gil Breitenfeld
    O plano de estudos do Mestrado Integrado em Ciências Farmacêuticas (MICF) contempla dois tipos de estágio em ambiente profissional: os estágios observacionais nas áreas de farmácia comunitária, hospitalar e análises clinicas e os estágios profissionais ativos em farmácia hospitalar e comunitária. Os estágios observacionais, não menos importantes que os ativos, permitiram além de um primeiro contato com a realidade profissional, o despertar os sentidos para o trabalho ativo e a aplicação de todos os conhecimentos que iria adquirir ao longo dos anos de estudo. Por outro lado, o estagio em ambiente profissional teve como objetivo o culminar e principalmente integrar os conhecimentos técnico-científicos adquiridos em ambiente académico com o ambiente profissional. O estágio ativo em farmácia comunitária decorreu na Farmácia Diamantino no Fundão, com duração de 480h (12 semanas) e em farmácia hospitalar, no Hospital Sousa Martins, na Guarda, com a duração de 320h (8 semanas). Do trabalho desenvolvido nos estágios acima referidos, resultaram os relatórios incluídos neste documento dividido em 2 capítulos, sendo o primeiro referente ao relatório do estágio em farmácia comunitária e o segundo referente ao estágio decorrente no Hospital. Não posso deixar de salientar os seguintes aspetos que me parecem pertinentes evidenciar e que advêm da experiencia vivida e sentida ao longo destas 20 semanas: No decorrer do estágio senti algumas lacunas na minha formação, que pretendo referir não como uma crítica, mas algo passível de melhorar para os anos que vêm, nomeadamente ao nível da legislação, ao nível da gestão, tanto hospitalar como comunitária (compras, gestão de recursos, etc) e ainda aprofundar conhecimentos ao nível de vacinas (Plano Nacional de Vacinação, indicações a grupos específicos, etc), medicamentos biológicos e Nutrição assistida (entérica e parentérica). No entanto, ao longo destas 20 semanas constatei que no geral estava bem preparada para o trabalho que desenvolvi, respondendo bem aos desafios que me foram surgindo, sem grande dificuldade e com conhecimento e rigor técnico e científico adquirido. Considero que as competências que adquiri nestes estágios vieram complementar a experiencia adquirida anteriormente na área da investigação e às quais me foram conferidas equivalências (mestrado e doutoramento).
    2014-7-14Dissertação de mestrado Acesso aberto Ver mais
  • Ciliopathy in cholangiocytes of biliary atresia patients – association with ischemic cholangiopathy, patterns of ductular reaction and disease severity
    Publication . Quelhas, Patrícia Alexandra Silveira ; Santos, Jorge Luiz dos; Vieira, Sandra Maria Gonçalves; Lusquinos, José Ignacio Verde
    Biliary 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.
    2025-12-11Tese de doutoramento Acesso aberto Ver mais
  • Depressão pós-AVC
    Publication . Freitas, Miriam Selma Telo; Leitão, Carlos Manuel de Moura Martins
    Distúrbios do humor, nomeadamente a depressão, são a complicação psiquiátrica mais comummente observada após acidentes vasculares cerebrais. Uma recente meta-análise e uma revisão sistemática feita por Luis Ayerbe et al identificaram uma incidência cumulativa de depressão de 52% nos primeiros cinco anos após o acidente vascular cerebral.(1) Uma revisão sistemática anterior, publicada em 2005, identificou uma incidência de 33% com um pico de incidência nos primeiros seis meses.(2) Apesar destes valores elevados, vários estudos demonstram que a depressão pós-acidente vascular cerebral é diagnosticada em apenas 10% dos casos. A depressão tem sido descrita como fator com impacto importante no processo de reabilitação funcional e cognitiva do doente pós-acidente vascular cerebral. Esta, quando não diagnosticada ou tratada, está associada à redução da participação ativa do doente no seu processo de reabilitação, diminuição da qualidade de vida e aumento da mortalidade, com consequente incremento económico para o sistema nacional de saúde. Esta revisão bibliográfica pretende analisar de forma crítica as publicações e estudos efetuados sobre a depressão pós-acidente vascular cerebral, tendo em especial atenção o contexto multifatorial no qual a depressão surge e identificar e analisar as hipóteses etiológicas estudadas até à data que apontam para uma correlação bidirecional entre os eventos vasculares e a depressão.
    2014-7-7Dissertação de mestrado Acesso aberto Ver mais
  • Development of DNA nanovaccines based on functionalized RALA and Chitosan nanoparticles bearing HPV-16 oncogenes
    Publication . Giusti, Andressa Moreira; Sousa, Ângela Maria Almeida de; Eusébio, Dalinda Isabel da Silva; Cui, Zhengrong
    Cervical cancer (CC), a leading cause of cancer mortality among women, is mainly caused by persistent high-risk human papillomavirus infections, particularly HPV-16 and -18. These viruses possess the oncoproteins E6 and E7, which interfere with p53 and retinoblastoma protein (pRB), respectively, contributing to tumorigenesis. Current vaccines prevent infection but have no therapeutic effect. Moreover, the aggressiveness and lack of specificity of current treatments require innovative targeted therapeutics. DNA vaccines targeting the E6 and E7 oncogenes can be a safer and more promising option for CC eradication, providing preventive and therapeutic effects. The optimal DNA vaccine scenario includes the use of minicircle DNA (mcDNA), a safer and efficient vector than the conventional plasmid DNA (pDNA). So, in this study, we explored the complexation of mcDNA encoding one or both mutated HPV-16 oncogenes (E7mut or E6mut) with biocompatible materials, such as cellpenetrating peptides (CPPs) like RALA, and chitosan (CS). These delivery systems were functionalized with ligands of R8-mannose (R8M), which can enhance DNA delivery and targeting to antigen-presenting cells (APCs). Additionally, we investigated the powder conversion of the DNA/CS-based vaccine using thin-film freeze-drying (TFFD) to enhance vaccine stability. Pure mcDNA (2 µg) was used to optimize the amine-to-phosphate (N/P) ratios of RALA, with and without R8M, and nanoparticles (NPs) were characterized for size, polydispersity index (PDI), zeta potential, complexation efficiency (CE), stability, morphology, and Fourier transform infrared spectroscopy (FTIR). In vitro studies assessed biocompatibility and gene expression in JAWSII dendritic cells after 24 h transfection. For CS-based systems, NPs were prepared with CS, sodium tripolyphosphate (TPP), and 2 µg of parental plasmid (PP)/pDNA or mcDNA encoding both genes, and R8M was also included. The same characterization and biocompatibility assays were performed. Powder conversion of CS NPs was done using TFFD, optimizing conditions for suitable and stable powders. For RALA-based NPs, a N/P ratio of 1.25 was optimal, resulting in homogeneous NPs under 150 nm, negative surface charge, and high CE (>97%). Their morphology was spherical/oval, and incorporation of components was confirmed by FTIR. The NPs were stable under cell culture conditions and biocompatible with JAWSII cells. Expression of the E6 gene was significantly higher in RALA-mannosylated systems, with no differences between the E6mut and multigenic vectors’ gene expression. For CS-based NPs, promising characteristics were obtained, with sizes under 120 nm, homogeneity, positive charges (>20 mV), and CE >98%. Incorporation of PP or mcDNA and R8M did not affect NP properties, indicating feasibility for formulation. The NPs exhibited spherical/oval morphology, and FTIR confirmed the presence of all components. The systems were stable under cell culture conditions and biocompatible with JAWSII cells. Powder conversion of CS-based NPs was optimized with sucrose (1% solid content) as a lyoprotectant, yielding the best results with 0.5 mL per vial. For scale-up with higher NP batches, a solid content of 5.05% with sucrose and leucine was optimal, where R8M incorporation and mcDNA usage showed consistent results, with no changes in NP properties after TFFD. The powders had porous, brittle matrices typical of TFFD. Stability tests over 14 days showed the best result of the powder vaccine at 4°C, supporting improved vaccine storage and distribution in low-resource settings. In conclusion, R8M functionalization enhanced cellular transfection and gene expression in RALA-based systems. Both peptide- and polymer-based delivery systems, with and without R8M, exhibited suitable physicochemical characteristics and biocompatibility. TFFD successfully converted liquid vaccines into stable powders while preserving NPs properties and producing highly porous powders with the potential for good aerosol properties. These findings support further studies exploring intranasal administration for cervical cancer immunization.
    2025-10-03Dissertação de mestrado Acesso embargado Ver mais