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- Burnout e Vinculação em Oncologia e Fim de VidaPublication . Gonçalves, Florbela dos Santos; Viana, Joaquim Manuel da Silva; Sousa, Miguel Castelo Branco Craveiro deIntroduction 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.
- Chronotherapy of Brain Diseases: Assessment of the Circadian Rhythms of Efflux Transporters at the Blood-cerebrospinal Fluid BarrierPublication . Furtado, André Filipe Lino ; Paixão, Telma Alexandra Quintela; Santos, Cecília Reis Alves; Gallardo Alba, Maria EugéniaThe 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.
- Development of DNA nanovaccines based on functionalized RALA and Chitosan nanoparticles bearing HPV-16 oncogenesPublication . Giusti, Andressa Moreira; Sousa, Ângela Maria Almeida de; Eusébio, Dalinda Isabel da Silva; Cui, ZhengrongCervical 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.
- O estudo dos humanos tornando-se máquinas: Criação de uma rede neuronal artificial para prevenção do risco da doença cardiovascularPublication . Mendes, Francisca Cornelio; Souza, José António Menezes Felippe de; Pombo, José Álvaro NunesAtualmente, os programas de Inteligência Artificial (IA) aplicados à medicina baseiam-se em modelos simbólicos que classificam doenças e as suas relações com os fatores do doente e as suas manifestações clínicas. Entre os sistemas mais utilizados, encontram-se softwares especializados que integram o conhecimento médico e realizam tarefas específicas, permitindo raciocinar a partir de dados dos utentes. Estes softwares procuram imitar o comportamento humano, fornecendo soluções especializadas em áreas médicas específicas. O principal objetivo desta dissertação consiste em analisar as técnicas que em vista a melhoria da qualidade de vida das pessoas através do desenvolvimento de órgãos artificiais avançados, membros, próteses e sangue artificial. O objetivo específico pretende investigar os fatores de risco associados ao desenvolvimento de doenças cardiovascular e criar uma aplicação prática, utilizando redes neuronais artificiais através do software Matlab. Esta aplicação visa identificar a probabilidade de um indivíduo desenvolver doenças cardiovasculares, facilitando a implementação de estratégias preventivas e tratamentos personalizados. O presente estudo incluí a utilização da rede neuronal, uma ferramenta poderosa em áreas da Ciência e da Engenharia, com foco na criação e manipulação de redes neuronais. Foram abordados comandos para operações matemáticas e análise de dados, bem como a implementação de redes neuronais com diferentes arquiteturas. Este estudo explorou também funções pré-definidas e personalizadas da rede neuronal, utilizadas para cálculos, processamento de sinais e simulação, com especial atenção para a visualização dos dados obtidos. Os resultados basearam-se numa amostra de 121 indivíduos, maioritariamente residentes na Covilhã. A maioria dos inquiridos encontrava-se na faixa etária dos 26-35 anos e era do sexo masculino. Quanto ao peso, por um lado, cerca de 19% apresentava excesso de peso e, por outro, a maioria tinha peso normal. Cerca de metade dos participantes tinham um nível médio de conhecimento sobre doenças cardiovasculares, e a maioria não apresentava histórico familiar dessas condições. Fatores de risco, como a hipertensão, colesterol elevado e obesidade, foram comuns entre os participantes. O consumo excessivo de álcool foi reportado por 55% da amostra, enquanto 11% eram fumadores. A prática de exercício físico era irregular, e quase metade dos inquiridos consome frequentemente gorduras saturadas. Concluiu-se que a população inquirida, maioritariamente jovem, apresenta fatores significativos para o desenvolvimento de doenças cardiovasculares. A hipertensão arterial, hipercolesterol, hábitos alcoólicos e tabágicos, sedentarismo e a ingestão insuficiente de frutas e vegetais foram os fatores identificados e considerados como os fatores mais relevantes no decorrer deste estudo. Estes resultados são cruciais para a compreensão da saúde cardiovascular dos jovens adultos.
- Fatores de risco genético para adenomas hipofisários: Uma análise nacional, multicêntrica, genética e clínicaPublication . Gaspar, Leonor Isabel Mesquita ; Lemos, Manuel Carlos Loureiro de; Gonçalves, Catarina Inês Nunes PiresOs adenomas hipofisários representam, aproximadamente, 10-15% do total dos tumores intracranianos. A prevalência destes tumores foi estimada em 1:1000 na população geral, sendo mais frequentemente diagnosticados entre os 40-60 anos de idade. Estes tumores são monoclonais, tipicamente benignos e de crescimento lento, no entanto podem estar associados a um aumento da morbilidade e mortalidade através da sobreprodução hormonal e dos efeitos de massa resultantes da compressão das estruturas adjacentes ao tumor. Os tumores hipofisários mais frequentes são os prolactinomas, seguido pelos adenomas hipofisários não funcionantes. Os mecanismos subjacentes à tumorigénese hipofisária não são ainda totalmente conhecidos, pelo que uma melhor compreensão desta questão ajudará a gerir a doença. O aumento do risco associado a mutações em genes como o AIP, MEN1, CDKN1B e PRKAR1A, fornece evidências de uma predisposição genética para adenomas hipofisários familiares. A grande maioria dos adenomas hipofisários (cerca de 95%) ocorre num contexto esporádico e na ausência de predisposição genética conhecida. No entanto, três polimorfismos (rs2359536, rs10763170 e rs17083838) foram significativamente associados a adenomas hipofisários esporádicos na população Chinesa Han. O objetivo geral desta tese foi realizar um estudo de âmbito multicêntrico nacional, acerca dos fatores de risco genético para o desenvolvimento de adenomas hipofisários familiares e esporádicos, de forma a ampliar o conhecimento sobre a tumorigénese hipofisária. Numa primeira fase desta tese, foi construída uma base de dados com todas as variantes germinativas identificadas no gene AIP publicadas em casos esporádicos e familiares de adenomas hipofisários, até à data, a nível mundial. Nesta revisão, foram identificadas e avaliadas, ao nível da sua patogenicidade, um total de 158 mutações germinativas entre 562 doentes com adenomas hipofisários esporádicos ou familiares. Estas variantes estavam localizadas em toda a região codificadora e nas regiões de splicing do gene AIP. A patogenicidade de todas as variantes germinativas publicadas foi categorizada de acordo com os critérios da American College of Medical Genetic and Genomics (ACMG), utilizando todos os dados disponíveis. Do número total de doentes, 35,4% apresentavam variantes patogénicas e 24,0% apresentavam variantes provavelmente patogénicas. Na segunda fase desta tese foi determinada a frequência de mutações germinativas do gene AIP em doentes portugueses com macroadenomas hipofisários esporádicos de início precoce. Para isso, foi sequenciado o gene AIP em 218 doentes com macroadenomas hipofisários esporádicos diagnosticados antes dos 40 anos. Foram identificadas variantes raras em heterozigotia neste gene em 18 (8,3%) doentes. No entanto, apenas quatro (1,8%) doentes apresentavam variantes patogénicas. Estas variantes compreendiam duas mutações já conhecidas (p.Arg81* e p.Leu115Trpfs*41) e duas mutações novas (p.Ser53Thrfs*36, e p.Glu246*). Estes quatro doentes tinham sido diagnosticados com somatotrofinoma em idades compreendidas entre os 14 e os 25 anos. A frequência de variantes patogénicas no gene AIP em doentes com idade inferior a 30 anos foi de 3,4% e com idade inferior a 18 anos foi de 5%, respetivamente. A frequência de mutações no gene AIP nesta coorte de doentes portugueses foi inferior à de outros estudos. A identificação de novas variantes no gene AIP expande o espetro das causas genéticas dos adenomas hipofisários e pode ajudar a compreender o papel das mutações neste gene nos mecanismos moleculares subjacentes à tumorigénese hipofisária. A terceira fase desta tese consistiu em identificar mutações germinativas num conjunto específico de 29 genes, descritos na literatura como tendo mutações germinativas em doentes com adenomas hipofisários, numa coorte de doentes portugueses diagnosticados com adenomas hipofisários esporádicos de início precoce. Para isso, foi feita a sequenciação completa do exoma em 225 doentes com macroadenomas hipofisários esporádicos diagnosticados até aos 40 anos de idade. Foram identificadas 154 variantes raras em 25 dos 29 genes. Destas foram identificadas três variantes patogénicas e 13 variantes provavelmente patogénicas, nos genes AIP, CDH23, MEN1, MSH2, PMS2, SDHB, TP53 e VHL, em 7,1% dos doentes. Nos doentes diagnosticados com idades inferiores a 30 e 18 anos, a frequência de mutações foi de 9,0% e 12%, respectivamente. Esta é, até à data, a maior análise multigénica de doentes com macroadenomas hipofisários esporádicos de início jovem. Confirmámos que o AIP é o gene mais frequentemente envolvido, mas também descobrimos causas genéticas mais raras de adenomas hipofisários, incluindo a primeira confirmação independente de um papel do gene CDH23. Na última fase desta tese foi avaliada a associação de três polimorfismos comuns próximos dos genes NEBL (rs2359536), PCDH15 (rs10763170) e CDK8 (rs17083838) à suscetibilidade a adenomas hipofisários esporádicos na população portuguesa. Foram determinadas as frequências genotípicas e alélicas de 570 casos e 546 controlos. O alelo minor CDK8 rs17083838 (alelo A) foi significativamente associado a adenomas hipofisários esporádicos. As variantes NEBL rs2359536 e PCDH15 rs10763170 não foram associadas a risco geral para a doença, embora tenha sido observada uma associação significativa entre o alelo minor PCDH15 rs10763170 (alelo T) e somatotrofinomas. Estes resultados sugerem que a variante CDK8 rs17083838, e possivelmente a variante PCDH15 rs10763170, podem aumentar a suscetibilidade a adenomas hipofisários esporádicos na população portuguesa. Concluindo, diferentes estratégias foram desenvolvidas e implementadas, ao longo desta tese, de forma a determinar quais os fatores de risco genético mais associados ao desenvolvimento de adenomas hipofisários esporádicos e familiares. Estes resultados são importantes sob o ponto de vista científico não só para uma melhor compreensão do panorama genético dos adenomas hipofisários, como também abrem portas para novas estratégias de rastreio genético direcionadas, oferecendo conhecimentos fundamentais para a gestão personalizada dos macroadenomas hipofisários de início precoce.
- From Hypoxia to Healing: Optimizing Stem Cell Therapy for Neonatal Hypoxic-Ischemic Brain InjuryPublication . Serrenho, Inês Isabel Pires; Baltazar, Graça Maria Fernandes; Manadas, Bruno José Fernandes Oliveira; Grãos, Mário Martins RodriguesNeonatal hypoxic-ischemic encephalopathy (HIE), caused by oxygen and blood flow deprivation to the neonatal brain, is a leading cause of mortality and long-term neurological disabilities in children under five. Its complex pathophysiology— comprising excitotoxicity, inflammation, oxidative stress, and delayed cell death—poses significant therapeutic challenges. Moreover, current management with therapeutic hypothermia (TH) shows limited efficacy, particularly in severe cases, and excludes many neonates due to strict enrollment criteria. To overcome these limitations, preclinical research has explored stem cell therapies (SCT), particularly using stem cells from umbilical cord blood (UCB) and tissue (UC). These therapies show promise in reducing brain injury and improving outcomes, but challenges remain in optimizing doses, delivery methods, and scalability. Building on previous work, this thesis evaluated several approaches to optimize SCT for neonatal HIE. First, administration of UCBCs alone and in combination with TH in a rat model of HIE, reduced brain lesion size and improved functional outcomes more effectively than TH alone, while also reducing glial reactivity—an effect not observed with TH alone. These findings establish UCBCs as a potential therapy, particularly when TH is unavailable or insufficient. Nonetheless, the use of these cells can present disadvantages: autologous use, limited culture and expansion, thus not being possible to manipulate with preconditioning strategies. Thus, we focused on the use of UC-derived MSCs that overcome some of the limitations previously mentioned. Also, since preconditioning strategies are being explored as ways to potentiate MSCs therapeutic effects, we hypothesized that hypoxic preconditioning could enhance UC-MSC efficacy and what explored the potential mechanisms behind neurological recovery. Proteomic analysis revealed that HIE rats treated with hypoxiapreconditioned MSCs had an enrichment of pathways related to synapse function, brain connectivity, and energy metabolism. Moreover, administration of UC-MSCs preconditioned with short hypoxia induced a greater functional recovery in HIE rats than administration of UC-MSCs preconditioned mild hypoxia. Stem cell therapy requires high doses of stem cells, which limits its clinical translatability. To optimize SCT and significantly reduce effective doses of MSCs, we evaluated the impact of two delivery methods and the combination of different strategies on the efficacy of UC-MSCs. Intranasal administration of UC-MSCs was more effective in reducing the infarct volume enhancing motor and cognitive recovery than intravenous administration, restoring myelination in the corpus callosum, and mitigating glial reactivity. Intranasal administration of half the dose of hypoxia-preconditioned UCMSCs reduced neurological deficits associated with neonatal HIE. Strikingly, intranasal administration of secretome from hypoxia-preconditioned MSCs demonstrated similar therapeutic efficacy, offering a promising cell-free alternative. Finally, to address the limitations associated with the expansion of UC-MSCs and donor variability on the therapeutic potency of these cells, we aimed to determine if induced pluripotent stem cell-derived MSCs (iMSCs) could hold the same potential in the neonatal HIE model. Intranasal administration of iMSCs or their secretome improved motor and cognitive recovery, reduced brain lesion size, modulated glial reactivity, and enhanced neurogenesis in the hippocampus of HIE rats. The comparable outcomes between iMSCs and their secretome underscore the critical role of secreted factors, presenting a scalable, cell-free option for clinical application. In summary, this thesis advances the field of regenerative medicine for neonatal HIE by identifying innovative strategies to optimize stem cell therapies. The findings demonstrate the potential of intranasal delivery and hypoxic preconditioning to reduce the required cell doses while maintaining efficacy, making these therapies more feasible for clinical use. Furthermore, secretome-based approaches offer a scalable alternative that addresses logistical challenges. Beyond neonatal HIE, these strategies may have broader implications for other neurodevelopmental and neurological disorders, as well as for veterinary medicine. While significant progress has been made, future research should aim to elucidate the mechanisms underlying MSC and secretome efficacy and advance these approaches toward clinical trials, paving the way for innovations in regenerative medicine.
- Hair as a monitoring tool for psychoactive substancesPublication . Simão, Ana Aysa Rocha da; Alba, Maria Eugénia Gallardo; Barroso, Mário Jorge Dinis; Andraus, Maristela HaddadThe global prevalence of substance use and its severe health, social, and economic consequences remain significant public health concerns. The continuous emergence of new psychoactive substances (NPS), alongside the widespread use of traditional drugs, exacerbates the challenges faced by forensic and clinical toxicologists. These substances often evade conventional detection methods due to their rapid evolution, posing significant risks to users and complicating monitoring efforts. Understanding substance use patterns and their impacts is essential for informing prevention strategies, shaping public policies, and implementing harm reduction initiatives. Hair analysis has emerged as a powerful tool in toxicological investigations, offering unique advantages over traditional biological matrices such as blood and urine. Its extended detection window enables the assessment of chronic and historical drug use, while its resistance to adulteration ensures reliability. As a non-invasive collection method, hair analysis facilitates large-scale studies, making it particularly suitable for exploring poly-drug use and evaluating the prevalence of both classical drugs and NPS across diverse populations. Quantitative hair analysis complements self-reported data, addressing discrepancies and enhancing the accuracy of substance use research. [...]
- Identificação de causas genéticas do hipogonadismo hipogonadotrófico congénito utilizando a sequenciação de nova geraçãoPublication . Carriço, Josianne Nunes; Lemos, Manuel Carlos Loureiro de; Gonçalves, Catarina Inês Nunes PiresO desenvolvimento pubertário é um processo fisiológico complexo que resulta da interação entre o sistema neuroendócrino, fatores genéticos e ambientais. Inicia-se com a reativação do eixo Hipotálamo-Hipófise-Gónadas, levando à produção de gonadotrofinas, hormonas sexuais e consequente desenvolvimento dos carateres sexuais secundários. O Hipogonadismo Hipogonadotrófico Congénito (HHC) é uma doença endócrina rara que, na adolescência, se manifesta clinicamente com ausência completa ou parcial do desenvolvimento dos caracteres sexuais secundários. Na idade adulta, os sintomas incluem ausência de virilização e disfunção erétil no sexo masculino, amenorreia primária no sexo feminino, ausência de líbido e infertilidade em ambos os sexos. O HHC caracteriza-se pela ausência ou diminuição da produção, secreção ou ação da hormona libertadora de gonadotrofinas. Em 50 a 60% dos casos associa-se a anosmia/hiposmia (Síndrome de Kallmann) ou a outras anomalias congénitas. O diagnóstico do HHC baseia-se na anamnese, observação de sinais clínicos, levantamento de sintomas e exames laboratoriais e imagiológicos. Contudo, desde o início do século XXI, o conhecimento sobre as causas genéticas do HHC tem avançado na mesma medida que o desenvolvimento das tecnologias de sequenciação genética. A disponibilidade das tecnologias de sequenciação de nova geração (NGS) proporcionou grandes avanços no diagnóstico molecular destes doentes. Este estudo teve como objetivo investigar as causas genéticas do HHC em doentes portugueses, utilizando a NGS, ferramentas in silico e técnicas de biologia molecular. Foram estudados 81 doentes com HHC e 263 controlos portugueses. O ADN genómico foi extraído dos leucócitos do sangue periférico e submetido à sequenciação do exoma completo. A partir das bases de dados OMIM® e Pubmed, identificaram-se 169 genes relacionados com o fenótipo de HHC, que foram incluídos num painel virtual. As variantes encontradas nesses genes foram filtradas com base na frequência populacional e localização no genoma, sendo classificadas conforme os critérios do American college of medical genetics and genomics e da Association for molecular pathology (ACMG-AMP) em cinco níveis, de benignas a patogénicas. Para avaliar o impacto de variantes com potencial para alterar o splicing, utilizou-se o método de minigene. A análise das variantes germinativas permitiu identificar a causa genética do HHC em 24 dos 81 doentes estudados (29,6%). As variantes causais estavam distribuídas por 10 dos 169 genes presentes no painel genético virtual analisado. Os genes GNRHR, FGFR1, ANOS1 e CHD7 foram os mais frequentemente afetados. Notavelmente, a ampliação do painel genético não resultou num aumentou considerável da taxa de diagnóstico. No entanto, é importante destacar que este estudo expandiu o espectro mutacional da doença, identificando várias variantes até então nunca descritas. A maioria das variantes encontradas foi classificada como de significado indeterminado (VUS), mas a taxa de VUS não diferiu significativamente entre doentes e controlos, com exceção do gene EGF, onde foram encontradas VUS exclusivamente nos doentes. Concluímos que a maioria dessas variantes raras não estão diretamente relacionadas com o fenótipo estudado, refletindo provavelmente o fundo genético da população portuguesa. Em relação à oligogenia, observou-se uma frequência de 6,2% entre os doentes (5/81), um valor estatisticamente mais elevado quando comparado com os controlos. O estudo funcional de uma variante identificada no gene CHD7 (NM_017780.4: c.4354G>T, p.Val1452Leu), resultou no aumento da taxa de diagnóstico para 30,9%, demonstrando a importância dos estudos funcionais na reclassificação de VUS. Este trabalho enfatizou a complexidade genética do HHC, reforçando a importância de uma compreensão aprofundada da doença, seja para estabelecer o diagnóstico e/ou o prognóstico, para ajudar no aconselhamento genético e tratamento, seja para o esclarecimento dos processos biológicos, que potencialmente poderão contribuir para o desenvolvimento de terapias dirigidas no futuro.
- O plexo coroide como porta de entrada para metástases cerebraisPublication . Capoti, Érica de Sousa; Santos, Cecília Reis Alves dos; Duarte, Ana Catarina AbreuO plexo coroide (PC) é uma estrutura vascularizada localizada nos ventrículos cerebrais que desempenha um papel crucial na produção e regulação do líquido cefalorraquidiano (LCR). Essa função é essencial para a proteção do sistema nervoso central (SNC), regulação da homeostase cerebral e transmissão de sinais neuronais. O PC é composto por células epiteliais que formam a BCSFB, caracterizada por junções apertadas que controlam seletivamente o fluxo de moléculas e células entre o sangue e o LCR. Anatomicamente, o PC apresenta configurações distintas nos diferentes ventrículos cerebrais, estende-se ao longo do assoalho ventricular nos ventrículos laterais e forma aglomerados no teto do terceiro e quarto ventrículos. A estrutura básica do PC inclui vasos sanguíneos envolvidos por tecido estromal e uma camada epitelial rica em junções de oclusão, constituídas por ocludinas e claudinas, que desempenham um papel crucial na manutenção da integridade da BCSFB. Além das funções na produção de LCR e na BCSFB, o PC atua na proteção antioxidante do SNC contra o estresse oxidativo, utilizando enzimas como superóxido dismutase e glutationa peroxidase. Esses sistemas enzimáticos são fundamentais para neutralizar espécies reativas de oxigênio que podem ser prejudiciais ao cérebro. Em contextos patológicos, como o cancro cerebral, o PC pode desempenhar um papel importante na propagação de metástases cerebrais, embora esse processo ainda não esteja completamente compreendido. Há indícios de que células tumorais, como as de cancro de pulmão, mama e melanoma, possam utilizar a via do PC para entrar no SNC, atravessando a BCSFB. No entanto, a capacidade de invasão por meio desta via e as interações moleculares envolvidas, como aquelas mediadas por E-caderina e ICAM2 nas células endoteliais do PC, ainda carecem de estudos mais aprofundados. Por isso, investigar esse mecanismo é crucial para esclarecer o papel do PC na invasão de células tumorais no cérebro. Para a realização deste trabalho foram utilizadas linhas celulares HIBCPP (que mimetizam a BCSFB) e A549 de cancro do pulmão para modelar a BCSFB in vitro e estudar a transmigração celular. As células foram cultivadas em meio DMEM/F12 suplementado com soro fetal bovino e antibióticos, mantidas em condições de 37°C e 5% de CO2. Os inserts foram revestidos com colagénio para melhorar a adesão das células HIBCPP, cuja integridade da barreira foi avaliada pela medição da resistência elétrica transepitelial (TEER). Ensaios de transmigração com células A549 marcadas com CellTracker Green CMFDA foram feitos com o intuito de investigar os possíveis efeitos na função da BCSFB e poder contribuir para a compreensão das interações celulares em condições fisiológicas e patológicas do SNC.
- SGLT2i and GLP1RA: Safety and Effectiveness Profiles Evaluation using Real-World Patient DataPublication . Lopes, António Luís Martins Coutinho Cabral e; Morgado, Manuel Augusto Nunes Vicente Passos; Lourenço, Olga Maria Marques; Roque, Maria de Fátima dos Santos MarquesType 2 Diabetes Mellitus (T2DM) is a highly prevalent chronic disease associated with substantial morbidity and mortality, largely driven by cardiovascular (CV) complications, chronic kidney disease (CKD), and obesity. The global burden of T2DM continues to rise, fueled by aging populations, urbanization, and lifestyle-related risk factors. In this context, newer therapeutic classes such as sodium-glucose cotransporter2 inhibitors (SGLT2i) and glucagon-like peptide-1 receptor agonists (GLP1RA) have transformed the management of T2DM by providing benefits that extend far beyond glycemic control. Robust evidence demonstrates that these agents reduce the risk of major adverse cardiovascular events (MACE), slow CKD progression, promote weight loss, and improve metabolic parameters. As a result, both drug classes have been increasingly repositioned and approved for new clinical indications, broadening their use to diverse and often complex patient populations. Despite substantial evidence from randomized controlled trials, real-world data on the safety and effectiveness of SGLT2i and GLP1RA remain essential, particularly in older individuals, polymedicated patients, and those with multiple comorbidities—groups often underrepresented in clinical trials. Post-marketing safety signals, including genitourinary infections with SGLT2i and gastrointestinal adverse effects with GLP1RA, underscore the need for continued pharmacovigilance. Understanding how these medicines perform in routine care settings is critical to optimizing therapeutic strategies and supporting clinical decision-making. This doctoral thesis aimed to evaluate the safety and effectiveness profiles of SGLT2i and GLP1RA using real-world data from hospital settings and pharmacovigilance reports. Retrospective observational studies were conducted across emergency departments, inpatient hospitalizations, and outpatient consultations, complemented by an analysis of adverse drug reaction (ADR) reports from the EudraVigilance (EV) database. Key findings highlight the effectiveness of SGLT2i in improving renal function, reducing systolic blood pressure, and lowering hospitalization rates for heart failure (HF). GLP1RA were particularly effective in achieving significant weight loss and reducing CV risk scores. Both drug classes demonstrated substantial reductions in glycated hemoglobin (HbA1c), consistent with clinical trial data. Safety analyses revealed low incidences of acute kidney injury (AKI) and major CV events, with gastrointestinal side effects being the most frequently reported ADRs for GLP1RA. These results underscore the critical role of SGLT2i and GLP1RA in comprehensive T2DM management, particularly for high-risk populations with multiple comorbidities. The findings also emphasize the need for ongoing pharmacovigilance and tailored therapeutic strategies to optimize patient outcomes. Future research should focus on mitigating adverse effects and validating these findings across diverse populations and healthcare settings.