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Percorrer Departamento de Ciências Médicas por Objetivos de Desenvolvimento Sustentável (ODS) "03:Saúde de Qualidade"

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  • ARIA-Italy managing allergic rhinitis and asthma in a changing world: The role of the Pharmacist
    Publication . Paoletti, Giovanni; Giua, Corrado; Marti, Alessandro; Baio, Matteo Alberto; Valli, Nicolò; Ridolo, Erminia; Ventura, Maria Teresa; Passalacqua, Giovanni; Puggioni, Francesca; Lourenço, Olga ; Bousquet, Jean; Canonica, Giogio Walter; Heffler, Enrico; Lombardi, Carlo
    Allergic rhinitis (AR) and asthma are common respiratory disorders that often occur together, affecting quality of life and increasing healthcare expenses of patients. These chronic illnesses are often managed without medical supervision, creating distinct challenges. A lack of resources can limit regular follow-up, which in turn promotes disease mismanagement and an increased reliance on self-medication, including the inappropriate use of corticosteroids and nasal decongestants. Community pharmacies could serve as critical primary healthcare providers, facilitating AR and asthma management by promoting therapy adherence, minimizing drug misuse, and improving symptom monitoring using digital tools. The evolving role of pharmacists as vital healthcare team members is highlighted by their involvement in screening, prevention, and patient education, particularly in underserved communities. Strengthening the partnerships between pharmacists, physicians, and patients may lead to more tailored and effective management strategies. This collaborative approach has demonstrated promise in enhancing disease outcomes and reducing healthcare costs.
    2025-04Estudo clínico Acesso aberto Ver mais
  • Beeswax-enriched tricalcium phosphate/hydroxyapatite/sodium alginate/ thymol 3D-printed scaffolds for application in bone tissue engineering
    Publication . Francisco, Martinho Jorge ; Cabral, Cátia Solange Duarte; Calvinho, Paula Cristina Nunes Ferreira ; Correia, Ilídio Joaquim Sobreira ; Moreira, André Ferreira
    Tissue engineering, particularly bone tissue engineering (BTE), continues to pose significant challenges to modern medicine. In this work, a rapid prototyping technique was explored to create 3D scaffolds using a Fab@Home 3D-Plotter extruder. For that purpose, a novel composite mixture containing tricalcium phosphate (TCP), hydroxyapatite (HAp), sodium alginate (SA), beeswax (BW), and thymol (TM) was formulated. BW and TM resulted in 3D scaffolds with rougher surfaces and moderate hydrophilic profiles, properties crucial for mediating cell adhesion and proliferation. Moreover, the 3D scaffolds containing BW displayed a significant increase in compressive strength and Young modulus, being comparable to those exhibited by trabecular bone. TM loading prevented the establishment of Staphylococcus aureus and Escherichia coli infections, inhibiting bacterial adhesion and proliferation at the scaffolds' surface. Additionally, the cytocompatibility of the scaffolds was confirmed over 21 days, with the adhesion and proliferation of Human osteoblasts (hOB) at the scaffold's surfaces. Simultaneously, calcium and phosphate ions accumulated at the scaffolds' surface, forming apatite crystals. Therefore, this improved composite mixture showed promising results for being applied in BTE, not only facilitating hOB cell adhesion and proliferation but also avoiding bacterial infection, addressing a critical challenge in implant-based therapies.
    2025-08-05Artigo de investigação 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
  • Characterization of Effector-Memory CD8+ T cells and their Association with Human Cognitive Function
    Publication . Esgalhado, André João Gabriel ; Arosa, Fernando Aguilar; Uhrberg, Markus
    Human effector-memory CD8+ T cells consist of highly differentiated cells that differ in the expression of the tyrosine phosphatase isoform CD45RA, being designated as CD8+ TEM and CD8+ TEMRA cells. These highly heterogeneous and polyfunctional cells possess cytotoxic, regulatory, and suppressive features, and are capable of migrating to non-lymphoid tissues and organs, including the brain under certain conditions. Expansions of CD8+ TEM and CD8+ TEMRA cells have been described in chronic inflammatory diseases, tumors and viral infections, as well as in healthy elderly individuals, including centenarians. Over the past few decades, the role of CD8+ T cells, particularly CD8+ TEMRA cells, has been the subject of several studies in the context of aging, cognition, and neurodegeneration, where they have been generally regarded as detrimental to the central nervous system (CNS), though recent investigations have challenged this view. These highly differentiated CD8+ T cells are known to arise through TCR-dependent and TCR-independent mechanisms, such as cytokine-driven proliferation via interleukin (IL)-15. Intriguingly, chronic antigenic stimulation has been shown to drive the generation of CD8+ T cells expressing low levels of the CD8β chain, though antigen-independent mechanisms remain poorly understood. Herein, we have performed a comprehensive characterization of peripheral blood mononuclear cells (PBMC) as well as of human leukocyte antigen (HLA) molecules in a cohort of elderly volunteers differing in their cognitive status. A detailed analysis of the level of expression of CD45RA in the CD8+ TEMRA compartment revealed the presence of two distinct populations: CD8+ TEMRAlow and CD8+ TEMRAhigh cells. Notably, CD8+ TEMRAhigh cells formed a well-defined and sharply delineated population that was significantly expanded in cognitively impaired volunteers, whereas cognitively unimpaired volunteers were enriched in CD8+ TEMRAlow cells. Further analysis of CD8α and CD8β expression also identified the existence of two distinct CD8+ T cells subsets based on the expression of CD8β: CD8αβlow and CD8αβhigh T cells, with the former being more prevalent among cognitively unimpaired individuals. Moreover, stimulation with PMA and Ionomycin revealed significantly increased IFN-γ production by CD4+ T cells from cognitively impaired elderly. Noteworthy, all but one of the volunteers studied were cytomegalovirus (CMV) seropositive. Finally, a higher prevalence of the HLA-B8 serotype, which belongs to the ancestral haplotype HLA-A1, Cw7, B8, DR3, DQ2, was found among the cognitively impaired elderly. Additionally, we assessed the impact of IL-15 on the cell surface expression of CD8β using CFSE-labeled purified human naïve CD8+ T cells cultured for 12 days. IL-15 induced a robust proliferation and differentiation, resulting in a cell cycle-dependent down-modulation of CD8β from the cell surface, while CD8α expression remained stable or increased slightly. This led to the generation of CD8αβlow and CD8αβ– (i.e., CD8αα) T cells. In contrast, IL-2 and IL-7 alone were unable to replicate this effect. Determination of mRNA levels for CD8α and CD8β isoforms by qPCR revealed that IL-15 promoted a significant decrease in mRNA levels of the CD8β M-4 isoform, while increasing the levels of the M-1 and M-2 isoforms alongside with CD8α. Remarkably, analysis of the level of the tyrosine kinase Lck showed a significant increase in CD8+ T cell blasts after culture of CD8+ T cells with IL-15, when compared to CD8+ T cells at the beginning of the culture. Our findings show an association with certain CD8+ T cell subsets that is compatible with a protective role in cognition and neurodegenerative diseases by identifying novel markers that define discrete subsets of highly differentiated CD8+ T cells expanded in cognitively unimpaired elderly individuals and identify IL-15 as a factor involved in the generation of these subsets. In-depth phenotypic, functional, and transcriptomic characterization of ex vivo and in vitro obtained CD8+ T cell subset is warranted to further elucidate their unique functional properties.
    2026-01-16Tese de doutoramento Acesso aberto 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
  • Contributo para a melhoria do rastreio do cancro do colo do útero em Portugal
    Publication . Caeiro, Vitor Manuel Branco e Silva; Moutinho, José Alberto Fonseca; Granadeiro, Luiza Augusta Tereza Gil Breitenfeld
    O cancro constitui, neste século, um dos principais problemas de saúde pública, com implicações sociais e económicas significativas. Globalmente, é responsável por aproximadamente 16,8% de todas as mortes e por 22,8% das mortes atribuídas a doenças não transmissíveis. O cancro do colo do útero (CCU) é um dos problemas de saúde pública mais significativos para a população feminina em todo o mundo, sendo o quarto cancro mais comum em termos de incidência e mortalidade nas mulheres, com uma estimativa de 662.301 novos casos e 342.000 mortes em todo o mundo em 2022, segundo a World Health Organization (WHO) 2023. Caracterizado pela sua elevada incidência e mortalidade, particularmente em países de Índice de Desenvolvimento Humano (IDH) baixo ou médio, este tipo de cancro está maioritariamente associado à infeção persistente pelo vírus do papiloma humano (HPV), e estima-se que mais de 90% dos casos de CCU sejam atribuídos aos genótipos de alto risco deste agente viral (HPV-hr). O CCU é um problema de saúde significativo também em Portugal, onde em 2020, foi determinada uma incidência e mortalidade padronizada por idades de 10,7 e 3,2/100 000 mulheres-ano, respetivamente. De acordo com os dados do The Global Cancer Observatory (IARC/WHO) 2024, a Incidência e Mortalidade em Portugal por CCU em 2022, foram de 897 e 459, respetivamente, tendo sido nesse ano, o 8.º cancro mais frequente entre as mulheres e o 3.º cancro mais frequente entre as mulheres entre os 15 e os 44 anos. A taxa de mortalidade por CCU em Portugal, situa-se nos níveis muitos altos de IDH. A WHO recomenda a implementação de programas de rastreio organizados, baseados na população, como a estratégia mais eficaz para a prevenção do CCU. No entanto, o rastreio oportunístico continua a desempenhar um papel importante na prevenção secundária do CCU em muitos países, incluindo Portugal, complementando os esforços do programa de rastreio, colmatando as dificuldades de acesso ao rastreio organizado, assim como possibilitando a liberdade de decisão e a oportunidade da escolha das mulheres na sua vigilância e cuidados de saúde. A WHO reconhece atualmente três métodos principais para o rastreio do CCU: a citologia convencional (teste de Papanicolaou), a inspeção visual com ácido acético (VIA) e o teste de deteção do DNA do HPV. Cada um destes métodos tem as suas especificidades, vantagens e limitações. O contexto epidemiológico e os recursos de cada país determinam a escolha e a implementação de uma estratégia de rastreio adequada. A WHO estabeleceu metas globais para a eliminação do CCU como problema de saúde pública, incluindo: 90% das meninas vacinadas contra o HPV até os 15 anos 70% das mulheres rastreadas com um teste de alta precisão aos 35 e aos 45 anos 90% das mulheres identificadas com doença cervical recebendo tratamento Portugal implementou em 2017 um programa nacional de rastreio baseado na pesquisa de HPV, como teste de rastreio primário, para mulheres com idades dos 25 aos 60 anos. No entanto, este programa não está totalmente implementado em todas as regiões, o que contribui para as dificuldades que o programa atravessa, com apenas 64 % das mulheres elegíveis a participarem no Rastreio em 2022, pelo que é lícito afirmar que o rastreio oportunístico (ou de conveniência) tem um papel importante na prevenção do CCU. Os trabalhos efetuados, de que resultaram os artigos científicos publicados, expressão dos estudos levados a cabo com a experiência do programa de rastreio da ULSCB (das mulheres que foram incluídas no programa de rastreio oportunístico na Consulta de Ginecologia do Hospital da Covilhã), são a base do contributo que se pretende deixar para a melhoria da eficácia da prevenção secundária do CCU em Portugal. Pretenderam esses estudos, apesar das suas limitações, nomeadamente a dimensão da amostra: i) avaliar o risco de HSIL em mulheres com teste de HPV-hr (não 16 e/ou 18) positivos repetidos e com citologia NILM; ii) verificar se é válido dispensar a realização da citologia nos casos de teste de HPV positivo para HPV 16 e HPV 18; iii) e desenvolver uma técnica laboratorial, que permita uma deteção adequada e pouco dispendiosa, para a determinação do genótipo do HPV em material parafinado. Em conclusão, Portugal tem um programa de rastreio organizado baseado no teste de HPV, mas o rastreio oportunístico continua a ser uma ferramenta importante na prevenção secundária do CCU, especialmente para alcançar populações não cobertas pelo programa organizado. A integração eficaz de ambas abordagens, juntamente com a vacinação contra o HPV, afigura-se crucial para atingir as metas da OMS e reduzir significativamente a incidência e mortalidade por CCU no país.
    2026-02-27Tese de doutoramento 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
  • Dual-crosslinked injectable in situ forming Alginate/CaCl2/Pluronic F127/ α-Cyclodextrin hydrogels incorporating Doxorubicin and graphene-based nanomaterials for cancer chemo-photothermal therapy
    Publication . Gonçalves, Joaquim; Melo, Bruna Daniela Lopes ; Pouso, Manuel António do Rosário ; Correia, Ilídio Joaquim Sobreira ; de Melo-Diogo, Duarte
    Injectable in situ forming hydrogels have been emerging due to their capacity to perform the direct delivery of therapeutics into the tumor site with minimal off-target leakage. Particularly, physical crosslinked injectable in situ forming hydrogels are appealing due to their straightforward preparation that exploits the native jointing capabilities of specific polymers/materials. However, the features of these hydrogels (e.g., injectability, degradation, swelling) are strongly pre-determined by the physical interactions available on the selected polymers/ materials, occasionally yielding undesired outcomes. Thus, the combination of multiple physical crosslinking cues may allow the preparation of hydrogels with enhanced properties. In this work, a dual-crosslinked injectable in situ forming hydrogel was engineered by combining Pluronic F127/α-Cyclodextrin and Alginate/CaCl2 (i.e., combination of host-guest and electrostatic interactions), being loaded with Doxorubicin (chemotherapeutic drug) and Dopamine-reduced Graphene Oxide (photothermal nano-agent) for application in cancer chemophotothermal therapy. When compared to the single-crosslinked hydrogels, the dual-crosslinking contributed to the assembly of formulations with suitable injectability and improved degradation and water absorption behaviors. Moreover, the dual-crosslinked hydrogels presented a good photothermal capacity (ΔT ≈ 14 ◦C), leading to a 1.18-times enhanced Doxorubicin release. In in vitro cell-based studies, the dual-crosslinked hydrogels exhibited an excellent cytocompatibility towards healthy (normal human dermal fibroblasts) and breast cancer (MCF-7) cells. As importantly, the dual-crosslinked hydrogels were able to mediate a chemophotothermal effect that diminished the cancer cells’ viability to just 23 %. Overall, the developed dualcrosslinked injectable in situ forming hydrogels incorporating Doxorubicin and Dopamine-reduced Graphene Oxide are a promising macroscale system for breast cancer chemo-photothermal therapy.
    2025-09-11Artigo de investigação Acesso aberto Ver mais
  • O estudo dos humanos tornando-se máquinas: Criação de uma rede neuronal artificial para prevenção do risco da doença cardiovascular
    Publication . Mendes, Francisca Cornelio; Souza, José António Menezes Felippe de; Pombo, José Álvaro Nunes
    Atualmente, 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.
    2025-01-20Dissertação de mestrado Acesso embargado Ver mais
  • Fatores de risco genético para adenomas hipofisários: Uma análise nacional, multicêntrica, genética e clínica
    Publication . Gaspar, Leonor Isabel Mesquita ; Lemos, Manuel Carlos Loureiro de; Gonçalves, Catarina Inês Nunes Pires
    Os 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.
    2025-06-04Tese de doutoramento Acesso aberto Ver mais