FCB1-Teses
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- Interplay between mineralization and inflammation in osteoarthritis: gla-rich protein and marine bioactive compounds as new therapeutic approachesPublication . Araújo, Nuna Cláudia Peixoto de; Simes, Dina; Viegas, Carla; Vermeer, CeesOsteoarthritis (OA) is a prevalent joint disorder with significant global impact, characterized by limited treatment options and challenges in early diagnosis. This research aimed to address a deeper understanding of the onset and progression of OA, while exploring novel therapeutic strategies. Pathologic calcification and inflammation, associated with degradation of cartilage extracellular matrix, are prominent features observed in OA. Gla-Rich Protein (GRP), a vitamin K-dependent protein, has recently shown promising potential in possessing anti-inflammatory and anti-mineralization properties in articular cells, suggesting its role in the interplay between inflammation and mineralization in OA. Based on that, we developed chitosan-based nanoparticles to encapsulate GRP, aiming to enhance its bioavailability and thereby facilitating its application in functional assays. These nanoparticles effectively delivered GRP and retained its anti-inflammatory activity in human activated macrophages (THP-1 MoM) and chondrocytes. The novel nanoformulation demonstrated promising potential for therapeutic applications in chronic inflammatory diseases. Furthermore, we developed an experimental pipeline in order to evaluate potential OA-modifying compounds and their effects on inflammation and mineralization. The pipeline involved a series of activity assessments, starting from a simpler cell-based model of OA and progressing to a more complex co-culture model based on human cartilage. This progressive approach allowed us to examine potential drugs as mediators of inflammation and mineralization, mimicking the early disease stages and providing valuable insights into the complex pathological processes involved in OA progression. In the search for new active compounds to treat OA, we investigated the utilization of a marine bioactive compound, amentadione (YP), in the context of OA treatment, using the established pipeline. Amentadione exhibited strong anti-inflammatory effects, by downregulating key inflammatory mediators such as cyclooxygenase 2 (COX-2) and interleukin 6 (IL-6), and also demonstrated the ability to regulate nuclear factor κB (NF-κB) signaling pathways. By decreasing matrix metalloproteinase-3 (MMP3) levels and chondrocyte hypertrophic differentiation factors, Col10 and Runx2, YP contributed to cartilage homeostasis. These findings highlight the high therapeutic potential of amentadione as a cartilage protective factor in OA. In summary, this research highlights the therapeutic potential of both GRP and marine bioactive compound YP in the management of inflammatory responses in OA. The utilization of novel drug delivery systems, including chitosan-based nanoparticles, along with the comprehensive evaluation of compounds using the established experimental pipeline, offers the possibility of better understanding the underlying disease mechanisms and to explore new potential active compounds in OA.
- Generation of a novel Cockayne syndrome B neuronal culture model from patient-derived induced pluripotent stem cellsPublication . Madeira, Cristiana Raquel Rodrigues; Matos, Carlos; Calado, SofiaCockayne syndrome type B (CSB) is a rare genetic, multisystem disorder, which results from accumulation of DNA damage that arises when the associated gene, ERCC6, is dysfunctional. This leads to premature aging and neurodegeneration. Studies on CSB pathophysiology are lacking, and pre-clinical research of potential therapeutic strategies is nonexistent. Patients with CSB are left without any chance of disease cure or delay, experiencing premature death. Patient-derived induced pluripotent stem cells (iPSCs) offer a powerful tool to investigate disease mechanisms while generating possibilities for treatment. As a result, our goal was to generate iPSCs from the fibroblasts of CSB patient, as an in vitro model, and differentiate them into neural cell cultures that exhibit CSB-related neuropathological alterations. To achieve this goal, human patient fibroblasts were reprogramed into CSB-iPSCs with the CytoTune iPS 2.0 Sendai Reprogramming Kit. CSB-iPSCs and fibroblasts genetic profile analysis confirmed relatedness between cell lines. The ERCC6 gene was sequenced demonstrating to have the expected mutation, and Western blot analysis further confirmed the presence of a pathogenic mutation. PCR showed that CSB-iPSCs are free of reprograming virus at passage 10 and cultured cells did not contain mycoplasma contamination. Through immunohistochemistry and flow cytometry, CSB-iPSCs demonstrated to express pluripotency markers and capacity to differentiate into the three germ layers. CSB fibroblasts displayed an accumulation of dysfunctional mitochondria, and their DNA repair capacity was affected, upon UV light exposure, as observed through flow cytometry and immunocytochemistry, respectively. This suggests that CSB cells have a dysfunctional transcription-coupled nucleotide excision repair (TC-NER) and mtDNA repair. CSB-iPSCs were successfully differentiated into neurons and CSB-Neurons appeared to have longer neuronal prolongations that neurons derived from a healthy donor, indicating premature maturation. Our study suggests that the in vitro CSB model generated can help understand this disorder and contribute to the pre-clinical testing of disease-modifying therapies.
- Demonstrating a role of paxillin in adherens junction strengthening and in premalignant to malignant progressionPublication . Rodrigues, Vanessa; Janody, Florence; Carvalhal, SaraBreast cancer poses a substantial threat to women worldwide, often preceded by premalignant lesions that hold potential for progression to invasive cancer. However, accurately predicting which premalignant lesions will progress remains a significant challenge. This study addresses this issue by unravelling the intricate interplay between adherens junctions (AJs) and focal adhesions (FAs) in the progression of premalignant breast cells to invasive cancer. Both AJs and FAs are complex, multifunctional protein assemblies linked to the actin cytoskeleton. Recent advancements in mass spectrometry and adhesion-specific enrichment techniques have elucidated hundreds of proteins constituting integrin and cadherin adhesomes. Nevertheless, these adhesomes compositions are highly variable across cell types and experimental conditions. Despite their traditional distinction, AJs and FAs share numerous signalling molecules, including Paxillin, and are mechanosensitive structures, capable of transducing mechanical cues into transcriptional regulation of cellular processes. Utilizing a human mammary epithelial cell line with conditional Src protooncogene activation, which recapitulates molecular processes in breast cancer progression, the CRC group demonstrated that before undergoing Epithelial to Mesenchymal transition, premalignant MCF10A-ER-Src cells experience a transient state characterized by actomyosin stress fiber accumulation associated with larger FAs, exclusively to cells with E-cadherin/P-cadherin-positive adherens junctions. This mechanical stiffening promotes cell proliferation and amplifies Src activation, propelling cells toward a fully transformed state. In this project we aimed to investigate the influence of Src activation on FA dynamics in premalignant MCF10A-ER-Src cells connected through AJs and to investigate the role of FA component Paxillin, in the progression from premalignant to malignant. The findings obtained are directly aligned with our previous research as the measurements performed on FAs unveils that Src activation triggers the assembly of larger FAs associated with polarized stress fibers in premalignant MCF10A-ER-Src cells connected through AJs. Alongside, we showed that knocking down Paxillin could be an effective tool to weaken FAs strengthening.
- Associação entre o infiltrado linfoide e expressão de KI-67 com diversos fatores clínico-patológicos em cancro da mama luminalPublication . Almeida, Érica Filipa Pinto de; Borralho, Paula; Tavares, ÁlvaroAtualmente o cancro da mama continua a ter uma grande importância a nível mundial, particularmente no que respeita à sua incidência e mortalidade. Neste contexto, o diagnóstico correto e precoce apresentam um valor imensurável, contribuindo assim para a cura deste tumor. Muitos são os estudos realizados que procuram encontrar fatores preditivos e de prognóstico, de modo a auxiliar a tarefa árdua de diagnosticar, orientar a terapêutica e avaliar o prognóstico do cancro da mama. No entanto, atualmente este tema ainda carece de respostas para muitas questões. Com o intuito de encontrar algumas respostas, no presente estudo foram analisadas correlações entre várias variáveis clínicas, histológicas e biológicas, a fim de validar determinados biomarcadores, como fatores preditivos e de prognóstico. Neste projeto, foi nosso objetivo comparar duas coortes de carcinoma da mama de tipos histológicos diferentes (carcinoma lobular invasivo e carcinoma sem tipo especial), mas com grau de diferenciação e estádios clínicos e patológicos sobreponíveis. Foram selecionados 60 casos de carcinoma da mama, dos quais 30 carcinomas lobulares e 30 carcinomas de tipo NST e foram recolhidas as características específicas de cada tipo de tumor, bem como os dados clínicos relevantes. Foram comparadas variáveis clínicas (idade, a presença ou ausência de metástases, recidiva e por fim, seguimento) e variáveis histológicas e biológicas (expressão de recetores hormonais, amplificação de HER2, expressão de Ki-67 e densidade da infiltração linfocitária) entre as duas coortes e correlacionadas estas variáveis entre si e com os dados de seguimento. Um total de 8 correlações foram identificadas, em ambos os carcinomas, entre os vários parâmetros de interesse. Evidencia-se a correlação da expressão de Ki-67 com a infiltração linfocitária peritumoral e intratumoral, que revelaram relações ligeiramente superiores às restantes correlações efetuadas.
- Caracterização da expressão do gene ZFP36L1 associado a mecanismos de patogénese da osteoartrose e osteoporosePublication . Lázaro, Mafalda Guela; Simão, MárcioO organelo sem membrana, designado como grânulo TIS, é caracterizado pela interação da proteína ZFP36 ring finger protein like 1 (ZFP36L1), também conhecida como TIS11B, com mRNAs que codificam proteínas membranares. ZFP36L1 promove o enriquecimento ou exclusão de transcritos, dependendo do tamanho dos seus 3'UTR e número de elementos ricos em adeninas (A) e uracilos (U) (AREs). A associação de grânulos TIS à superfície do retículo endoplasmático cria um microambiente que favorece a tradução e localização de proteínas específicas na superfície da membrana das células. ZFP36L1 também é conhecido por regular a estabilidade e degradação de transcritos de vários genes alvo, como é o caso de vários intermediários inflamatórios, o que sugere um papel duplo para o ZFP36L1, favorecendo a transcrição de proteínas membranares e destabilizando outros alvos. Para além dos marcadores de inflamação, também regula os intermediários da via de sinalização das BMPs e está envolvido em mecanismos de senescência o impacto do ZFP36L1 nas células precursoras de osteoblastos e de condrócitos e na sua diferenciação terminal é praticamente desconhecido. Recentemente, o ZFP36L1 tem sido implicado na promoção da diferenciação de osteoblastos e a sua expressão foi identificada como sendo diminuída em ratinhos mais idosos, resultando numa diferenciação preferencial das células mesenquimais para a linhagem adipocitária. Também foi publicado que o aumento da expressão de ZFP36L1 está associado aos mecanismos de progressão da osteoartrose. Este projeto tem como objetivo a identificação, compilação e discussão de estudos publicados que descrevem a expressão de ZFP36L1 em contextos patológicos associados ao desenvolvimento de osteoporose e osteoartrose, focando a discussão dos resultados obtidos no impacto que ZFP36L1 pode ter no metabolismo do osso e da cartilagem. Através da pesquisa da base de dados Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geoprofiles) e bibliografia associada vai-se procurar compilar e comparar dados relativos à expressão de ZFP36L1 em experiências usando tecidos de humano e de ratinho. Com base nesses resultados vai-se procurar contextualizar a expressão desta proteína nos mecanismos associados ao desenvolvimento de osteoartrose e osteoporose. Serão igualmente estudadas (i) as relações filogenéticas destes genes nas principais espécies utilizadas como modelos biológicos em ciências biomédicas, e (ii) a presença de polimorfismos no gene que codifica para a ZFP36L1 e possam ser associados a patologias no sistema musculoesquelético e contextualizar os resultados relativamente às funções das proteínas codificadas.
- Estabelecimento de modelos celulares de Síndrome de Down: uma nova ferramenta para o estudo do desenvolvimento neuronalPublication . Ventura, Igor; Araújo, Inês; Simão, SóniaA síndrome de Down (DS) é comumente causada pela trissomia do cromossoma 21, e caracteriza-se por deficit cognitivo, alterações na morfologia craniofacial e várias comorbilidades. Desequilíbrios na sinalização neurogénica durante períodos críticos da neurogénese são capazes de afetar a proliferação, migração e diferenciação das células estaminais neuronais, causando microcefalia e perturbação do desenvolvimento cerebral, responsáveis pelo deficit cognitivo. Assim, é de extrema importância compreender como o desenvolvimento cerebral é afetado na DS. Para tal, é necessário desenvolver ferramentas celulares que nos permitam estudar em detalhe o neurodesenvolvimento em células humanas. Deste modo, neste projeto pretendeu-se estabelecer e caracterizar linhas de células estaminais pluripotentes induzidas (iPSCs) obtidas a partir de fibroblastos da pele de pacientes com trissomia 21 (AG06922) reprogramadas com os factores de transcrição de Yamanaka. Para a caracterização da linha, foram utilizados os critérios propostos pela Sociedade internacional de investigação em células estaminais (ISSCR). Dentro dos quais inclui imunocitoquímica, citometria de fluxo, análise da fosfatase alcalina, deteção viral por PCR, capacidade de diferenciação nas 3 camadas germinativas, entre outros. Com a realização deste trabalho foi possível estabelecer com sucesso a linha iPSC proveniente de um paciente com DS. Esta ferramenta celular irá permitir de futuro estudar a diferenciação neuronal e avaliar vias de sinalização que possam estar alteradas nesta patologia.
- Revisão da medicação de idosos institucionalizados em estruturas residenciais para pessoas idosas no AlgarvePublication . Andrade, Amanda de Oliveira; Leitão, Helena Santos; Nascimento, TâniaO avanço da medicina e a melhoria dos cuidados de saúde possibilitam que os indivíduos vivam mais e com melhor qualidade de vida. O envelhecimento é um processo natural que conduz a alterações anatomofisiológicas e metabólicas que modificam a farmacocinética e farmacodinâmicas dos medicamentos num idoso. Além destas alterações, o avanço da idade diminui a autonomia e a perceção de autocuidado, sendo necessário recorrer a instituições de cuidados, como as Estruturas Residenciais para Pessoas Idosas (ERPI). A revisão da medicação é um processo eficaz que procura a otimização e gestão correta da medicação, além da identificação de problemas relacionados com uso de medicamentos, como interações medicamentosas e reações adversas. O principal objetivo deste trabalho foi a caracterização do perfil farmacoterapêutico, por meio da revisão da medicação, de idosos institucionalizados em ERPI, no Algarve. Foram analisados os perfis terapêuticos dos 96 pacientes, com média de idade de 86.57±7.86 anos. Os idosos utilizavam em média 9.13±4.15 medicamentos e mais de 90% eram polimedicados. A revisão terapêutica ainda revelou que mais de 44% dos pacientes apresentavam elevada complexidade farmacoterapêutica e 91.7% possuíam potenciais interações medicamentosas. Mais de 86% fazia uso de pelo menos um Medicamento Potencialmente Inapropriado, segundo a EU(7)-PIM List, sendo os medicamentos para o sistema nervoso central os mais utilizados. A população idosa institucionalizada possui inúmeros problemas de saúde e é tendencialmente polimedicada e com problemas relacionados à medicação. A revisão farmacoterapêutica é uma ferramenta completa que pode minimizar a ocorrência destes obstáculos, buscando melhorar a qualidade de vida dos pacientes, além de facilitar e potencializar o regime terapêutico.
- Modulation of a RBP as potential therapeutic strategy for SCA2Publication . Couto, Filipa Lopes; Nóbrega, ClévioSpinocerebellar ataxia type 2 (SCA2) is one of the nine polyglutamine disease (polyQ), characterized by an abnormally long expansion of the adenine-cytosine-guanine (CAG) trinucleotide within each respective disease-associated gene. PolyQ diseases are dominantly inherited neurodegenerative disorders in which, affected people experience a myriad of highly debilitating motor symptoms. SCA2 arises from a mutation within the coding region of the ATXN2 gene, which is translated into ataxin-2 protein bearing an abnormally long polyQ tract. This mutant protein is prone to aggregate and known to gain toxic function, which is in the basis for disrupting several molecular pathways, which ultimately lead to neuronal death, within specific brain region, including the pons and the cerebellum. Among other, these disrupted pathways might include aberrant RNA metabolism or even loss of cellular proteostasis. Unfortunately, there are no therapeutic options capable of preventing such pathways to became dysregulated, which mean that people affected by SCA2 have no therapies able to cure nor delay the disease progression, ultimately suffering a premature death. To understand whether reestablishing cellular proteostasis could, in fact mitigate SCA2-associated pathology we sought to evaluate the impact of expressing HSPA8, a crucial chaperone involved in the mechanism of proteome homeostasis. In this work we used both in vitro and in vivo approaches to assess whether HSPA8 expression was able prevent SCA2 pathology. We observed that in in vitro models of SCA2 and in an in vivo lentiviral model of this disease, HSPA8 expression was able to both prevent the number of ataxin-2 aggregates and at the same time preserve the neuronal tissue. This work suggests that the expression of HSPA8, a chaperone involved in the mechanism of cellular proteostasis, was able to mitigate SCA2-associated disease hallmarks. Therefore, HPSPA8 could represent a novel therapeutic target for SCA2.
- Engineering immunotherapies for thyroid cancerPublication . Silva, Francisca Borges Dias Monteiro da; Tavares, Álvaro; Deckers, JeroenThyroid cancer affects millions of people worldwide, particularly women. While most thyroid cancer patients have a good long-term prognosis, advanced anaplastic thyroid cancer is one of the most aggressive human malignancies. For most patients suffering from anaplastic thyroid cancer, treatment options are severely limited as tumors are resistant to conventional treatments. Anaplastic thyroid cancers typically have a highly immunosuppressive, pro-tumorigenic phenotype, characterized by high levels of immune checkpoint expression and the accumulation of tumor-associated macrophages that can account for more than 50% of the tumor volume. These immune-mediated mechanisms that facilitate rapid tumor growth represent attractive new treatment opportunities, particularly combinations of immunotherapies targeting T-cells and macrophages. The concept of innate immune memory, also known as trained immunity, has recently gained significant attention in the study of the innate immune system. It has also been shown that the induction of trained immunity can help overcome immunosuppression and can be beneficial in the context of cancer. Given this fact, we proposed trained immunity as a therapy target to overcome the characteristic immunosuppressive environment of anaplastic thyroid cancer. The research was conducted to determine the best conditions for inducing trained immunity in bone marrow-derived macrophages. We focused on commonly used primary stimuli such as β-glucan, bacillus Calmette-Guérin (BCG) vaccine, and MDP and secondary bacterial stimuli. Furthermore, tumor culture medium was used as a secondary stimulus. Through measurements of cytokine production, we detected the induction of trained immunity in bone marrow-derived macrophages trained with BCG and we observed that factors released by tumor cells can have a comparable effect as bacterial stimuli. We also examined the effect of a tumor on the immune system, looking at the cytokine production of splenocytes and bone marrow-derived macrophages of tumor-bearing mice. At the same time, we analyzed immune cell populations of tumor-bearing mice using flow cytometry, focusing on myeloid, lymphoid, and progenitor cell populations. In summary, this study provides fundamental new knowledge on how to target the bone marrow and induce trained immunity as a new treatment paradigm for anaplastic thyroid cancer.
- Combined baclofen with pregabalin administration as a potential therapy for spinal cord injuryPublication . Ferreira, Marta Raquel Lima; Salgado, António; Sousa, Nídia de; Araújo, InêsSpinal cord injury (SCI) is a neurological impairment that hampers the communication between the brain and the rest of the body, resulting in permanent loss of motor function and sensory perception. After the injury, it is initiated a cascade of biological and biochem ical processes. No effective therapy for SCI has yet been developed, however several studies in distinct areas has been developed. The comorbidities that result from a SCI decrease the patient’s quality of life N evertheless, some approved drug can help to control some of these problems, such as baclofen and pregabalin. B aclofen and pregabalin are effective in controlling spasticity and neuropathic pain in people with SCI, but recently they have also been used to as potential therapies to treat the conseque nces induced by SCI. In these studies , it was found that baclofen could improve the locomotor function and act as a neuroprotector. Regarding to pregabalin, it was observed that this drug improve motor function, but could also induce axonal regeneration, a nd protect the spinal cord after an insult. The aim of these study was to evaluat e the effect of a combine baclofen with pregabalin administration after a transected SCI in mice. To do that , it was performed an in vivo experiment during six week, where it was assessed the animals´ behaviour and histological analysis. No motor improvements were observed, however treated animals achieved weight support. On this study one of the treated group s do not exhibit allodynia after SCI, confirmed by v on Frey test. Through the spinal cord tissue analysis, it was observed that the treatment not only promotes a better neuroprotection and an axonal regeneration after SCI, but also appears to induce a more controlled systemic inflammatory response. Overall, o ur study suggests that combined administration of baclofen with pregabalin leads to a neuroprotection and a neuroregeneration after SCI, but further studies need to be done to a better understanding of these strategy.