Role of glial cells as contributors to the onset and propagation of als disease

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2017Amyotrophic lateral sclerosis (ALS) is a motor neuron (MN) disease comprehending critical neuroinflammatory pathways, where microglia and astrocytes play a crucial role. ALS onset events are largely unknown and identification of disease steps during progression and dissemination, including the possible role of exosomes, are not clarified. Several models were used to improve data validity and deepen knowledge in ALS. We identified innovative targets to regulate microglia M1 polarization, including NLRP3-inflammasome, HMGB1 alarmin and MFG-E8/lactadherin, and demonstrated the sorting of microglial microRNA(miR) 155/miR-146a into exosomes. We showed that ALS NSC-34 MNs and their exosomes are enriched in miR-124, which are captured and drive early N9-microglia M1 polarization, with later development of M1/M2 subpopulations containing increased miR-124/miR-146a/miR-155. Moving from in vitro models to the spinal cord of the SOD1G93A ALS mouse model, we observed that depressed intercellular communication and increased miR-155 were early disease events preceding the inflammatory status of the symptomatic stage. Upregulated CX3CL1-CX3CR1, connexin-43/pannexin-1 and miR-124/miR-125b/miR-146a/miR-21 emerged as candidate targets for pathological neuroinflammation. Reduced MN number, together with aberrant/reactive astrocytes showing deficient glutamate transporters and GFAP, additionally characterized such state. Differently deregulated profiles of microglia isolated from the spinal cord of 7-day old SOD1G93A mice, after short- and long-term cultures, highlighted that cells present transient phenotypes accordingly to ALS environmental progression-stimuli and ultimately acquire a less responsive phenotype to stimulation. Astrocytes isolated from these mice promoted diverse inflammatory polarized subtypes in wild-type and ALS microglia, thus accounting to microglia heterogeneous populations, while strengthened deregulated microglia-astrocyte cross-talk as part of ALS neurodegenerative mechanisms. Our studies in ALS models reveal early promising biomarkers and novel targets to control excessive neuroinflammation and spread, including exosomal microRNAs. Due to multiple microglia phenotypes induced by MNs and their exosomes, and by reactive astrocytes, in the ALS disease, differentiated and combined therapeutic approaches may be recommended.Santa Casa da Misericórdia de Lisboa, programa de Investigação Científica em Esclerose Lateral Amiotrófica, projeto ELA-2015-002, The EU Joint Programme-Neurodegenerative Disease Research (JPND), projeto JPCOFUND/003/201

E agora, Shakti? : como formular uma nova lei ambiental em uma sociedade politicamente dividida?

O estudo de caso analisa o dilema enfrentado pela presidente de uma república fictícia na elaboração do Novo Código Florestal de seu país. O enfoque está centrado na tarefa árdua de conciliar desenvolvimento econômico e proteção ambiental numa única legislação. O debate se dá em torno da trama política que envolve a tomada de decisão em momentos de crise e na necessidade de se discutir coletivamente temas sensíveis para a sociedade. Como conciliar direito ao desenvolvimento com proteção ambiental num país marcado por lutas? Como decidir em meio à pressão política de grupos divergentes? O estudo de caso centra os debates em torno da gestão do meio ambiente num contexto de expansão econômica baseada no agronegócio. O material didático pode ser utilizado em disciplinas relacionadas a Direito Ambiental, Direito Internacional Público, Direitos Humanos e Administração Pública.Número de páginas: 15 p.Administração PúblicaDireito AmbientalA nota pedagógica deste estudo de caso é de acesso restrito e exclusivo a professores e coordenadores de ensino cujos cadastros tenham sido autorizados pela coordenação da Casoteca de Gestão Pública. Se você é professor ou coordenador de ensino e deseja ter acesso à íntegra das notas pedagógicas que alguns estudos de caso possuem, solicite sua autorização de uso pleno no site da Casoteca de Gestão Pública, na opção “Cadastre-se” (http://casoteca.enap.gov.br/index.php?option=com_user&view=register&Itemid=14)

Chama da fé, luz da razão : o ideário de frei Servando Teresa de Mier no contexto das independências hispano-americanas

Dissertação (mestrado)—Universidade de Brasília, Departamento de História, 2006.Esta dissertação visa a compreensão da realocação de ideários europeus e norte-americanos para o contexto hispano-americano de formação dos estados nacionais independentes, na passagem do século XVIII para o início do século XIX, momento em que as correntes de pensamento do Iluminismo fluíam para o universo colonial, tomando-se por base os trabalhos do frei dominicano mexicano Servando Teresa de Mier (1763-1827). Por ter sido testemunha privilegiada, ao percorrer lugares de onde emanavam as ondas reformistas, como a Europa e os Estados Unidos, e após ter sofrido pena de expatriação por oferecer explicação política ao milagre guadalupano, Mier aparece como típico representante criollo, cujo protagonismo alia conceitos da modernidade ilustrada, com seus ideais racionais e científicos, ao arcabouço cultural e espiritual vivido pelo México colonial. Este trabalho discute o limite do alcance das doutrinas iluministas nas obras de frei Servando, sua contribuição para a construção do estado mexicano independente, bem como para a formação da identidade nacional, utilizando-se as categorias históricas de memória, identidade e representação para melhor compreensão deste fenômeno. Conclui-se que o ideário elaborado por frei Servando é expoente hispano-americano da corrente intelectual reformista presente na história ocidental do período analisado e fundamental para a compreensão da História das Idéias na Hispano América. ____________________________________________________________________________ ABSTRACTThis dissertation aims at comprehending the changes which took place in the way of thinking in the Hispanic-American context of formation of the national independent states, during the transition from the 18th to the 19th century, when Illuminism ideas reached the colonies, becoming the basis of Mexican priest Servando Teresa de Mier (1763-1827) ideas. For being privileged witness, wandering around places from where reforming forces were strong, like in the United States of America and Europe, and after suffering expatriation for relating political reasons to Guadalupe’s miracle, Mier appears as a typical representation of the criollos, which protagonism associates concepts of the illustrated modernity, with its rational and scientific ideals, to the Colonial Mexico cultural and spiritual background. This work discuss the boundaries of the Illuminist doctrine in the work of Servando, his contribution to the establishment of the Mexican Independent State, as well as to the formation of a national identity making use of the historical categories of memory, identity and representation to explain this phenomenon better. It is concluded that Servando´s ideal is a Hispanic-American exponent of the reformist intellectual trend present in Western history of the period which was analyzed, being essential to the comprehension of the History of Ideas in Hispanic-America. Key words: History of Ideas; Hispanic-America; Illuminism; Independent National State; memory; identity; representation; Servando Teresa de Mier

Exploring motor neuron degeneration in ALS - prevention by glycoursodeoxycholic acid and signaling to microglia

Dissertação para obtenção do Grau de Mestre em Genética Molecular e BiomedicinaAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects mainly motor neurons. Neuronal pathology involves glial cells, in particular microglia. However it is not known how these cells interact with motor neurons. This is particularly important because till now no therapy has shown efficacy in ALS treatment. Here, we aim (i) to evaluate the suitability of NSC-34, a hybrid cell line of neuroblastoma and motor neurons, as a model of ALS, (ii) to explore the reactivity of microglia to the neuronal released factors and (iii) to assess the efficacy of glycoursodeoxycholic acid (GUDCA), which already has shown beneficial effects in several neurodevelopmental and neurodegenerative diseases. For that, we used NSC-34 cells transfected with human superoxide dismutase 1 (hSOD1), either wild type or mutated in G93A and the microglial N9 cell line. We observed mitochondrial dysfunction, energy impairment, NO production and metalloproteinase-9 activation, with consequent apoptosis in NSC-34/hSOD1G93A cells after 4 days of differentiation, in comparison to NSC-34/hSOD1wt cells. In addition, we established GUDCA as an anti-apoptotic and anti-inflammatory agent, able to prevent all the above mentioned features. Finally, released neuronal factors induced N9 microglia apoptosis and decreased their phagocytic ability. Overall, our results emphasize NSC-34/hSOD1G93A cells as a good ALS model, highlight GUDCA as having beneficial effects and point to microglia neuroprotective failure as a determinant mechanism of ALS pathogenesis.Fundação para a Ciência e Tecnologia - PTDC/SAU-FAR/118787/2010, entitled “Are microglia a new potential target in amyotrophic lateral sclerosis (ALS)?” to DB and PEst- OE/SAU/UI4013/2011 to iMed.U

A deep learning solution for real-time human motion decoding in smart walkers

Dissertação de mestrado integrado em Engenharia Biomédica (especialização em Eletrónica Médica)The treatment of gait impairments has increasingly relied on rehabilitation therapies which benefit from the use of smart walkers. These walkers still lack advanced and seamless Human-Robot Interaction, which intuitively understands the intentions of human motion, empowering the user’s recovery state and autonomy, while reducing the physician’s effort. This dissertation proposes the development of a deep learning solution to tackle the human motion decoding problematic in smart walkers, using only lower body vision information from a camera stream, mounted on the WALKit Smart Walker, a smart walker prototype for rehabilitation purposes. Different deep learning frameworks were designed for early human motion recognition and detec tion. A custom acquisition method, including a smart walker’s automatic driving algorithm and labelling procedure, was also designed to enable further training and evaluation of the proposed frameworks. Facing a 4-class (stop, walk, turn right/left) classification problem, a deep learning convolutional model with an attention mechanism achieved the best results: an offline f1-score of 99.61%, an online calibrated instantaneous precision higher than 97% and a human-centred focus slightly higher than 30%. Promising results were attained for early human motion detection, with enhancements in the focus of the proposed architectures. However, further improvements are still needed to achieve a more reliable solution for integration in a smart walker’s control strategy, based in the human motion intentions.O tratamento de distúrbios da marcha tem apostado cada vez mais em terapias de reabilitação que beneficiam do uso de andarilhos inteligentes. Estes ainda carecem de uma Interação Humano-Robô avançada e eficaz, capaz de entender, intuitivamente, as intenções do movimento humano, fortalecendo a recuperação autónoma do paciente e reduzindo o esforço médico. Esta dissertação propõe o desenvolvimento de uma solução de aprendizagem para o problema de descodificação de movimento humano em andarilhos inteligentes, usando apenas vídeos recolhidos pelo WALKit Smart Walker, um protótipo de andarilho inteligente usado para reabilitação. Foram desenvolvidos algoritmos de aprendizagem para o reconhecimento e detecção precoces de movimento humano. Um método de aquisição personalizado, incluindo um algoritmo de condução e labelização automatizados, foi projetado para permitir o conseguinte treino e avaliação dos algoritmos propostos. Perante a classificação de 4 ações (parar, andar, virar à direita/esquerda), um modelo convolucional com um mecanismo de atenção alcançou os melhores resultados: f1-score offline de 99,61%, precisão instantânea calibrada online de superior a 97 % e um foco centrado no ser humano ligeiramente superior a 30%. Com esta dissertação alcançaram-se resultados promissores para a detecção precoce de movimento humano, com aprimoramentos no foco dos algoritmos propostos. No entanto, ainda são necessárias melhorias adicionais para alcançar uma solução mais robusta para a integração na estratégia de controlo de um andarilho inteligente, com base nas intenções de movimento do utilizador

The Importance on Self-Expression Through Clothing and Fashion: A view on Digital Identity and Digital Fashion

As a vast theme with a variety of studies in different scientific areas, like philosophy and sociology, Identity poses as a multi-disciplinary subject (Kawamura, 2005). This way, this investigation had in consideration both these fields of study, connecting them. It began by analysing the role that Fashion portraits in Identity and at what point do they influence each other, as well as the importance of self-expression through clothing. The investigation’s focus was the development and impact that the globalization phenomena and the digital era have, how they influence and mould users and their perception, and what potential Fashion has in constructing a personal Identity, in envoirnments such as Virtual Worlds and the Metaverse. Moreover, Digital Fashion is also introduced as to understand this relationship between Fashion and the virtual world and what it might bring into the universe of Identity, and more specifically Digital Identity. This research used types of method designs to pursue its goals. A qualitative method, the literature review, where studies and author’s work were revised, to explore the themes and form hypothesis, and a quantitative one, a public survey where those hypotheses were determined either true or untrue. The conclusions show that technology is at its peak with increasing usage and engagement, and although there isn’t clear information to deduct Digital Fashion will become a major success within the Fashion industry, it is possible that it could mean a lot to freedom of wear.Sendo um vasto tema com uma larga variedade em diferentes áreas de estudo, como a filosofia e sociologia, a Identidade apresenta-se como um tema multidisciplinar (Kawamura, 2005). Desta forma, esta investigação teve em consideração ambos estes campos de estudo, conectando-os. Começando por analisar o papel que a Moda tem na Identidade e até que ponto se influenciam mutuamente, bem como qual a importância da auto-expressão através do vestuário. O foco desta investigação foi o desenvolvimento e impacto que o fenómeno da globalização e a era digital têm, como influenciam e moldam os seus utilizadores e a sua perceção, e o potencial que a Moda pode vir a ter na construção de uma Identidade pessoal, em espaços como Mundos Virtuais e o Metaverso. Assim, a Moda Digital é também introduzida de forma a perceber esta relação entre Moda e o mundo virtual e o que poderá vir a trazer ao universo da Identidade, mais especificamente à Identidade digital. Este estudo utilizou duas formas distintas de design de método para atingir os seus objetivos. Um método qualitativo, a revisão de literatura, onde estudos e autores são revisados, de modo a explorar temas e formar hipóteses, e mais tarde um método quantitativo, um questionário público onde essas hipóteses serão determinadas verdadeiras ou falsas. As conclusões mostram que a tecnologia está no seu pico com utilização e engagement a crescer, e embora não tenha havido informação necessária para deduzir que a Moda Digital poderá ter um papel maior na indústria da Moda no futuro, é possível que possa significar mais liberdade de vestir

Mesenchymal stromal cell (MSC)- based control of angiogenesis and inflammation in cartilage formation

The overall goal of my PhD studies was to analyze how the regulation of angiogenesis, a key factor that plays very important roles in tissue repair, could ultimately influence cartilage formation by mesenchymal stromal cells (MSC) and by differentiated nasal chondrocytes (NC) as a proof of concept. Due to their multipotency and great self-renewal capacity, MSC is a highly attractive cell source for cartilage formation and regeneration. MSC have immunomodulatory properties and are in direct contact with inflammatory cells (monocytes) in the cartilage repair environment. By directly regulating the inflammatory response of monocytes and by regulating their phenotype, MSC could significantly benefit from their ability to control inflammatory events during cartilage repair processes. Thus, we also assessed whether MSC could modulate the properties and the phenotype of monocytes in order for monocytes to actively aid MSC in cartilage repair. In chapter 1, we sought a proof of principle to confirm that the blocking of angiogenesis does indeed improve cartilage formation when using genetically-modified nasal chondrocytes (NCs) or antiangiogenic peptides associated with NCs. For this purpose, NCs were genetically-modified to express mouse soluble VEGF receptor-2 (sFlk-1) or were associated with an antiangiogenic peptide in order to have their chondrogenic capacity assessed in vitro and in vivo. Improved cartilage regeneration could be observed after in vivo implantation of NCs in an ectopic nude mouse model. Whereas the anti-angiogenic approaches did not improve chondrogenesis in vitro, frank chondrogenesis occured in vivo only in the constructs generated by NCs expressing sFlk-1 or treated with the peptide. Blood vessel ingrowth was significantly hampered in the anti-angiogenic experimental groups when compared with naïve NCs, which correlated with chondrogenis improvement. Strikingly, the anti-angiogenic effect was even more evident when NC donors with low chondrogenic capacity were used, and no-preculture with chondrogenic soluble factors was performed prior to in vitro implantation of the constructs. In chapter 2, we investigated how angiogenesis control by genetically-modified bone marrow-derived MSC could augment their chondrogenic potential in vivo. These MSC were genetically modified to release sFlk-1, the soluble version of VEGF receptor 2 (VEGFR2), which acted as a decoy receptor and could sequester VEGF from the immediate surroundings of MSC. Importantly, no external morphogens were supplemented in order for MSC chondrogenic differentiation to occur. Moreover, the in vivo chondrogenic capacity of sFlk-1-releasing MSC was assessed up to 12 weeks by an ectopic nude mouse model, in which collagen sponges seeded with MSC were implanted subcutaneously. Angiogenesis, as analyzed by blood vessel invasion, was markedly reduced in the constructs seeded with sFlk-1-releasing MSC. Frank and stable cartilage formation was only achieved once VEGF was blocked. In chapter 3, we aimed at investigating whether MSC can instruct monocytes to acquire traits of mesenchymal progenitors or tissue repair macrophages when in direct or indirect contact with the latter. Thus, MSC could instruct monocytes to assist in the tissue-repairing process. MSC and monocytes were cocultured either in 3D collagen sponges or in transwells for a period of five consecutive days. We demonstrated that MSC instruct monocytes into acquiring a hybrid macrophage-mesenchymal phenotype, which varies greatly depending on which kind of contact with MSC the monocytes were exposed to (whether direct cell-to-cell contact or through the release of soluble factors by MSC). However, future studies will be needed to elucidate the mechanism in which MSC could instruct monocytes into differentiating into this hybrid state, and how one could better control this process in order for monocytes to optimally aid MSC in cartilage repair