Дисертації з теми "Balance inflammatoire"

Le traitement des patients atteints de Maladies Inflammatoires Chroniques de l'Intestin (MICI) représente un enjeu d'avenir. La réponse inflammatoire dans l'intestin impliquent la libération de plusieurs médiateurs, comme les protéases. Dans cette thèse, nous avons montré qu'il y avait un déséquilibre entre ces protéases et leurs inhibiteurs au cours des MICI. D'une part, les tissus coliques de ces patients libéraient davantage d'activité protéolytique en comparaison aux tissus sains. D'autre part, les patients atteints de MICI exprimaient dans la muqueuse intestinale une quantité réduite d'ARN messager de l'élafine, un inhibiteur de protéase. Nous avons donc émis l'hypothèse que l'inflammation peut être réduite en rééquilibrant cette balance grâce à l'administration d'élafine. Nous avons utilisé des souris transgéniques pour l'expression de l'élafine de façon constitutive. Puis, nous avons utilisé un virus et des bactéries lactiques pour exprimer de façon transitoire cette molécule dans le côlon. En parallèle, nous avons développé des approches in vitro pour connaître le rôle de l'élafine dans la physiologie des cellules épithéliales de l'intestin. Grâce à ces différentes approches, nous avons montré que l'élafine réduisait : les symptômes de la maladie dans différents modèles, la libération de cytokines pro-inflammatoire, l'infiltration des cellules immunitaire et restaurait l'homéostasie de l'épithélium intestinal au cours de l'inflammation. Ces résultats nous ont permis de proposer que l'utilisation d'inhibiteurs de protéases dans l'intestin au cours de l'inflammation constitue une nouvelle piste thérapeutique contre les MICI
Treatment of Inflammatory Bowel Disease (IBD) represents a major medical challenge. Inflammatory processes in the gut are induced by several cells and mediators. Among them, serine proteases are mediators involved in many pathways leading to inflammation in the gut. During this thesis, we have shown that serine proteases and their inhibitors were dysregulated during IBD. On one hand, colonic biopsies from IBD patients released higher amount of proteolytic activity than healthy controls did. On the other hand, the expression of elafin mRNA (i. E. A protease inhibitor) was downregulated in the mucosa of patients suffering from IBD. We have hypothesized that gut inflammation could be reduced by re-equilibrating that balance in the gut, using elafin inhibitor. We have developed several in vivo approaches to evaluate the therapeutic properties of elafin. We used transgenic mice expressing elafin constitutively, we have used recombinant viral vectors and recombinant lactic acid bacteria to express transiently elafin in the gut during colitis. We have also evaluated in vitro the role of elafin in the physiology of human intestinal epithelial cells. Using those different approaches, we have demonstrated that elafin reduced the clinical score of colitis in different models in mice, reduced the release of pro-inflammatory cytokines, reduced immune cell infiltration and also restored epithelium homeostasis during inflammation. Those results led us to think that protease inhibitors have a promising therapeutic potential for the treatment of IBD

Cette thèse, consacrée aux myopathies inflammatoires, démontre le rôle dans les maladies auto-immunes des Toll-like récepteurs (TLRs), véritable passerelle entre immunité innée et adaptative, et plus spécifiquement dans le muscle, le rôle fondamental de la cellule musculaire elle-même. Après une présentation globale des myopathies inflammatoires et des différents aspects immunopathologiques, la réponse immunitaire adaptative est abordée en rapportant notamment dans le muscle des myopathies inflammatoires une accumulation de cellules dendritiques matures, et la présence des lymphocytes Th1 et Th17, avec un profil prépondérant Th1. L'implication de l'immunité innée est démontrée in vivo par l'expression musculaire des TLR3 et 7, et des C-type lectin récepteurs, spécifique des myopathies inflammatoires. In vitro, l'activation de la voie TLR3 induit la production par les cellules musculaires d'IL6, de la βchémokine CCL20, contribuant au recrutement et à la différentiation des cellules dendritiques et lymphocytes T, et de l'IFNβ qui participe à la surexpression des antigènes HLA de classe I. Les mécanismes de régulation impliquent une balance cytokinique Th1 et Th17. Finalement, l'importance des précurseurs musculaires immatures est soulignée. Contrairement au tissu musculaire normal, une surexpression des antigènes HLA de classe I, des TLRs, des auto-antigènes et de l'IFNβ, par les précurseurs musculaires immatures, est caractéristique des myopathies inflammatoires. Le rôle central de ces cellules musculaires immatures à potentiel de régénération pourrait expliquer un défaut de réparation associé au processus auto-immun de destruction musculaire.

La protéase C1s est un élément central dans l’initiation de la voie classique du système du complément. Elle était auparavant considérée comme ciblant exclusivement les protéines C2 et C4 dans cette cascade protéolytique. Des découvertes récentes ont cependant mis en lumière la présence de C1s libre constitutivement active dans certaines pathologies, suggérant un rôle plus large de cette protéase au-delà de l'activation du complément. Parmi les cibles non-canoniques identifiées de C1s figure la protéine HMGB1, initialement décrite comme une protéine nucléaire impliquéedans la condensation de la chromatine et l'expression des gènes. Des études récentes ont démontré que HMGB1 peut également être localisée dans différents compartiments cellulaires et qu'elle joue un rôle crucial dans l'inflammation lorsqu'elle est libérée dans le milieu extracellulaire. Ce projet de thèse avait pour objectif principal d'élucider le rôle du clivage de HMGB1 par C1s dans la modulation de la réponse inflammatoire. Nos travaux ont démontré que les fragments de digestion de HMGB1 possèdent des effets distincts de la protéine entière sur l'activation du complément et laréponse cytokinique des macrophages. Nous avons notamment confirmé que la protéine entière active la voie classique du complément lorsqu’elle est fixée à une surface et qu’elle favorise la polarisation M1 des macrophages en réponse aux LPS. En revanche, le fragment f2 est capable d'activer la voie classique du complément même lorsqu’il est en solution, tandis que le fragment f3 inhibe la sécrétion de cytokines pro-inflammatoires dans les études cellulaires. De plus, nous avons exploré l'impact de l'état d'oxydo-réduction des cystéines sur les effets de HMGB1 et de ses fragmentsen utilisant des mutants mimétiques. La digestion de HMGB1 est restreinte lorsque la protéine est sous forme disulfure, suggérant un rôle important du pont disulfure dans l’accès aux sites de digestion par C1s. Les formes redox de la protéine entière ne semblent pas affecter sa capacité à activer le complément, tandis que le fragment f2 oxydé pourrait perdre sa capacité d'activation en solution. Ces résultats révèlent que le clivage de HMGB1 par C1s agit comme un chronomètre de l’inflammation, orchestrant la réponse inflammatoire via la transition d’une phase d’amplification pro-inflammatoireà une phase de résolution. Ces découvertes ouvrent de nouvelles perspectives pour la compréhension des mécanismes complexes de l'inflammation et le développement de thérapies pour le traitement de pathologies inflammatoires
C1s protease is a central component in the initiation of the classical pathway of the complement system. It was originally believed to exclusively target proteins C2 and C4 in this proteolytic cascade. However, recent discoveries have highlighted the presence of constitutively active free C1s in certain pathologies, suggesting a broader role for this protease beyond complement activation. Among the non-canonical targets identified for C1s is the HMGB1 protein, initially described as a nuclear protein involved in chromatin condensation and gene expression.Recent studies have shown that HMGB1 can also be localized in different cellular compartments and plays a crucial role in inflammation when released into the extracellular environment. The main objective of this thesis project was to elucidate the role of C1s cleavage of HMGB1 in modulating the inflammatory response. Our work has shown that HMGB1 digestion fragments have distinct effects from the whole protein on complement activation and macrophage cytokine responses.In particular, we confirmed that the whole protein activates the classical complement pathway when bound to a surface and promotes M1 macrophage polarization in response to LPS. In contrast, fragment f2 is capable of activating the classical complement pathway, even when in solution, while fragment f3 inhibits the secretion of pro-inflammatory cytokines in cell studies. In addition, we explored the impact of cysteine redox state on the effects of HMGB1 and its fragments using mimetic mutants. HMGB1 digestion is restricted when the protein is in disulfide form, suggesting an important role of the disulfide bridge in access to the C1s digestion site. The redox forms of the whole protein do notappear to affect its ability to activate complement, while oxidized fragment f2 may lose its ability to activate it in solution. These results reveal that C1s cleavage of HMGB1 acts as an inflammation timer, orchestrating the inflammatory response through the transition from a pro-inflammatory amplification phase to a resolution phase. These findings open new perspectives for understanding the complex mechanisms of inflammation and the development of therapies for the treatment of inflammatory diseases

The aim of this study was to investigate the balance of pro- and anti-inflammatory cytokines in patients undergoing cardiac and hip fracture surgery in relation to the development of postoperative acute kidney injury (AKI). 400 patients undergoing elective cardiac surgery and 237 patients undergoing emergency hip fracture surgery were recruited. For each patient blood and urine samples were analysed preoperatively and postoperatively to determine how cytokine balance alters in those patients who develop postoperative AKI. In both patients groups, the balance of pro- and anti-inflammatory cytokines in the blood was maintained, regardless of whether or not the patient developed postoperative AKI. This demonstrates that the underlying process responsible for AKI in these patients was not located within the systemic circulation. In the cardiac surgery patients who developed postoperative AKI, it was found that there was a local imbalance of pro- and anti-inflammatory cytokines in the urine, indicating that the pathological cause of AKI is located within the kidney. What was observed was an inadequate anti-inflammatory response to the pro-inflammatory insult of surgery, thus, leaving the kidney vulnerable to the pro- inflammatory onslaught and, subsequently renal injury. A different picture was seen in the hip fracture patients, where elevated pro- and anti-inflammatory mediators were observed preoperatively due to the trauma that occurred in sustaining the hip fracture. This showed an important finding that the hip fracture patients had been undergone cytokine preconditioning as a result of trauma, prior to surgery. In this group the cytokines behaved quite differently to the cardiac surgery group. In the absence of preconditioning, postoperative AKI is associated with an inadequate anti- inflammatory cytokine response to the pro-inflammatory rise associated with surgery. However, when preconditioning is present, the cytokine levels must be analysed more carefully with the clinical context in mind.

Les rhumatismes inflammatoires juvéniles sont des maladies chroniques débutant avant l’âge de 16 ans. Comprennent des pathologies classées dans un continuum, allant de la dérégulation de l’immunité innée à la dérégulation de l‘immunité adaptative. L’arthrite juvénile idiopathique (AJI) reste la plus fréquemment diagnostiqué. Les options thérapeutiques se sont élargies à partir des années 2000 avec le développement des thérapies ciblées, les biothérapies, associés aux traitements standard utilisés en rhumatologie pédiatrique (ex : anti-inflammatoires non stéroïdiens, corticostéroïdes, méthotrexate, et autres immunosuppresseurs). L’objectif de ce travail de thèse était d’estimer la relation bénéfice-risque des biothérapies utilisés dans les rhumatismes inflammatoires juvéniles à partir des essais cliniques randomisés (ECR) et d’explorer la tolérance à long cours à partir des essais observationnels. Premièrement, en utilisant une approche méta-analitique, les données des ECR en double aveugle contre placebo ou en ouvert dans l’AJI ont été analysées pour modéliser la relation bénéfice-risque des biothérapies avec le bénéfice net. Pour cela, l’efficacité clinique, mesuré par un score composite clinique et biologique (ACRped30), a été confronté à la tolérance clinique pendant la phase randomisée des ECR. Le critère de tolérance était la survenue d’un évènement indésirable (EI) grave (EIG). La balance bénéfice-risque reste favorable pour les biothérapies. Cependant, ces résultats sont limités par le suivi de faible durée, qui peut sous-estimer l’incidence des EI. Deuxièmement, nous avons conduit une étude observationnelle pour étudier la tolérance à moyen et long-terme des biothérapies utilisant les EI et les EIG décrits dans une base de données multicentrique rétrospective. La sécurité globale des biothérapies a été acceptable chez les enfants atteints de rhumatismes inflammatoires. Nous avons observé une variation des EIG au cours du temps et que la prescription concomitante des immunosuppresseurs a représenté un risque indépendant pour la survenue des EI. Afin d’explorer ces éléments et connaître la tolérance au long-terme, une méta-analyse des études observationnelles a été fait. Nous avons utilisé les EIG pour étudier précisément la tolérance à court et à long terme
Juvenile inflammatory rheumatism is a chronic disease that begins before the age of 16. Includes conditions classified along a continuum, ranging from the deregulation of innate immunity to the deregulation of adaptive immunity. Juvenile idiopathic arthritis (JIA) remains the most frequently diagnosed disease. Therapeutic options have expanded since the 2000s with the development of targeted therapies: biological agents (BAs). They can be combined with standard treatments used in paediatric rheumatology (e.g. non-steroidal anti-inflammatory drugs, corticosteroids, methotrexate, and other immunosuppressive drugs). The objective of the work of this thesis was to model the benefit-risk balance of BAs used in JIA from randomized clinical trials (RCTs) and to explore long-term tolerance from observational trials. First, using a meta-analytical approach, the data from double-blind, placebo-controlled or open RCTs in JIA were analysed for modelling the benefit-risk balance of BAs. For this purpose, the efficacy measured by a composite clinical and biological score (ACRped30), was compared with clinical safety during the randomized phase of RCTs. Safety criterion was the occurrence of adverse events (AEs). The risk-benefit balance remains favourable for biotherapies. However, these results are limited by the short follow-up period, which may underestimate the incidence of AEs. Second, we conducted an observational study to investigate the medium- and long-term safety of biotherapies using AEs and serious AEs described in a retrospective multicentre database. The overall safety of biotherapies has been acceptable in children with inflammatory rheumatic diseases. We observed a variation in the SAEs over time and that the concomitant prescription of immunosuppressants represented an independent risk for the occurrence of AEs. In order to explore these elements and long-term safety, a meta-analysis of observational studies was conducted. We used the SAEs to study precisely the short and long-term tolerance

L'endothélium artériel pulmonaire joue un rôle déterminant dans la régulation du tonus vasomoteur en libérant des substances vasodilatatrices et vasoconstrictrices. Toute perturbation des fonctions endothéliales entraîne une perte de l'équilibre physiologique très finement régulé entre réponse vasoconstrictrice et vasodilatatrice, avec perte conjointe du contrôle de la prolifération des cellules musculaires lisses vasculaires et de l'effet antiagrégant plaquettaire. Ces modifications conduisent à un remodelage du lit vasculaire pulmonaire avec une augmentation des résistances vasculaires parfois à l'origine d'une hypertension pulmonaire (HTP) irréversible. Le rôle de l'hypoxémie n'y est pas exclusif. Le réseau bronchique est soumis quant à lui à des agressions physiques par les particules inhalées. Celles-ci peuvent entraîner un remodelage bronchique et une perturbation du tonus bronchique et de la broncho-réactivité, dont les médiateurs peuvent être en partie partagés par la dysfonction vasculaire.Le but de ce travail était d'évaluer dans un premier temps l'existence d'une dysfonction endothéliale, en tant que précurseur potentiel d'une HTP, dans deux modèles de bronchopathies chroniques à des stades opposés de la sévérité de l'atteinte respiratoire : la mucoviscidose terminale et le tabagisme avec peu ou pas d'impact sur la fonction respiratoire.Nous avons ainsi montré à partir d'explants pulmonaires qu'une dysfonction endothéliale était fréquente au cours de la mucoviscidose au stade pré-greffe et qu'elle était au-moins en partie liée à une surexpression vasculaire pulmonaire de l'ET-1 et de ses récepteurs ET-A.Nous avons montré par ailleurs que 25% environ des sujets fumeurs à fonction respiratoire normale ou peu altérée présentaient également une telle dysfonction. Nous en avons étudié ici deux voies physiopathologiques potentielles, celle de l'ET-1 et celle qui gouverne la synthèse du NO : la balance NO Synthases (NOS)/arginases. Nous avons mis en évidence une surexpression des récepteurs ET-A de l'ET-1 chez les sujets avec dysfonction ainsi qu'une corrélation inverse entre cette expression et la réponse vasoactive à l'Acétylcholine (Ach). Par ailleurs, si le NO a entre autres un effet anti-prolifératif sur les cellules musculaires lisses, la voie des arginases - système enzymatique compétitif avec les NOS - mène quant à elle à la réparation et au remodelage. Nous avons étudié l'expression vasculaire de ces différentes enzymes ainsi que l'effet pharmacologique d'inhibiteurs des NOS ou des arginases sur la réponse vasoactive à l'Ach. Nous avons montré qu'il n'existait pas de déficit d'expression des NOS et qu'il ne semblait pas y avoir d'effet délétère des arginases dans la dysfonction endothéliale dans ce modèle.Parallèlement aux mécanismes sous-tendant le remodelage vasculaire au cours du tabagisme nous avons recherché l'existence d'une « dysfonction » bronchique chez ces fumeurs, qui pourrait précéder le remodelage bronchique qui les caractérise. La présence d'une hyperréactivité bronchique est un marqueur prédictif de remodelage des voies aériennes et de l'obstruction bronchique qui en découle. Le rôle de la balance NOS/arginases dans le contrôle du tonus bronchique est encore méconnu. Notre premier objectif était d'évaluer l'expression de cette balance enzymatique dans le tissu bronchique de ces patients et le second d'étudier les effets de l'inhibition des NOS et des arginases sur la réponse bronchoconstrictrice à l'Ach. Nous avons montré qu'une augmentation d'expression bronchique de la NOS 2 chez les fumeurs était impliquée dans la régulation du tonus bronchique et dans l'obstruction bronchique, tandis qu'une augmentation de l'activité des arginases était impliquée dans la sensibilité bronchique
Pulmonary arteriel endothelium has a key role in the regulation of vascular tone by the release of dilating and constrictive mediators. Impairment of endothlium functions leads to a loss of the physiological equilibrium between vasoconstriction and vasodilation, together with the loss of vascular smooth muscle cells (SMC) proliferation. These alterations induce pulmonary vascular remodeling and elevation of vascular resistance which can lead to an irreversible pulmonary hypertension (PH). The role of hypoxemia is not exclusive. Airways are exposed to physical aggressions by inhaled particles, which can lead to bronchial remodeling and impaired bronchial tone and reactivity, the mediators of which can be partly shared by endothelial dysfunction.Our goal was to evaluate the existence of endothelial dysfunction as a precursor of PH in 2 models of chronic bronchopathy of opposite stages of disease severity: end-stage cystic fibrosis (CF) and tobacco smoking with or without impaired lung function. We showed that in end-stage CF pulmonary explants, endothelial dysfunction is frequent and that it was at least partly due to a vascular upregulation of the endothelin (ET)-1 pathway.Furthermore, approximately ¼ of smokers with normal or poorly impaired lung function also displayed a similar endothelial dysfunction. We studied therein 2 potential physiopathological pathways, that of ET-1 and that which governs nitric oxide (NO) synthesis: the NO synthases (NOS)/arginases balance. We showed an upregulation of ET-A receptor expression and an inverse correlation between this expression and the vasoactive response to acetylcholine (Ach). If NO has an anti mitogenic effect on SMC, the arginases pathway-competitive with the NOS- leads to tissue repair and remodeling.We studied the vascular expression of these enzymes and the pharmacological effect of NOS and arginases inhibitors on response to Ach. We showed that the expression of NOS was not deficient and that arginases did not seem to have a deleterious effect on endothelial function in this model.Concomitantly to these mechanisms leading to vascular remodeling, wr searched for a bronchial dysfunction in smokers which could antecede bronchial remodeling, a well known feature of tobacco smoking. Bronchial hyperresponsiveness is a predictive marker of airway remodeling and subsequent bronchial obstruction. The role of the NOS/arginases pathway in the control of bronchial tone is still unknown. We evaluated the bronchial expression of the NOS/arginases balance in smokers and the effects of NOS and arginases inhibitors on bronchoconstrictive response to Ach. We found that an upregulation of NOS2 expression in COPD patients is involved in airway tone regulation and functional airflow limitation, whereas increased arginase activity is involved in airway sensitivity

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2015-07-27T14:45:59Z No. of bitstreams: 1 Fernando Oliveira Costa.pdf: 1199928 bytes, checksum: f721f323b7b101614061ea44e7dd6cb3 (MD5)
Made available in DSpace on 2015-07-27T14:45:59Z (GMT). No. of bitstreams: 1 Fernando Oliveira Costa.pdf: 1199928 bytes, checksum: f721f323b7b101614061ea44e7dd6cb3 (MD5) Previous issue date: 2014-02-21
The metabolic syndrome (MetS) consists of a combination of conditions that tend to cluster together, and increase the risk of type 2 diabetes and cardiovascular disease. The components of the metabolic syndrome include central (abdominal) obesity, elevated fasting glucose, dyslipidemia (abnormally high triglycerides and low high-density lipoprotein cholesterol), and elevated blood pressure. MetS is also associated with proinflammatory and prothrombotic states, non-alcoholic liver steatosis, obstructive sleep apnea and reproduction disorders. Although a common unifying physiopathological mechanism is not known, central obesity and inflammation play a major role in MetS and upon each of its components. The MetS has reached epidemic proportions and to date there are no proven pharmacological interventions that simultaneously target all of the components of this syndrome. Inflammation plays an important role in the pathogenesis of the MetS. Recently, it was discovered that inflammation can be regulated by neural, cholinergic mechanisms and a cholinergic drug, the acetylcholinesterase inhibitor galantamine suppresses abnormal inflammation and alleviates MetS pathologies in rodents. The fact that galantamine is an approved drug, used to treat patients with Alzheimer´s disease with a known safety profile, will facilitate its clinical application in another situations. We hypothesize that treatment of subjects with the MetS with galantamine will result in alleviation in the MetS clinical conditions and inflammation. The objective of our study was to initiate an investigation on the safety profile of galantamine in MetS patients, with special attention on autonomic, hemodynamic and cognitive parameters. A randomized, double-blind, prospective study evaluated clinical, autonomic, hemodynamic and cognitive variables of patients with MetS in two moments: before treatment (basal state) and after 28 days of treatment with galantamine 8 mg daily. There was a statistical tendency in reducing systolic blood pressure in the HRV with Finometer® in patients under galantamine (124.4 ± 4 vs 119.7 ± 3.7 mmHg, basal and 28 days values, respectively) and also a reduction in diastolic blood pressure (72.5 ± 1.3 vs 67.2 ± 1.7 mmHg, basal and 28 days values, respectively). Paradoxically, an increase in the sympathetic modulation of the heart was observed with the HRV study measuring the LF (nu) value (46.2 ± 3.8 vs 57.1 ± 3.4 basal and 28 days, respectively) and a decrease in the parasympathetic modulation HF (nu) value (53.8 ± 3.8 vs 43.0 ± 3.4, basal and 28 days, respectively). We did not observe any significant change in cognitive domains. Our conclusion is that treatment with galantamine 8 mg exhibits a safe clinical profile and can be used in MetS patients.
A síndrome metabólica consiste na combinação de condições agrupadas e aumentam o risco para diabetes tipo 2 e doença cardiovascular. Seus componentes incluem obesidade central, níveis aumentados de glicose, dislipidemia caracterizada por aumento de triglicérides e baixos níveis de HDL e aumento da pressão arterial. Também está associada a um estado proinflamatório, a um estado protrombótico, a esteatose hepática não-alcoólica, apnéia obstrutiva do sono e a desordens reprodutivas. Apesar da não determinação de um mecanismo fisiopatológico unificador, obesidade central e inflamação parecem ser centrais na síndrome metabólica e nos seus componentes individuais. A síndrome metabólica tem alcançado proporções epidêmicas universais e até o presente não há intervenção farmacológica comprovada que atue simultaneamente em todos os seus componentes. Sabe-se hoje que o processo inflamatório tem um papel importante na patogenia da síndrome. Recentemente foi evidenciado que a inflamação pode ser regulada por mecanismos neurais colinérgicos, e que a galantamina, um inibidor da acetilcolinesterase, suprime a inflamação e atua nos componentes da síndrome diminuindo a patogenia em roedores. O fato de a galantamina ser uma droga já aprovada e de perfil seguro em portadores de demência facilita seu uso em outras situações clínicas. Considerando a hipótese de que a galantamina causará melhora da inflamação e dos outros distúrbios relacionados, o objetivo deste estudo foi iniciar a investigação sobre o perfil de segurança da galantamina em pacientes com síndrome metabólica, em especial, em parâmetros hemodinâmicos, autonômicos e de cognição. Realizamos um estudo prospectivo, duplo-cego e randomizado, que avaliou os dados clínicos e os parâmetros descritos, no momento basal e após 28 dias de uso de galantanima (8mg por dia), em portadores de síndrome metabólica. Houve uma tendência à redução da PAS, avaliada batimento-a-batimento com o Finometer no grupo que usou galantamina (124,4 ± 4 vs 119,7 ± 3,7 mmHg, respectivamente basal e após 28 dias de uso, p=0,04), o mesmo ocorrendo com a PAD (72,5 ± 1,3 vs 67,2 ± 1,7, p=0,005), respectivamente basal e após 28 dias de uso). De forma paradoxal, ocorreu um aumento da atividade simpática na modulação autonômica para o coração, avaliada por meio do estudo da variabilidade da freqüência cardíaca como atestado por um valor LF (nu) (46,2 ± 3,8 vs 57,1 ± 3,4 , p=0,0005)), e redução da modulação parassimpática, representada pelo valor do HF (nu) (53,8 ± 3,8 vs 43,0 ± 3,4, p=0,0005) respectivamente basal e após 28 dias de uso. Não observamos alterações significativas nos testes que avaliam o domínio cognitivo dos indivíduos. Concluímos que a dose utilizada de galantamina tem um perfil de segurança clínica que permite expandir seu uso em pacientes portadores de síndrome metabólica.

O estudo consistiu de dois experimentos com o objetivo de avaliar os efeitos da acidose ruminal e restrição nutricional na ocorrência de processos inflamatórios nos animais e relacionar com as alterações na produção e composição do leite. Foram utilizadas doze vacas Holandês e Mestiças Holandês Jersey. Experimento 1: Na fase de adaptação, os animais receberam uma dieta formulada para atender 100% das necessidades nutricionais de energia e proteína. Na indução foi administrada uma dieta com restrição de 50% das necessidades em energia e proteína. Na recuperação os animais receberam uma das três dietas experimentais, para recuperar a estabilidade do leite: (1) suprimento somente de energia; (2) suprimento somente de proteína; (3) suprimento de energia e proteína. A restrição nutricional em energia e/ou proteína afeta negativamente a produção de leite, o peso vivo e o escore de condição corporal. Além de reduzir a eficiência de utilização de proteína da dieta e provocar uma maior instabilidade do leite ao teste do álcool. Entretanto, não altera o perfil sanguíneo e metabólico. Experimento 2: Os animais foram divididos em dois grupos (1) controle e (2) acidose. O delineamento experimental foi reversível simples com dois tratamentos e dois períodos experimentais. Foram analisados parâmetros referente às características físico-quimica, saúde da glândula mamária, medidas fisiológica, perfil metabólico e parâmetros sanguíneos. A indução da acidose ruminal subaguda (SARA) causou redução da produção e estabilidade do leite ao teste do álcool, pH urinário, pH fecal, pH ruminal. Entretanto, a indução a SARA não alterou os parâmetros sanguíneos avaliados. A SARA altera as características físico-químicas do leite, sem influenciar nas concentrações proteínas de fase aguda, caracterizando uma resposta inflamatória. A SARA pode acometer os animais sem apresentar mudanças no perfil sanguíneo dos mesmos.
The study consisted of two experiments with the aim of evaluating the effects of ruminal acidosis and nutritional restriction on the occurrence of inflammatory processes in animals and correlate with changes in milk production and composition. Twelve Holstein and cross bred Holstein and Jersey cows were used. In the first study, in the adaptation phase, the animals received a diet formulated to supply 100% of the nutritional needs of energy and protein. In the induction, a diet composed by 50% restriction of energy and protein requirements was administered. In the recuperation, the animals received one of the three experimental diets to recover milk stability: (1) only energy supply; (2) supply only of protein; (3) supply of energy and protein. The nutritional restriction in energy and / or protein can affects negatively milk production, weight and condition score body. In addition to reduce the efficiency of protein utilization of the diet and cause greater instability of the milk to the alcohol test. However, it does not changed the blood and metabolic profile. In second study, the animals were divided into two groups (1) control and (2) acidosis. The experimental design was simple reversible with two treatments and two experimental periods. Physiochemical characteristics, health of the mammary gland, physiological measures, metabolic profile and blood parameters were analyzed. Losses in milk production, reduction of alcohol stability test, urinary pH, fecal pH, ruminal pH were caused by Subacute ruminal acidosis (SARA) induction. However, induction of SARA did not changed the blood parameters evaluated. SARA changes the physical-chemical characteristics of the milk, without influencing the acute phase proteins concentrations, characterizing an inflammatory response. SARA can affect the animals without demostrate changes in the blood profile of the animals.

Les mycotoxines sont des métabolites secondaires des moisissures qui peuvent naturellement contaminer de nombreux supports (céréales, fruits, papiers peints, compost). Dans ces travaux de thèse, nous avons focalisé notre intérêt sur deux mycotoxines produites principalement par les champignons du genre Fusarium, appartenant toutes deux au même groupe des trichothécènes, le déoxynivalénol (DON) et la toxine T-2 (T-2). Les objectifs de cette thèse ont été de déterminer les effets de ces deux toxines sur la mise en place des réponses immunitaires au sein de la muqueuse respiratoire et intestinale. L’ensemble de ces études a été réalisé chez le porc, espèce cible de ces contaminants et animal modèle pour l’Homme. Les résultats de ces travaux ont montré que ces deux toxines affectent la réponse immunitaire. Au niveau du tractus respiratoire, une faible dose de toxine T-2 altère l’activation des macrophages alvéolaires lorsqu’ils sont stimulés par les agonistes des TLRs -4, -2/6 (lipopolysaccharides et acides lipoteichoïques, respectivement) en diminuant la synthèse du composé antimicrobien NO et des cytokines pro-inflammatoires IL-1β et TNF-α. Cette immunosuppression pourrait alors conduire à une susceptibilité plus accrue des porcs à des infections opportunistes. Au niveau du tractus intestinal, à l’état basal, nous avons mis en évidence que le DON comme la toxine T-2 induit une forte réponse inflammatoire innée associée à la stimulation de la voie IL-17 en inhibant le développement des cellules T régulatrices. Des études mécanistiques ont permis de déterminer que les cytokines associées à la voie IL-17 suite à une exposition aux trichothécènes, sont produites par une des sous-populations de cellules T innées, les cellules Tγδ productrices d’IL-17. La troisième partie de ce travail a porté sur l’utilisation de la souche Lactobacillus sobrius DSM 16698T dans le but de lutter contre les effets immunomodulateurs générés lors d’une exposition aux trichothécènes. Les résultats de ce travail ont montré que cette souche bactérienne est capable de réduire les effets inflammatoires IL-17 et de rétablir les paramètres impliqués dans les fonctions de la barrière intestinale, suite à une exposition ex vivo et in vivo au DON. En revanche, cette souche a peu d’effet contre la toxine T-2. L’ensemble de ce travail de thèse suggère donc qu’une exposition à de faibles doses de trichothécènes pourrait accroître la susceptibilité des animaux aux perturbations de nature infectieuse ou inflammatoire
Mycotoxins are fungi secondary metabolites that can contaminate a lot of environments worldwild such as cereals, fruits, wallpapers, and compost heaps. Throughout this phD work, we focused on two mycotoxins mainly produced by Fusarium species, both belonging to the trichothecenes group: the deoxynivalenol (DON) and the T-2 toxin (T-2). The aims of this study were to determine the effects of these two toxins on the immune response implementation in respiratory and intestinal mucosa. Studies were performed on swine being a target species of these contaminants and a model species for Humans. The results of these works proved that these two mycotoxins can affect the immune response. In the respiratory tract, a low dose of T-2 toxin alters the activation of the alveolar macrophages when they are stimulated by the agonists of TLRs -4 and -2/6 (lipopolysaccharides and lipoteichoic acids, respectively). This alteration is due to the decrease of the synthesis of the anti-microbial compound NO and the pro-inflammatory cytokines such as IL-1β and TNF-α. This immunosuppression can induce the emergence of opportunist infections in pig. In the intestinal tract, in background level, we demonstrated that DON as well as T-2 toxin induces a strong inflammatory immune response associated with stimulation of IL-17 pathway by inhibiting of the development of regulatory T cells. Mechanistic studies were used to determine the production origin of the cytokin associated to the IL-17 pathway. This cytokine is produced by one of the subpopulations of Tregs, the Tγδ cells IL-17 producing when exposed to trichothecenes. The third part of this work was about the use of Lactobacillus sobrius DSM 16698T strain to counteract the immunomodulatory effects induced after trichothecen exposure. The results of this study showed that this bacterial strain is able to reduce IL-17 inflammatory effect and is also able to re-etablish the parameters involved in the intestinal barrier functions in ex vivo and in vivo response to DON. Nevertheless, this strain is less effective against the T-2 toxin. Taken together, results of this phD suggest that an exposure to low doses of trichothecens could be intensify the susceptibility of animal to infectious or inflammatory disease

Tese de mestrado, Ciências Biofarmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2017
Amyotrophic Lateral Sclerosis (ALS) is the third most common neurodegenerative disease, mostly sporadic, with limited identified targets, biomarkers and therapeutic options. The most widely used animal model and experimental cellular models to study ALS pathological mechanisms are based on mutations in the anti-oxidant protein SOD1, particularly that of G93A. ALS affects mainly motor neurons, but it is widely accepted that immune unbalance plays a crucial role in the ALS disease, and microglial dysfunction is described to be associated with neuronal injury influencing disease onset and progression. As the immune cells of the central nervous system, microglia produce inflammatory responses towards an insult by secreting pro-inflammatory mediators to the extracellular milieu in the form of soluble factors, or in membrane-bound vesicles called exosomes, an important component in intercellular communication and in disease dissemination. In this thesis we aimed to better understand the role of microglia in ALS disease using the mutant SOD1G93A microglia, and assessing their reactivity upon the immunostimulation by lipopolysaccharide (LPS), and immunomodulation by glycoursodeoxycholic acid (GUDCA) and vinyl sulfone (VS), having in mind the goal of fighting ALS neurodegeneration. For that, we assessed microglia function/dysfunction and reactivity after human SOD1 overexpression in the N9 cell line, either wild type (hSOD1WT) or mutated in G93A (hSOD1G93A), alone or treated with LPS, and when exposed to GUDCA and VS, known for their potential anti-inflammatory effects. Data showed that overexpression of hSOD1WT in N9 cells leads to a decrease in all analyzed pro- and anti-inflammatory markers, whereas hSOD1G93A increases both pro-inflammatory TNF-α, IL-1β, MHCII and HMGB1 gene expression levels, together with anti-inflammatory Arg1 and SOCS1 indicators, and reduces iNOS, Fizz1, IL-10, TLR4, miR-125b and miR-21. Interestingly we found an elevated cargo of miR-155 and miR-146a in hSOD1G93A microglia-derived exosomes. Upon LPS exposure, all cells switched from ramified into amoeboid morphology. LPS-treated transgenic microglia showed equivalent pro-inflammatory markers, when compared to LPS-treated naïve cells. However, they revealed decreased levels of the anti-inflammatory Arg1, Fizz1 and IL-10, thus reducing the ability to later balance the microglia reactivity to the insult. Surprisingly, cells also evidenced reduced miR-155 expression, what may even compromise an adequate pro-inflammatory response. In contrast with hSOD1WT cells, SOD1G93A microglia displayed decreased gene expression of S100B and equal of TNF-α mRNA, when compared to naïve cells. Additionally, the ability of ingesting a high number of beads (≥ 11) was found diminished. Treatment with GUDCA or VS decreased the cell body area of reactive microglia, and SOCS1 and Arg1 mRNA expression. Nevertheless, both immunomodulators increased TLR4, as well as reduced IL-1β and S100B gene expression, which may represent benefits for response to selected insults, while protecting from destructive secondary damage, respectively. In addition, though it decreased cellular MFG-E8 and enhanced miR-125b in exosomes, GUDCA markedly increased the cellular gene expression of the anti-inflammatory IL-10. On the other hand, VS was the only one able to reduce the pro-inflammatory MMP-9 activity and to elevate the exosomal cargo in the anti-inflammatory miR-21. In conclusion, this work demonstrates the advantageous hSOD1WT overexpression in balancing pro- and anti-inflammatory mediators in microglial cells, but overall that upregulation of hSOD1G93A increases their reactivity and may have a detrimental role in reducing their wound repair ability after insult, thus causing homeostatic imbalance between anti-inflammatory and pro-inflammatory gene expression mediators. In addition, the study also highlights that, although with different potential roles, both VS and GUDCA may have benefits over specific hSOD1G93A polarized microglia subtypes.
A Esclerose Lateral Amiotrófica (ELA) é a terceira doença neurodegenerativa mais comum, sendo maioritariamente esporádica, e limitada em termos de alvos, biomarcadores e opções terapêuticas. Os modelos animais e celulares mais usados no estudo dos mecanismos envolvidos na patogénese da ELA consideram mutações na enzima antioxidante SOD1, particularmente, a mutação G93A. A ELA afeta maioritariamente neurónios motores. No entanto, é considerado que existe uma desregulação inflamatória nesta doença que contribui para a sua progressão. A disfunção de células microgliais é associada ao dano neuronal, o que consequentemente leva ao início e progressão da doença. No Sistema Nervoso Central (CNS), as células da microglia são responsáveis pela produção da resposta inflamatória em consequência da presença de moléculas estranhas no ambiente extracelular. Esta resposta baseia-se na secreção de mediadores pro-inflamatórios para o meio extracelular sob a forma de fatores solúveis ou incorporados em vesículas membranares denominadas de exossomas, um importante meio de comunicação intercelular na disseminação da patologia. Na presente tese, pretendeu-se compreender melhor o papel da microglia na ELA, utilizando células da microglia sobreexpressando SOD1G93A, e avaliando a sua reatividade após estimulação com lipopolissacárido (LPS), e após tratamento com os imunomoduladores ácido glicoursodesoxicólico (GUDCA) e vinil sulfona (VS), com o objetivo de combater a neurodegeneração na ELA. Para isso, avaliámos a função/disfunção e reatividade microglial após a sobreexpressão da enzima SOD1 na linha celular N9, na conformação WT (hSOD1WT) ou mutada em G93A (hSOD1G93A) da enzima, em células sem tratamento ou tratadas com LPS. Adicionalmente, avaliámos o potencial anti-inflamatório dos compostos GUDCA e VS nas células sobreexpressando hSOD1G93A. Os nossos resultados demonstraram que a sobreexpressão de hSOD1WT em células N9 leva a uma diminuição de todos os parâmetros pro- e anti-inflamatórios analisados, enquanto que da sobreexpressão de hSOD1G93A leva a um aumento da expressão génica dos marcadores pro-inflamatórios TNF-α, IL-1β, MHCII e HMGB1 em conjunto com os marcadores anti-inflamatórios Arg1 e SOCS1, reduzindo iNOS, Fizz1, IL-10, TLR4, miR-125b e miR-21. Curiosamente, exossomas derivados de microglia sobreexpressando hSOD1G93A revelaram transportar maiores quantidades de miR-155 e miR-146a. Após exposição ao LPS, todas as células modificaram a sua morfologia ramificada para uma forma ameboide. Células N9 hSOD1G93A tratadas com LPS demonstraram marcadores pro-inflamatórios com níveis equivalentes ao das células controlo. No entanto, revelaram também uma diminuição dos marcadores pró-inflamatórios Arg1, Fizz1 e IL-10, reduzindo assim a capacidade da microglia de resposta ao insulto. Surpreendentemente, estas células demonstraram ainda uma diminuição de miR-155, o que pode sugerir uma resposta pró-inflamatória adequada. Ao contrário de células sobreexpressando hSOD1WT, microglia SOD1G93A apresentou uma diminuição nos níveis de expressão génica de S100B e igual expressão de TNF-α quando comparadas ao controlo. Adicionalmente, estas células evidenciaram uma diminuição da capacidade de ingestão de um elevado número de beads [≥11]. O tratamento com GUDCA ou VS demonstrou diminuir a área do corpo celular das células reativas da microglia, em conjunto com uma diminuição da expressão génica de SOCS1 e Arg1. Contudo, ambos os imunomoduladores aumentaram a expressão de TLR4, diminuindo a expressão de IL-1β e S100B, o que pode sugerir o efeito benéfico destes compostos na resposta a insultos, protegendo contra efeitos secundários destrutivos, respetivamente. Adicionalmente, apesar da diminuição da expressão de MFG-E8 e aumento da expressão de miR-125b em exossomas, o composto GUDCA evidenciou um aumento significativo da expressão do marcador anti-inflamatório IL-10. Por outro lado, apenas o tratamento com VS foi bem-sucedido na diminuição da atividade da MMP-9 e aumento do transporte do anti-inflamatório miR-21 em exossomas. Em conclusão, este trabalho demonstra o benefício da sobreexpressão de hSOD1WT no equilíbrio de marcadores pro- e anti-inflamatórios nas células da microglia, enquanto a sobreexpressão de hSOD1G93A aumenta a reatividade microglial, podendo ter um papel prejudicial na redução da sua capacidade de resposta a estímulos externos, causando assim um desequilíbrio na expressão génica de marcadores pro- e anti-inflamatórios. Adicionalmente, este estudo foca ainda que, apesar de com diferentes funções, os compostos GUDCA e a VS que podem ser benéficos para as células da microglia hSOD1G93A com diferentes polarizações.
The studies presented in this master thesis were performed in the Neuron Glia Biology in Health and Disease Group, at the Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, University of Lisbon, under the supervision of Dora Brites, Ph.D. (group leader) and Ana Rita Vaz, Ph.D.
Work presented in this master thesis was supported by Santa Casa da Misericórdia de Lisboa [Ela Project 2015-002 (DB)] and in part by Fundação para a Ciência e Tecnologia [project Pest-UID/DTP/04138/2013 iMed.ULisboa project].

碩士
國立陽明大學
臨床護理研究所
91
The purpose of this study was to examine the effect of systemic inflammatory response on energy expenditure and nitrogen balance of surgical critically ill patients. In respect to the existence of systemic inflammatory response syndrome (SIRS) or infection, fifty-six patients were classified into three groups: Non-SIRS, Nonseptic SIRS and SEPSIS. The resting energy expenditure (REE) was determined by indirect calorimetry. On the day of the energy measurement, the 24-hour urine samples were collected and the dietary intake was recorded. Nonseptic SIRS and SEPSIS groups had significantly higher REE than the Non-SIRS group. However, the REE between the Nonseptic SIRS and SEPSIS groups was not significantly different. The estimated basal energy expenditure (EBEE) calculated by using Harris-Benedict equation was greater than the REE in the Non-SIRS group. In contrast, the EBEE was lower than the REE in both Nonseptic SIRS and SEPSIS groups. Negative nitrogen balance was observed in all three groups despite of comparable amount of energy supply, indicating these patients were in the hypermetabolic state. The REE was negatively correlated with nitrogen balance, whereas the energy intake, the ratio of energy intake to REE and protein intake were positively correlated with nitrogen balance in all SIRS patients with or without infection. No such correlations were found in the Non-SIRS group. Results of the present study suggest that systemic inflammatory response is responsible for the higher REE observed in the Nonseptic SIRS and SEPSIS groups. Nitrogen balance is a valuable index in evaluating the energy metabolism and demands of surgical critically ill patients.

My research project aims to evaluate the possible beneficial effects of Omega (ω)-3 polyunsaturated fatty acids (PUFA) administration in an experimental animal model. I performed the research activity with two different experimental approaches. In the first experimental project, it was used an animal model with obesity-diet induced. Male Wistar rats were fed, for 6 weeks, with three different diets: standard diet (control); HFD enriched with saturated fatty acids (lard); HFD enriched with ω-3 (PUFA) derived from fish oil. In the second experimental project, the rats were fed with standard diet for 4 weeks and two groups received a supplementation with two different milk: the first group were supplemented with low forage milk (LFM), the second group was supplemented with milk low in ω6:ω3 ratio and high conjugated linoleic acid (CLA) levels a high forage milk (HFM). In both experimental projects, at the end of the experimental period was assessed the effects of these dietary treatments on energy balance, lipid metabolism and inflammatory state. Since the skeletal muscle shares with the liver the capacity to influence energy metabolism throughout the body, this work investigated the ability of PUFA to affects lipid metabolism and inflammatory state by modulating the mitochondrial function, ROS emission and Nrf-2 activation in these tissues. Diet supplementation with ω-3 (PUFA) results in a significant decrease in whole body weight, lipid utilization and inflammation with an increased energy expenditure due to the modulation of skeletal muscle and liver mitochondrial efficiency, and to an increased AMPK activation with a consequent reduction of oxidative stress. Moreover, the increased mitochondrial respiration rates were related to an increased mitochondria biogenesis as shown by the increase in PGC1-α and –β expression levels in ω-3 fish oil treated rat models. Furthermore, my data provided the first evidence of HFM beneficial effects on rat metabolism, and suggested that these effects are mediated by the high ω6/ω3 ratio and high content of CLA of HFM.

AIM : Balanitis xerotica obliterans (BXO) is a chronic inflammatory skin disorder of unclear etiology, it is considered the male genital variant of lichen sclerosus. The etiology and the exact molecular mechanisms underlying the disease are still unknown. The histopathology of BXO is characterized by alterations in the epidermis and dermis as well as inflammatory cell infiltration. In this regard several factors may be involved in underling molecular changes associated with inflammatory response. Antiinflammatory drugs are commonly used in pre-operative setting to decrease the inflammatory/autoimmune response and for proper wound healing. Olive Oil Ozonides are used in several medical applications for its anti-inflammatory, anti-microbial and skin restoring properties. The human Transglutaminases (TGs) family consists of several proteins with catalytic activity essential for biological processes such as skin barrier formation and extracellular matrix assembly but can also contribute to the pathophysiology of various inflammatory , autoimmune and degenerative conditions. Given the key role played by TGs in the maintenance of epidermis integrity and extracellular matrix stability, the aim of this study was to investigate transcript levels of three TGs in foreskin of children underwent to surgery for phimosis without or with histologically confirmed BXO, to characterize the relationship between changes of TG levels and BXO and furthermore we evaluated the anti-inflammatory effects of treatment by assessing their effects on foreskin of patients with BXO. M&M. Thirty children with acquired phimosis were enrolled. The removed foreskins were sent both for the histological diagnosis and for the analysis of transcript levels of keratinocyte TG (TG1), tissue TG (TG2), epidermal TG (TG3) and IFN-γ, these were evaluated by quantitative Real-Time PCR .Furthermore, fifteen patients with BXO and previously treated by pediatrician with topical olive oil ozonides were compared with fifteen without any treatment. The foreskins were used to evaluate the transcript levels of pro-inflammatory cytokines, transglutaminase 2 (TG2), inducible nitric oxide synthase (NOS2), enzymes involved in the inflammatory process, as well as Vascular endothelial growth factor (VEGF) by Real-Time PCR. Results. We observed a significant increase in IFN-γ and TG2 mRNA levels by 2.8 and 2.9 folds (p<0.001), respectively, and a decrease in TG1 and TG3 transcripts by about 70% (p<0.001) in foreskin from patients with BXO (n=15) in comparison with patients without BXO (n=15). We observed a reduction by about 70% (p<0.001) in mRNA levels of cytokines, such IL-1β, TNF-α and INF–γ, as well as a decrease higher than 90% (p<0.001) in TG2 and NOS2 gene transcripts in tissues from Ozonides group. We also found an increase of 17 folds (p<0.001) in VEGF transcript in tissues treated with ozonides in comparison to untreated patients. Conclusion The reduced expression of TG1 and TG3 is associated with altered structure of foreskin in BXO and can be a consequence of damage to keratinocytes. Increased expression of TG2 and IFN-γ can be the result of chronic inflammation. TG2 overexpression can play a pivotal role in triggering and maintaining the inflammatory response in BXO patients. Our results suggest that pre-operatory treatment with Ozonides could provide a considerable benefit in terms of reduction of the inflammatory response in tissues with BXO. Additionally, the increased production of VEGF could stimulate a faster recovery and promote the healing process in patients undergoing surgery