Obesity over the last several has become a major health concern in our country as well as the world. Obesity is also one of the risk factors which lead to several inflammatory complications such as diabetes, artherosclerosis, etc. Two leading factors involved in the causes of inflammatory complications include leptin and low dose endotoxin lipopolysaccharide (LPS). However, the mechanism underlying the involvement of these two mediators is not clearly understood. The purpose of this study is to understand the mechanism underlying inflammatory complications caused by leptin and low dose endotoxin most recently coined metabolic endotoxemia. Interleukin-Receptor Associated Kinase 1 (IRAK-1) is an intracellular signaling component shown to activate NFκB which leads to the induction of proinflammatory mediators. Deletion of IRAK-1 in mice has beneficial effects in alleviating inflammatory complications and human variations in IRAK-1 gene are correlated with higher risks for inflammatory diseases. Therefore, we hypothesized that IRAK-1 is critically involved for the induction of proinflammatory mediators induced by leptin and low dose LPS. IL-6 mRNA levels were measured in THP-1 (human monocytic cells) and wild type and IRAK-deficient bone marrow derived macrophages (BMDM) challenged with different combinations of leptin and LPS. Data shows that leptin alone will not induce inflammatory mediators. However, increased induction of IL-6 was observed in a synergistic manner involving both LPS and leptin in an IRAK-1 dependent manner causing a robust inflammatory response. With regard to the effect of low dose LPS, we observed that human monocytic cells treated with low concentrations of LPS showed a mild yet sustained induction of proinflammatory cytokines, which is contrast to the robust and transient induction of cytokines by a high dose LPS. To further determine the molecular mechanisms, we measured several key signaling molecules that include IRAK-1, IKKepsilon, and C/EBPdelta. Our study revealed a novel mechanism that appears to be distinct from the traditional NFï «B pathway responsible for the effect of low dose LPS. / Ph. D.

The metabolic activity of cartilage cells (chondrocytes) is regulated by mechanical forces which act on them. Chondrocytes can respond to these forces through synthesis or degradation of extracellular matrix and changes in gene expression. The overall objective of this study was to investigate the effects of mechanical compression on gene regulation, proteoglycan (PG) synthesis and activation of signaling pathways. To achieve this goal a simple oscillatory displacement controlled device was designed to provide uniaxial unconfined strain to cell constructs. Static compression and dynamic compression with various waveforms are utilized with a stroke range of 0.25 mm to 4 mm and a frequency range of 0.1 Hz to 3 Hz. Poly-L-lactic acid (PLLA/)alginate disks and alginate disks with equine chondrocytes embedded in them were developed and showed unchanged viability for 24 hr under static and dynamic compression. Testing to relate the strains applied to forces experienced in cell constructs was completed and the simple procedure outlined for companion use with our device. Quantitative reverse transcription polymerase chain reaction (QRT-PCR) revealed changes in expression of collagen II and matrix metalloproteinase-3 under dynamic compression for 24 hr. Equine chondrocytes compressed for 48 hr showed lower PG synthesis for both static and dynamic compression when compared to uncompressed samples in replicate experiments. Repeatability of this experiment was problematic possibly due to decreased viability and inefficient extraction. Different patterns of extracellular signal regulated kinase (ERK) activation with time were found for uncompressed and compressed samples (static at 15% strain and dynamic at 15% strain, 1 Hz) and protein kinase B (also called Akt) was not regulated by compression. Results from experiments involving frequency and strain for dynamic compression were inconclusive. These studies show that regulation of gene expression, PG synthesis and intracellular signaling can be studied with our device but optimization of the experimental procedure is still needed. To our knowledge these studies are the first to show these types of studies utilizing equine chondrocytes. Despite issues encountered, our studies provide valuable insights into the effects of compression on equine chondrocytes and detail a simple device for use in a wide variety of compression studies. / Ph. D.

Les cellules dendritiques plasmacytoïdes sont les productrices majeures d’IFN de type I dans l’organisme humain. Durant les infections virales chroniques, telles que l’infection par le Virus de l’Hépatite C, les pDCs sont fonctionnellement altérées. L’efficacité dans plus de 50% des cas du traitement par IFN-α, utilisé jusqu’à récemment, suggère que la modulation de la fonction des pDCs serait une cible intéressante pour le traitement HCV. Les pDCs reconnaissent l’ARN du HCV par les récepteurs Toll-like, et disposent de plus d’un set de récepteurs dits régulateurs qui régulent la production d’IFN-I. L’activation de ces RR inhibe la production d’IFN-I par les pDCs stimulées par les agonistes de TLR7/9. Nous montrons ici que la glycoprotéine d’enveloppe E2 du HCV est un nouveau ligand des RR BDCA2 et DCIR des pDCs, et que cette liaison est responsable de l’inhibition d’IFN-I via l’activation de la voie de signalisation BCR-like. Nous avons ensuite voulu restaurer la production d’IFN-I dans les pDCs en ciblant les kinases décrites de la voie BCR-like, Syk et Mek. En inhibant Syk, l’IFN-I n’a été que partiellement restauré par les concentrations subliminales de l’inhibiteur; les concentrations élevées de cet inhibiteur ont bloqué la production d’IFN-I, suggérant l’implication de Syk dans la voie TLR7/9 comme montré pour l’activation des TLR dans les macrophages. En inhibant MEK, la restauration d’IFN-I est efficace. Les mécanismes de cette restauration sont explorés. Le ciblage pharmacologique de la signalisation BCR-like constituerait une nouvelle approche intéressante pour étudier les mécanismes de modulation de l’activation des pDCs dans les conditions physiopathologiques. / Plasmacytoid dendritic cells are major producers of type I IFN in human organism. During chronic viral infections, such as Hepatitis C Virus infection, pDCs are functionally impaired. More than 50% efficiency of IFN-α treatment, until recently used, suggested that modulation of pDC function could be an important target for HCV treatment. pDCs recognize HCV RNA by Toll-like receptors, and dispose of a set of so-called regulatory receptors that regulate IFN-I production. Crosslinking of these RR such as BDCA-2 and ILT7 has been shown to inhibit IFN-I production by pDCs stimulated with TLR7/9 agonists. In this work we show that HCV envelope glycoprotein E2 is a novel ligand of pDC RR, BDCA-2 and DCIR, and that this binding is responsible for IFN-I inhibition via the activation of the BCR-like pathway. Then we assayed to restore IFN-I in pDCs with crosslinked RR by targeting well-known kinases of BCR-like pathway, Syk and Mek. When inhibiting Syk, IFN-I was only partially restored by subliminal concentrations of Syk inhibitor; high concentrations of Syk inhibitor effectively blocked IFN-I production, suggesting involvement of Syk in the TLR7/9 pathway as it was already demonstrated in TLR activation in macrophages. When inhibiting MEK, the restoration of type I IFN was effective. The underlying mechanisms leading to the restoration are further explored. Pharmacological targeting of BCR-like signaling may constitute an attractive new approach to study mechanisms of modulation of pDC activation in pathophysiological conditions.

Doctor of Philosophy / Department of Biochemistry / Anna Zolkiewska / Muscle satellite cells are the primary stem cells of postnatal skeletal muscle. Quiescent satellite cells become activated and proliferate during muscle regeneration after injury. They have the ability to adopt two divergent fates: differentiation or self-renewal. The Notch pathway is a critical regulator of satellite cell activation and differentiation. Notch signaling is activated upon the interaction of a Notch ligand present in a signal-sending cell with a Notch receptor present in a signal-receiving cell. Delta-like 1 (Dll1) is a mammalian ligand for Notch receptors. In this study, we found that Notch activity is essential for maintaining the expression of Pax7, a transcription factor associated with self-renewing satellite cells. We also demonstrated that Dll1 represents a substrate for several ADAM metalloproteases. Dll1 shedding takes place in a pool of Pax7-positive self-renewing cells, but Dll1 remains intact in differentiated myotubes. Inhibition of Dll1 shedding with a dominant-negative form of ADAM12 leads to elevated Notch signaling, inhibition of differentiation and expansion of the pool of self-renewing cells. We propose that ADAM-mediated shedding of Dll1 helps achieve an asymmetry in Notch signaling in initially equivalent myogenic cells and helps sustain the balance between differentiation and self-renewal. Pax7 plays a key role in protecting satellite cells from apoptosis. The mechanism of Pax7 protecting muscle satellite cells from apoptosis is not well understood. In the second part of this study, we show that Pax7 up-regulates manganese superoxide dismutase (MnSOD) at the transcriptional level, suggesting the involvement of MnSOD in Pax7-mediated cell survival. A specific chromosomal translocation involving the Pax7 gene and generation of a fusion protein Pax7-FKHR is found a childhood cancer, rhabdomyosarcoma. Furthermore, the level of the wild-type Pax7 is down-regulated in rhabdomyosarcomas. In the third part of this dissertation, we investigated the dominant-negative effect of Pax7-FKHR fusion protein on the wild-type Pax7, and found that the Pax7 protein level is down-regulated by Pax7-FKHR expression while the Pax7 mRNA level is not affected. We propose a specific microRNA-mediated inhibition of Pax7 mRNA translation by the oncogenic Pax7-FKHR fusion protein.

L’apurinic/apyrimidic endonuclease 1 (APE1) est une protéine multifonctionnelle qui joue un rôle important dans la voie de réparation de l’ADN par excision de base. Elle sert également de coactivateur de transcription et est aussi impliquée dans le métabolisme de l’ARN et la régulation redox. APE1 peut cliver les sites AP ainsi que retirer des groupements, sur des extrémités 3’ créées suite à des bris simple brin, qui bloquent les autres enzymes de réparation, permettant de poursuivre la réparation de l’ADN, puisqu’elle possède plusieurs activités de réparation de l’ADN comme une activité phosphodiestérase 3’ et une activité exonucléase 3’→5’. Les cellules de mammifères ayant subi un knockdown d’APE1 présentent une grande sensibilité face à de nombreux agents génotoxiques. APE1 ne possède qu’une seule cystéine située au 65e acide aminé. Celle-ci est nécessaire pour maintenir l’état de réduction de nombreux activateurs de transcription tels que p53, NF-κB, AP-1, c-Jun at c-Fos. Ainsi, elle se retrouve impliquée dans la régulation de l’expression génique. APE1 passe également à travers au moins 4 types de modifications post-traductionnelles : l’acétylation, la désacétylation, la phosphorylation et l’ubiquitylation. La façon dont APE1 est recrutée pour accomplir ses différentes fonctions biologiques demeure un mystère, bien que cela puisse être relié à sa capacité d’interaction avec de multiples partenaires différents. Sous des conditions de croissance normales, il a été démontré qu’APE1 interagit avec de nombreux partenaires impliqués dans de multiples fonctions. Nous émettons l’hypothèse que l’état d’oxydation d’APE1 est ce qui contrôle les partenaires avec lesquels la protéine interagira, lui permettant d’accomplir des fonctions précises. Dans cette étude nous démontrons que le peroxyde d’hydrogène altère le réseau d’interactions d’APE1. Un nouveau partenaire d’interaction d’APE1, Prdx1, un membre de la famille des peroxirédoxines responsable de récupérer le peroxyde d’hydrogène, est caractérisé. Nous démontrons qu’un knockdown de Prdx1 n’affecte pas l’activité de réparation de l’ADN d’APE1, mais altère sa détection et sa distribution cellulaire à l’intérieur des cellules HepG2 conduisant à une induction accrue de l’interleukine 8 (IL-8). L’IL8 est une chimiokine impliquée dans le stress cellulaire en conditions physiologiques et en cas de stress oxydatif. Il a été démontré que l’induction de l’IL-8 est dépendante d’APE1 indiquant que Prdx1 pourrait réguler l’activité transcriptionnelle d’APE1. Il a été découvert que Prdx1 est impliquée dans la régulation redox suite à une réponse initiée par le peroxyde d’hydrogène. Ce dernier possède un rôle important comme molécule de signalisation dans de nombreux processus biologiques. Nous montrons que Prdx1 est nécessaire pour réduire APE1 dans le cytoplasme en réponse à la présence de H2O2. En présence de Prdx1, la fraction d’APE1 présent dans le cytoplasme est réduite suite à une exposition au peroxyde d’hydrogène, et Prdx1 est hyperoxydé suite à l’interaction entre les deux molécules. Cela suggère que le signal, que produit le peroxyde d’hydrogène, sur APE1 passe par Prdx1. Un knockdown d’APE1 diminue la conversion de la forme dimérique de Prdx1 vers la forme monomérique. Cette observation implique qu’APE1 pourrait être impliquée dans la régulation de l’activité catalytique de Prdx1 en accélérant son hyperoxydation. / Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein, which play important roles in base excision repair (BER) pathway and serve as transcriptional co-activator. APE1 is also involved in RNA metabolism and redox regulation. APE1 can cleave abasic sites and process 3’-blocking termini into 3’-OH for DNA repair replication as it posseses several DNA repair activities including AP endonuclease, 3’-phosphodiesterase and 3’ to 5’-exonuclease. Mammalian cells knockdown for APE1 are very sensitive to various DNA damaging agents. APE1 has a unique cysteine C65, which is required to maintain the reduced state of several transcriptional activators such as p53, NF-кB, AP-1, c-Jun, and c-Fos and therefore is involved in the regulation of gene expression. APE1 also undergoes at least four types of post-translational modifications that include acetylation, deacetylation, phosphorylation and ubiquitylation. How APE1 is being recruited to execute the various biological functions remains a challenge, although this could be directly related to its ability to interact with multiple different partners. Under normal growth conditions, APE1 has been shown to interact with a number of proteins that are involved in various functions. We propose that the oxidative state of APE1 governs its interacting partners thereby allowing the protein to perform specific functions. In this study we find that APE1 interactome alters in response to hydrogen peroxide. One novel APE1 interacting partner Prdx1, a member of the peroxiredoxin family that can scavenge hydrogen peroxide is characterized. We demonstrate that knockdown of Prdx1 did not impair APE1 DNA repair activity, but alters APE1 detection, and subcellular distribution in HepG2 cells leading to the induction of interleukin 8 (IL-8). IL-8 is a pro-inflammatory chemokine involved in cellular stress, under physiological and iv oxidative stress conditions. It has been shown that the induction of IL-8 is dependent on APE1 indicating Prdx1 may regulate APE1 transcriptional activity. Prdx1 has been discovered to be involved in the redox regulation of cell signaling initiated by hydrogen peroxide, which has important roles as a signaling molecule in the regulation of a variety of biological processes. Prdx1 exists as a dimer in the cells and we show that Prdx1 is required to reduce APE1 in the cytoplasm in response to H2O2. During this process, the dimeric form of Prdx1 is converted to the oxidized monomeric form. Interestingly, the H2O2-induced conversion of Prdx1 to the monomeric form is dependent upon the presence of APE1. These observations imply that there is a tight regulatory network existing between APE1 and Prdx1.

L’infarctus du myocarde, problème majeur de santé publique, est caractérisé par une nécrose cardiomyocytaire. Des séries d’occlusions-reperfusions courtes, réalisées avant l’ischémie (Préconditionnement (PréC) ischémique) ou au moment de la reperfusion (Postconditionnement (PostC) ischémique), protègent le coeur contre des lésions d’ischémie-reperfusion (IR). Les mécanismes intracellulaires impliqués restent obscurs. Nous avons étudié la signalisation intracellulaire du PréC et du PostC, et la cardioprotection qui en découle, sur un modèle de coeur isolé perfusé de souris. Le PréC ischémique peut être mimé par une activation directe du canal potassique mitochondrial ATP dépendant (mitoKATP), entraînant la mise en place d’une boucle d’auto-amplification incluant l’activation d’Akt, l’inhibition de GSK-3β et l’ouverture du mitoKATP. Cette réponse est liée à la production modérée d’espèces dérivées de l’oxygène par le mitoKATP et aboutie à une cardioprotection. La voie de développement Wnt est capable de moduler le PréC via GSK-3β. La voie de survie mTOR, cible de GSK-3β est aussi impliquée et pourrait induire des modifications traductionnelles lors de la réponse adaptative à l’IR. Le PostC ischémique peut également être mimé par activation directe du mitoKATP lors de la reperfusion, engendrant une protection du coeur et la mise en place d’une boucle d’auto-amplification similaire à celle du PréC, comprenant Akt, GSK-3β et le mitoKATP. Le PostC est dépendant de GSK-3β, mais contrairement au PréC, il n’impliquerait pas les voies Wnt et mTOR. Cette étude est la première démontrant que le PréC implique les voies de survie mTOR et de développement Wnt avec un rôle central de GSK-3β. / Myocardial infarction is a major problem of public health, whose prognosis is related to the extent of the infarcted territory. Transient episodes of ischemia/reperfusion before ischemia (ischemic PreConditioning (PreC)), or at the onset of reperfusion (ischemic PostConditioning (PostC)) confer myocardium resistance to lethal ischemia. However the exact mechanism of PreC and PostC remains obscure. Our objectives were to examine signaling events during PreC and PostC and their effects on cardioprotection in an isolated mouse heart model. We provide evidence that pharmacological PreC by direct activation of mitoKATP, like ischemic PreC, involve an amplification loop involving ROS production and resulting in a sustained down-regulation of GSK-3β via Akt activation and a constant opening of mitoKATP. The mTOR pathway is a target of this loop and participates to cardioprotection. Disruption of Wnt pathway by sFRP1 modulates this loop inducing GSK-3β activation. This is the first evidence that PreC involves both a pro-survival mTOR pathway and an embryonic developmental Wnt pathway targeting GSK-3β. During ischemic and pharmacological PostC, the same amplification loop is activated, including Akt, GSK-3β and the mitoKATP. Unlike PreC, PostC did not induce the mTOR survival pathway: neither phosphorylation of mTOR nor of its targets p70S6K and 4E-BP1 were observed. However, cardiac overexpression of a Wnt antagonist, impairing PreC through GSK3-β, was unable to abolish cardioprotection afforded by PostC. PostC signaling differs from the preC pathway. Despite these discrepancies, GSK-3β plays a key role in both types of cardioprotection.

Les bactéries Pectobacterium sont classées parmi les agents pathogènes les plus importants économiquement pour la culture de la pomme de terre. Au cours des dernières années, une augmentation des maladies dues à ces bactéries a pu être observée. Le but de ce travail de thèse était d’analyser certains aspects de la diversité liés aux Pectobacterium sp à savoir : (i) la diversité génétique liée aux régulateurs du pouvoir pathogène de la bactérie (ii) la diversité de virulence et d’agressivité des souches de Pectobacterium vis-à-vis de leurs hôtes et (iii). Le rôle et la diversité des effets induits par le lipopolysaccharides (LPS), composants de la surface bactérienne de bactéries phytopathogène ou non phytopathogène. Ce travail de thèse souligne également le rôle potentiel que pourrait jouer ces molécules LPS dans le biocontrôledes Pectobacterium sp. Différentes expérimentations cellulaires et moléculaires allant de l’identification de la bactérie à la compréhension des voies de signalisation ont été utilisées. Les résultats obtenus nous ont permis de montré, en premier lieu que, le séquençage du gène pmrA, gène connu pour être impliqué dans la régulation du pouvoir pathogène desPectobacterium, est un outil moléculaire complémentaire d’identification de sous espèces de Pcc et pourrait être aussi un moyen efficace d’évaluation de la diversité génétique intraspécifique. Dans un second temps, nous avons montré que les cultures cellulaires d’Arabidopsis thaliana pourraient être un modèle végétal d’évaluation de l’agressivité des Pectobacterium. Ceci a été obtenu par quantification des cinétiques de trois paramètres associés à la pathogénie de ces bactéries à savoir : l’activité des pectate-lyases, déterminant majeur du pouvoir pathogène des Pcc, la fuite des électrolytes, considérée comme un marqueur précoce de la mort cellulaire, et la mort cellulaire des cultures elle-même. Enfin nous avons également montré que les effets induits par les LPS chez les cellules d’Arabidopsis thaliana sont dépendant du type bactérien. En effet Les résultats obtenus nous ont permis de mettre en évidence trois types de réponses différentes aux LPS en fonction de leur origine: les réponses identiques (régulation des flux d’ions), des réponses communes mais présentant des intensités et de cinétiques différentes (production de ROS, induction de gènes de défense) et des réponses spécifiques (induction d’une PCD, alcalinisation du milieu). Ces résultats indiquent que différentes voies de signalisation pourraient être activées chez Arabidopsis thaliana. Ils nous ont permis également de mieux comprendre l’implication des LPS dans le biocontrôle contre les Pectobacterium sp. / Pectobacterium are classified among the most economically important pathogens of culture of potato. Recently, an increase in diseases caused by these bacteria was observed. The work presented in this thesis has allowed highlighting some aspects on diversity associated with Pectobacterium sp namely: (i) genetic diversity related with the pathogenicity of thebacterium (ii) the diversity in virulence and aggressiveness of Pectobacterium strains on its hosts and (iii) the role and the diversity of effects induced by lipopolysaccharide (LPS), bacterial surface components of phytopathogenic and non- phytopathogenic bacteria. First, we have demonstrated that sequencing the pmrA gene, known to be involved in the regulation of pathogenicity of Pectobacterium, is an additional molecular tool for identification of Pectobacterium subspecies. Moreover, pmrA gene could be an effective tool for evaluation of genetic diversity within species. In a second time, we have showed that cell cultures of Arabidopsis thaliana, could be used as an alternative system to evaluate rapidly and efficiently the virulence of different Pectobacterium strains. This was achieved by quantification of different parameters associated to the pathogenesis of these bacteria namely, the activity of pectate lyases, major determinant of the pathogenicity of Pcc, the electrolyte leakage, considered an early marker of cell death and the cell death itself. Finally, our data further suggest the effects induced by LPS from different origin on Arabidopsis thaliana cells, could be different. Indeed, three different types of responses to LPS have been shown: the identical responses (regulation of ion flux), the common responses but having different intensities and kinetics (ROS production, induction defense genes) and specific responses (induction of PCD, alkalinization of the medium). These results indicate that different signaling pathways could be involved in Arabidopsis thaliana in response to LPS and allowed us to highlight the potential role of LPS in the biocontrol of Pectobacterium sp.

Long-chain fatty acids (LCFA) are the primary energy source in the myocardium and an imbalance in the LCFA and glucose utilization could cause cardiovascular diseases. More than 50% of LCFA uptake by the heart is mediated by the fatty acid translocase CD36 and disruption of its function has been shown to impair cardiovascular functions. The spontaneously hypertensive rat (SHR) harbors a deletion variant of the Cd36 gene that results in reduced LCFA transport into myocytes. Therefore, the main aim of this thesis was to investigate the importance of a functional CD36 to sustain normal physiological functions of the heart. We used SHR and two genetic modified SHR strains, the congenic SHR-4 and the transgenic SHR-Cd36, with fully functional CD36. They differ in the CD36 expression and in the manner how they were derived from the SHR. CD36 has been proven to play a role in the pathogenesis of insulin resistance. Therefore we analyzed the effect of a functional CD36 on insulin resistance and protein kinase C (PKC) expression, which is known to be involved in the mechanism of insulin resistance, in the heart of SHR-4 and SHR. We showed that the SHR-4 had lower serum free fatty acids (FFA) and triacylglycerols (TAG) concentrations, indicating improved insulin sensitivity. Furthermore, SHR-4 had increased...

Os carboidratos apresentam-se envolvidos em diversos eventos celulares, tais como geração de energia, sustentação celular, reconhecimento celular, processos de sinalização, etc. A OGlcNAcilação, uma das alterações proteicas pós-traducionais relacionada à adição de Nacetilglicosamina a resíduos de serina ou treonina em proteínas citoplasmáticas ou nucleares, vem demonstrando ser uma das alterações recíproca a O-fosforilação de proteínas e pode estar envolvida no desencadeamento de patologias como câncer, diabetes tipo II, e doenças neurodegenerativas. Tendo em vista à relevância da O-GlcNAcilação e a necessidade de ferramentas para seu estudo, temos como objetivo, desenvolver uma rota sintética para a obtenção de moléculas modificadas derivadas de N-acetilglicosamina, contendo o átomo de flúor ligado ao grupo N-acetil. As moléculas correspondem aos derivados glicopiranosídeo de metila 1 e glicoaminoácidos de serina 2 e treonina 3. Uma vez padronizada, os intermediários finais da rota serão utilizados para futura marcação com o 18F, o qual poderá ser empregado em estudos do processo de sinalização celular por O-GlcNAcilação, e no diagnóstico de câncer por PET. Assim, foram propostas, inicialmente, duas rotas sintéticas, uma para a síntese do derivado glicopiranosídeo de metila 1 e outra para os glicoaminoácidos 2 e 3, ambas utilizando o reagente cloridrato de 2-amino-2-desoxi-D-glicose (4) como percursor. A síntese do derivado 1 foi conduzida por meio da proteção do grupo amino do composto 4 pela formação de carbamato 5, e sequencial reação de glicosilação de Fischer (6 e 7), per-Oacetilação 8 e remoção do grupo protetor do amino 9. As etapas sequenciais relacionadas à condensação com o ácido bromoacético 10, e finalmente halogenação e desproteção do carboidrato 11 estão em andamento. A rota sintética proposta para a obtenção dos glicoaminoácidos 2 e 3 foi fundamentada na obtenção de um doador glicosídico contendo o grupo tricloroacetimidato e as hidroxilas protegidas com grupos benzílicos 16, e na utilização do aceptor glicosídico serina 17 e treonina 18, contendo os grupos amino e carboxila protegidos com 9-fluorenilmetóxicarbonil (Fmoc) e benzil (Bn), respectivamente. Até o momento não foi possível a síntese dos glicoaminoácidos 2 e 3 empregando o doador glicosídico inicialmente proposto (tricloroacetimidato), e mesmo após a variação do doador glicosídico, empregando tioaçucares per-O-benzilado 28, ou per-O-acetilado 26, não foi possível a obtenção do produto desejado. As dificuldades observadas na obtenção dos compostos, conduziram a elaboração de novas estratégias sintéticas que possuem o átomo de cloro como grupo abandonador na posição anomérica e a) uma amida {[(4- metilfenil)sulfonil]oxi}acético (33) em C-2, a qual exerce assistência anquimérica em C-1, e permite a funcionalização com o flúor e b) um azido em C-2, preparado a partir de glicais per- O-acetilados, desprovido de participação em C-1. Ambas as rotas sintéticas estão em andamento. / Carbohydrates are involved in many cellular events, such as energy source, sustenance, recognition, signaling processes, etc. O- GlcNAcylation is a post- translational proteins\' alteration related to the addition of N-acetylglucosamine to residues of serine or threonine in cytoplasmic or nuclear protein which has proven to be one of the reciprocal changes to Ophosphorylation of proteins and may be involved in the onset of pathologies such as cancer, type II diabetes, and neurodegenerative diseases. Given the relevance of O- GlcNAcylation and the importance of tools required for the study of this event, we aim to develop a synthetic route to obtain modified molecules derived from N-acetylglucosamine containing fluorine atom attached to the N -acetyl group. The molecules correspond to methyl glucopyranoside derivatives 1 and gluco-amino acids derived from serine 2 and threonine 3. Once the synthetic route is established, the final intermediates of the route will be used for further labeling with 18F, which may be employed in studies of cell signaling processes, involving OGlcNAcylation, and cancer diagnosis by PET. Thus, two synthetic routes were initially proposed: one for the preparation of the methyl glucopyranoside derivative 1 and another one for the gluco-amino acids 2 and 3, both using glucosamine hydrochloride (4) as a precursor. The synthesis of derivative 1 was conducted by protecting the amine group of compound 4 to form the carbamate 5, and sequential Fischer glycosylation (6, 7), per-O-acetylation (8) and removal of the protecting group from the amine (9). The sequential steps related to condensation with bromoacetic acid (10), halogenation and deprotection of the carbohydrate 11 are in progress. The proposed synthetic route for the preparation of 2 and 3 was based on a glycosidic donor containing trichloroacetimidate group, the protection of the hydroxyl groups with benzyl group (16) and glycoside acceptors serine 17 and threonine 18 containing amine and carboxyl groups protected with 9- fluorenylmethoxycarbonyl (Fmoc) and benzyl (Bn), respectively. Hitherto, the synthesis of gluco-amino acids 2 and 3 was not achieved using the glycosidic donor initially proposed (trichloroacetimidate), even after the change of the glycosidic donor using per-O-benzylated (28) or per-O-acetylated (26) thiosugars. The difficulties encountered for the synthesis of the target compounds led the design of new synthetic strategies which comprise a chlorine atom as leaving group in the anomeric position and either: a) an amide acetic acid (33) at C-2, which exerts anchimeric assistance at C-1, and allows functionalization with fluorine and b) an azide group at C- 2, prepared from per-O-acetylated glucal, with no participation at C-1. Both synthetic routes are in progress.

L’hypertension artérielle pulmonaire (HTAP) est une maladie grave caractérisée par une obstruction progressive des petites artères pulmonaires, conduisant à une augmentation progressive des résistances vasculaires pulmonaires (RVP) et à terme à une défaillance cardiaque droite et au décès du patient. La vasoconstriction, le remodelage vasculaire pulmonaire, la thrombose in situ et la dysfonction endothéliale sont autant de facteurs qui contribuent au développement et à la progression de la maladie. Ces travaux de doctorat ont visé à préciser et mieux comprendre le phénotype anormal de la cellule endothéliale (CE) pulmonaire de patients HTAP afin d’identifier de nouvelles cibles thérapeutiques adéquates pour corriger et restaurer la dysfonction endothéliale associée à l’hypertension pulmonaire. Plusieurs perturbations fonctionnelles liées à des anomalies moléculaires ont pu être identifiées. Les CE HTAPi sont caractérisées par une prolifération, une migration, une survie et une réponse excessive à différents facteurs de croissance : PDGF, EGF, et FGF2. Parmi les anomalies moléculaires à l’origine de ces perturbations fonctionnelles, nous avons retrouvé une production et une libération anormale de FGF2, de MMP2, de MIF, d’IL-6 et de MCP-1, mais aussi une activité constitutive de la MAPK, une surexpression des facteurs anti-apoptotiques BCL2 et BCL-xL, de FGFR2, de p130cas et de CD74. Cette meilleure compréhension de la CE-HTAPi a déjà permis d’identifier plusieurs cibles qui pourraient être évaluées et à aussi permis de soulever un certain nombre de questions qui requiert des études plus poussées. / Pulmonary arterial hypertension (PAH) is a severe disease characterized by progressive obstruction of small pulmonary arteries, leading to a progressive increase in pulmonary vascular resistance and to right heart failure and death. Vasoconstriction, pulmonary vascular remodeling, in situ thrombosis and endothelial dysfunction are all factors that contribute to the development and progression of the disease. These work aimed to clarify and better understand the abnormal phenotype of the pulmonary endothelial cell (P-EC) of PAH patients to identify new therapeutic targets to correct and restore the endothelial dysfunction associated with pulmonary hypertension. Several functional disturbances related to molecular abnormalities have been identified. The IPAH-P-EC is characterized by proliferation, migration, survival and an excessive response to various growth factors: PDGF, EGF, and FGF2. Among the molecular abnormalities responsible for these functional disturbances, we found an abnormal production and release of FGF2, MMP2, MIF, IL-6 and MCP-1, but also a constitutive activity of MAPK, overexpression of anti-apoptotic factors BCL2 and BCL-xL, of FGFR2, of p130Cas and CD74. This better understanding of the EC-IPAH has already identified several targets that could be evaluated and also helped raise a number of issues requiring further study.