Le trouble bipolaire est une maladie invalidante caractérisée par une alternance d’épisodes maniaques et dépressifs. Malgré des efforts de recherche notables, la physiopathologie et les mécanismes d’action des traitements du trouble bipolaire demeurent peu connus. La protéine kinase C (PKC) est récemment apparue comme une cible moléculaire potentielle pour le traitement du trouble bipolaire. Dans ce travail de thèse, nous avons cherché à étudier le rôle de la PKC dans les phases maniaque et dépressive du trouble bipolaire. Nous avons montré que l’inhibition de la PKC a un effet antimaniaque non seulement chez le rat naïf, mais aussi dans un modèle de manie basé sur une privation de sommeil, que nous avons validé au cours de notre étude. De plus, les inhibiteurs de la PKC sont capables de rétablir les déficits de prolifération cellulaire hippocampique que présentent les rats privés de sommeil. Ces effets prolifératifs et antimaniaques seraient indépendants, puisque le blocage de la prolifération cellulaire n’abolit pas l’efficacité antimaniaque des inhibiteurs de la PKC dans le modèle de privation de sommeil. En parallèle, nous avons montré que l’activation de la PKC a un effet antidépresseur chez le rat naïf, alors que son inhibition provoque un phénotype pseudodépressif qui s’accompagne d’une diminution de la prolifération cellulaire hippocampique. L’ensemble de ces données révèle une implication de la PKC dans les deux phases du trouble bipolaire, et soutient l’hypothèse qu’une suractivation du système PKC serait à l’origine des perturbations de neuroplasticité associées à la manie. / Bipolar disorder is a devastating long-term disease characterized by alternate episodes of mania and depression. Despite extensive research, the molecular and cellular underpinnings of bipolar disorder remain to be fully elucidated. Protein kinase C (PKC) has emerged as a potential molecular target for the treatment of bipolar disorder. The present study investigated the role of PKC in manic- and depressive-like behaviors. Our results showed that PKC inhibition produced an antimanic-like effect not only in naive rats, but also in an animal model of mania based on sleep deprivation, that we have validated in our study. Interestingly, PKC inhibitors rescued the hippocampal cell proliferation deficits displayed by sleep-deprived animals. These proliferative and antimanic effects were independent, since blockade of cell proliferation did not abolish the antimanic efficacy of PKC inhibitors in the sleep deprivation model. At the same time, we showed that PKC activation had an antidepressant-like effect in naive rats, whereas its inhibition caused a depressive-like phenotype accompanied by a decrease in hippocampal cell proliferation. Taken together, our results demonstrate the involvement of the PKC system in regulating opposite facets of bipolar disorder, and support the hypothesis that an overactivation of the PKC signaling system may be crucial for the deficits of neuroplasticity associated with mania.

La myopathie de Duchenne est une maladie génétique qui se caractérise principalement par une dégénérescence progressive des muscles squelettiques dont la cause est l’absence de dystrophine fonctionnelle dans les muscles. A ce jour, il n’existe toujours pas de traitement efficace contre ces maladies. Comme le plus grand gène connu chez l’Homme, la dystrophine code pour une protéine de 427kDa. La protéine connecte l’actine avec le DAPC (Dystrophin Associated Protein Complex) dans les muscles striés. Pour l’instant, il y a 3 hypothèses concernant le mécanisme du DMD. L’absence de la dystrophine peut supprimer le lien physique entre les protéines structurales de la membrane basale (laminines) et les protéines structurales du cytosquelette (filaments intermédiaires et actine), ou la distribution et la fonction des canaux ioniques, ou des voies de signalisation nécessaires à la survie du muscle. Caenorhabditis elegans ne possède qu’un homologue du gène de la dystrophine humaine, le gène dys-1. La protéine DYS-1 présente 37% d’homologie avec la dystrophine humaine. Le double mutant dys-1(cx18) ; hlh-1(cc561) présente une forte dégénérescence musculaire. Comme le sarcomère de C. elegans ressemble au sarcomère de mammifère, C. elegans est modèle pertinent d’étude la maladie. En vue de comprendre la raison du DMD chez les mammifères et chez les vers, le groupe L. SEGALAT a effectué des cribles pour identifier les molécules et les gènes qui peuvent supprimer la dégénérescence musculaire. On a trouvé un gène pkc-2 qui est capable de supprimer la dégénérescence musculaire chez C. elegans. La protéine PKC-2 est l’orthologue de la Protein Kinase C Alpha (PKC) humaine et appartient à la famille du serine/threonine protéine kinase. Afin d’étudier la fonction du gène pkc-2, on a analysé l’expression du gène avec les construits différents in vivo et a utilisé la technique de double-hybride dans la levure. De plus, le crible par EMS (éthane méthyle sulfonâtes) a identifié une molécule sérotonine (5-HT) qui est un neuromédiateur, et supprime partiellement la dégénérescence musculaire des doubles mutants dys-1; hlh-1. La sérotonine a aussi un effet fort sur le mutant pkc-2(ok328), puisqu’elle provoque un phénotype blister. Ça nous permet de rechercher le lien entre la signalisation sérotoninergique et pkc-2. Le crible génétique peut contribuer à la connaissance du rôle pkc-2. […]. Elle sert aussi de plate-forme de voie de signalisation intracellulaire. L’identification de Y59A8A.3 propose la possibilité que pkc-2 modifie la filamin A par l’intermédiaire de la filamin A interacting protéine 1. Le crible génétique par EMS pour rechercher des suppresseurs de l’effet blister de la sérotonine sur les mutants pkc-2(ok328) a donné 8 candidats sur 5000 F1s : cx253, cx254, cx259, cx263, cx267, cx268, cx270, cx276. Les mutations ont été localisées sur les chromosomes par SNP mapping avec une souche de C. elegans très polymorphe, mais le temps a manqué pour leur identification exacte. L’expérience valide notre approche à étudier le lien entre la signalisation sérotoninergique et pkc-2. En résumé, le but de la thèse était de rechercher la fonction du gène pkc-2 dans les mécanismes moléculaires conduisant à la nécrose musculaire en absence de dystrophine. Les résultats présentés dans la thèse apportent des réponses aux questions fondamentales sur pkc-2 et aussi demandent des expériences supplémentaires afin de élucider plus avant les mécanismes de la dégénérescence musculaire dystrophine-dépendante. / Duchenne Muscular Dystrophy (DMD) is an X-linked progressive muscle disease which is caused by mutations in the dystrophin gene. Until now, there is no effective therapy for DMD. As the largest gene in human beings, it produces a 427-kDa cytoskeleton protein: Dystrophin. Dystrophin links actin and dystrophin associated protein complex (DAPC) in muscles. Currently, there are 3 hypotheses to explain the mechanisms of DMD. They suggest that the absence of dystrophin could lead to periodic muscle cell membrane ruptures, or affect the distribution and function of ion channels, or perturb signal transduction pathways. In Caenorhabditis elegans, there is only one homologue of mammalian dystrophin gene named dys-1, and the nematode protein DYS-1 presents 37% similar to the human one. The double mutant dys-1; hlh-1 exhibits a severe progressive muscle degeneration. The protein composition of the sarcomere has been studied and it has revealed a high degree of similarity with mammalian sarcomere. These allow C. elegans be a relevant animal model to study DMD.To understand why the lack of dystrophin induces muscle degeneration in mammals and worms, and to find new drugs that might help in reducing muscle degeneration, L. Ségalat and his coworkers performed several screens for drugs and genes suppressing muscle degeneration. An interesting gene pkc-2 came out and was considered as a possible regulator in the process of muscle degeneration in C. elegans. The protein that is encoded by this gene in C. elegans is an orthologous of the human gene Protein Kinase C Alpha (PKC), which belongs to the family of serine/threonine specific protein kinases. To study the function of pkc-2, we generated different recombinant constructs, analyzed the expression pattern of pkc-2 with immunocytochemistry, and performed yeast two-hybrid to search for PKC-2 binding partners. In addition, a neurotransmitter serotonin (5-HT) was found by drug screening to be an active blocker of striated muscle degeneration. As C. elegans lacking PKC-2 displays a severe blister phenotype in exogenous 5-HT, studying the correlation between PKC-2 and 5-HT therefore seems to be an opportunity to explore the reasons of muscle degeneration. A genetic screen with EMS (ethane methyl sulfonate) to search serotonin resistant mutant in strain pkc-2 (ok328) would help us study further about the role of pkc-2.In this thesis, different clones myo3::pkc-2 and pkc-2::gfp were made to inject into wild-type animals. The results revealed that pkc-2 expressed intensely in neurons and pharynx, but was not found in body-wall muscles. Mutants dys-1;hlh-1 fed with pkc-2 RNAi did not reduce muscle degeneration statistically comparing to triple mutant pkc-2;dys-1;hlh-1. This indicated that PKC-2 may be dominantly acting in neurons. A yeast two-hybrid screen identified the gene Y59A8A.3, which is a homologue to mammalian filamin A interacting protein 1 isoform 3, as a binding partner of PKC-2. Filamin A is a cytoskeleton protein, anchoring various trans-membrane proteins to the actin cytoskeleton and may also function as an important signaling scaffold. The result suggested that PKC-2 may therefore modulate filamin A activity through the filamin interacting protein 1. Genetic screen by EMS presented 8 candidates named cx253, cx254, cx259, cx263, cx267, cx268, cx270, cx276, which were mapped on chromosomes by SNP mapping using a polymorphic C. elegans strain, but time was too short to identify these genes formally. The experiment also offered possibilities of searching links between PKC-2 and serotonin pathways.In summary, this work studied the gene pkc-2 in order to reveal the function of PKC-2 and its involvement in muscle degeneration. The present results answered some questions about pkc-2, and needed further researches to elucidate the in vivo role of PKC-2 protein and its interaction with other proteins in the mechanism of muscle dystrophy in C. elegans.

L'amygdale centrale (EAc) est un macrosystème du cerveau antérieur qui joue un rôle important dans la peur, l'anxiété et la douleur. Les deux composants clés, le noyau latéral du lit de la strie terminale (STL) et l’amygdale centrale (CeA), possèdent des caractéristiques neurochimiques, hodologiques et fonctionnelles très similaires. En dépit de cette vision simplifiée du STL et du CeA, de nombreuses questions résident quant à l'organisation mésoscopique des entrées et des sorties des subdivisions de l''EAc chez la souris. En outre, il reste à déterminer si ces similitudes de connexion sont également partagées au niveau cellulaire. Dans ce travail, nous avons abordé ces questions de manière comparative chez la souris. Nous avons trouvé de riches afférences et efférences préférentielles pour les différentes subdivisions de l'EAC, ainsi que des afférences convergentes et divergentes. Nous avons également mis en évidence deux groupes distincts de cellules exprimant la protéine kinase C delta (PKCδ) ou la somatostatine (SOM) qui sous-tendent des circuits neuronaux spécifiques parallèles dans le STL et le CeA, ainsi qu'entre les deux structures. Enfin, des données préliminaires suggèrent que les neurones exprimant la PKCδ dans le STL et le CeA pourraient être impliqués dans la douleur tonique. Ces organisations structurales parallèles, mais aussi différentielles, des circuits neuronaux dans le EAc pourraient sous-tendre des aspects fonctionnels similaires et dissociables de l'anxiété, de la peur et de la douleur. / Central extended amygdala (EAc) is a forebrain macrosystem that plays important roles in fear, anxiety and pain. The two key components, the lateral bed nucleus of stria terminalis (STL) and central nucleus of amygdala (CeA), are highly similar in their neurochemical, connectional, and functional features. Despite this simplified view of STL and CeA, much remains elusive of the mesoscopic inputs and outputs of EAc subdivisions in mouse model. Also, it is not known whether the connectional similarities are also shared at cellular level. Here, we addressed these question in comparative ways in mice. We found rich preferential inputs and outputs to different subdivisions of EAc, as well as convergent and divergent inputs. We also found two non-overlapping cell groups expressing either protein kinase C delta (PKCδ) or somatostatin (SOM) organize the parallel cell-type specific neuronal circuits in STL and CeA. Finally, preliminary data suggest that PKCδ in STL and CeA might be implicated in tonic pain. These parallel but also differential structural organizations of neuronal circuits in EAc might underlie similar and dissociable functional aspects of anxiety, fear and pain.

L'allodynie mécanique est un symptôme cardinal des douleurs persistantes. Elle est due à l’activation de circuits, habituellement bloqués, des couches superficielles de la corne dorsale spinale ou du sous-noyau caudal du trijumeau (Sp5C), par lesquels les afférences mécaniques à bas seuil peuvent accéder aux neurones nociceptifs de projection de la couche I. Un élément déterminant de ces circuits est une classe d’interneurones excitateurs de la couche II interne (IIi) exprimant l'isoforme gamma de la protéine kinase C (PKCγ), et recevant des afférences des mécanorecepteurs à bas seuil. La modulation de l’inhibition tonique de ces interneurones PKCγ contribue à l’apparition de l’allodynie mécanique. Cependant la morphologie, les propriétés électrophysiologiques et les caractéristiques des afférences excitatrices et inhibitrices de ces interneurones PKCγ ne sont toujours pas connues. Utilisant des techniques d’électrophysiologie (enregistrements patch-clamp) et d'immunohistochimie sur tranches de Sp5C, nous avons caractérisé les propriétés des interneurones PKCγ de la couche IIi du Sp5C chez le rat adulte et comparé ces propriétés avec celles d’interneurones voisins n’exprimant pas la PKCγ.Cette étude révèle que l’arborisation neuritique des interneurones PKCγ s’étend largement au sein de la couche IIi, et peut se prolonger du coté dorsal jusqu’à la couche II externe, sans jamais atteindre la couche I. En outre, en fonction de cette extension neuritique, au moins deux sous-populations d'interneurones PKCγ peuvent être dissociées – centrales et radiales – qui s’avèrent être aussi fonctionnellement différentes. Comparés aux autres neurones non-PKCγ de la conche IIi, les interneurones PKCγ, dans leur ensemble, présentent un seuil de déclenchement des potentiels d’action plus bas et, souvent associée, plus fréquemment une réponse tonique à un courant dépolarisant, indiquant ainsi qu’ils sont plus facilement excitables. Cependant, ils reçoivent inversement une excitation synaptique plus faible. Quant aux afférences inhibitrices, la plupart des interneurones PKCγ expriment des synapses mixtes associant récepteurs GABAAergiques (GABAAR) et récepteurs glycinergiques (GlyR). Seul un petit nombre d’entre eux exprime des synapses uniquement GABAAR ou GlyR. Pourtant, tous les interneurones PKCγ reçoivent non seulement des mIPSCs mixtes GABAAR-GlyR, mais aussi des mIPSCs uniquement GABAAR ou uniquement GlyR. / Mechanical allodynia, a cardinal symptom of persistent pain, is associated with the unmasking of usually blocked local circuits within the superficial spinal or medullary dorsal horn (MDH), through which low-threshold mechanical inputs can gain access to the lamina I nociceptive output neurons. Key determinants of these circuits are lamina II (IIi) excitatory interneurons that selectively concentrate the gamma isoform of protein kinase C (PKCγ) and receive low-threshold mechanical receptor (LTMR) inputs. Tonic inhibition of PKCγ interneurons is thought to gate circuits underlying mechanical allodynia. However, the morphology, electrophysiological properties and excitatory and inhibitory synaptic inputs on these PKCγ interneurons are still unknown. Using whole-cell patch-clamp recordings and immunohistochemical techniques in slices of adult rat MDH, we characterized these lamina IIi PKCγ interneurons and compared them with neighboring non-PKCγ interneurons. Our results reveal that the neurites of PKCγ interneurons arborize extensively within lamina IIi, can spread dorsally into lamina IIo, but never reach lamina I. In addition, according to cell bodies and the orientation and extent of dendritic arbors, at least two morphologically different classes of PKCγ interneurons can be identified – central and radial – which appear to be also functionally different. Compared with neighboring lamina IIi non-PKCγ interneurons, PKCγ interneurons exhibit a lower threshold for action potentials, consistent with a more frequent tonic spike discharge to depolarizing step current, indicating that they are more excitable than other lamina IIi neurons. On the other hand, they receive a weaker excitatory synaptic drive. According to inhibitory inputs, most PKCγ interneurons display mixed-GABAA (GABAAR) and glycine (GlyR) receptor synapses with only very few of them displaying also GABAAR-alone or GlyR-alone synapses. Interestingly, all PKCγ interneurons exhibit mixed GABAAR–GlyR as well as GABAAR-only and GlyR-only mIPSCs. Altogether, this study indicates that PKCγ interneurons within lamina IIi of MDH are different from other lamina IIi neighboring neurons according to morphology, electrophysiological properties and synaptic inputs. This is consistent with their specific role in the gating of dorsally directed circuits within the MDH underlying mechanical allodynia. Moreover, we have identified two morphological and functional subclasses of PKCγ interneurons which might thus differently contribute to this gating.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Le paludisme constitue la première maladie parasitaire au monde, responsable d’environ 800 000 morts chaque année. Il peut entraîner plusieurs complications sévères aggravant le pronostique vital, et pouvant conduire à la mort. Dans ce contexte, l'objectif de ma thèse a été d'étudier, pour une majeur partie, la pathologie cérébrale et dans une moindre mesure la forme pulmonaire. Cette étude a plus particulièrement porté sur le rôle de certaines voies d’activation des lymphocytes T. Mes résultats ont mis en évidence la fonction primordiale, dans le développement du neuropaludisme, de trois molécules impliquées dans l’activation des cellules T : le récepteur LTβR, la protéine PKC-θ et la sous unité β2 du récepteur à l’IL-12. Grâce à l’utilisation d’un modèle murin délété en ces gènes, nous avons pu déterminer par des techniques non invasives (IRM, ARM) ou invasives (test de perméabilité vasculaire, cytométrie en flux) que ces souris étaient résistantes à la forme cérébrale. En revanche elles ne sont pas essentielles à la pathologie pulmonaire. / The malaria is the first parasitic pathology, responsible of 800 000 death a year. It can cause several severe complications which might deteriorating vital forecast and lead to the death. In this context, my thesis objective was to study, for major point, the cerebral pathology and in a lesser measure the lung pathology. This study concernes the role of some signaling pathways of T cells activation. My results show the involvement of three molecules in cerebral malaria development LTβR receptor, PKC-θ kinase and IL-12Rβ2 receptor subunit. With the use of knockout mice for these genes, we proved by non invasive approaches (MRI and MRA) or invasive approaches (vascular permeability test, flow cytometry) that these mice are resistant to cerebral malaria. By contrast they are not protected against lung pathology.

Plaquetas apresentam um papel importante no desenvolvimento de metastases tumorais. Os eventos que levam à ativação plaquetária, como agragação e secreção de proteínas, podem significar etapas importantes neste papel. O agonista plaquetário trombospondina está envolvido no processo de agragação plaquetária. Com o intuito de clonar o receptor de trombospondina GpIIIb, produziu-se um soro policlonal contra uma banda eluída de SDS PAGE de extrato proteico de plaquetas, que apresentava peso molecular igual ao de GpIIIb (denominada banda 80kD). Uma biblioteca de cDNA de endotélio de cordão umbilical humano construída em lambda gt11 foi varrida com este soro anti-80kD. Dois clones diferentes foram isolados, seus insertos foram subclonados no vetor pGEM-3Z e sequenciados. Através de consulta ao Genbank observou-se que um dos clones não apresentou homologia significativa com nenhuma proteína até então clonada. O outro clone, por sua vez, apresentou 100% de homologia com a proteína 80K-H, substrato de proteína quinase C. Levando em consideração o fato de que as vias detransdução de sinal que utilizam PKC apresentam extrema importância nos processos de ativação plaquetária decidiu-se prosseguir com a caracterização de 80K-H. Para isto foi produzido um soro policlonal contra a proteína de fusão 80K-H, que foi utilizado em ensaios bioquímicos e imunoquímicos que permitiram caracterizar a proteína 80K-H quanto a alguns aspectos como distribuição em diferentes tipos celulares, localização celular e fosforilação. Além de estar presente em plaquetas, a proteína 80K-H foi encontrada em todas as linhagens celulares testadas, parecendo portanto ser uma proteína ubíqua. Os dados obtidos indicaram que, apesar de apresentar uma sequência N-terminal que é clivada \"in vivo\" muito semelhante a um peptídeo sinal, 80K-H não é secretada nem é de membrana plasmática, mas sim citoplasmática. Em ensaios de fosforilação \"in vivo\" não se detectou fosforilação de 80K-H. Portanto, apesar de 80K-H ser um bom substrato para PKC \"in vitro\", ela não o é \"in vivo\", ao menos nas células analisadas, ou é fosforilada de uma forma extremamente rápida e transiente. / Abstract not available.

A O-glicosilação com N-acetil-glucosamina (O-GlcNAc) é uma modificação pós-translacional altamente dinâmica que modula diversas vias de sinalização. O processo de O-GlcNAc é controlado por duas enzimas: a enzima OGT é responsável por catalisar a adição de N-acetil-glucosamina no grupo hidroxila dos resíduos de serina e treonina, enquanto a OGA catalisa a remoção de O-GlcNAc das proteínas modificadas. Proteínas com importante papel na função vascular são alvo de O-GlcNAc e o aumento da expressão de proteínas modificadas por O-GlcNAc promove aumento da reatividade vascular para estímulos contráteis. Um dos mecanismos de extrema importância no controle do tônus vascular está ligado à regulação da concentração de cálcio (Ca2+) intracelular, onde destacamos a participação do sistema STIM1/Orai1. As moléculas de interação estromal (STIM) atuam como sensores dos estoques intracelulares de Ca2+ e as proteínas Orai representam as subunidades que formam os canais de Ca2+ ativados pela liberação de Ca2+ (CRAC). Neste estudo investigamos a hipótese de que o aumento dos níveis vasculares de proteínas glicosiladas aumenta a resposta contrátil em aorta de ratos, por mecanismos relacionados ao controle da concentração intracelular de Ca2+.Em nossos experimentos, utilizamos aortas torácicas de ratos incubadas com PugNAc (inibidor seletivo da OGA, ), por 24h. Utilizando protocolo experimental que permite avaliar contrações induzidas pelo influxo de Ca2+ e liberação de Ca2+ intracelular, demonstramos que a incubação com PugNAc aumentou a resposta contrátil à PE bem como a contração durante o período de influxo de Ca2+, induzida pela reintrodução de solução fisiológica contendo Ca2+ (1,56 mM). O bloqueio dos canais CRAC com 2-APB (100 ) e gadolíneo (Gd3+, 100 ) diminuiu significativamente as contrações induzidas pelo influxo de Ca2+ em aortas incubadas com PugNAc. Além disso, estas aortas apresentaram aumento da expressão protéica de STIM1, o que resultaria em maior influxo de Ca2+. A contração induzida por cafeína (20 mM) e serotonina (10 ), a qual reflete a capacidade funcional do retículo sarcoplasmático (RS) em captar Ca2+, foi maior em aortas incubadas com PugNAc. O papel da Ca2+-ATPase (SERCA) foi avaliado com a utilização de tapsigargina, bloqueador da SERCA. O efeito da tapsigargina foi semelhante em artérias incubadas com PugNAc e veículo, apesar do aumento de expressão proteica da SERCA em aortas incubadas com PugNAc. Como a proteína cinase C (PKC) é ativada por aumentos de Ca2+ intracelular, determinamos se a atividade de proteínas alvo da PKC estavam aumentadas. A incubação com PugNAc aumentou a expressão das formas fosforiladas da CPI-17, MYPT-1 e MLC. Em conjunto, estes resultados sugerem que a ativação de STIM1/Orai1, aumento da liberação de Ca2+ intracelular e ativação da via de sinalização da PKC podem representar mecanismos que modulam as alterações vasculares em resposta ao aumento de proteínas glicosiladas por O-GlcNAc. / Glycosylation with O-linked -N-acetyl-glucosamine (O-GlcNAc) is a highly dynamic post-translational modification. The process of O-GlcNAc is controlled by two enzymes: the OGT enzyme catalyses the addition of N-acetyl-glucosamine to the hydroxyl group of serine and threonine residues of a target protein, while OGA catalyzes the cleavage of O-GlcNAc from post-translationally-modified proteins. Proteins with an important role in vascular function are targets of O-GlcNAc and increased levels of proteins modified by O-GlcNAc increase vascular reactivity to contractile stimuli. The regulation of intracellular calcium (Ca2+) concentration, including the activation of STIM1/Orai1, is key in the control of vascular tone. The stromal interaction molecules (STIM) act as sensors of intracellular Ca2+ stores whereas the Orai proteins represent subunits of the Ca2+ release-activated Ca2+ channels (CRAC). We hypothesized that increased levels of vascular O-GlcNAc proteins augment vascular contractile responses by altering mechanisms that regulate the intracellular Ca2+. Rat thoracic aortas were incubated with PugNAc (OGA selective inhibitor, ) for 24h. Using an experimental protocol that evaluates contractions induced by Ca2+ influx and release, we demonstrated that incubation with PugNAc increases contractile responses to phenylephrine (PE) as well as the contraction induced by Ca2+ influx, after depletion of intracellular Ca2+ stores. The CRAC channel blockers, 2-APB (100 ) and gadolinium (Gd3+, 100 ), significantly reduced the contractions induced by Ca2+ influx in aortas incubated with PugNAc. Furthermore, these aortas showed increased STIM1 protein expression, which could result in increased influx of Ca2+ and, in turn, increase vascular contraction. The contraction induced by the release of intracellular Ca2+ stores, stimulated by caffeine (20 mM) and serotonin (10 ), was increased in aortas incubated with PugNAc. The Ca2+-ATPase (SERCA) inhibitor thapsigargin produced similar effects in arteries incubated with PugNAc or vehicle, despite the increased SERCA protein expression in aortas incubated with PugNAc. Since PKC is activated by increases in intracellular Ca2+ and arteries incubated with PugNAc show activation of PKC, we determined whether the activity of proteins that are targets of PKC was increased in PugNAc-treated aortas. Incubation with PugNAc increased the expression of phosphorylated forms of CPI-17, MYPT-1 and MLC. Together, these results suggest that activation of STIM1/Orai1, increased release of intracellular Ca2+ and PKC activation may represent mechanisms that modulate vascular responses upon increased O-GlcNAc proteins.

A O-glicosilação com N-acetil-glucosamina (O-GlcNAc) é uma modificação pós-translacional altamente dinâmica que modula diversas vias de sinalização. O processo de O-GlcNAc é controlado por duas enzimas: a enzima OGT é responsável por catalisar a adição de N-acetil-glucosamina no grupo hidroxila dos resíduos de serina e treonina, enquanto a OGA catalisa a remoção de O-GlcNAc das proteínas modificadas. Proteínas com importante papel na função vascular são alvo de O-GlcNAc e o aumento da expressão de proteínas modificadas por O-GlcNAc promove aumento da reatividade vascular para estímulos contráteis. Um dos mecanismos de extrema importância no controle do tônus vascular está ligado à regulação da concentração de cálcio (Ca2+) intracelular, onde destacamos a participação do sistema STIM1/Orai1. As moléculas de interação estromal (STIM) atuam como sensores dos estoques intracelulares de Ca2+ e as proteínas Orai representam as subunidades que formam os canais de Ca2+ ativados pela liberação de Ca2+ (CRAC). Neste estudo investigamos a hipótese de que o aumento dos níveis vasculares de proteínas glicosiladas aumenta a resposta contrátil em aorta de ratos, por mecanismos relacionados ao controle da concentração intracelular de Ca2+.Em nossos experimentos, utilizamos aortas torácicas de ratos incubadas com PugNAc (inibidor seletivo da OGA, ), por 24h. Utilizando protocolo experimental que permite avaliar contrações induzidas pelo influxo de Ca2+ e liberação de Ca2+ intracelular, demonstramos que a incubação com PugNAc aumentou a resposta contrátil à PE bem como a contração durante o período de influxo de Ca2+, induzida pela reintrodução de solução fisiológica contendo Ca2+ (1,56 mM). O bloqueio dos canais CRAC com 2-APB (100 ) e gadolíneo (Gd3+, 100 ) diminuiu significativamente as contrações induzidas pelo influxo de Ca2+ em aortas incubadas com PugNAc. Além disso, estas aortas apresentaram aumento da expressão protéica de STIM1, o que resultaria em maior influxo de Ca2+. A contração induzida por cafeína (20 mM) e serotonina (10 ), a qual reflete a capacidade funcional do retículo sarcoplasmático (RS) em captar Ca2+, foi maior em aortas incubadas com PugNAc. O papel da Ca2+-ATPase (SERCA) foi avaliado com a utilização de tapsigargina, bloqueador da SERCA. O efeito da tapsigargina foi semelhante em artérias incubadas com PugNAc e veículo, apesar do aumento de expressão proteica da SERCA em aortas incubadas com PugNAc. Como a proteína cinase C (PKC) é ativada por aumentos de Ca2+ intracelular, determinamos se a atividade de proteínas alvo da PKC estavam aumentadas. A incubação com PugNAc aumentou a expressão das formas fosforiladas da CPI-17, MYPT-1 e MLC. Em conjunto, estes resultados sugerem que a ativação de STIM1/Orai1, aumento da liberação de Ca2+ intracelular e ativação da via de sinalização da PKC podem representar mecanismos que modulam as alterações vasculares em resposta ao aumento de proteínas glicosiladas por O-GlcNAc. / Glycosylation with O-linked -N-acetyl-glucosamine (O-GlcNAc) is a highly dynamic post-translational modification. The process of O-GlcNAc is controlled by two enzymes: the OGT enzyme catalyses the addition of N-acetyl-glucosamine to the hydroxyl group of serine and threonine residues of a target protein, while OGA catalyzes the cleavage of O-GlcNAc from post-translationally-modified proteins. Proteins with an important role in vascular function are targets of O-GlcNAc and increased levels of proteins modified by O-GlcNAc increase vascular reactivity to contractile stimuli. The regulation of intracellular calcium (Ca2+) concentration, including the activation of STIM1/Orai1, is key in the control of vascular tone. The stromal interaction molecules (STIM) act as sensors of intracellular Ca2+ stores whereas the Orai proteins represent subunits of the Ca2+ release-activated Ca2+ channels (CRAC). We hypothesized that increased levels of vascular O-GlcNAc proteins augment vascular contractile responses by altering mechanisms that regulate the intracellular Ca2+. Rat thoracic aortas were incubated with PugNAc (OGA selective inhibitor, ) for 24h. Using an experimental protocol that evaluates contractions induced by Ca2+ influx and release, we demonstrated that incubation with PugNAc increases contractile responses to phenylephrine (PE) as well as the contraction induced by Ca2+ influx, after depletion of intracellular Ca2+ stores. The CRAC channel blockers, 2-APB (100 ) and gadolinium (Gd3+, 100 ), significantly reduced the contractions induced by Ca2+ influx in aortas incubated with PugNAc. Furthermore, these aortas showed increased STIM1 protein expression, which could result in increased influx of Ca2+ and, in turn, increase vascular contraction. The contraction induced by the release of intracellular Ca2+ stores, stimulated by caffeine (20 mM) and serotonin (10 ), was increased in aortas incubated with PugNAc. The Ca2+-ATPase (SERCA) inhibitor thapsigargin produced similar effects in arteries incubated with PugNAc or vehicle, despite the increased SERCA protein expression in aortas incubated with PugNAc. Since PKC is activated by increases in intracellular Ca2+ and arteries incubated with PugNAc show activation of PKC, we determined whether the activity of proteins that are targets of PKC was increased in PugNAc-treated aortas. Incubation with PugNAc increased the expression of phosphorylated forms of CPI-17, MYPT-1 and MLC. Together, these results suggest that activation of STIM1/Orai1, increased release of intracellular Ca2+ and PKC activation may represent mechanisms that modulate vascular responses upon increased O-GlcNAc proteins.

<p><em>Panax ginseng</em> is a traditional herb that has been used for over 2000 years to promote health and longevity. Active components of ginseng include ginsenosides, polysaccharides, flavonoids, polyacetylenes, peptides, vitamins, phenols and enzymes, of which the ginsenosides are considered to be the major bioactive constituents. Although widely used, the exact mechanisms of ginseng and its compounds remain unclear. In this thesis we use melanophores from <em>Xenopus laevis</em> to investigate the effects of <em>Panax ginseng</em> extract G115 and its constituents on organelle transport and signalling. Due to coordinated bidirectional movement of their pigmented granules (melanosomes), in response to defined chemical signals, melanophores are capable of fast colour changes and provide a great model for the study of intracellular transport. The movement is regulated by alterations in cyclic adenosine 3’:5’-monophosphate (cAMP) concentration, where a high or low level induce anterograde (dispersion) or retrograde (aggregation) transport respectively, resulting in a dark or light cell. Here we demonstrate that <em>Panax ginseng</em> and its constituents ginsenoside Rc and Rd and flavonoid quercetin induce a concentration-dependent anterograde transport of melanosomes. The effect of ginseng is shown to be independent of cAMP changes and protein kinase A activation. Upon incubation of melanophores with a combination of Rc or Rd and quercetin, a synergistic increase in anterograde movement was seen, indicating cooperation between the ginsenoside and flavonoid parts of ginseng. Protein kinase C (PKC) inhibitor Myristoylated EGF-R Fragment 651-658 decreased the anterograde movement stimulated by ginseng and ginsenoside Rc and Rd. Moreover, ginseng, but not ginsenosides or quercetin, stimulated an activation of 44/42-mitogen activated protein kinase (MAPK), previously shown to be involved in both aggregation and dispersion of melanosomes. PKC-inhibition did not affect the MAPK-activation, suggesting a role for PKC in the ginseng- and ginsenoside-induced dispersion but not as an upstream activator of MAPK.</p> / <p><em>Panax ginseng </em>är ett av de vanligaste naturläkemedlen i världen och används traditionellt för att öka kroppens uthållighet, motståndskraft och styrka. Ginseng är ett komplext ämne bestående av ett antal olika substanser, inklusive ginsenosider, flavonoider, vitaminer och enzymer, av vilka de steroidlika ginsenosiderna anses vara de mest aktiva beståndsdelarna. Flavonoider (som finns i till exempel frukt och grönsaker) och ginseng har genom forskning visat sig motverka bland annat hjärt-och kärlsjukdomar, diabetes, cancer och demens. Trots den omfattande användningen är dock mekanismen för hur ginseng verkar fortfarande oklar. I den här studien har vi använt pigmentinnehållande celler, melanoforer, från afrikansk klogroda för att undersöka effekterna av <em>Panax ginseng</em> på pigment-transport och dess maskineri. Melanoforer har förmågan att snabbt ändra färg genom samordnad förflyttning av pigmentkorn fram och tillbaka i cellen, och utgör en utmärkt modell för studier av intracellulär transport. Förflyttningen regleras av förändringar i halten av cykliskt adenosin-monofosfat (cAMP) i cellen, där en hög eller låg koncentration medför spridning av pigment över hela cellen (dispergering) eller en ansamling i mitten (aggregering), vilket resulterar i mörka respektive ljusa celler. Här visar vi att <em>Panax ginseng</em>, ginsenosiderna Rc och Rd samt flavonoiden quercetin stimulerar en dispergering av pigmentkornen. När melanoforerna inkuberades med en kombination av ginsenosid Rc eller Rd och quercetin, kunde en synergistisk ökning av dispergeringen ses, vilket tyder på en samverkan mellan ginsenosid- och flavonoid-delarna av ginseng. Ett protein som tidigare visats vara viktigt för pigmenttransporten är mitogen-aktiverat protein kinas (MAPK), och här visar vi att också melanoforer stimulerade med ginseng, men dock inte med ginsenosider eller quercetin, innehåller aktiverat MAPK. Genom att blockera enzymet protein kinas C (PKC) (känd aktivator av dispergering), minskade den ginseng- och ginsenosid-inducerade dispergeringen, medan aktiveringen av MAPK inte påverkades alls. Detta pekar på en roll för PKC i pigment-transporten men inte som en aktivator av MAPK.</p>