During development, neurons must find their way to and make connections with their appropriate targets. Growth cones are dynamic, motile structures that are integral to the establishment of appropriate connectivity during this wiring process. As growth cones migrate through their environment, they encounter guidance cues that direct their migration to their appropriate synaptic targets. The gaseous messenger nitric oxide (NO), which diffuses across the plasma membrane to act on intracellular targets, is a signaling molecule that affects growth cone motility. However, most studies have examined the effects of NO on growth cone morphology when applied in large concentrations and to entire cells. In addition, the intracellular second messenger cascade activated by NO to bring about these changes in growth cone morphology is not well understood. Therefore, this dissertation addresses the effects that a spatially- and temporally-restricted application of physiological amounts of NO can have on individual growth cone morphology, on the second messenger pathway that is activated by this application of NO, and on the calcium cascades that result and ultimately affect growth cone morphology. Helisoma trivolvis, a pond snail, is an excellent model system for this type of research because it has a well-defined nervous system and cultured neurons form large growth cones. In the present study, local application of NO to Helisoma trivolvis B5 neurons results in an increase in filopodial length, a decrease in filopodial number, and an increase in the intracellular calcium concentration ([Ca2+]i). In B5 neurons, the effects of NO on growth cone behavior and [Ca2+]i are mediated via sGC, protein kinase G, cyclic adenosine diphosphate ribose, and ryanodine receptor-mediated intracellular calcium release. This study demonstrates that neuronal growth cone pathfinding in vitro is affected by a single spatially- and temporally-restricted exposure to NO. Furthermore, NO acts via a second messenger cascade, resulting in a calcium increase that leads to cytoskeletal changes. These results suggest that NO may be a signal that promotes appropriate pathfinding and/or target recognition within the developing nervous system. Taken together, these data indicate that NO may be an important messenger during the development of the nervous system in vivo.

The KATP channels play an important role in the membrane excitability and vascular tone regulation. Previous studies indicate that the function of KATP channels is disrupted in oxidative stress seen in a variety of cardiovascular diseases, while the underlying mechanism remains unclear. Here, we demonstrate S-glutathionylation to be a modulation mechanism underlying the oxidant-mediated vascular KATP channel inhibition, the molecular basis for the channel inhibition and the alleviation of the channel inhibition by vasoactive intestinal peptide (VIP). We found that an exposure of isolated mesenteric rings to H2O2 impaired the KATP channel-mediated vascular dilation. In whole-cell recordings and inside-out patches, micromolar H2O2 or diamide caused a strong inhibition of the vascular KATP channel (Kir6.1/SUR2B) in the presence, but not in the absence, of glutathione (GSH), indicating S-glutathionylation. By co-expressions of Kir6.1 or Kir6.2 with SUR2B subunits, we found that the oxidant sensitivity of the KATP channel relied on the Kir6.1 subunit. Systematic mutational analysis revealed three cysteine residues (Cys43, Cys120 and Cys176) to be important. Among them, Cys176 was prominent, contributing to >80% oxidant sensitivity. Biochemical pull-down assay with biotinylated glutathione ethyl ester (BioGEE) showed that mutations of Cys176 impaired the oxidant-induced incorporation of GSH to the Kir6.1 subunit. Simulation modeling of Kir6.1 S-glutathionylation revealed that after incorporation to residue 176, the GSH moiety occupied a space between slide helix and two transmembrane helices. This prevented the necessary conformational change of the inner helix for channel gating, and retained the channel in its closed state. VIP is a potent vasodilator, and is shown to have protective role against oxidative stress. We found that the channel was strongly augmented by VIP and the channel activation relied on PKA phosphorylation. These results therefore indicate that 1) the vascular KATP channel is strongly inhibited in oxidative stress, 2) S-glutathionylation underlies the oxidant-mediated KATP channel inhibition, 3) Cys176 in the Kir6.1 subunit is the major site for S-glutathionylation, and 4) the Kir6.1/SUR2B channel is activated in a PKA-dependent manner by VIP that has been previously shown to alleviate oxidative stress.

La Proteína Quinasa C (PKC) juega un papel fundamental en la regulación del crecimiento celular. Estas proteínas están implicadas en diferentes vías intracelulares que son consideradas como dianas para el tratamiento contra el cáncer. Atendiendo a las propiedades enzimáticas, las PKC se clasifican en tres grandes subfamilias: clásicas, nuevas y atípicas. En las PKC clásicas, el dominio C2 es un motivo regulador que responde a señales de Ca2+ intracelulares. Este dominio presenta un motivo denominado región rica en lisinas que interacciona con fosfolípidos acídicos. Los resultados de esta tesis demuestran que la afinidad de este dominio por fosfolípidos como el PIP2 es mayor frente a otros de la misma naturaleza. El dominio C2 de las PKC nuevas se une a fosfolípidos cargados negativamente de modo Ca2+-independiente. Nuestro estudio demuestra que la interacción de este dominio con las membranas es principalmente electrostática con una pequeña contribución de interacciones hidrofóbicas. Por otra parte, el estudio de la estructura secundaria del dominio catalítico de la PKC mostró una elevada proporción de hélice . La adición de Mg2+-ATP provocó un mayor efecto protector frente a la desnaturalización térmica. / Protein kinase C (PKC) is a family of related protein kinases that plays an important role in regulating cell growth. These protein kinases are involved in several intracellular pathways that end in transcription and are considered to be potential targets for anticancer therapy. The mammalian isoenzymes have been grouped into three subfamilies according to their enzymatic properties: classical, novel and atypical.The C2 domain is a regulatory sequence motif and is a targeting domain that responds to intracellular Ca2+ signals in classical protein kinases. This domain presents a motif named the lysine-rich cluster that interacts with acidic phospholipids. The results demonstrate that PIP2 interacts with the C2 domain of PKCα in a different way to that described for other phospolipids.C2 domain in novel PKC binds to negatively charged phospholipid vesicles in a Ca2+-independent manner. Our study confirms that the main way in which C2-PKC interacts with membranes is electrostatic in nature, with a very small contribution on the part of hydrophobic interactions.The secondary structure of catalytic domain from atypical PKC showed a high contribution of -helix component. In addition, Mg2+-ATP significantly altered the denaturation pattern of this domain because it protected against denaturation.

En esta Tesis Doctoral se ha estudiado el efecto de una serie de ácidos grasos como el araquidónico, docosahexaenoico, eicosapentaenoico y oleico, así como un conjunto de derivados de DAG-lactonas en la activación de diferentes isoenzimas de PKC. El sistema modelo de estudio utilizado han sido diferentes líneas celulares de cáncer de mama (BT-474, MCF-7 y MDA-MB-231). Mediante técnicas de biología molecular y celular, microscopía confocal, ARN de interferencia y microarrays de expresión diferencial de genes se ha estudiado la función de la PKCalpha en la capacidad proliferativa, invasiva y de migración de las células modelo de cáncer de mama, revelando nuevos mecanismos moleculares por los que la PKCalpha se localiza en la membrana plasmática y se activa en dichas células. También se pone de manifiesto el efecto antiproliferativo e inductor de apoptosis de los diversos ácidos grasos estudiados, así como la implicación directa de la PKCalpha en la capacidad proliferativa, migr atoria e invasiva de dichas células / In this Doctoral Thesis, the role of several fatty acids like arachidonic, docosahexaenoic, eicosapentaenoic and oleic, as well as some DAG-lactones derivatives, on the activation of different PKC isoforms has been studied. Some breast cancer cell lines, specifically BT-474, MCF-7 and MDA-MB-231, have been used as a model. The role of PKC in proliferation, invasion and migration has been studied by means of cellular and molecular techniques, confocal microscopy, siRNA and gene expression microarrays. The results obtained reveal new molecular mechanisms of PKCalpha; localization and activation in breast cancer cell lines. It is also interesting the anti-proliferative and pro-apoptosis role of several fatty acids tested, as well as the direct involvement of PKCalpha; in proliferation, migration and invasion of breast cancer cells

Multiple modulating genes and environmental factors have been implicated in the pathogenesis of high-altitude pulmonary edema (HAPE). However, at the present time, there exists an incomplete understanding of the molecular mechanisms and pathways which underlie constitutional susceptibility. Genome-wide measurements of gene expression in peripheral blood mononuclear cells (PBMCs) were performed using microarray technology. Comparison of gene expression profiles of HAPE-susceptible and resistant individuals resulted in the identification of several previously undescribed candidate genes. RhoA and Rho-kinase (ROCK), regulators of vascular smooth muscle contraction, were differentially regulated in the HAPE-susceptible cohort, as compared to both HAPE-resistant patients with acute mountain sickness (AMS+) and healthy controls (p=0.0014; p=0.0020). Furthermore, biological pathways involving RhoA and Rho-kinase were strongly upregulated in subjects with HAPE. These findings represent the first description of the RhoA/Rho-kinase signaling pathway in HAPE. Currently, few pharmacologic therapies have been demonstrated to be effective in the prevention and treatment of HAPE. The results of this study provide early evidence that Fasudil, a selective Rho-kinase inhibitor, may represent a novel therapeutic intervention effective in the prevention and/or treatment of high-altitude pulmonary edema.

Σκοπός της παρούσας διατριβής ήταν η μελέτη των σηματοδοτικών μονοπατιών που εμπλέκονται στην επαγόμενη από IL-1β παραγωγή της MMP-1, μιας από τις κυριότερες μεταλλοπρωτεϊνάσες του εξωκυτταρικού χώρου, από ινοβλάστες ανθρώπου και η διερεύνηση του μηχανισμού της κατασταλτικής επίδρασης του TGF-β1 στην επαγόμενη από IL-1β παραγωγή της μεταλλοπρωτεϊνάσης αυτής. Η μελέτη πραγματοποιήθηκε σε ινοβλάστες αρθρικού υμένα ασθενών με οστεοαρθρίτιδα ή ρευματοειδή αρθρίτιδα, ρινικού πολύποδα και πνεύμονα. Βρέθηκε ότι η επαγωγική δράση της IL-1 πραγματοποιείται μέσω του μονοπατιού της PKC, ενώ σημαντικό ρόλο φαίνεται να παίζουν η ενεργοποίηση του NF-kB, αλλά και των μεταγραφικών παραγόντων της οικογένειας AP-1, μέσω των MAP κινασών. Το μονοπάτι, επίσης των κινασών τυροσίνης φαίνεται να συμμετέχει. Τα μονοπάτια αυτά φαίνεται ότι είναι κοινά στις ινοβλάστες, ανεξάρτητα από την προέλευση. Η δράση αυτή της IL-1β βρέθηκε ότι καταστέλλεται από το μονοπάτι cAMP/PKA στις ινοβλάστες αρθρικού υμένα και ρινικού πολύποδα όχι όμως και πνεύμονα. Ο TGF-β1 βρέθηκε ότι στις ινοβλάστες αρθρικού υμένα και ρινιού πολύποδα είχε κατασταλτική επίδραση στην επαγόμενη από IL-1β παραγωγή της MMP-1 ενώ σε αυτές από πνεύμονα είχε συνεργειακή. Η κατασταλτική δράση του TGF-β1 βρέθηκε ότι πραγματοποιείται μέσω ενεργοποίησης της PKA, κατά τρόπο ανεξάρτητο της παραγωγής cAMP. / The aim of present work was the study of signal tranduction pathways, which are implicated in IL-1β-induced production of MMP-1, one from the predominant metalloproteinases of extacellular matrix, from human fibroblasts, and the investigation of mechanism of suppressive effect of TGF-β1 on IL-1β-induced production of this metalloproteinases. The study was performed in fibroblasts with osteoarthrits or rheumatoid arthritis, nasal polyps and lung. It was found that the IL-1β-induced production of MMP-1 from fibroblasts, independently of their origin, is carried out via PKC pathway, while the activation of transcription factors NF-kB and AP-1, via MAP kinases, seems to play significant role. The tyrosine kinases pathway may also contributes. The IL-1β effect is suppressed from the cAMP/PKA pathway and nasal polyps fibroblasts but not in lung fibroblasts. TGF-β1 is able to antagonize the IL-1β-induced production of MMP-1 from synovial and nasal polyps fibroblasts, while in lung fibroblasts it exhibits synergistic effect. This suppressive effect of TGF-β1 is carried out via PKA activation, independently of cAMP production.

No description available.

No description available.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

La connexine 43 (Cx43) est l’unité protéique de base dans la formation des canaux des jonctions gap (JG) responsables des échanges intercellulaires. Toutefois, elle forme aussi des canaux non-jonctionnels à large conductance, nommés hémicanaux (Hc), qui fournissent un accès entre l’intérieure des cellules et le milieu extracellulaire. Bien qu’ils soient beaucoup moins étudiés que les JG, on estime que les Hc restent normalement à l’état fermé, et ce, grâce à la phosphorylation des connexines qui les forment. Suite à un stress ischémique, les Cx43 se déphosphorylent et entraînent ainsi l’ouverture des Hc de Cx43 (HcCx43), un effet qui compromet la survie des cellules. La protéine kinase C (PKC) est l’enzyme de phosphorylation qui possède le plus grand nombre de sites de phosphorylation sur la Cx43 en comparaison avec les autres kinases. Ses fonctions dépendent de la mise en jeu d’un répertoire d’au moins 12 isoformes distinctes. Dans les cardiomyocytes, les isoformes de PKC participent au développement des réponses adaptées ou mésadaptées au stress ischémique. Malgré que la régulation des canaux de Cx43 par la PKC lors d’une ischémie soit bien documentée, il n’existe pas à l’heure actuelle de connaissances sur les effets fonctionnels spécifiques qu’exercent des différentes isoformes de PKC sur les HcCx43, ni sur la valeur thérapeutique de la modulation de ses derniers. Dans ce contexte, nous avons proposé que les HcCx43 sont régulés sélectivement et différentiellement par les différentes isoformes de PKC et que l’inhibition spécifique de ces hémicanaux peut protéger le coeur lors d’un événement ischémique. Le présent travail comporte trois études qui ont été entreprises spécialement dans le but de valider ces hypothèses. Dans la première étude, nous avons profité de l’expertise du laboratoire du Dr Baroudi dans la dissection des isoformes de PKC pour étudier le rôle fonctionnel de chacune d’elles dans la régulation des HcCx43 en utilisant une gamme unique de peptides synthétiques inhibiteurs et activateurs spécifiques des isoformes de PKC, en combinaison avec la technique du patch-clamp. Nous avons démontré, entre autre, que les HcCx43 sont particulièrement inhibés par l’isoforme PKC epsilon, connue pour son effet cardioprotecteur contre les dommages ischémiques lors d’un préconditionnement ischémique. Dans la deuxième étude, nous avons caractérisé l’effet d’un peptide synthétique mimétique structural de la Cx43 sur la fonction des HcCx43. En plus d’avoir élucidé ces effets sur les propriétés fonctionnelles du canal, nous avons démontré d’une manière directe et indéniable que le peptide Gap26 inhibe et spécifiquement les HcCx43 et que son administration in vitro (cardiomyocytes isolés) et ex vivo (coeur intact) confère à ces modèles expérimentaux une résistance importante contre le stress ischémique. Dans la troisième étude, nous avons investigué pour la première fois in vivo le potentiel de deux peptides uniques mimétiques structuraux de la Cx43, Gap26 et Gap27, dans la cardioprotection contre les lésions ischémiques lorsqu’ils sont administrés à basse dose sous forme d’un bolus intraveineux unique. Nous avons démontré que l’injection de ces peptides avant ou après la survenue de l’ischémie réduit significativement la taille de l’infarctus qui en résulte.En conclusion, l’ensemble de ces résultats révèlent le rôle bénéfique de l’inhibition des HcCx43 lors d’une ischémie et dévoilent un potentiel thérapeutique prometteux des mimétiques structuraux de Cx43 dans la prévention et le traitement de l’infarctus du myocarde. / Connexin 43 (Cx43) is the basic unit in the composition of Gap junction channels but also of the non-junctional unapposed hemichannels (Hc). Gap junction channels play key roles in cardiac function by allowing conduction of electrical impulses and exchange of biologically important molecules between cells. The unapposed Hc, however, perform functions different from those achieved by Gap junction channels mainly by providing pathways between the cytosol and the extracellular space allowing movement of ions and other small metabolites. Although they are much less studied than Gap junction channels, Hc are believed to remain normally in a closed state and that phosphorylation is an important factor promoting their closure. Under ischemic stress,the amount of non-phosphorylated Cx43 increases resulting in increasing hemichannels opening, an effect that can lead to irreversible tissue injury and cell death. Protein kinase C (PKC) possesses the largest number of phosphorylation sites on Cx43 and exerts significant control on Cx43 channels. Its function depends on the involvement of at least 12 distinct isoformes. Various PKC isoforms exert specific cellular and cardiovascular functions, nonetheless the functional role of PKC isoforms in the modulation of the unapposed Cx43 hemichannels has never been assessed, neither has the therapeutic potential of Cx43Hc modulation in the protection of ischemic heart. In this context, three studies have been performed, they form the body of this thesis. In the first study, a unique set of synthetic PKC isoform-selective activator and inhibitor peptides was utilised. In combination with the patch clamp technique, we have demonstrated that Cx43Hc conductance is strongly inhibited by, among many isoforms, epsilon PKC isoforme, known for its cardioprotective effect against ischemic injury. In the second study, we characterized the effect of a synthetic structural mimetic peptide of Cx43. Using patch clamp technique, we have demonstrated that the peptide Gap26 inhibits directly and specifically Cx43Hc, we also showed that Gap26 can confer resistance to cardiomyocytes (in vitro) and intact heart (ex vivo) against ischemia. In the third study, we investigated for the first time in vivo the capability of a unique pair of structural Cx43 mimetic peptides, Gap26 and Gap27, to protect heart from ischemic injury when administered in single low-dose intravenous boluses. We demonstrated that administration of either one or both peptides, before or after the onset of ischemia renders heart more resistant to ischemia and reduces significantly the size of myocardial infarct. Altogether, our results revealed salvatory effect of Cx43Hc inhibition during ischemia and uncovered therapeutique potentials of the synthetic structural mimetic peptides of Cx43 in ischemic heart disease.