Au cours de ces années de Doctorat, nous avons étudié les effets ototoxiques de certains médicaments et les moyens de prévenir les surdités neuro-sensorielles quils peuvent induire. Parmi ces molécules, nous nous sommes concentrés sur les plus couramment utilisées en pratique clinique : les antibiotiques de la famille des aminoglycosides et le cisplatine, un agent anti-cancéreux. Lintroduction de notre travail replace la surdité dans son contexte de santé publique. En particulier, nous décrivons pourquoi les médicaments ototoxiques sont utilisés et dans quelles circonstances. Nous présentons la structure de loreille interne et nous tentons dexpliquer sa vulnérabilité aux molécules ototoxiques. Nous abordons ensuite les moyens de prévention et/ou de traitement de ces atteintes neuro-sensorielles pharmaco-induites. Outre les moyens classiques de prévention, que sont les facteurs trophiques et les antioxydants, nous décrivons de nouvelles voies dapproche que sont les voies de signalisation impliquant la protéine kinase C ou la cascade dactivation RhoA/ROCK. La présentation de notre travail original sarticule autour de deux parties. Dans la première partie, nous rapportons les résultats obtenus au cours de notre étude de la toxicité des aminoglycosides et du cisplatine chez la souris et le cobaye in vivo. Nous avons mis en évidence une différence de vulnérabilité significative entre ces deux espèces face à lagression ototoxique. Cette différence existe au niveau fonctionnel, mis en évidence par létude des potentiels évoqués auditifs, et au niveau anatomique, étudié en histologie et en immunohistochimie. Nous en discutons les implications en recherche et en pratique clinique. Dans la seconde partie, nous étudions les moyens de prévenir cette surdité in vivo et in vitro. Nous avons utilisé un modèle de surdité par aminoglycoside chez le cobaye. Nous avons testé et validé une technique de perfusion intra-cochléaire in vivo. Nous avons observé les effets de deux molécules expérimentales : la Bryostatine 1, un activateur de la protéine kinase C, et un inhibiteur de la voir RhoA-ROCK. Leffet protecteur de ces molécules est actuellement limité au ganglion spiral, dont la survie est essentielle à tout traitement dimplantation prothétique et de réadaptation. Nous discutons des perspectives en médecine humaine dans notre conclusion. In this work, we focused our attention on the effects of main ototoxic drugs i.e. aminoglycosides and cisplatin in mammals. We identified new avenues for the prevention of this toxicity. In the introduction, we described how and why ototoxic drugs are used. We then described potential otoprotective strategies in neurosensory deafness. Among them, trophic factors and antioxidant molecules have been widely used. New otoprotective approaches do exist, implying the protein kinase C or RhoA/ROCK signalling. Our original work was presented in two parts. In the first part, we reported the in vivo effects of aminoglycosides and cisplatin in two mammalian species: mice and guinea pigs. Contrarily to guinea pigs, evidence of mice resistance to ototoxicity was found at a functional level, assessed by auditory brainstem responses, and at an anatomical level, studied by immunohistochemistry. We discussed the implication of such differences in research and in clinical practice. In the second part, we studied the effect of two potential otoprotective molecules: Bryostatine 1, an activator of the protein kinase C, and Y-27632, a Rho kinase inhibitor. We showed that these molecules are protecting spiral ganglion neurons both in vitro and in vivo. Survival of spiral ganglion neurons is crucial in the management and rehabilitation of deafness. The potential perspectives of these results in human medicine were discussed.

Malignant pleural mesothelioma (MPM) is an aggressive, treatment-resistant tumour. Cisplatin (cis-diamminedichloroplatinum (II)) is the best single-agent chemotherapy for MPM, but platinum-based combination therapies give the best overall response rates. However, cisplatin use is limited by resistance and severe side effects. This thesis has increased the knowledge concerning cisplatin-induced cell death in MPM by describing a novel potential therapeutic target, and three novel phenotypes of cisplatin-resistance in a human MPM cell line (P31) and its cisplatin-resistant sub-line (P31res1.2). The novel potential therapeutic target, and one of the novel phenotypes, was cisplatin-resistant pro-apoptotic BH3-only proteins. In the P31 cells, cisplatin transiently increased pro-apoptotic BH3-only proteins during 6 h of exposure. This response was almost completely abrogated in the P31res1.2 cells. De-regulated caspase activity and activation was the second novel phenotype identified. The P31res1.2 cells had earlier, possibly mitochondria-independent, caspase-3 activation, increased basal caspase-3 activity and increased basal cleavage of caspase-8 and -9. Despite these differences, 6-h equitoxic cisplatin exposures rendered 50-60% of the cells apoptotic in both cell lines. The third novel phenotype was abrogated Na+K+2Cl--cotransporter (NKCC1) activity. Although NKCC1 activity was dispensable for cisplatin-induced apoptosis, balanced potassium transport activity was essential for P31 cell survival. Finally, the survival signalling protein Protein Kinase B (PKB or Akt) isoforms α and γ were constitutively activated in a PI3K-independent manner in P31 cells. In the P31res1.2 cells, PKBα and γ activities were increased, and there was PI3K-dependent activation of PKBβ. However, this increase in PKB isoform activity was not strongly associated to the cisplatin-resistance of the P31res1.2 cells.

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.

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