ABSTRACT Chronic activation of the sympathetic nervous system, via β-adrenoreceptor (AR) stimulation, contributes toward progressive heart failure. However, in this regard there are some outstanding issues which require clarity. First, in addition to contributing toward progressive heart failure, it is not clear whether chronic β-AR activation can also initiate cardiac decompensation. If so, the mechanisms of this effect also need to be determined. Second, the role of functional variants of β-AR genes as determinants of either the development or progression of heart failure requires elucidation. Moreover, whether there is any practical value in genotyping of patients for these variants has yet to be determined. These questions were addressed in the present thesis. With respect to the question of whether chronic β-AR activation initiates cardiac decompensation, the mechanisms responsible for the transition from compensated cardiac hypertrophy to heart failure in pressure overload states, such as hypertension, are uncertain. In this thesis I explored whether chronic sympathetic nervous system activation, produced by daily administration of a β-AR agonist, could promote the transition to cardiac pump failure in spontaneously hypertensive rats (SHR) with compensated cardiac hypertrophy. After 5 months of daily administration of a β-AR agonist, SHR developed marked left ventricular pump dysfunction, whereas normotensive control rats maintained pump function. The pump dysfunction noted in SHR was attributed to marked chamber dilatation with wall thinning, whilst myocardial contractile function appeared to be intact. The changes in cardiac structure and function noted after chronic β-AR activation in SHR were similar to those noted in SHR with advanced heart failure. These data provided the first evidence to indicate that chronic β- AR activation can promote the transition to decompensated cardiac hypertrophy in pressure overload states, and that this effect is principally mediated by adverse structural remodeling of the cardiac chamber. iii The mechanisms responsible for the effect of chronic β-AR activation on cardiac chamber dilatation were subsequently studied. The identified mechanisms included activation of an enzyme that degrades myocardial collagen (matrix metalloproteinase 2) and an increase of myocardial collagen of the type that is susceptible to collagen degradation (non-cross-linked collagen). I also excluded alternative potential mechanisms such as necrosis, apoptosis and an accumulation of type III collagen. However, previous studies have indicated that increases in myocardial collagen concentrations determine myocardial stiffness and not cardiac chamber dilatation. Hence, I performed a study to examine whether the impact of increases in myocardial collagen concentrations on cardiac structure and function depends on the qualitative changes in myocardial collagen. Indeed, using a variety of models of pressure overload hypertrophy associated with increases in myocardial collagen concentrations, I was able to provide evidence to support the theory that increases in myocardial collagen of the cross-linked phenotype will promote myocardial stiffness, whereas increase in myocardial collagen of the non-cross-linked phenotype promotes cardiac dilatation. With respect to the question of whether functional variants of β-AR genes contribute toward either the development or progression of heart failure, I studied the role of both functional β1-AR and β2-AR (together with a α2C-AR) gene variants in black South Africans with idiopathic dilated cardiomyopathy (IDC). In a prospective study I obtained data to indicate that the relationship between functional β2-AR genotypes and the progression to hospitalization, death or transplantation; a reduced exercise capacity, and left ventricular functional responses to b-blocker therapy, as described by other groups, is unlikely to be attributed to an independent effect of genotype on cardiac chamber dimensions and pump function. Moreover, I was able to show that contrary to what had previously been suggested, genotyping black subjects for functional α2C-AR iv and β1-AR gene variants is of little use when predicting the development or severity of IDC in this population group.

Th ocular bilateral hypotensive effect after unilateral topical administration of medetomidine and 4 analogs was demonstrated in a dose-response study (0.5%-2%) in NZW rabbits (bilateral IOP-lowering efficacy: medetomidine>detomidine and MPV-1440>MPV-1441 and MPV-305BIII).

Biologically controlled mineralization implies that organisms devote a part of their physiological activity to build up a specific mineralized skeleton. A preliminary comprehensive general view of the morphology and physiology of a given organism is therefore required before trying to understand where and how its biomineralizing system functions. Furthermore, the entire biomineralization sequence is not mediated by purely inorganic mineralogical rules but rather by a cellular machinery. Accordingly, a mineralogical characterization should be linked to a histological and cytological investigation of mineralizing cells to understand how a skeleton is produced. In the present thesis, we developed such a multi-disciplinary approach of some biomineralization processes of the massive basal skeleton in a few Recent hypercalcified sponges, likely survivors from Palaeozoic and early Mesozoic seas.<p>The three first chapters of this thesis are dedicated to the Mediterranean Calcarea Petrobiona massiliana, a conveniently accessible living hypercalcified sponge whereas all other Recent hypercalcified species are tropical and less easily reached. This model species permitted an initial morphological approach followed by an integrated biological and mineralogical study of biomineralization mechanisms. The fourth chapter aims at the comparative mineralogical study of the basal skeleton of eight tropical Recent hypercalcified demonsponges.<p>In the first chapter, important modifications and/or morphogenesis at the tissular or cellular level in response to life cycle phases and environmental conditions were depicted in specimens of Petrobiona massiliana. A survey of “storage cells” filling trabecular tracts, which are specific to P. massiliana, suggested that these cells may provide energy and a pool of toti- or pluripotent cells able to restructure the aquiferous system and repopulate cell types like pinacocytes. This potentiality of "storage cells would allow the sponge to sustain important physiological activities, like calcification, along its life cycle. Furthermore, basopinacocytes, cells delineating basally the soft tissue from the underlying basal skeleton, were identified through ultrastructural observations as the most probable cell type involved in the formation of the basal skeleton.<p>In the second chapter, the skeleton was found to be composed of ca. 50 to 100 nm crystallized grains as the smallest skeletal units, likely initially deposited in a mushy amorphous state. TEM and SEM observations further highlighted that these submicronic grains were assembled in clusters or fibres, the later even laterally associated into bundles. A model of crystallization propagation through amorphous submicronic granular units is proposed to explain the single-crystal feature of these micron-scale structural units, as demonstrated by selected area electron diffraction (SAED) in TEM. Finally, these units were assembled into a defined microstructure forming flattened growth layers called "sclerodermites", which superposed to produce the massive basal skeleton. In addition, X-ray diffraction (XRD) and energy electron loss spectroscopy (EELS) analyses highlighted respectively heterogeneous concentration and spatial distribution of Mg and Ca ions in the skeleton and structural units. This characterization highlighted mineralogical features, not conforming to the inorganic principles, and presuming a highly biologically regulated construction of the basal skeleton.<p>Accordingly, in the third chapter, it arose that the endomembrane system of basopinacocytes might play a dual function in the production and transport of both mineralizing ions and organic matrices. Combining partial decalcification methods with histochemical dyes and observing ultra-thin sections of the mature basal skeleton in TEM, very spatially and functionally diverse organic matrix components were found to occur in growing and mature portions of the skeleton. The following model of biomineralization was proposed for Petrobiona massiliana: basopinacocytes would use the endomembrane system pathway to produce and carry organic-coated submicronic amorphous grains in a mushy state within intracellular vesicles. These would then be released through the basal cell membrane toward the growing layer of the skeleton, where a highly structured gel-like organic framework, rich in sulfated/acidic GAGs-rich macromolecules, secreted by basopinacocytes, would ensure their assemblage into oriented fibres or clusters.<p>In the fourth chapter, the basal skeleton of eight tropical Recent hypercalcified species belonging to demosponges: Acanthochaetetes wellsi, Willardia caicosensis, Astrosclera willeyana, Ceratoporella nicholsoni, Goreauiella auriculata, Hispidopetra miniana, Stromatospongia norae and Calcifibrospongia actinostromarioides, were compared. Some mineralogical nano- to submicronic patterns already observed in the Calcarea P. massiliana, appeared as general features: the occurrence of submicronic granular units, their coherent assemblage into monocrystalline fibres and bundles and the likely presence of organic material around all structural units. Additional features brought new insights in our comprehension of biomineralization mechanisms in hypercalcified sponges. Among them, micro-twin and stacking-fault planes aligned with the fibres/bundles axis and crossing over submicronic granular units characterized the skeleton of most aragonitic species. This highly supports the crystallization propagation model proposed for P. massiliana, although it additionally suggests that it should occur only after the oriented assemblage of submicronic grains. Furthermore, lighter transverse striations separated by few nanometres occurred systematically in fibres and bundles of the eight basal skeletons investigated, suggesting the involvement of nanoscale intracrystalline fibrils in the biological control.<p>In conclusion, this comparative study of nine Recent hypercalcified sponges belonging to phylogenetically distant taxa resulted in the proposition of a shared biomineralization model based on the production of micron and submicron-scale structural units to build up macro-scale basal skeletons under a high biological control. We suggest that the cellular toolkit used for the biologically controlled biomineralization in these sponges is very ancient<p>and was already developed by their early Palaeozoic ancestors. Furthermore, this model supports recent concepts of calcium carbonate biomineralization developed for example in corals, molluscs and echinoderms, suggesting an even more universal and ancestral character of initial biomineralization mechanisms in all Metazoa producing a calcium carbonate skeleton.<p><p>La minéralisation biologiquement contrôlée implique qu’un organisme consacre une partie de son activité physiologique à l'élaboration de son squelette. La connaissance de sa morphologie et de sa physiologie est donc une étape préliminaire indispensable pour comprendre les mécanismes de formation de celui-ci. L’entièreté du processus de biominéralisation ne dépend pas simplement de principes fondamentaux issus de la minéralogie inorganique mais aussi de mécanismes cellulaires particuliers. La caractérisation minéralogique d'un squelette devrait donc être systématiquement liée à une étude histologique et cytologique des cellules impliquées dans la formation du biominéral. La thèse présentée ici a suivi une telle approche multidisciplinaire de certains mécanismes de biominéralisation du squelette basal de plusieurs éponges hypercalcifiées actuelles, considérées comme reliques d'espèces plus anciennes du Paléozoïque et Mésozoïque.<p>Les trois premiers chapitres de cette thèse concernent l'espèce calcaire de Méditerranée, Petrobiona massiliana, une éponge hypercalcifiée actuelle plus accessible que d'autres principalement distribuées dans les mers tropicales. Une approche de sa morphologie générale a été réalisée en préliminaire à une étude de ses mécanismes de biominéralisation, intégrant une caractérisation minéralogique et biologique. Le quatrième chapitre compare d’un point de vue minéralogique le squelette basal de huit autres espèces hypercalcifiées tropicales appartenant aux démosponges.<p>Au cours du premier chapitre, d'importantes modifications morphogénétiques à l'échelle tissulaire et cellulaire, liées à certaines phases du cycle biologique et aux conditions environnementales, ont ainsi été mises en évidence chez Petrobiona massiliana. Par l'observation de modifications de l'organisation et de l'ultrastructure des cellules de réserves remplissant les cordons trabéculaires, structures spécifiques de l'espèce, un rôle dans l'approvisionnement nutritif des cellules de l'éponge ainsi qu'un caractère toti- ou pluripotent leur ont été conférés. Les fonctions potentielles de ces cellules dites de réserves pourraient permettre à l'éponge de maintenir des activités physiologiques importantes, telles que la calcification, au cours de son cycle vital. Finalement, l'analyse ultrastructurale des tissus de P. massiliana a permis d'identifier les basopinacocytes, cellules délimitant les tissus mous du squelette basal, comme le type cellulaire ayant le plus de probabilité d'être impliqué dans la formation de ce dernier.<p>Dans le deuxième chapitre, des granules de 50 à 100 nm de diamètre se sont avérés les plus petites unités structurales du squelette basal de Petrobiona massiliana, probablement déposées initialement dans un état amorphe à consistance molle. Des observations en MEB et MET ont mis en évidence l'assemblage de ces granules en amas ou fibres, ces dernières étant elles-mêmes latéralement associées en faisceaux. Un modèle impliquant la propagation de la<p>cristallisation au travers de ces assemblages de granules submicroniques a été établi pour expliquer le caractère monocristallin des unités microstructurales, démontré par diffraction électronique en MET. Leur assemblage en une microstructure particulière produisant des couches\ / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Introduction: The discovery of the endothelium as a regulator of vascular tone, and the subsequent discovery of nitric oxide (NO) as the major endothelium-derived relaxing factor (EDRF), has opened up vast possibilities in the continued efforts to prevent and manage cardiovascular disease. Endothelial dysfunction (ED) is defined as reduced NO bioavailability and hence the reduced ability of the endothelium to maintain vascular homeostasis. ED represents the first, reversible step in the initiation of atherosclerotic disease and is thus regarded as a strong predictive tool of ischaemic heart disease (IHD). ED and its underlying mechanisms have been largely under-investigated in myocardial capillary-derived endothelial cells (cardiac microvascular endothelial cells, CMECs), and this study aimed to address this gap in the literature. Oleanolic acid (OA) is a bioactive triterpenoid derived from leaf extracts of African medicinal plants such as Syzigium cordatum (Water berry tree), and has been reported to elicit vasodilatory, hypoglycaemic and hypolipidaemic properties. However its effects particularly on CMECs and its putative role in reversing ED remain unclear, and this study aimed to investigate such effects. Aims: The aims of this study were to: (1) Establish an in vitro model of ED in cultured myocardial capillary-derived CMECs by developing protocols for the induction of ED. (2) Asses ED induction by measurement of the following biomarkers: (i) intracellular NO production, (ii) superoxide (O2-) production, (iii) nitrotyrosine expression and (iv) NADPH oxidase expression. (3) Investigate underlying cellular mechanisms of our ED model by measuring and comparing eNOS and PKB/Akt expression and activation in control and dysfunctional CMECs. (4) Investigate the effects of OA derived from leaf extracts obtained from Syzigium cordatum (Hochst.) [Myrtaceace], in both control and dysfunctional CMECs. Methods: (1) To induce ED, hyperglycaemia and inflammation were simulated by incubation with 25 mM glucose (24 hours) and 1 ng/ml TNF-á (24 hours) or 5 ng/ml TNF-á (6 and 24 hours) respectively. Reduced intracellular NO production was used as the main indicator of ED. NO production and cell viability were quantified by FACS analysis of the fluorescent probes, DAF-2/DA and propidium iodide (PI) / Annexin V respectively. Cellular mechanisms were investigated by measurement of O2- levels via FACS analysis of DHE fluorescence, and measurement of total and activated PKB / Akt and eNOS, p22-phox, nitrotyrosine expression via Western blotting. (2) Effects of OA on CMECs were investigated by pre-treatment with 30 or 40 ìM OA for 5 and 20 min followed by NO production and cell viability measurements. To investigate the effects of OA on ED, CMECs were pre-treated with 40 ìM OA 1 hour prior ED induction followed by NO, cell viability, and eNOS expression / activation measurements. Results: (1) 25 mM glucose (24hours), 1 ng/ml TNF-á (24 hours) and 5 ng/ml TNF-á (6 hours) failed to induce ED as verified by an increase in NO production in the treated cells. A model of ED was successfully achieved by incubating CMECs with 5 ng/ml TNF-á (24 hours), as verified by a significant decrease in NO production. Investigations into cellular mechanisms underlying our TNF-á-induced ED model, showed that activated eNOS and PKB / Akt levels were reduced. Furthermore, O2- levels remained unchanged, however p22-phox (NADPH) expression was significantly increased suggesting oxidative stress. Nitrotyrosine levels (an oxidative / nitrosative stress marker and indirect measure of eNOS uncoupling) remained at control levels. (2) Investigations into the effects of OA on CMECs showed that 30 ìM OA increased NO production after 5 and 20 min of incubation whereas 40 ìM increased NO production after 20 min only. Pre-treatment with 40 ìM OA significantly reversed ED by restoring NO production back to control levels. Data from cellular mechanism investigations showed that 40 ìM OA significantly increased eNOS activation in both normal and dysfunctional CMECs. Cellular viability was not negatively affected by any of the above interventions. Discussion and Conclusions: Based on our findings, reduced activation of the PKB / Akt-eNOS pathway appears to be the primary mechanistic pathway of the TNF-á-induced model of ED. Though O2- levels remained at control levels, the significant increase in p22-phox is indicative of increased expression of the O2- producing enzyme, NADPH oxidase, thus suggesting oxidative stress. However, based on our nitrotyrosine expression data, there was no strong evidence of eNOS uncoupling in our ED model. OA significantly stimulated NO production in our model of CMECs. Furthermore, our findings showed that OA is able to reverse ED. The NO production stimulatory effects of OA in our cells appear to be achieved via the increased activation of eNOS. We have, for the first time as far as we are aware, developed a TNF-á-induced model of ED in myocardial capillary-derived endothelial cells. It appears that reduced activation of the PKB/Akt-eNOS pathway is the primary mechanism leading to decreased NO production in this model. However, we did find some evidence of elevated oxidative stress, which led us to believe that eNOS uncoupling cannot be excluded as a mechanism of ED in our model. In this study, we report for the first time convincing evidence that OA has powerful NO-increasing properties in myocardial capillary-derived CMECs. Our study also show novel data, which suggest that OA is able to reverse ED in this model. Follow-up investigations could shed more light on the exact mechanisms underlying OA.s effects in this model. / AFRIKAANSE OPSOMMING: Inleiding: Die ontdekking dat endoteel 'n reguleerder van vaskulêre tonus is, en die gevolglike ontdekking dat stikstofoksied (NO) die belangrikste endoteel-afgeleide verslappingsfaktor (EDRF) is, het verskeie moontlikhede in aangaande pogings om kardiovaskulêre siektes te voorkom en hanteer, ontsluit. Endoteel-disfunksie (ED), word gedefineer as verlaagde NO biobeskikbaarheid en dus 'n ingekorte vermoë van die endoteel om vaskulêre homeostase te handhaaf. ED verteenwoordig die eerste, omkeerbare stap in die ontstaan van aterosklerotiese siekte en word dus beskou as 'n sterk instrument waarmee isgemiese hartsiekte voorspel kan word. Studies oor ED en sy onderliggende meganismes, veral in miokardiale kapillêre-afgeleide endoteelselle (kardiale mikrovaskulêre endoteelselle, CMECs), word redelik afgeskeep in die literatuur, en hierdie studie het dit ten doel gehad om die gaping in die literatuur aan te spreek. Oleanoliese suur (OA) is 'n bio-aktiewe triterpenoïede wat gevind word in blaar ekstrakte van inheemse medisinale plante soos bv. Syzigium cordatum (Waterbessie boom). OA het bewese vasodilatoriese, hipoglukemiese en hipolipidemiese eienskappe. OA se effekte op CMECs, en sy moontlike rol in die omkering van ED, is egter onbekend, en hierdie studie het dit ten doel gehad om sulke effekte te ondersoek. Doelwitte: Die doelwitte van hierdie studie was: (1) Die vestiging van 'n in vitro model van ED in gekultuurde CMECs afkomstig van miokardiale kapillêre deur protokolle vir die induksie van ED te ontwikkel. (2) Die evaluering van ED induksie deur die volgende bio-merkers te meet: (i) intrasellulêre NO produksie, (ii) superoksied (O2-) produksie, (iii) nitrotirosien uitdrukking en (iv) NADPH oksidase uitdrukking. (3) Die ondersoek na onderliggende sellulere meganismes van ED in ons model deur die meting en vergelyking van eNOS and PKB/Akt uitdrukking en aktivering in kontrole en disfunksionele CMECs. (4) Ondersoek na die effekte van OA afkomstig van blaar ekstrakte verkry van Syzigium cordatum (Hochst.) [Myrtaceace], in beide kontrole en disfunksionele CMECs. Metodes: (1) Daar was gepoog om ED te induseer deur hiperglukemie en inflammasie te simuleer met onderskeidelik 25 mM glukose (24 uur) en 1 ng/ml TNF-a (24 uur) of 5 ng/ml (6 en 24 uur) inkubasie. Verlaagde intrasellulere NO produksie was ingespan as die hoof indikator van ED. NO produksie en sellewensvatbaarheid was gekwantifiseer deur vloeisitometriese analises (FACS) van die fluoresserende agense, DAF-2/DA en propidium jodied (PI) / Annexin V onderskeidelik. Sellulere meganismes was ondersoek deur O2- vlakke via FACS analise van DHE fluoressensie te meet, asook die meting van totale en geaktiveerde PKB / Akt en eNOS, p22-phox, nitrotirosien uitdrukking via Western blot tegnieke. (2) Effekte van OA op CMECs was ondersoek deur vooraf-behandeling met 30 of 40 µM OA vir 5 en 20 min gevolg deur NO produksie en sellewensvatbaarheid metings. Resultate: (1) 25 mM glukose (24 uur), 1 ng/ml TNF-a (24 uur) and 5 ng/ml TNF-ƒaa (6 uur) kon nie daarin slaag om ED te induseer nie, soos blyk uit die verhoogde NO produksie waargeneem in die behandelde selle. 'n Model van ED was suksesvol verkry deur CMECs met 5 ng/ml TNF-a (24 uur) te inkubeer, soos waargeneem deur verlaagde NO produksie. Ondersoek na sellulere meganismes onderliggend tot ons TNF-a-geinduseerde ED model, het getoon dat geaktiveerde eNOS en PKB / Akt vlakke verlaag was. Verder is gevind dat O2- vlakke onveranderd gebly het hoewel p22-phox (NADPH) uitdrukking betekenisvol toegeneem het, wat 'n aanduiding van oksidatiewe skade is. Nitrotirosien vlakke (.n oksidatiewe / nitrosatiewe stres merker en indirekte maatstaf van eNOS ontkoppeling) het onveranderd rondom kontrole vlakke gebly. (2) Ondersoek na die effekte van OA op CMECs het getoon dat 30 µM OA tot verhoogde NO produksie na 5 en 20 min inkubasie gelei het, terwyl 40 µM slegs na 20 min NO-verhogende effekte gehad het. Vooraf behandeling met 40 µM OA het ED betekenisvol omgekeer deur NO terug na kontrole vlakke te laat herstel. Ondersoek na sellulere meganismes het getoon dat 40 µM OA eNOS aktivering betekenisvol verhoog het in beide normale en disfunksionele CMECs. Sellulere lewensvatbaarheid was nie negatief geaffekteer deur enige van bogeneemde ingrepe nie. Bespreking en afleidings: Gebaseer op ons bevindinge, blyk verlaagde aktivering van die PKB/Akt-eNOS pad die primere meganistiese pad in ons TNF-a-geïnduseerde model van ED te wees. Alhoewel O2- vlakke rondom kontrole vlakke gebly het, was die betekenisvolle toename in p22-phox .n aanduiding van verhoogde uitdrukking van die O2- produserende ensiem, NADPH oksidase, wat dus suggererend van oksidatiewe stres was. Aan die ander kant was daar nie sterk bewyse van eNOS ontkoppeling in ons ED model nie, gebaseer op die nitrotirosien uitdrukking data. OA het duidelik NO produksie in ons model van CMECs gestimuleer. Verder wys ons resultate dat OA in staat is om ED om te keer. Die NO produksie-stimulerende effekte van OA in ons selle blyk die gevolg te wees van verhoogde aktivering van die PKB / Akt-eNOS pad. Ons het hier vir die eerste keer, sover ons bewus is, 'n TNF-a-geinduseerde model van ED in CMECs afkomstig van miokardiale kapillere gevestig. Dit blyk dat verlaagde aktivering van die PKB/Akt-eNOS pad die primere meganisme was waardeur verlaagde NO produksie in ons model veroorsaak was. Ons het egter wel bewyse van verhoogde oksidatiewe stress gevind, wat ons laat glo dat eNOS ontkoppeling nie heeltemal as .n meganisme van ED in ons model uitgesluit kan word nie. In hierdie studie toon ons vir die eerste maal oortuigende bewyse dat OA kragtige NO-verhogende eienskappe in miokardiale kapillere-afgeleide CMECs het. Ons studie bring ook nuwe data na vore, wat suggereer dat OA in staat is om ED in hierdie model om te keer. Opvolgstudies sal meer lig kan werp op die onderliggende meganismes van OA in hierdie model.

Les protéines kinases sont importantes dans de nombreux processus biologiques comme la prolifération, la différenciation, la migration, l’apoptose cellulaire et dans le maintien de l’intégrité de l’ADN. Dans de nombreuses pathologies comme le cancer, ces kinases sont anormalement suractivées. Pour ces raisons, plusieurs inhibiteurs de tyrosine kinase (ITK) ont été développés comme le dasatinib (Sprycel®), un ITK ciblant principalement le BCR-Abl et la famille SRC, qui montre une grande efficacité contre la leucémie myéloïde chronique. Cependant, le dasatinib présente aussi d’importants effets indésirables qui se manifestent au niveau pulmonaire, comme le développement d’hypertension artérielle pulmonaire (HTAP) et l’apparition d’épanchements pleuraux dont l’incidence est respectivement de 0,45% et de 15-35%. Ces effets secondaires représentent donc un problème majeur de santé publique. Les mécanismes impliqués dans ces effets indésirables ne sont pas connus.Mon projet de thèse entre donc dans l’étude des mécanismes par lesquels le dasatinib peut induire ces effets indésirables. Dans ce contexte, nous avons testé l’hypothèse que l’utilisation du dasatinib est associée à l’inhibition inappropriée d’une ou de plusieurs kinases qui jouent un rôle central dans l’intégrité de l’endothélium pulmonaire.Mon travail s’est articulé autour de trois grands axes : (1) Le premier axe a visé à déterminer le rôle des atteintes de la perméabilité endothéliale par le dasatinib dans l’apparition d’épanchement pleural ; (2) Le deuxième axe a consisté à étudier les mécanismes impliqués dans l’HTAP induite par le dasatinib ; (3) Le troisième axe a permis d’identifier le rôle de l’inhibition de la protéine kinase c-Abl dans l’intégrité de la cellule endothéliale pulmonaire.L’ensemble de nos données montre que le dasatinib altère, à forte dose, les fonctions des cellules endothéliales pulmonaires et ainsi fragilise l’intégrité vasculaire. Tout d’abord, nous avons en effet pu mettre en évidence qu’un traitement quotidien à fortes doses de dasatinib chez le rat augmente la perméabilité endothéliale et conduit à l’apparition d’épanchements pleuraux. De manière intéressante, nous avons pu démontrer que cette augmentation de perméabilité endothéliale est liée à une génération de dérivés oxygénés capable de redistribuer les protéines de jonctions intercellulaires. Deuxièmement, nous avons pu mettre en évidence que le dasatinib, à forte dose, induit une apoptose des cellules endothéliales (CE) pulmonaires, un phénomène lié en partie à la génération d’un stress oxydant d’origine mitochondriale. De plus, nous avons noté que des prétraitements de rats avec du dasatinib conféraient une prédisposition à l’hypertension pulmonaire expérimentale par la mise en place d’une dysfonction endothéliale. Ces dernières observations n’ont pas été retrouvées avec d’autres ITK proches du dasatinib comme l’imatinib. Enfin, nous avons pu identifier que c-Abl est une protéine kinase clef de l’intégrité de la cellule endothéliale pulmonaire. En effet, son inhibition par certain ITK conduit à un défaut de réparation des cassures ADN et ainsi à une altération de certaines fonctions des cellules endothéliales.En conclusion, les effets indésirables pulmonaires associés au dasatinib semblent directement liés à une perte de l’homéostasie de l’endothélium pulmonaire par des mécanismes faisant appel à la génération de stress oxydatif mitochondrial, à l’induction d’apoptose et à la perte de perméabilité vasculaire. / By catalysing reversible phosphorylation of their substrates, protein kinases play central role in regulating a multitude of cellular processes, including division, proliferation, apoptosis, and differentiation as well as in the maintenance of DNA integrity. Since deregulation of different protein kinases contribute to several human disorders, a multitude of tyrosine kinase inhibitors (TKIs) inhibiting various kinases have been developed and some of them are currently approved for different indications, and many more are under development. Dasatinib (Sprycel®), an orally available short-acting dual ABL/SRC tyrosine kinase inhibitor (TKI), is an effective treatment for Philadelphia-positive chronic myelogenous leukemia (CML) and for all phases of Philadelphia-positive CML with resistance or intolerance to prior therapy, including imatinib. However, dasatinib treatment is associated with the development of pulmonary arterial hypertension (PAH; 0.45%) and pleural effusion (15-35%). These serious pulmonary adverse events represent a serious public health problem.Therefore, my PhD project is seeking to identify the underlying molecular mechanisms responsible for these pulmonary adverse events induced by dasatinib. My work has followed three different strategic axes seeking to: 1) Determine whether dasatinib alters endothelial integrity, resulting in increased pulmonary vascular endothelial permeability and pleural effusion; 2) Study the mechanisms involved in the long-term development of dasatinib-induced PAH; 3) Precise the role of c-Abl protein kinase inhibition in pulmonary endothelial cell DNA integrity.First, our data indicate that dasatinib can lead to pulmonary endothelial dysfunction and thus affect vascular integrity. Interestingly, we demonstrated that this increased endothelial permeability is a reactive oxygen species (ROS)-dependent mechanism in vitro and in vivo. Second, we also found that high doses of dasatinib induce pulmonary endothelial cell apoptosis by increasing mitochondrial oxidative stress and cause endothelial cell apoptosis. Consistent with these observations, we found that pretreatment of rats with dasatinib leads pulmonary endothelial dysfunction and confers increased susceptibility to experimental pulmonary hypertension (PH) in contrast to rats pretreated with imatinib or vehicle. Finally, we identified c-Abl as a key protein kinase in pulmonary endothelial cells, since its inhibition by dasatinib and ponatinib leads to impaired DNA repair in human pulmonary endothelial cells.Taken together, my PhD results demonstrate the importance played by damage to the pulmonary endothelium in the onset of dasatinib-induced PAH and pleural effusion.