Les cellules dendritiques jouent un rôle prépondérant dans le développement des réponses immunitaires ; les nombreuses activités fonctionnelles de ces cellules dont leur importante capacité de phagocytose leur confèrent le pouvoir d'activer les différents mécanismes de défense de l'organisme qu'ils fassent parties de l'immunité innée ou adaptative. Or de nombreux travaux ont montré que lors du développement des réponses immunitaires, les mécanismes oxydatifs ont un rôle essentiel et sont donc étroitement contrôlés. Dans le cas des cellules dendritiques, la production de radicaux oxygénés est en très grande partie relié au fonctionnement d'une NADPH Oxydase (NOX2) que notre laboratoire a été le premier à identifier dans ces cellules, il y a quelques années. Ce complexe enzymatique à une position centrale dans la production en premier des radicaux superoxydes puis des différentes formes réactives de l'oxygène (FRO) qui en découlent. Au cours de ma thèse, j'ai tout d'abord mis en place un protocole permettant l'étude de la production des FRO dans les cellules dendritiques. Les différentes méthodes employées m'ont permis de démontrer que d'autres protéines du système oxydant tels NOX1 et SOD3, étaient également produites par les cellules dendritiques. Nos résultats montrent que ces protéines, à l'instar de ce qui avait été montré pour NOX2, jouent un rôle dans la formation et le contrôle des FRO. J'ai également étudié la localisation intracellulaire de ces protéines, ce qui m'a permis de mieux comprendre les rôles respectifs de ces dernières dans le contrôle de la production et de l'activité des FRO dans les cellules dendritiques. Il avait été préalablement montré que la dégradation des pathogènes dans les cellules dendritiques, après qu'ils aient été phagocytés, était en partie liée à l'activité NOX2. Le travail réalisé dans le cadre de cette thèse nous permet d'envisager un impact des FRO résultant de l'activité de NOX1 sur le contrôle de l'apoptose des cellules dendritiques ou sur certaines voies de signalisation au niveau de noyau. De plus, la dismutation du superoxyde en peroxyde (H2O2) pourrait ne pas être une réaction spontanée comme plusieurs auteurs l'ont proposé, mais pourrait être lié à la présence de SOD3 qui interviendrait dans les compartiments d'endocytose. / Dendritic cells play an important role in the development of immune responses; the numerous functional activities of DC, among them their important phagocytic potential, give to these cells the capacity to activate both innate and acquire immune responses. Many authors have shown that during the development of these processes, oxidative mechanisms have a very important role and consequently must be strictly controlled. In dendritic cells, oxygen radicals production is mainly related to an NADPH Oxidase activity (NOX2), that our laboratory was the first to identify in these cells few years ago. This enzymatic complex has an important function in the production of firstly oxygen superoxide radicals and secondly, various reactive oxygen species (ROS) that are produced like hydrogen peroxide. During my PhD, I have developed various protocols, which enabled us to analyse ROS production by dendritic cells. The different results allowed me to demonstrate that other proteins belonging to the family of the Red/Ox controlling elements are produced by dendritic cells: NOX1 and SOD3. My results showed that these proteins play an important role in ROS formation and control, in complement to what was previously demonstrated for NOX2. I have studied also their intracellular localisation, which permitted a better understanding of their respective role in dendritic cells. It was already shown that, after phagocytosis, pathogen degradation in dendritic cells was partially related to NOX2 activity. The work performed in this thesis let us consider a potential impact of NOX1 derived ROS on the control of the apoptosis of dendritic cells or on signalling pathways at nucleus level. Moreover, superoxyde anion dismutation in hydrogen peroxide (H2O2) could be due to the presence of SOD3 that may be found in endocytic compartments rather than the result of spontaneous reactions most often proposed by authors.

Reactive oxygen species (ROS) induce oxidative stress and although are primarily recognized for playing a deleterious biological role, they can be beneficial to cell systems. ROS are extremely short-lived and normally tightly regulated by antioxidant defence systems. Cells react to oxidative stress in different ways, which primarily depends on cell type, stress severity, or both. There is a general limitation in extrapolating to humans conclusions drawn from in vitro and animal studies because of important species-specific differences. Therefore, the general aim of this thesis was to study the influence of oxidative stress on human muscle and bone in vivo. In paper I we presented a one-step HPLC method optimized for the simultaneous determination of purine degradation products in small microdialysis samples. The clinical utility of the method was successfully tested in a patient with traumatic brain injury. In paper II we evaluated microdialysis as an in vivo method to characterize the relative kinetics of ROS-related metabolites in human skeletal muscle exposed to ischaemia-reperfusion. Results indicated that microdialysis was feasible and safe to use in monitoring metabolic events during tourniquet-assisted surgery. In paper III we investigated the association between an oxidative stress marker (urinary 8-iso-PGF2α) and bone mineral density (BMD) and whether α-tocopherol modified the association. The main finding was the negative association between 8-iso-PGF2α and BMD and that the association was further dependent on serum α-tocopherol level. In paper IV we performed a randomized controlled trial to evaluate the influence of Q10 supplementation on exercise performance and metabolites of muscular damage. We did not observe any effects on exercise capacity after 8 weeks of Q10 administration. Nor did we find a significant effect on serum markers related to oxidative stress. In conclusion we have studied the influence of oxidative stress on muscle and bone in vivo in humans. The oxidative stress was triggered by four different causes (trauma, ischemia-reperfusion, ageing, and exercise exhaustion).

Gesteigerter oxidativer Stress ist ein typisches Zeichen in der Pathogenese der Herzinsuffi-zienz. Anhand von 2 Rattenmodellen sollen charakteristische Veränderungen des enzymatischen antioxidativen Systems, repräsentiert durch die Glutathionperoxidase (GSH-Px) und Superoxiddismutase (SOD) sowie Änderungen im Hitzeschockproteinsystem (Hsp) untersucht werden, für das stellvertretend Hsp25 und Hsp72 bestimmt wurden. Daneben wurde die Lipidperoxidation durch die Konzentration von Thiobarbitursäure-reaktiven Substanzen (TBARS) quantifiziert. Im ersten Rattenmodell, der Hypertonie-induzierten Herzinsuffizienz durch permanente Renin-Angiotensin-System-Aktivierung in doppelt transgenen Ratten konnte eine linksventrikuläre Hypertrophie mit CK-Isoenzym-Shift mit vermehrter Expression von CK-MB und -BB beobachtet werden. Es kam in beiden Ventrikeln der transgenen Tiere zu einer Zunahme der Lipidperoxidation begleitet von einer Erniedrigung der Serum-Vitamin E-Konzentration durch Verbrauch. SOD und Hsp72 blieben unverändert. Durch die biventrikuläre Zunahme der GSH-Px-Aktivität bei linksventrikulär erhöhtem Hsp25-Gehalt konnte die Toleranz gegenüber Hypoxie/Reoxygenierungsstress erhöht werden. In der 2. Teilstudie - der Myokardinfarkt-induzierten Herzinsuffizienz - wurden die Parameter oxidativer Schädigung und antioxidativen Schutzes im Akutstadium (14-16h) und 3, 6 und 9 Wochen nach experimentellem Infarkt (MI) bestimmt. In der Akutphase zeigten sich in beiden Ventri-keln und im Papillarmuskel gesteigerte GSH-Px- und SOD-Aktivitäten, ohne dass kardiale Hypertrophie vorlag. Hsp72 und Hsp25 waren während der Akutphase nach MI im Papillar-muskel und im linken Ventrikel erhöht. In der Folge resultiert eine verbesserte kontraktile Funktion bei experimentellem Hypoxie/Reoxygenierungsstress. Damit gelang es erstmals eine Selbstprotektion des Myokards während eines akuten MI nachzuweisen. Erst ab der 6. Woche trat kardiale Hypertrophie auf, begleitet vom charakteristischen CK-Isoenzym-Shift. Während die SOD und die Hitzeschockproteine nach der akuten Phase auf das Niveau der Kontrollen absanken, blieb die hohe GSH-Px-Aktivität im linken Ventrikel über den gesamten Untersuchungszeitraum bestehen, bei zunehmender Toleranz des Herzens gegenüber Hypoxie/Reoxygenierungsstress. Die vorgelegten tierexperimentellen Untersuchungen zeigen, dass das Herz sowohl unter akut als auch unter chronisch gesteigertem oxidativen Stress Mechanismen zur Selbstprotektion aktivieren kann, die eine Prävention bzw. Minimierung von Schädigungsreaktionen durch Sauerstoffradikale ermöglichen. / Elevated oxidative stress is typical in the pathogenesis of heart failure. Characteristical changes in antioxidant enzyme status, represented by glutathionperoxidase (GSH-Px) and superoxide dismutase (SOD), and changes in heat shock protein status (Hsp), denoted by Hsp25 and Hsp72, have been revealed in two different rat-models. Lipidperoxidation was quantified by the concentration of thiobarbituric acid reactive substances (TBARS). In the first model of heart failure caused by permanent activation of renin-angiotensin system in double transgenic rat, leftventricular hypertrophy accompanied by a shift of creatine kinase (CK) isoenzyme pattern to higher concentration of fetale CK-MB an -BB-isoforms was found. Higher TBARS concentrations and lower alpha-tocopherol levels caused by consumption have been measured. SOD and Hsp72 remained unchanged. The tolerance against experimental hypoxia/reoxygenation was improved by higher levels of GSH-Px and Hsp25 in both right and left ventricular tissue. In the second study of heart failure caused by experimental myocardial infarction (MI) the parameters of oxidant demolishing and antioxidant defence were evaluated under acute condi-tions (14-16h) and 3, 6 and 9 weeks after infarction. In the acute period higher activities of GSH-Px and SOD in non-hypertrophied left and right ventricular tissue and papillary muscle have been reported. Leftventricular and papillary muscle Hsp25 and Hsp72 content showed higher levels 14-16 hours after MI compared with controls, improving the contractile function in hypoxia/reoxygenation experiments. These findings suggest for the first time a myocardial self-protection after acute myocardial infarction. 6 and 9 weeks after MI leftventricular hyper-trophy occurred attended by the characteristic CK-isoenzymeshift. While SOD-activity and Hsp-content decreased to the levels of the controls, GSH-Px remained on higher altitude in leftventricular tissue in all examined periods after MI. This was accompanied by better toler-ance against hypoxia/reoxygenation stress. These findings of experimental-induced heart failure show the activation of myocardial self protecting mechanisms under acute and chronic oxidative stress conditions, minimizing or even preventing demolishing reactions of oxygen radicals.

<p>Maternal type 1 diabetes is associated with an increased risk for foetal malformations. The mechanism by which diabetes is teratogenic is not fully known. Previous studies have demonstrated that radical oxygen species can contribute to the teratogenicity of glucose and diabetes. The aim of the present work was to study different aspects of free radical damage and antioxidant defence in experimental diabetic pregnancy.</p><p> The activity of the antioxidant enzyme catalase and the mRNA levels of antioxidant enzymes in embryos of normal and diabetic rats of two strains were measured. The catalase activity was higher in embryos of a malformation-resistant strain than in a malformation-prone strain, the difference increased further when the mother was diabetic. Maternal diabetes increased embryonic mRNA levels of catalase and manganese superoxide dismutase in the malformation-resistant strain, but not in the malformation-prone strain. Embryos of the malformation-prone rat thus had lower antioxidative defence than embryos of the malformation-resistant strain.</p><p> Administration of either vitamin E or vitamin C has previously been shown to protect embryos from maldevelopment in experimental diabetic pregnancy. The vitamins were used together in this thesis to yield protection in both the lipid and aqueous phase. The protective effect was not higher than what had been achieved using the vitamins individually. No synergistic effect was thus found using the two antioxidants together. </p><p> The urinary excretion of the lipid peroxidation marker 8-iso-PGF_2á was increased in pregnant dia-betic rats compared with non-diabetic controls, as was the plasma content of carbonylated proteins. Carbonylated proteins and TBARS concentrations were increased in foetal livers in diabetic pregnancy. However, no increased concentration of 8-iso-PGF_2á was found in the amniotic fluid of pregnant diabetic rats. Both lipids and proteins were thus oxidatively modified in experimental diabetic pregnancy. It is concluded that experimental diabetic pregnancy is associated with increased oxidative stress and that the embryonic antioxidant defence is likely to be of importance for normal development in a diabetic environment.</p>

Maternal type 1 diabetes is associated with an increased risk for foetal malformations. The mechanism by which diabetes is teratogenic is not fully known. Previous studies have demonstrated that radical oxygen species can contribute to the teratogenicity of glucose and diabetes. The aim of the present work was to study different aspects of free radical damage and antioxidant defence in experimental diabetic pregnancy. The activity of the antioxidant enzyme catalase and the mRNA levels of antioxidant enzymes in embryos of normal and diabetic rats of two strains were measured. The catalase activity was higher in embryos of a malformation-resistant strain than in a malformation-prone strain, the difference increased further when the mother was diabetic. Maternal diabetes increased embryonic mRNA levels of catalase and manganese superoxide dismutase in the malformation-resistant strain, but not in the malformation-prone strain. Embryos of the malformation-prone rat thus had lower antioxidative defence than embryos of the malformation-resistant strain. Administration of either vitamin E or vitamin C has previously been shown to protect embryos from maldevelopment in experimental diabetic pregnancy. The vitamins were used together in this thesis to yield protection in both the lipid and aqueous phase. The protective effect was not higher than what had been achieved using the vitamins individually. No synergistic effect was thus found using the two antioxidants together. The urinary excretion of the lipid peroxidation marker 8-iso-PGF2á was increased in pregnant dia-betic rats compared with non-diabetic controls, as was the plasma content of carbonylated proteins. Carbonylated proteins and TBARS concentrations were increased in foetal livers in diabetic pregnancy. However, no increased concentration of 8-iso-PGF2á was found in the amniotic fluid of pregnant diabetic rats. Both lipids and proteins were thus oxidatively modified in experimental diabetic pregnancy. It is concluded that experimental diabetic pregnancy is associated with increased oxidative stress and that the embryonic antioxidant defence is likely to be of importance for normal development in a diabetic environment.

Therapeutic hypothermia (TH) is the only clinically used intervention that suppresses nearly all manifestations of ischemia-reperfusion injury after cardiac arrest. Experimental models has proven that exogenous antioxidants have positive impact on ischemia-reperfusion injury and it is able to prevent it as well. Results in this thesis are based on application of high dosages of N-acetylcystein (NAC) on ischemia-reperfusion injury after experimental cardiac arrest in a porcine model. It was used as a form of monoteraphy or in combination with TH. During the experiment animals were randomized into 5 groups: administration of NAC and TH (group A), administration of NAC during cardiac arrest (group B), induction of TH (group C), without any intervention (group D) and administration of NAC after return of spontaneous circulation (group E). We were not able to confirm additive effect of NAC in combination with TH. Administration of NAC during cardiac arrest led to statistically important reduction of oxidative stress but in the same time anafylactic reaction led to higher mortality in group B and changes in hemodynamical parameters in group E. Key words: Therapeutic hypothermia, cardiac arrest, N-acetylcysteine, ischemia-reperfusion injury, cardiopulmonary resuscitation, oxidative stress, oxygen radicals

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