Endothelial dysfunction is a pathologic state characterized by an altered equilibrium among vasodilatory and antithrombotic mediators and vasoconstrictive and prothrombotic mediators produced by the vascular endothelium. Multiple factors induce impaired production or increased consumption nitric oxide (NO), the key mediator of vascular homeostasis, produced by the nitric oxide synthase enzymes (NOS). Endothelial dysfunction represents one of the initial steps in the development of atherosclerosis, a chronic inflammatory disease of the vascular wall. The inducible enzyme heme oxygenase 1 (HO-1) represents one of the main cellular defense mechanisms against increased oxidative stress and decreased NO bioavailability accompanying endothelial dysfunction and atherosclerosis. We studied the genetic determinants of endothelial dysfunction and atherosclerosis by evaluating the association of the G894T endothelial NOS (eNOS) polymorphism and the HO-1 (GT)n promoter polymorphism with coronary artery atherosclerosis severity and risk profile and their evolution during hypolipidaemic treatment. In addition, we searched for genetic variations in exons 25 and 26 of eNOS gene, encoding the C-terminal part of the protein, deemed crucial for proper enzyme function and the 3'- untranslated region crucial for eNOS...

Na lesão renal aguda, os mecanismos de defesa atuam como genes protetores, como a proteína heat shock 32 (HSP 32), também conhecida como heme oxigenase-1 (HO-1). A polimixina B (PmxB) é um antimicrobiano nefrotóxico. O objetivo deste estudo foi caracterizar a participação da enzima HO-1 na toxicidade da PmxB em células LLC-PK1. As células foram submetidas aos seguintes tratamentos: Controle (CTL- 0µM); Hemin (indutor de HO-1, 25µM); Hemin II (250M), Protoporfirina de zinco (ZnPP - inibidor de HO-1, 10M,); Nitro-L-arginina-metilester (L-NAME - inibidor de iNOS, 0,1mM); PmxB (375µM); PmxB + Hemin (25µM de Hemin uma hora antes da PmxB); PmxB + ZnPP (10M de ZnPP uma hora antes da PmxB); PmxB + Hemin + L-NAME (25M de Hemin e 0,1mM de L-NAME uma hora antes da PmxB). Os grupos foram avaliados em 24 e 72 horas. Foram analisados os seguintes parâmetros: desidrogenase láctica (DHL), peroxidação lipídica (MDA), expressão gênica da HO-1 por RT-PCR, síntese protéica da HO-1 por imunofluorescência, óxido nítrico (NO) pelo método de Griess e expressão protéica da HO-1 e da iNOS por western blotting. Os resultados mostraram que a PmxB elevou o DHL com aumento dos níveis de MDA. O Hemin e a ZnPP elevaram as variáveis DHL, MDA e óxido nítrico (NO). O indutor de HO-1 incrementou a expressão protéica da HO-1 e da iNOS. A PmxB se confirmou como citotóxica e a HO-1 intensificou a lesão por mecanismos oxidativos. O efeito da HO-1 na lesão celular parece ser mediado pelo NO / In the acute kidney injury, the mechanisms of defense act as protector genes, as the protein heat shock 32 (HSP 32), also known as heme oxygenase-1 (HO-1). The polymyxin B (PmxB) is a nephrotoxic antimicrobial. The aim of this study was to distinguish the role of the HO-1 enzyme in the PmxB toxicity in LLC-PK1 cells. The cells were submitted to the following treatments: Control (CTL- 0µM); Hemin (inhibitor of HO-1, 25µM); Hemin II (250M), Zinc protoporphyrin (ZnPP - inhibitor of HO-1, 10M,); NG-nitro-L-arginine methyl ester (L-NAME - inhibitor of iNOS, 0,1mM); PmxB (375µM); PmxB + Hemin (25µM of Hemin one hour before the PmxB); PmxB + ZnPP (10M of ZnPP one hour before the PmxB); PmxB + Hemin + L-NAME (25M of Hemin and 0,1mM of L-NAME one hour before the PmxB). All groups were evaluated in 24 and 72 hours. The following parameters were analysed: lactate dehydrogenase (LDH), lipid peroxidation (MDA), genic expression of HO-1 by RT-PCR, protein syntesis of HO-1 by immunofluorescence, nitric oxide (NO) by Griess method and protein expression of HO-1 and of iNOS by western blotting. The results showed that PmxB increased the LDH and the levels of MDA. Hemin and ZnPP also increased the LDH variables, MDA and nitric oxide (NO). The inducer of HO-1 improved the protein expression of HO-1 and of iNOS. The PmxB was confirmed as a cytotoxic and the HO-1 intensified the failure by oxidative mechanisms. The effect of HO-1 in the cell injury seemed to be mediated by NO

Bien que les résultats métaboliques de la transplantation d’îlots chez le patient diabétique de type 1 soient désormais bien démontrés, ils sont contrebalancés par les effets indésirables des traitements immunosuppresseurs et la perte de fonctionnalité du greffon à long terme.Au cours de cette thèse, nous avons étudié deux approches complémentaires offrant la perspective de s’affranchir du traitement immunosuppresseur tout en protégeant les îlots de l’apoptose et de la perte de fonctionnalité du greffon induites par les mécanismes d’isolement, de culture et de transplantation : l’immunoisolation des îlots dans des capsules de biomatériaux et la co-transplantation avec des cellules souches mésenchymateuses (CSM).Au sein du projet européen de pancréas artificiel BIOCAPAN, nous avons évalué in vitro, la biocompatibilité de différents biomatériaux et mis en évidence un effet combiné de la présence de CSM et des tripeptides RGD sur le maintien de la viabilité et de la fonctionnalité des îlots encapsulés. L’évaluation ultérieure de la biocompatibilité et de l’effet ajouté de la capsule BIOCAPAN sur des animaux diabétiques permettra la validation de la capsule qui sera proposée à des tests d’essais cliniques.Nous avons également démontré, dans un modèle de co-culture d’îlots avec des CSM dans des conditions de culture classiques et exposées à des cytokines pro-inflammatoires, que les CSM régulaient les capacités sécrétrices des îlots probablement via la régulation de l’hème oxygénase 1 (HO-1). L’identification des facteurs de transcription régulant HO-1 ainsi que des médiateurs permettant la communication entre les deux types cellulaires sont des perspectives de développement.Ce travail a souligné l’intérêt, au sein d’une approche immuno-isolante, de la reconstitution d’un environnement favorable au sein de la capsule permettant la préservation de l’îlot notamment via l’utilisation de CSM. / Although, the metabolic results of islets transplantation for patient with type 1 diabetes are now well documented, they are counteracted by the adverse effects of immunosuppressive therapies and the long-term loss in graft functionality.During this thesis, we worked on two complementary approaches offering the perspective of avoiding immunosuppressive treatment while protecting islets from apoptosis and loss of functionality induced by the mechanisms of isolation, culture and transplantation. These two tools are islet immunoisolation in capsules composed of specific biomaterials and islets co-transplantation with mesenchymal stem cells (MSCs) described for their immunomodulatory, proangiogenic and cytoprotective properties.In the european project of bioartificial pancreas BIOCAPAN, we have evaluated in vitro the biocompatibility of several biomaterials and we have highlight a combined effect of the presence of MSCs and tripeptides RGD on the viability and the functionality maintenance of the encapsulated islets. Subsequent in vivo validation of the biocompatibility and the added effect of the BIOCAPAN capsule on diabetic animals will allow the final validation of the capsule to be proposed for clinical trials.We also demonstrated, in an islet co-culture model with MSCs under conventional culture conditions and exposed to pro-inflammatory cytokines, that MSCs regulate the secretory capacity of islets probably via the regulation of heme oxygenase 1 (HO-1) described for its antioxidant and anti-inflammatory properties. The identification of transcription factors regulating HO-1 as well as mediators, allowing communication between the two cell types, are development perspectives.This work underlined the interest, within an immuno-isolation approach, of the reconstitution of a favorable environment within the capsule allowing the preservation of islet physiology thanks to the use of MSCs.

Endothelial dysfunction is a pathologic state characterized by an altered equilibrium among vasodilatory and antithrombotic mediators and vasoconstrictive and prothrombotic mediators produced by the vascular endothelium. Multiple factors induce impaired production or increased consumption nitric oxide (NO), the key mediator of vascular homeostasis, produced by the nitric oxide synthase enzymes (NOS). Endothelial dysfunction represents one of the initial steps in the development of atherosclerosis, a chronic inflammatory disease of the vascular wall. The inducible enzyme heme oxygenase 1 (HO-1) represents one of the main cellular defense mechanisms against increased oxidative stress and decreased NO bioavailability accompanying endothelial dysfunction and atherosclerosis. We studied the genetic determinants of endothelial dysfunction and atherosclerosis by evaluating the association of the G894T endothelial NOS (eNOS) polymorphism and the HO-1 (GT)n promoter polymorphism with coronary artery atherosclerosis severity and risk profile and their evolution during hypolipidaemic treatment. In addition, we searched for genetic variations in exons 25 and 26 of eNOS gene, encoding the C-terminal part of the protein, deemed crucial for proper enzyme function and the 3'- untranslated region crucial for eNOS...

Die Hämoxygenase-1 (HO-1) ist das geschwindigkeitsbestimmende Enzym des Hämabbaus und ist wichtiger Regulator inflammatorischer Prozesse. Der Verlauf einer experimentellen akuten Pankreatitis (AP) konnte im Tiermodell durch HO-1 Induktion abgemildert werden. Die Aktivierung und Proliferation pankreatischer Stellatum Zellen (PSC) wird durch eine experimentelle HO-1 Induktion inhibiert und kann so möglicherweise vor der Fibrosierung des Pankreasparenchyms bei chronischer Pankreatitis (CP) schützen. Die Transkription der HO-1 wird durch einen GT-Repeat beeinflusst, der im Promoter lokalisiert ist. Diese Arbeit untersuchte, ob Varianten des GT-Repeat oder weitere genetische Varianten der HO-1 mit verschiedenen Pankreatitisformen assoziiert sind. Der GT-Repeat und der SNP rs2071746 wurden mit fluoreszensmarkierten Primern bzw. mit Schmelzkurvenanalyse bei 285 Patienten mit AP, bei 208 Patienten mit alkoholischer CP (ACP), bei 207 mit idiopathischer/hereditärer CP (ICP/HCP), 147 Patienten mit Alkoholischer Leberzirrhose (ALZ) und bei 289 Kontrollen untersucht. Bei den ACP Patienten wurde die GT-Repeat Analyse auf insgesamt 446 Patienten erhöht. Zusätzlich wurden die kodierenden HO-1 Abschnitte mittels DNA-Sequenzierung bei 145 Patienten mit ACP, 138 Patienten mit ICP/HCP, 147 Patienten mit ALZ und bei 151 Kontrollen analysiert. Das Exon 3 wurde darüber hinaus bei zusätzlichen ICP/HP Patienten und Kontrollen untersucht. Die Längenverteilungen des GT-Repeat, die Allelverteilung des SNP rs2071746 und die Verteilung der bei der DNA-Sequenzierung gefundenen synonymen und nicht synonymen Varianten waren bei allen untersuchten Gruppen nicht signifikant unterschiedlich. Obwohl die funktionellen Daten einen Einfluss von HO-1 Varianten auf die Pathogenese der verschiedenen Pankreatitis-Formen nahelegen, konnte unsere umfangreiche genetische Analyse keine Assoziation nachweisen. Genetische Varianten der HO-1 haben keinen Einfluss auf die Entwicklung einer AP, ACP, ICP/HCP und ALZ.:Inhaltsverzeichnis Vorbemerkung ..................................................................................................... 3 Bibliographische Beschreibung.......................................................................... 4 Abkürzungen/Abbildungen ................................................................................ 6 1. Einleitung........................................................................................................9 1.1 Akute Pankreatitis ......................................................................................................................... 9 1.2 Chronische Pankreatitis ............................................................................................................... 11 1.3 Genetische Aspekte der Chronischen Pankreatitis ...................................................................... 12 1.3.1 Kationisches Trypsinogen (PRSS1) ...................................................................................... 12 1.3.2 Anionisches Trypsinogen (PRSS2) ....................................................................................... 14 1.3.3 Serinproteaseinhibitor, Kazal Typ1 (SPINK1)..................................................................... 14 1.3.4 Chymotrypsin C (CTRC) ...................................................................................................... 15 1.3.5 CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) ...................................... 15 1.4 Hämoxygenase-1 ......................................................................................................................... 16 1.4.1 Physiologische Bedeutung der Hämoxygenase-1 (HO-1) .................................................... 16 1.4.2 Genetische Varianten der Hämoxygenase-1 ........................................................................ 18 1.4.3 Hämoxygenase-1 und Pankreatitis....................................................................................... 20 1.5 Hypothese/Fragestellung ............................................................................................................. 21 2. Publikation ..................................................................................................... 22 3. Zusammenfassung der Arbeit ...................................................................... 23 4. Literaturverzeichnis...................................................................................... 28 5. Danksagung.................................................................................................... 35 6. Erklärung über die eigenständige Abfassung der Arbeit .......................... 36 7. Lebenslauf ...................................................................................................... 37

L’hème oxygénase-1 (HO-1), enzyme antioxydante, est capable de prévenir l’initiation tumorale tandis qu’elle promeut la progression de certaines tumeurs et l’angiogenèse. Ce travail a recherché si HO-1 peut moduler les microARNs et régule le développmemnt du carcinome pulmonaire humain non à petites cellules (NSCLC). La surexpression stable de HO-1 dans les cellules du NSCLC NCI-H292 accroit la production globale des miARNs et diminue significativement l’expression des oncomirs et angiomirs, tandis qu’elle augmente les miARNs suppresseurs de tumeurs. Le plus amplement diminué est le miR-378. Dans les cellules surexprimant HO-1 la p53 est aussi augmentée, Ang-1 et MUC5AC diminuées, prolifération migration et potentiel angiogéniques réduits. Les effets de HO-1 sur la prolifération tumorale, la migration et et l’expression de miR-378 sont modulées par CO. Au contraire, la surexpression stable de miR-378 décroit celle de HO-1 et de p53 tandis qu’elle accroît celle de MUC5AC, VEGF, IL-8 et Ang-1 et en conséquence accroit la prolifération, migration la stimulation des cellules endothéliales. L’ajout de HO-1 à des cellules surexprimant miR-378 réverse l’effet de miR-378 sur la prolifération et la migration des cellules cancéreuses. In vivo, les tumeurs surexprimant HO-1 sont de taille réduite, moins vascularisées et oxygénées et moins métastatiques tandis que la surexpression de miR-378 produit les effets inverses. Conformément, chez les patients NSCLC, l’expression de HO-1est réduite dans les métastases lymphatiques par rapport à la tumeur primaire tandis que miR-378 n’est pas modifié de manière significative. En conclusion, les résultats in vitro et in vivo indiquent que l’action coordonnée entre HO-1 et miR-378 module de manière significative la progression et l’angiogenèse du carcinome humain pulmonaire non à petites cellules. / Heme oxygenase-1 (HO-1), an antioxidant enzyme can prevent tumor initiation while it has been demonstrated to promote various tumors progression and angiogenesis. Here it was investigated whether HO-1 can modulate microRNAs and regulate human non-small cell lung cancer (NSCLC) development. Stable HO-1 overexpression in NSCLC NCI-H292 cells enhanced global production of miRNAs and significantly diminished expression of oncomirs and angiomirs, whereas upregulated tumor suppressive miRNAs. The most potently downregulated was miR-378. HO-1 overexpressing cells displayed also upregulated p53, downregulated Ang-1 and MUC5AC, reduced proliferation, migration and diminished angiogenic potential. CO was a mediator of HO-1 effects on tumor cells proliferation, migration and miR-378 expression. In contrast, stable miR-378 overexpression decreased HO-1 and p53 while enhanced expression of MUC5AC, VEGF, IL-8 and Ang-1 and consequently increased proliferation, migration and stimulation of endothelial cells. Adenoviral delivery of HO-1 to miR-378 overexpressing cells reversed miR-378 effect on proliferation and migration of cancer cells. In vivo, HO-1 overexpressing tumors were smaller, less vascularized and oxygenated and less metastatic, whereas miR-378 overexpression exerted the opposite effects. Accordingly, in patients with NSCLC, HO-1 expression was lower in metastases to lymph nodes than in primary tumors while miR-378 did not differ significantly. To conclude, in vitro and in vivo data indicate that interplay between HO-1 and miR-378 significantly modulates NSCLC progression and angiogenesis.

Die Hämoxygenase 1 (HO-1) ist ein Stressprotein mit antientzündlichen, immunsupprimierenden und zytoprotektiven Eigenschaften, welche in vielen Tiermodellen nachgewiesen wurden. Die zugrunde liegenden Mechanismen sind wenig bekannt. Diese Arbeit demonstriert erstmalig, dass die physiologische Induktion von HO-1 wichtig für die Limitierung von T-Zell-abhängigen Hautentzündungen ist. So führt der HO-1-Inhibitor, Zinn-Protoporphyrin IX (Sn-PPIX), zu einer verstärkten Hautentzündung im Mausmodell. Die pharmakologische Induktion von HO-1 durch Kobalt-Protoporphyrin IX, Co-PPIX, hemmt dagegen die Entzündung in DNFB- bzw. TMA-induzierten murinen Kontaktallergiemodellen sowohl bei Verabreichung von Co-PPIX während der Sensibilisierung als auch vor der Auslösung. Bemerkenswerterweise hemmt eine Co-PPIX-Behandlung die Antigen-induzierte T-Zellproliferation ex vivo in Milzzellen von behandelten Mäusen und in vitro in humanen mononukleären Zellen des peripheren Blutes. Da eine HO-1-Induktion durch Co-PPIX nur in Monozyten und in aus Monozyten abgeleiteten myloischen Dendritischen Zellen (MDDC), nicht aber in T-Zellen, beobachtet wurde, fokussierten alle weiteren Untersuchungen auf Antigen-präsentierende Zellen. HO-1-Induktion durch Co-PPIX reduziert die Expression von MHC-Klasse II und akzessorischen Molekülen und steigert die Phagozytose und den oxidativen Burst von Monozyten. Die immunphänotypische Differenzierung und Maturierung von MDDC wird gehemmt. Funktionsteste zeigen eine Reduktion der Expression und Sekretion von proinflammatorischen und immunstimulatorischen Zytokinen, während die Sekretion des antientzündlichen Zytokins IL-10 gesteigert ist. Die Fähigkeit der MDDC zur Antigenpräsentation gegenüber T-Helferzellen ist für Allo- und Recallantigene stark herabgesetzt. Mittels adenoviraler HO-1-Transduktion von MDDC konnte die Spezifität der Effekte bestätigt werden. Diese Daten zeigen, dass eine verstärkte HO-1-Aktivität die Dendritischen Zellen zu einem unreifen und immunkompromittierten Phänotyp verändert und weisen darauf hin, dass die HO-1-Induktion einen wichtigen Ansatz für die Hemmung der zellulären Immunität und für die Behandlung von T-Zell-abhängigen Hautentzündungen darstellt. / Heme oxygenase 1 (HO-1) is an antiinflammatory stress protein. Its immunosuppressive and cytoprotective activities have been demonstrated in several animal models. The underlying mechanisms, however, are poorly understood. This study demonstrates for the first time that the physiological induction of HO-1 is important for the limitation and resolution of T cell-dependent skin inflammation. So, the HO-1 inhibitor, tin protoporphyrin IX (Sn-PPIX), augments cutaneous inflammation in mouse model. Moreover, pharmacologic HO-1 induction by the potent HO-1 inducer, cobaltic protoporphyrin IX (Co-PPIX), inhibits inflammation when applied around sensitization or before challenge in murine DNFB- and TMA-induced contact hypersensitivity models. Remarkably, Co-PPIX treatment inhibits antigen-driven T cell proliferation both ex vivo in murine splenocytes and in vitro in human peripheral blood mononuclear cells. Since induction of HO-1 mRNA and protein was found in monocytes and monocyte-derived myeloid dendritic cells (MDDC) but not T cells, further investigations focused on antigen-presenting cells. HO-1 induction by Co-PPIX depresses monocytic MHC class II and accessory molecule expression whereas phagocytosis and respiratory burst activities are augmented. Moreover, HO-1 induction inhibits the immunophenotypic differentiation and maturation of MDDC. Functional analysis revealed a decreased proinflammatory cytokine production whereas secretion of the antiinflammatory cytokine IL-10 is increased. Remarkably, the antigen-presenting capacity of MDDC for T-helper cells is diminished both for allo- and for recall-antigens. Adenoviral HO-1 transduction of MDDC confirmed that the effects are mediated by HO-1. These data indicate that an enhanced HO-1 activity switches myeloid DCs to an immature and functionally compromised phenotype and suggest that HO-1 induction represents an important approach for depressing T cell immunity and for the treatment of T cell-dependent skin inflammation.

De l’isolement des îlots pancréatiques à leur implantation, l’inflammation est omniprésente au cours de la transplantation d’îlots pancréatiques. Le maintien d’une inflammation contrôlée est essentiel pour préserver la survie et la fonctionnalité du greffon à court et long terme. L’objectif de ce travail de thèse est d’identifier précisément les mécanismes inflammatoires à l’origine de la perte précoce des îlots et de déterminer des cibles thérapeutiques pour limiter ces réactions inflammatoires.Nous avons ainsi démontré que les conditions de culture induisent des réactions à l’origine du développement d’un phénotype pro-inflammatoire et pro-oxydant propre à l’îlot. Cette induction se caractérise par une élévation de la sécrétion de cytokines, de chimiokines pro-inflammatoires, une activation des voies de l’inflammation Toll-like récepteurs (TLRs)-dépendantes et une génération d’espèces réactives de l’oxygène (ROS). Toutefois, ce processus peut être prévenu par l’activation de l’Hème oxygénase-1 (HO-1), une enzyme anti-oxydante et anti-inflammatoire.Par l’étude des réactions inflammatoires sur un modèle animal de transplantation mimant les conditions de transplantation humaine, nous avons démontré qu’un changement des médiateurs plasmatiques de l’inflammation et du protéome hépatique s’opère 12 heures après transplantation. De plus, ces résultats sont associés à une infiltration des îlots par les cellules immunitaires qui s’organise 12 heures après transplantation. Nous avons également établi le rôle anti-inflammatoire de la rapamycine (une drogue immunomodulatrice) sur les îlots et les macrophages in vitro. Nous avons ainsi démontré que l’usage de la rapamycine avec la mise en place d’un pré-traitement des îlots et du receveur avant la greffe serait envisageable. Ces travaux ont permis de caractériser les mécanismes inflammatoires mis en oeuvre immédiatement avant et après transplantation. Ainsi, ces données offrent de nouvelles pistes thérapeutiques susceptibles de prévenir et/ou limiter l’inflammation au cours de la transplantation d’îlots pancréatiques. / From isolation of pancreatic islets to their implantation, the inflammation is ubiquitous in the pancreatic islet transplantation. Maintaining a controlled inflammation is essential to preserve the survival of the graft and the functionality in the short and long term. The objective of this work is to identify precisely the inflammatory mechanisms behind the early loss of islets and identify therapeutic targets to reduce these inflammatory reactions. We have demonstrated that culture conditions induce reactions causing the development of a specific proinflammatory and pro-oxydant phenotype islet. This induction is characterized by an increase in the secretion of cytokines, chemokines pro-inflammatory activation pathways of inflammation Toll-like receptors (TLRs) -dependent and generation of reactive oxygen species (ROS). However, this process can be prevented by the activation of Heme oxygenase-1 (HO-1), an antioxidant and anti-inflammatory enzyme.By studying the inflammatory responses in an animal model of transplantation mimicking the conditions of human transplantation, we demonstrated that a change of plasma mediators of inflammation and liver proteome occurs 12 hours after transplantation. Furthermore, these results are associated with infiltration of the islets by immune cells which organizes 12 hours after transplantation. We also determined the anti-inflammatory role of rapamycin (an immunomodulatory drug) on the islets and macrophages in vitro. We have thus demonstrated that the use of rapamycin with the establishment of a pre-treatment of islets and recipient before transplantation could be considered. These studies have characterized the inflammatory mechanisms implemented immediately before and after transplantation. Thus, these data provide new therapeutic approaches that can prevent and / or reduce inflammation during pancreatic islet transplantation

L’intoxication tabagique, source d’oxydants, est un facteur de risque important de développement de l’emphysème pulmonaire et du pneumothorax spontané primitif. Les macrophages alvéolaires contribuent pour une large part à l’inflammation pulmonaire au cours de ces pathologies en produisant des métalloprotéases et des espèces réactives de l’oxygène à l’origine du déséquilibre des balances protéase/anti-protéase et oxydant/antioxydant. L'hème oxygénase-1 (HO-1), exprimée principalement par les macrophages, est une enzyme clé des défenses anti-oxydantes pulmonaires. Nous avons initialement étudié l’expression et la localisation cellulaire de l’HO-1 et de ses régulateurs potentiels (Nrf2, Keap1, Bach1 et HIF-1a) dans les macrophages alvéolaires au cours de l’emphysème pulmonaire post-tabagique et du pneumothorax spontané primitif. Les voies de régulation de l’expression de ces protéines ont été analysées in vitro sur des macrophages dérivés de la lignée THP-1 exposés ou non au condensat de fumée de cigarette et à l’hypoxieréoxygénation visant à mimer une partie des effets de l’atélectasie-réexpansion observée lors de la prise en charge thérapeutique des pneumothorax récidivants. Les travaux présentés dans cette thèse nous ont permis de mettre en évidence une altération de l’expression de la voie Nrf2/Keap1-Bach1 associée à une diminution de l’expression des enzymes anti-oxydantes, dont l’HO-1, dans les macrophages alvéolaires au cours de l’emphysème pulmonaire sévère post-tabagique, malgré un stress oxydant important. In vitro, ces altérations pourraient être liées à une activation spécifique des MAPKinases ERK1/2 et JNK par le condensat de fumée de cigarette. Nous avons également montré que la stimulation du système de l’HO-1 était probablement orchestrée par la voie du facteur HIF-1a, et non par celle de Nrf2, dans les macrophages alvéolaires au cours du pneumothorax spontané primitif récidivant du sujet fumeur. Ces résultats pourraient contribuer à une meilleure connaissance de la physiopathologie de l’emphysème pulmonaire et permettre d’envisager de nouvelles approches thérapeutiques basées sur la préservation et/ou la restauration de l’équilibre Nrf2/Keap1-Bach1. Nos travaux suggèrent également que la physiopathologie du pneumothorax spontané primitif est différente chez les patients fumeurs et non fumeurs. Le pneumothorax du sujet fumeur est associé à un stress oxydant pulmonaire et à une induction de l’HO-1 probablement orchestrée par HIF-1a. Ces résultats, confirmés in vitro, mettent en évidence une interaction potentielle entre le stress oxydant et l’hypoxie-réoxygénation / Chronic cigarette smoking, a source of oxidants, is an important risk factor for lung emphysema and primary spontaneous pneumothorax development. Alveolar macrophages are mainly involved in lung inflammation observed in these pathologies through the production of metalloproteases and reactive oxygen species resulting to protease/anti-protease and oxidant/anti-oxidant imbalances. Heme oxygenase-1 (HO-1), mainly expressed in macrophages, is a key enzyme in pulmonary anti-oxidant defences. Therefore, the first aim of our studies was to investigate the expression and cellular localisation of HO-1 and its potential regulators (Nrf2, Keap1, Bach1 and HIF-1a) in alveolar macrophages from smoking related lung emphysema and primary spontaneous pneumothorax. Regulation pathways involved in expression of these proteins were assessed in vitro in macrophage cell line THP-1 exposed or not to cigarette smoke condensate and with or without hypoxia-reoxygenation mimicking parts of events induced by atelectasia-reexpansion during recurrent pneumothorax constitution and treatment. In these studies, we showed an altered expression of Nrf2/Keap1- Bach1 pathway associated with a reduced expression of anti-oxidants enzymes, like HO-1, in alveolar macrophages from smoking related lung emphysema patients, despite an important oxidative stress. These alterations might be related to cigarette smoke condensate activated ERK1/2 and JNK MAPKinases as observed in THP-1 cells. Furthermore, we showed that HO- 1 system induction was mediated by HIF-1a instead of Nrf2 pathway in alveolar macrophages from smoking related recurrent primary spontaneous pneumothorax. These findings may contribute to a better knowledge of the pathophysiology of lung emphysema and could provide new therapeutic approaches based on preservation and/or restoration of Nrf2/Keap1-Bach1 equilibrium. Our results also suggest that the pathophysiology of primary spontaneous pneumothorax could be different in smokers and non smokers. Spontaneous pneumothorax in smokers is associated with lung oxidative stress and the orchestrated induction of HO-1 probably via HIF-1a. These results provide a new link between oxidative stress and hypoxia/reoxygenation

Many neurodegenerative diseases are characterized by the accumulation of misfolded proteins that often share common morphological and biochemical features, and can similarly co-localize with several other proteins, including various chaperone proteins. Chaperone proteins, like heat shock protein 27 (HSP27), heme oxygenase 1 (HO-1) and clusterin, have been implicated as potent modulators of misfolded proteins, thus may play important roles in the pathogenesis of neurodegenerative diseases. The present study aims to investigate their roles in the pathogenesis of Frontotemporal lobar degeneration (FTLD), Alzheimer's disease (AD), Parkinson's disease (PD), and Motor neuron disease (MND) by determining their distribution and amount via immunohistochemical staining and western blotting in diseased and control subjects.There were distinct patterns of HSP27 and clusterin immunostaining in different brain regions. For HSP27, patients with AD and FTLD were in general more severely affected than were patients with MND and control subjects. For clusterin, patients with AD and FTLD were more severely affected than control subjects where neurons and glial cells were concerned, while patients with AD and control subjects were more severely affected than those with FTLD where diffuse and cored plaques were concerned. However, there were no obvious differences in the pattern of HO-1 immunostaining in various brain regions in patients with AD or FTLD relative to control subjects. Moreover, there was no association between HSP27, HO-1 and clusterin with disease or histological type, and the ‘classic’ neuropathological changes in FTLD, AD and MND were not immunoreactive to any of these proteins. There were significant correlations between the degrees of HO-1 and clusterin immunostaining in many brain areas for both AD and FTLD cases, and for all cases overall, but none between HSP27 and clusterin or HSP27 and HO-1. Present results suggest an involvement with ongoing cellular stress, misfolded or unfolded protein accumulation or the deficits/failure of other relevant protein quality control systems, in the pathogenesis of these neurodegenerative diseases. Present work may therefore have implications for the further development of ideas concerning the cause or treatment of neurodegenerative diseases where there is aberrant accumulation of misfolded, aggregated protein, and perhaps for conformational diseases in general. However, there are still many issues remain to be elucidated. Further research aimed at understanding the function and mechanisms of the chaperone system, and other protein quality control mechanisms, in the pathogenesis of neurodegenerative diseases is still needed.