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

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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.

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

O hipertireoidismo é uma patologia epidemiologicamente importante, que afeta o sistema cardiovascular de forma proeminente. O estado hipertireoideo pode afetar o metabolismo basal, consumo de O2 celular, sistema renina angiotensina, assim como, estimular a produção de espécies ativas de oxigênio. Estas alterações produzem consequências morfológicas, funcionais, bioquímicas e moleculares no tecido cardíaco. A hipertrofia cardíaca, decorrente do hipertireoidismo, instala-se devido a uma série de eventos que sinalizam à proliferação e sobrevivência celular, envolvendo as espécies ativas de oxigênio, a ativação do sistema renina angiotensina cardíaco e o sistema nervoso autonômico. Neste estudo, bloqueamos o receptor AT1 da angiotensina II para avaliarmos a influência do sistema renina angiotensina cardíaco sobre o desenvolvimento da hipertrofia cardíaca, a participação do balanço autonômico sobre o coração e o papel das espécies ativas de oxigênio neste processo, em modelo experimental de hipertireoidismo. Para isto, foram utilizados ratos Wistar machos, pesando cerca de 220g, divididos em 4 grupos experimentais: Controle (C), Losartan (L) (10 mg/Kg de peso corporal/dia, 28 dias, sonda intragástrica) , T4 (12mg/L água de beber, 28 dias), e T4+L. Foram avaliados a massa cardíaca, análise espectral do balanço simpato-vagal, a expressão protéica do receptor AT1 da Angiotensina II e da gp91phox, peróxido de hidrogênio (H2O2), Nrf-2 e Heme-oxigenase-1 (HO-1) no tecido cardíaco. A hipertrofia cardíaca e o desequilíbrio autonômico induzidos pelo hipertireoidismo foram atenuados no grupo T4+L. Os níveis de H2O2, Nrf-2, gp91phox e HO-1 foram elevados no grupo T4, e significativamente reduzidos no grupo T4+L, quando comparados ao grupo Controle. A expressão protéica do receptor AT1 esteve elevada nos dois grupos hipertireoideos. Os resultados obtidos sugerem que o bloqueio do receptor AT1 promove importante impacto sobre o balanço simpato-vagal e a hipertrofia cardíaca, no hipertireoidismo, sendo as espécies ativas de oxigênio e o sistema Nrf-2/HO-1 possíveis mediadores destas alterações. / Hyperthyroidism is an epidemiologic relevant pathology, which substantially affects the cardiovascular system. The hyperthyroid state may affect basal metabolism, O2 cell consumption, renin-angiotensin system, and increase reactive oxygen species production. Those alterations produce morphological, biochemical, functional and molecular consequences in cardiac tissue. Hyperthyroidism induced cardiac hypertrophy develops due to a set of events, which signals cell survival and proliferation, including reactive oxygen species, cardiac rennin-angiotensin system, and autonomic nervous system. In the present study, the role of cardiac renin-angiotensin system on development of hyperthyroidism induced cardiac hypertrophy, and the involvement of autonomic nervous system and reactive oxygen species, were assessed trough blockade of angiotensin II receptor AT1. For that, were used male Wistar rats, weighting about 220g, divided in 4 experimental groups,: Control (C), Losartan (L) (10mg/Kg body weight/day, 28 days, intragastric probe), T4 (12mg/L L-thyroxin in drinking water, 28 days), and T4+L. Cardiac mass, spectral analysis (autonomic balance), hydrogen peroxide (H2O2), and myocardial protein expression of angiotensin II receptor (AT1), NADPH oxidase, Nrf-2, and heme-oxygenase-1 (HO-1), were quantified. Cardiac hypertrophy and autonomic umbalance induced by thyroid hormones were attenuated in the T4+losartan group. The H2O2, as well as Nrf-2, gp91phox, AT1 and HO-1 immunocontent were elevated in T4 group. All these effects were attenuated by losartan, except AT1 levels. The overall results suggest that blockade of AT1 receptor lead to relevant impact on autonomic balance and cardiac hypertrophy, being ROS and Nrf-2/ HO-1 system possible mediators in this alterations in experimental hyperthyroidism.

Nutritional factors with antioxidant properties, such as those contained in edible algae or green plants, might be implicated in protection against cancer development. Chlorophyll and other tetrapyrrolic compounds, structurally related to heme and antioxidant bile pigment bilirubin, belong to important candidate molecules, which might be responsible for these effects. Based on our studies demonstrating antiproliferative effects of S. platensis edible alga extract on experimental model of human pancreatic adenocarcinoma we investigated in detail the effect of chlorophyll occurring abundantly in this alga. Since only scarce data exist on the antiproliferative effects of chlorophylls, the aim of our study was to assess these effects. The study was performed on experimental models of human pancreatic and prostate cancer. The inhibitory effects of chlorophylls (chlorophyll a, chlorophyll b, chlorophyllin and pheophytin a) on cell proliferation and cell viability were investigated in in vitro studies. Chlorophylls reduced the mRNA expression as well as activity of hemeoxygenase in tested pancreatic cancer cells. Simultaneously, chlorophylls played an important role in redox environment of studied cancer cell lines including modulation of mitochondrial membrane potential, reactive oxygen species (ROS)...

Background and aims: Treatment of acute fulminant liver damage arising as a result of various origins (ischemia-reperfusion injury, toxic shock, an infectious cause or cholestasis) still remains a major clinical problem. We currently do not have available clinically proven, pharmacologically effective and universal compound for the treatment of acute liver injury. The main aim of my research work was, therefore, to test the potential hepatoprotective effect of selected cytoprotective drugs and try to find out or suggest their mechanism of action, which we have examined in the systems for the intracellular gaseous signaling molecules NO and CO, where the key enzymes for their formation are NOS / HO respectively. My PhD study had two main directions: 1) Experimental study of the relationship between HO / CO and NOS / NO systems in the environment of hepatotoxic substances on isolated primary rat hepatocytes and in rat model, 2) Evaluation of ameliorative effect of selected substances in the hepatotoxicity models and to test the relationship of this effect on changes in some parameters of cytotoxicity / cytoprotection, antioxidant parameters, gene expression of mRNA for selected genes and histological changes in the state of cells / tissues / organs. Methods: We measured urea, bilirubin and liver...

This thesis focuses on the importance of the heme catabolic pathway in chronic hepatitis C (HCV). The aim is mainly to investigate, whether expresion/activity of key enzymes of the heme catabolic pathway, heme oxygenase (HMOX) and biliverdin reductase (BLVRA) in the liver and blood (study A) or promoter variations of HMOX1 and UDP- glucuronosyltransferase (UGT1A1) (study B) may be associated with the progression of fibrosis and may also predict antiviral treatment outcome in patients chronically infected with HCV. We set up a new sensitive method to quantify HMOX activity by reduction gas chromatography. We developed and extensively validated RealTime PCR assay for HMOX and BLVRA expression in the liver and peripheral blood leucocytes (PBL). The (GT)n and (TA)n dinucleotide variations in HMOX1 and UGT1A1 gene promoters, respectively, were determined by fragment analysis. No association was detected between either expression of HMOX/BLVRA or the HMOX1/ UGT1A1 promoter variants and the individual histological stages of liver disease in the HCV positive patients. A marked difference in BLVRA expression in PBL between the sustained responders (SVR) and patients with treatment failure (NVR) was detected before antiviral treatment and during the follow-up. Our data suggests, that BLVRA basal expression...