The inner surface of the stomach is lined by a mucous membrane known as the gastric mucosa. The integrity of the gastric mucosa is critical for protecting the stomach from the low pH and proteolytic environment within the lumen. Both clinically and experimentally, exposure of gastric mucosal cells to bile salts is known to cause injury. Bile salts present in duodenogastric reflux are thought to play a significant role in gastric ulcer formation and alkaline gastritis. In vitro, studies using physiologic concentrations of the secondary bile salt, deoxycholic acid, indicate that bile salts can induce apoptosis in cultured human gastric epithelial cells in a caspase-dependent manner. Therefore, there is interest in developing approaches that can protect gastric cells from bile salt-induced damage. It has been shown that induction of the stress protein, heme oxygenase-1, can provide protection against apoptosis. Therefore, the objective of this study was to test the hypotheses that heme oxygenase-1 expression could be induced in human gastric epithelial cells and that furthermore; this would provide protection from deoxycholic acid-induced apoptosis. Heme oxygenase-1 expression was induced pharmacologically or by introduction of a plasmid expressing heme oxygenase-1 into the gastric epithelial cell line, AGS. Induction of heme oxygenase-1 prior to challenge with deoxycholate reduced apoptotic-associated morphological changes, DNA fragmentation, the appearance of oligonucleosomes in the cytoplasm, and activation of caspase-3 and caspase-9. Based on these results, it was concluded that expression of heme oxygenase-1, or the introduction of its products, can provide protection to human gastric epithelial cells against sodium deoxycholic acid induced-apoptosis.

Hydrogen sulfide mediates various physiological functions and along with carbon monoxide and nitric oxide it is an important gaseous signaling molecule. Cellular targets for H2S are proteins, enzymes, transcriptional factors or ion channels. In many cases, the effect of H2S on the regulatory protein is mediated by modifications of its cystein residues. In hemeproteins, the regulation of catalytic activity is induced by formation of the Fe(III)-SH complex or by reduction of the heme iron with subsequent formation of Fe(II)-O2 complex. The effect of Na2S on model sensor heme-containing proteins is presented in this thesis. Protein, isolated from bacterium Anaeromyxobacter sp. strain FW109-5, containing a globine coupled sensor domain and a histidine kinase domain is one of the studied proteins, the second one is protein isolated from bacterium Escherichie coli, containing a globine coupled sensor domain and a diguanylate cyclase domain. The effect of Na2S on both model proteins and their mutants was studied by UV-Vis spectral analysis. Spectra of YddV-HD Y43A were very unique, because thery confirmed formation of a homogenous complex Fe(III)-SH in this protein, whereas only mixtures of varous heme complexes were detected in other tested proteins. Additionally the effect of Na2S on functional domain...

Hemoproteins represent very important components of many living organisms. Participation in the processes of oxygen transport and storage, electron transport or enzymatic catalysis of reactions involving oxygen or hydrogen peroxide are commonly known functions of hemoproteins. Recently, there has been discovered a new group of hemoproteins. The main feature of this new group of proteins is their ability to detect changes in heme concentration (heme-responsive proteins) or changes in diatomic gas concentration (gas-responsive heme-containing sensor proteins) in their vicinity. Detection of these concentration changes generates signals that induce structural changes of the respective sensor proteins. Finally, the structural changes of the respective sensor proteins affect their functions or activities. The subject of this diploma thesis is the preparation and characterization of the eukaryotic heme sensor Bach1. We especially focused on the ability of Bach1 to bind heme molecules and on the comparison of various Bach1 properties in its apoform and holoform. Determination of the exact amount of heme molecules that specifically interact with heme sensor Bach1 represents very important part of this thesis. We also studied the effect of different redox states of heme iron and the presence of interaction...

La porphyrie aiguë intermittente (PAI) est la plus fréquente des porphyries hépatiques aiguës. Elle est décrite comme une maladie autosomique dominante dont le trait génétique est estimé à 1/1675 en France avec une pénétrance faible et variable allant de 10% à 50% dans les familles connues de PAI. La PAI est due à des mutations réduisant le niveau d’activité de l’hydroxyméthylbilane-synthase (HMBS). Son déficit entraîne l’accumulation de précurseurs neurotoxiques responsables de la symptomatologie clinique. Dans le foie, la synthèse d’hème est contrôlée par l’enzyme ALA-Synthétase 1 (ALAS1) dont l’activité est régulée par un rétrocontrôle négatif par le produit final : l’hème. Le traitement consiste à freiner l’induction d’ALAS1 induit par la carence en hème, par l’administration d’hème exogène. Ce traitement de la crise aiguë est très efficace mais génère rapidement une dépendance physique avec apparition de crises récurrentes nécessitant l’administration chronique d’hème exogène. L’objectif principal de ce projet a été d’étudier les mécanismes physiopathologiques et génétiques liés à cette pathologie afin de traiter et conseiller au mieux les patients. Une partie du projet a consisté à explorer les facteurs génétiques modulateurs de la pénétrance de la maladie. Tout d’abord, une prévalence minimale du trait génétique dans la population générale a été estimée à 1/1299 permettant d’en déduire une pénétrance de l’ordre de 1% alors que celle dans les familles PAI suivies par le CFP est estimée à 22,9 %. Ensuite, concernant les facteurs pouvant expliquer cette différence, la présence d’une mutation type non-sens est plus fréquemment associée aux formes sévères et à une pénétrance plus élevée. De plus, les études de corrélation et d’héritabilité suggèrent plutôt une transmission de type oligogénique associée à des facteurs épigénétiques modulateurs de la pénétrance dont le facteur environnemental. Une autre partie a consisté à explorer les effets de l’administration d’hème exogène sur les patients et un modèle murin de PAI créé génétiquement. Chez l’homme, le traitement est associé à une augmentation des formes chroniques (1,7 % avant vs 7,5 % après l’introduction du celui-ci). Dans le modèle murin de PAI, les injections intrapéritonéales répétées induisent une augmentation paradoxale d’ALAS1 (3 fois), une augmentation de l’hème oxygénase 1 qui catabolise l’hème (HMOX1, 9 fois) ainsi que des voies de l’inflammation (analyse transcriptomique et protéomique hépatique) et une surcharge en fer. De plus, cette administration induit une altération des complexes de la chaine respiratoire mitochondriale responsable d’anomalies du métabolisme énergétique au niveau hépatique, cérébral et musculaire pouvant expliquer la symptomatologie neuroviscérale. En conclusion, ce travail a permis d’explorer les caractéristiques génétiques de la maladie (prévalence, pénétrance) en remettant en cause le mode de transmission autosomique dominant jusqu’ici admis, et d’explorer les mécanismes physiopathologiques associées à l’administration d’hème exogène faisant de cette thérapeutique un pharmakon / The biosynthesis of porphyrins is one of the most conserved pathways known. By associating different metals, porphyrins give rise to the "pigments of life". The formation of haem is accomplished by a sequence of eight dedicated enzymes encoded by different genes, some being active in ubiquitous as well as in erythroid isoforms. In humans, the genes for each of the haem synthetic enzymes may become the target of mutations that give rise to an impaired cellular enzyme activity called porphyrias. The acute porphyrias are characterized by attacks of neuropsychiatric symptoms, which may be due to a toxic surplus of the porphyrin precursor 5-aminolevulinic acid, or a consequence of a deficit of vital hemoproteins. Mutations of the gene encoding the third enzyme: hydroxymethylbilane synthase, are associated with the most frequent type of acute hepatic porphyria, acute intermittent porphyria. AIP is thought to display autosomal dominant inheritance with incomplete penetrance. In the classical form of AIP, HMBS activity is about 50% lower than normal in all tissues. These levels of activity in basal conditions are not sufficiently low to cause symptoms. However, factors increasing hepatic heme demand, resulting in an upregulation of hepatic aminolevulinate synthase (ALAS1, the first enzyme of the heme biosynthesis pathway), precipitate acute attacks. The treatment of the attack of AIP consists to repress ALAS1 and restores metabolic equilibrium. But this treatment leads side effects and dependency. The pathophysiological mechanism of the disease is partially known and difficult to explore because there is not an AIP model or prediction model of porphyrogenicity. We aimed to obtain further insight into the pathophysiological mechanism of AIP and into the genetic (prevalence and penetrance) of AIP, and the contribution of genetic factors to the variable clinical expression of HMBS mutations.We first calculated the penetrance of HMBS mutations in AIP patients seen at the French reference center for porphyria: 22.9%. We then used the Exome Variant Server (EVS) to estimate the prevalence of deleterious HMBS mutations in the general population: 1/1299; and the penetrance of the AIP genetic trait in France: 1%. Finally, we investigated further the genetic factors underlying the penetrance of AIP by analyzing genotype/phenotype correlations, and the pattern of familial correlations for the symptoms of the acute crises of AIP. Intrafamily correlation studies showed correlations to be strong overall and modulated by kinship and the era in which the person was living, demonstrating strong influences of genetic and environmental modifiers on inheritance suggesting that AIP inheritance does not follow the classical autosomal dominant model. Null alleles were associated with a more severe phenotype and a higher penetrance than for other mutant alleles.On the other hand, we explored the effect of heme administration. In human, the introduction of hemin into the pharmacopeia has coincided with a 4.4-fold increase in the prevalence of chronic patients. We show that repeated hemin infusions in mice trigger a high level heme oxygenase 1 response, induce a pro-oxidative iron accumulation, a complex pattern of liver inflammation with macrophage infiltration and an alteration of oxidative phosphorylation

5-Aminolevulinate synthase (ALAS; EC 2.3.1.37) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the first committed step of heme biosynthesis in animals, the condensation of glycine and succinyl-CoA yielding 5-aminolevuliante (ALA), CoA, and CO2. Murine erythroid-specific ALAS (mALAS2) variants that cause high levels of PPIX accumulation provide a new means of targeted, and potentially enhanced, photosensitization. Transfection of HeLa cells with expression plasmids for mALAS2 variants, specifically for those with mutated mitochondrial presequences and a mutation in the active site loop, caused significant cellular accumulation of PPIX, particularly in the membrane. Light treatment of HeLa cells expressing mALAS2 variants revealed that mALAS2 expression results in an increase in cell death in comparison to aminolevulinic acid (ALA) treatment producing a similar amount of PPIX. Generation of PPIX is a crucial component in the widely used photodynamic therapies (PDT) of cancer and other dysplasias. The delivery of stable and highly active mALAS2 variants has the potential to expand and improve upon current PDT regimes. Mutations in the C-terminus of human ALAS2 (hALAS2) can increase hALAS2 activity and are associated with X-linked erythropoietic protoporphyria (XLEPP), a disease phenotypically characterized by elevated levels or PPIX and zinc protoporphyrin in erythroblasts. This is apparently due to enhanced cellular hALAS2 activity, but the biochemical relationship between these C-terminal mutations and increased hALAS2 activity is not well understood. HALAS2 and three XLEPP variants were studied both in vitro to compare kinetic and structural parameters and ex vivo in HeLa and K562 cells. Two XLEPP variants, delAGTG, and Q548X, exhibited higher catalytic rates and affinity for succinyl-CoA than wild-type hALAS2, had increased transition temperatures, and caused porphyrin accumulation in HeLa and K562 cells. Another XLEPP mutation, delAT, had an increased transition temperature and caused porphyrin accumulation in mammalian cells, but exhibited a reduced catalytic rate at 37[deg]C in comparison to wild-type hALAS2. The XLEPP variants, unlike wild-type hALAS2, were more structurally responsive upon binding of succinyl-CoA, and adopted distinct features in tertiary and PLP cofactor-binding site. These results imply that the C-terminus of hALAS2 is important for regulating its structural integrity, which affects kinetic activity and stability. XLEPP has only recently been identified as a blood disorder, and thus there are no specific treatments. One potential treatment involves the use of the antibiotic isonicotinic acid hydrazide (isoniazid, INH), commonly used to treat tuberculosis. INH can cause sideroblastic anemia as a side-effect and has traditionally been thought to do so by limiting PLP availability to hALAS2 via direct inhibition of pyridoxal kinase, and reacting with pyridoxal to form pyridoxal isonicotinoyl hydrazone. We postulated that in addition to PLP-dependent inhibition of hALAS2, INH directly acts on hALAS2. Using FACS and confocal microscopy, we show here that INH reduces protoporphyrin IX accumulation in HeLa cells expressing either wild-type human hALAS2 or XLEPP variants. In addition, PLP and pyridoxamine 5'-phosphate (PMP) restored cellular hALAS2 activity in the presence of INH. Kinetic analyses with purified hALAS2 demonstrated non-competitive or uncompetitive inhibition with an apparent Ki of 1.5 uM. Circular dichroism studies revealed that INH triggers structural changes in hALAS2 that interfere with the association of hALAS2 with its PLP cofactor. These studies demonstrate that hALAS2 can be directly inhibited by INH, provide insight into the mechanism of inhibition, and support the prospective use of INH in treating patients with XLEPP and potentially other cutaneous porphyrias.

Porphyromonas gingivalis, a Gram negative anaerobe implicated in the progression of periodontal disease, is capable of surviving and causing infection despite high levels of reactive nitrogen species found in the oral cavity due to its efficient nitrosative stress response. HcpR is an important sensor-regulator that plays a vital step in the initiation of the nitrosative stress response in many Gram negative anaerobic bacteria. We employ a combination of X-ray crystallography, SAXS, resonance Raman spectroscopy, UV-Vis spectroscopy, and molecular biology techniques to better understand this key regulator. Knockout of the hcpR gene in W83 P. gingivalis results in the inability of the bacteria to grow in physiological concentrations of nitrite and complementation of hcpR using the novel plasmid Pg108 rescues this phenotype. HcpR causes a drastic, dose dependent upregulation of PG0893, a gene coding for a putative NO reductase, when exposed to nitrite or nitric oxide. Full transcriptome sequencing reveals that hcp is the only significantly upregulated gene when P. gingivalis is exposed to nitrite and knockout of hcp resulted in a phenotype that is similar to that of the hcpR deficient strain. HcpR directly regulates the expression of hcp via direct binding to an inverted repeat sequence in the promoter region of the hcp gene. We present a 2.6 Å crystal structure of the N-terminal sensing domain of HcpR and show that it is FNR-CRP regulator. A putative hydrophobic heme binding pocket was identified in the junction between the N-terminal domain and the dimerization helix. Mutation of two methionine residues (Met68 and Met145) in this pocket abrogates activation of HcpR thus verifying the binding site. Heme bound to HcpR exhibits heme iron as a hexa-coordinate system in the absence of nitric oxide (NO) and upon nitrosylation transitions to a penta-coordinated system. Finally, Small Angle X-ray Scattering experiments of the full length HcpR reveal that the C-terminal DNA binding domain of HcpR has a high degree of interdomain flexibility.

Le syndrome hémolytique et urémique atypique (SHUa) est une microangiopathie thrombotiquecomplément-dépendante dont l'atteinte majoritairement rénale reste, à ce jour, incomprise.L'objectif de ce travail était d'améliorer la compréhension de ce tropisme d’organe au moyen dedeux axes d'étude : i) la susceptibilité de l'endothélium glomérulaire à l'hémolyse, celle-ci étant à la fois la conséquence des microthromboses dans le SHUa et un amplificateur de la voie alterne du complément via l'hème libre, molécule issue des globules rouges lysés ; ii) le rôle potentiel du récepteur aux produits de glycation avancés ou RAGE. Ce récepteur membranaire aux fortes propriétés pro inflammatoires et pro thrombotiques a en effet été impliqué dans de nombreuses pathologies rénales, et sa liaison au C3a - anaphylatoxine libérée dans l'activation du complément - a été rapportée par une équipe.La première partie de ce travail a visé à expliquer la vulnérabilité de l'endothélium glomérulaire sous l'effet d'une hémolyse. Nous avons étudié plusieurs types de cellules endothéliales exposées +/- à l'hème, et mis au point un modèle murin traité par de l'hème. En conditions hémolytiques, plusieursfacteurs pouvant participer à cette susceptibilité endothéliale glomérulaire ont ainsi été mis en avant: i) une moindre liaison du facteur H, principal régulateur du complément, à sa surface ; ii) une faible expression de la thrombomoduline, protéine de la coagulation et régulatrice du complément ; iii) une faible expression de l'enzyme principale de dégradation de l'hème, l'hème-oxygénase 1. Ces deux derniers points étaient rattachés à une faible induction endothéliale glomérulaire de leurs facteurs de transcription, KFL2 et KLF4.La seconde partie de ce travail s'est concentrée sur le RAGE. N'ayant pas réussi à reproduire l'interaction RAGE/C3a, nous avons exploré l'hypothèse d'une liaison du RAGE à l'hème. En effet, le seul récepteur endothélial connu jusqu'à présent est le Toll Like Receptor 4 (TLR4), qui partage plusieurs ligands communs avec le RAGE (LPS - lipopolysaccharide, HMGB1 - high–mobility group box1). Nous avons découvert que le RAGE était un récepteur de l'hème, et identifié que le site de liaisonse trouvait sur le domaine V. A l'aide d'un modèle murin invalidé pour le RAGE et traité +/- par l'hème, nous avons mis en évidence que : i) l'invalidation de RAGE avait un effet protecteur en cas d’exposition à l'hème, marqué par une diminution de l'expression de gènes de l'inflammation (IL1β,TNFα) et du facteur tissulaire au niveau pulmonaire, organe exprimant le plus fortement RAGE ; ii)l'hème activait la phosphorylation des voies ERK1/2 et Akt via le RAGE.Par ces travaux, nous avons précisé les liens entre activation du complément, hémolyse et susceptibilité endothéliale glomérulaire dans le SHUa. Parallèlement, nous avons identifié le RAGE comme un nouveau récepteur à l'hème, dont la liaison à ce récepteur activerait différentes voies designalisation de l'inflammation. Le contrôle de l'hème et du RAGE pourrait ainsi constituer de nouvelles voies thérapeutiques dans le SHUa et les maladies hémolytiques. / The atypical haemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy of which the predominantly renal damage remains, to date, misunderstood. The aim of this work was to improve the understanding of this organ tropism by two axes of study: i) the susceptibility of the glomerular endothelium to hemolysis, which is the consequence of microthrombosis in aHUS, and also an complement pathway enhancer via free heme, hemoglobin-mediated hemolysis molecule, ii) the potential role of the receptor for advanced glycation end products, RAGE. Indeed, RAGE is described as an endothelial receptor with high proinflammatory and prothrombotic potential, involved in many kidney diseases; a team also reported that it was a receptor for the C3a molecule, anaphylatoxin released in complement activation.The first part of this work aimed to explain the vulnerability of the glomerular endothelium under the effect of hemolysis. We studied several types of endothelial cells exposed +/- to heme, and developed a murine model treated with heme. In hemolytic conditions, several factors that could participate in the endothelial glomerular susceptibility have been put forward: i) less binding of factor H, the main complement regulator, on its surface; ii) low expression of thrombomodulin, coagulation protein and complement regulator; iii) low expression of heme-oxygenase 1, the main heme degradation enzyme. These last two points were related to low induction, on glomerular endothelial cells, of transcription factors, KFL2 and KLF4.The second part of this work focused on RAGE. Having failed to reproduce the RAGE / C3a interaction, we explored the hypothesis of a linkage of RAGE to heme. Indeed, the only known endothelial receptor is Toll Like Receptor 4 (TLR4), which shares several common ligands (LPS - lipopolysaccharide, HMGB1 - high-mobility group box 1). We found that RAGE was a heme receptor, and identified that the binding site was on domain V. Using a mouse model knock out for RAGE and treated +/- with heme, we demonstrated that i) the invalidation of RAGE had a protective effect in case of exposure to heme, marked by a decrease in the expression of genes of inflammation (IL1β; TNFα; and tissue factor) at the pulmonary level, organ expressing most strongly RAGE, ii) the heme is an activator or the phosphorylation of ERK1 / 2 and Akt pathways via RAGE.Through this work, we have clarified the links between complement activation, hemolysis and glomerular endothelial susceptibility in aHUS. At the same time, we have identified RAGE as a new heme receptor, whose RAGE/heme bindind would activate different signaling pathways for inflammation. The control of heme and RAGE could constitute new therapeutic pathways in the aHUS, and hemolytic diseases.

Le système du complément est une cascade de défense immunitaire complexe et étroitement régulée, conduisant à des dommages tissulaires lorsqu’il est suractivé. L’hème, un motif moléculaire de danger dérivant de l’hémolyse, est capable d’activer le complément dans le sérum et à la surface des cellules endothéliales (CE) in vitro, suggérant un rationel pour examiner l’impact de l’activation du complément dans les maladies hémolytiques. L’objectif de ce projet était d’étudier si et comment l’hémolyse intravasculaire active le complément in vivo, et de comprendre les mécanismes sous-jacent conduisant à l’acquisition d’un phénotype activateur du complément par les CE afin d’identifier de nouvelles cibles thérapeutiques. Nous avons détecté des dépôts de complément, de C3 et de C5b-9, dans des reins de patients souffrants de nephropathie drépanocytaire ainsi que dans un modèle murin de drépanocytose. Nous avons mis en place un modèle murin d’hémolyse intravasculaire massive, déclenchée par la phénylhydrazine (PHZ), et caractérisé l’atteinte rénale. Nous avons détecté des dépôts de C3 au niveau des reins de ces souris. Cet effet a été inhibé par l’administration préventive du scavenger naturel de l’hème, l’hémopexine (Hx), et reproduit par des injections d’hème libre, démontrant une activation hème-dépendante in vivo. Les microvésicules d’érythrocytes (MVs) drépanocytaires représentent une source naturelle d’hème, de par leur concentration en hème trois fois supérieure à celle observée chez les donneurs sains. Nous avons démontré que les MVs drépanocytaires activent le complément dans le sérum et sur les CE, de manière en partie hème-dépendante. Ces résultats révèlent le rôle activateur de l’hème sur le complément dans les maladies hémolytiques. De plus, nous avons démontré que l’interaction de l’hème avec TLR4 peut en partie expliquer les dépôts de C3 sur l’endothélium in vivo et les CE in vitro. L’utilisation d’un inhibiteur de TLR4, le TAK-242, a réduit de 50% les dépôts de complément sur les CE, confirmé par une réduction des dépôts sur l’endothélium vasculaire chez des souris TLR4-/- traitées par PHZ ou hème. De plus, nous avons montré que ces dépôts hème/TLR4 dépendants sont liés à l’expression rapide de P-sélectine, qui recrute C3b et C3(H2O) à la membrane des CE, révélé par l’analyse des interactions protéiques en temps réel et l’utilisation d’un anticorps bloquant anti-P-sélectine. Ensemble, ce projet démontre que l’hème et les MVs sont les produits dérivés de l’hémolyse responsables de l’activation du complément. Au niveau cellulaire, l’induction par l’hème d’un phénotype activateur du complément des CE dépend de l’axe TLR4/P-sélectine, induisant des dépôts de C3 à la surface cellulaire. Ainsi, ces études soulignent les bénéfices potentiels de l’Hx et du TAK-242 contre l’activation du complément dans des pathologies associées à une hémolyse. / Complement system is a complex and tightly regulated innate immune defensive cascade, which can promote tissue damage, when overactivated. Hemolysis-derived danger associated molecular pattern heme is able to activate complement in serum and on endothelial cells (EC) in vitro, providing a rational for scrutinizing the impact of complement activation in hemolytic diseases. The objectives of this work were to study whether and how intravascular hemolysis induces complement activation in vivo, and to understand the underlying mechanism that leads to the acquisition of a complement activating phenotype of the endothelium in order to identify novel therapeutic strategies. We found complement deposits, including C3 activation fragments and C5b-9, within kidneys of patients with sickle cell disease (SCD) nephropathy (a prototypical hemolytic disease) as well as in a mouse model of SCD. We set up and characterized the renal injury of a mouse model of massive intravascular hemolysis, triggered by injection of phenylhydrazine (PHZ). We revealed C3 deposition within kidneys of the PHZ-treated animals. It was prevented by heme scavenging with hemopexin (Hx) and reproduced by injections of free heme, thus demonstrating the importance of heme for the complement activation in vivo. SCD erythrocytes microvesicles (MVs), are a pathologically relevant source of labile heme, since they carry three times more heme on their surface compare to MVs from healthy donors. We demonstrated that MVs, generated from SCD erythrocytes, activate complement in human serum and on EC surface, in part on a heme-dependent manner. These data highlight the importance of heme as a complement activator in hemolytic diseases. Further, we found that the C3 activation fragments deposits on endothelium in vivo and on EC in vitro can be in part explained by interaction of heme with TLR4. Indeed, the use of a specific inhibitor of TLR4, TAK-242, reduced about 50% the complement deposits on EC surface and such deposits on vascular endothelium in PHZ- or heme-injected mice were attenuated TLR4-/- mice. Moreover, we found that heme/TLR4-dependent complement deposition was mediated by the rapid expression of P-selectin, which in turn, recruited C3b and C3(H2O) on the EC surface, as evidenced by real time protein interaction analyses and using of blocking antibodies. Together our results demonstrated that heme and erythrocytes MVs are the hemolysis-derived products which promoted complement activation. At cellular level, heme induced complement-activating phenotype of EC by triggering TLR4/P-selectin axis and resulting in C3 activation fragments on cell surface. Together, these studies underline the potential benefits of Hx and TAK-242 against complement activation in pathologies related to hemolysis.

Quantum chemistry has nowadays become a powerful and efficient tool that can be successfully used for studies of biosystems. It is therefore possibleto model the enzyme active-site and the reactions undergoing into it, as well as obtaining quite accurate energetic profiles. Important conclusions can be drawn from such profiles about the  plausibility of different putative mechanisms. Density Functional Theory is used in the present thesis for investigation of the catalytic mechanism of dioxygenase metallo-enzymes and synthetic model complexes. Three enzymes were studied – Homoprotocatechuate 2,3-dioxygenase isolated from Brevibacterium fuscum (Bf 2,3-HPCD), Manganese-Dependent Homoprotocatechuate 2,3-Dioxygenase (MndD) and Homogentisate Dioxygenase (HGD). Models consisting of 55 to 208 atoms have been built from X-ray crystal structures and used in the calculations. The computed energies were put in energy curves and were used for estimation of the feasibility of the suggested reaction mechanisms. A non-heme [(L4Me4)Fe(III)]+3 complex that mimics the reactivity of intradiol dioxygenases, and a heme [T(o-Cl)PPFe] complex catalyzing the stepwise oxidation of cyclohexane to adipic acid, were also studied. For the enzymes and the non-heme biomimetic complex the reaction was found to follow a mechanism that was previously suggested for extradiol and intradiol dioxygenases – ordered substrates binding and formation of peroxo species, which further undergoes homolytic O-O bond cleavage. Different reaction steps appear to be rate limiting in the particular cases: proton transfer from the substrate to the peroxide in Bf 2,3-HPCD, the formation of the peroxo bridge in HGD and the biomimetic complex, and notably, spin transition in MndD. The catalytic oxidation of cyclohexane to adipic acid in the presence of molecular oxygen as oxidant was studied, a reaction of great importance for the chemical industry. Reaction mechanism is suggested, involving several consecutive oxidative steps. The highest calculated enthalpy of activation is 17.8 kcal/mol for the second oxidative step. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: In progress, Paper 5: In progress

A limited understanding of iron uptake mechanisms is available for Streptococcus pyogenes, a hemolytic human pathogen capable of using a variety of hemoproteins in addition to ferric and ferrous iron. This study characterizes the transporters of iron-complexes siuADBG (for streptococcal iron uptake) and siaABC (for streptococcal iron acquisition). These ABC-type transporters are encoded by iron regulated operons and their protein products are homologous to components of heme and siderophore transporters found in both Gram-positive and Gram-negative bacteria. Mutants of the membrane permeases siuG and siaB were constructed and characterized. Mutations in both transporters demonstrated growth reduction in comparison to the parent strain when grown in complex medium containing iron in the form of hemoglobin. The addition of heme to the growth medium inhibited ferric uptake by the wild-type while the addition of protoporphyrin IX did not, suggesting that heme utilization as an iron source is responsible for the inhibition of ferric accumulation. Inactivation of siuG reduced the ability of heme to inhibit ferric incorporation by the cells. Inactivation of siaB in addition to siuG had a cumulative effect, indicating that both siu and sia transporters are involved in heme utilization. We also demonstrated that purified rSiaA, the surface receptor of SiaABC, binds heme and hemoglobin in vitro, and we propose a mechanism of heme binding by SiaA. Studies in a zebrafish infection model revealed that the siuG mutant was attenuated in producing disease. While the siaB mutant also presented virulence attenuation, infection by this mutant was characterized by an increase in the host inflammatory response. These observations show that iron acquisition is important for S. pyogenes virulence. We propose that the SiaABC and SiuADBG, together with the multi-metal transporter MtsABC, are involved in iron acquisition from different iron sources present in the human body, thus contributing to the survival and pathogenesis of S. pyogenes.