University, Diss., 2003--Bonn.

Host defense against pathogenic organisms represents one of the most important and highly-conserved biological processes across the evolutionary timescale. The ability to detect, engulf and destroy particulates for either nutrition or host defense is conserved from single-celled protists to complex multicellular organisms. A central component of host defense is the recognition of invariant, conserved patterns on pathogenic organisms through the use of pattern recognition receptors (PRRs), such as macrophage receptor with collagenous structure (MARCO). MARCO modulates binding and phagocytosis of unopsonized microorganisms and particulates, tethers ligands to signalling complexes and enhances cellular adhesion. Current literature suggests these functions are mediated via the C-terminal scavenger receptor cysteine rich (SRCR) domain. The relative importance of this domain remains unclear, as other, closely-related scavenger receptors function independently of the SRCR domain via a shared lysine-rich motif. In chapter 3.1, we discovered and cloned a naturally-occurring transcript variant of MARCO which lacks the SRCR domain, termed MARCOII. We demonstrated that the SRCR domain is required for ligand binding and internalization and that MARCOII can form heteromeric complexes with MARCO and reduce receptor function. Furthermore, the SRCR domain enhanced TLR2/CD14-mediated pro-inflammatory responses to Streptococcus pneumoniae. Finally, it was demonstrated that the SRCR domain modulates MARCO-mediated cellular adhesion. In chapter 3.2, we used comparative phylogenetics to identify human specific mutations and residues undergoing positive selection in human MARCO. We demonstrated that humans possess a unique phenylalanine residue at position 282 that is polymorphic, with some humans encoding an ancestral serine residue. We also demonstrated that glutamine at position 452 is found in Denisovans, Neanderthals, and extant humans, but all other non-primate, terrestrial, and aquatic mammals possess an aspartic acid residue. We cloned the ancestral residues and loss-of-function mutations and demonstrated that these residues enhance ligand association and phagocytosis of Escherichia coli. / Thesis / Doctor of Philosophy (PhD) / Some of the most ancient mechanisms of host defense rely on invariant recognition of pathogens through the use of pattern recognition receptors, such as the macrophage receptor with collagenous structure (MARCO). MARCO plays an integral role to allow for specialized subsets of white blood cells to bind pathogens, activate signalling complexes and to bring pathogens inside the white blood cell for destruction. Current literature suggests the C-terminal Scavenger Receptor Cysteine Rich (SRCR) domain of MARCO is required for these processes. This remains under scrutiny, as other closely-related receptors have been shown to operate independently of the SRCR domain. Herein, we utilized a variant of MARCO which lacks the SRCR domain and patterns of evolution to confirm both that the SRCR domain is critical for receptor function and to discover novel sites within the human MARCO protein that play indirect, but important roles in receptor function.

Meningokokken gehören zu den wichtigsten Erregern bakterieller Sepsis und Meningitis. Der Schweregrad des Krankheitsverlaufs bei Meningokokkenerkrankungen korreliert mit der Konzentration an proinflammatorischen Zytokinen im Serum. Dendritische Zellen (DZ) bilden die erste Abwehr am humanen Epithel des Nasopharynx, welches die Eingangspforte von Neisseria meningitidis darstellt und sind eine wichtige Quelle proinflammatorischer Zytokine. In dieser Arbeit konnte gezeigt werden, dass bekapselte Meningokokken-Stämme bei DZ signifikant weniger proinflammatorische Zytokine als isogene Kapsel-defiziente Stämme oder obligat unbekapselte Stämme induzieren. Dieser Effekt ist unabhängig von der chemischen Zusammensetzung der Kapsel, da aufgereinigtes Kapselpolysaccharid der Serogruppe B nicht den reduzierenden Effekt der Zytokininduktion beeinflusste. Darüber hinaus spielt die Kapsel-O-Acetylierung bei Serogruppe C, W-135 und Y nur eine untergeordnete Rolle bei der Erkennung von Meningokokken durch DZ. Microarray Versuche zum Transkriptionsprofil von DZ, die mit dem konstitutiv unbekapselten Trägerisolat alpha 14, dem bekapselten MC58 oder dem isogenen unbekapselten Stamm MC58siaD durchgeführt wurden, zeigten nach 4 h ein identisches Profil von proinflammatorischen Zytokinen. Nur der phagozytierte unbekapselten Stamm MC58siaD zeigte eine differentielle Regulation von weiteren Zytokinen. Jedoch glich sich das Profil nach 18 h Infektion durch alle drei Stämme an. Der Scavenger Rezeptor der Klasse A (SR-A) wurde als Hauptrezeptor identifiziert, der die Erkennung und Phagozytose von Meningokokken durch DZ initialisiert. Eine Assoziation phagozytierter Meningokokken mit SR-A konnte mittels Elektronenmikroskopie bestätigt werden. Nach Infektion von THP-1 Makrophagen mit bekapselten Serogruppe B und C Stämmen, den isogenen Kapsel-defizienten Stämmen und dem obligat unbekapselten Stamm alpha 14 wurde auf Transkriptionsebene keine differentielle Regulation der SR-A nachgewiesen. Lediglich eine minimale Hochregulation des SR-A auf der zellulären Oberfläche konnte nach einer Stunde Infektion verzeichnet werden. Nach Infektion von DZ oder THP-1 Makrophagen mit MC58siaD kommt es zur Dephosphorylierung des SR-A. Unter Verwendung von globalen Phagozytose-Inhibitoren konnte gezeigt werden, dass für die maximale Induktion der proinflammatorischen Zytokine TNF-alpha, IL-6 und IL-1 Phagozytose von N. meningitidis benötigen wird, dies ist jedoch für die IL 8 Produktion nicht notwendig. Außerdem konnte gezeigt werden, dass mit dem spezifischen SR-A Inhibitor poly G eine Reduktion von TNF-alpha, IL-6, IL-1 und IL-8 zu verzeichnen war. Folglich ist die Phagozytose über SR-A nötig, um TNF-alpha, IL-6 und IL-1 zu induzieren, jedoch nur die Erkennung aber nicht die Phagozytose via SR-A die IL-8 Produktion initiiert. Die Aufnahme von Neisseria meningitidis über den SR-A durch DZ ist damit nicht nur für die Phagozytose und Abtötung verantwortlich sondern auch für die Zytokininduktion wichtig ist. Es gibt jedoch auch Meningokokken Stämme, die nicht vom SR-A erkannt werden. Mit alpha 14 konnte erstmals ein Meningokokken-Stamm identifiziert werden, der nicht an SR-A bindet. Die Induktion von Zytokinfreisetzung durch alpha 14 erfolgt dementsprechend unabhängig von SR-A und nach Kontakt von alpha 14 mit humanen DZ ist keine Veränderung der Phosphorylierungsstatus dieses Rezeptors zu beobachten. Die erhobenen Daten legen eine zentrale Rolle von SR-A in der Induktion von Immunität gegen N. meningitidis nahe. / Meningococci belong to the most important pathogens which cause bacterial sepsis and meningitis. The severity and outcome of meningococcal disease correlates with the concentration of proinflammatory cytokines in the serum. Dendritic cells (DC) build a first line of defence within the nasopharyngeal epithelium, which is the port-of-entry for Neisseria meningitidis and are an important source of proinflammatory cytokines. Infection of DC with encapsulated meningococcal strains induces significantly lower levels of proinflammatory cytokines than isogenic capsule-deficient mutants or constitutively unencapsulated strains. It could be shown that this effect does not depend on the chemical composition of the meningococcal capsule, because purified capsular polysaccharide of serogroup B is unable to modulate cytokine secretion. O-acetylation of serogroup C, W-135 and Y capsules plays a minor role in the recognition of meningococci by DC. Microarray studies on the expression of cytokine genes by DC infected with constitutively unencapsulated carriage isolate alpha 14, encapsulated serogroup B strain MC58 or the isogenic unencapsulated strain MC58siaD showed an identical pattern for proinflammatory cytokines after 4 h of infection. Only the unencapsulated strain MC58siaD which is easily phagocytosed induced additional cytokines. However, the cytokine profile after 18 h of infection was equal for all three strains. Scavenger receptor A (SR-A) was identified as the main receptor mediating recognition and phagocytosis of meningococci by DC. Association of phagocytosed meningococci with SR-A was visualized by electron microscopy. Infection of THP-1 macrophages with encapsulated serogroup B und C strains, their isogentic capsule-deficient muntants or the obligatory unencapsulated strain alpha 14 did not induce differential regulation of SR-A on the transcriptional level and only minor up-regulation of SR-A on cellular surface after one hour of infection. After infection of DC or THP-1 macrophages with MC58siaD dephosphorylation of SR-A can be detected. Using global phagocytosis inhibitors, it could be determined that the induction of the proinflammatory cytokines TNF-alpha, IL-6 and IL-1 require phagocytosis of N. meningitidis, whereas induction of IL-8 does not. In addition, the specific SR-A-inhibitor poly G also leads to a reduction of TNF-alpha, IL-6, IL-1 and IL-8 secretion. Taken together these data indicate that phagocytosis via SR-A is necessary for TNF-alpha, IL-6 and IL-1 secretion, whereas recognition but not phagocytosis via SR-A enhances IL-8 secretion. Therefore, the uptake of Neisseria meningitidis via SR-A is not only important for phagocytosis and killing but also involved in cytokine induction. However, not all meningococcal strains are recognized via SRA. The cytokine induction by DZ infected with alpha 14 is completely independent of SR-A and after contact of human DC with alpha 14 no changes in SR-A phosphorylation can be detected. These data suggest that SR-A plays a central role in the induction of immunity against N. meningitidis.

Würzburg, Universiẗat, Diss., 2008.

University, Diss., 2002--Münster (Westfalen).

L’entrée cellulaire du virus de l’Hépatite C (VHC) est un processus complexe qui met en jeu plusieurs facteurs cellulaires. L’un d’eux est un récepteur des lipoprotéines, le « Scavenger Récepteur B1 » (SR-BI). SR-BI a initialement été proposé comme récepteur viral de par son interaction avec la glycoprotéine (GP) E2 du VHC. L’importance de SR-BI pour l’entrée cellulaire du VHC n’était, jusqu’alors suggérée que par des preuves indirectes. En outre, les mécanismes par lesquels SR-BI permet l’entrée du VHC restent peu connus. Néanmoins, grâce à l’identification de cellules hépatiques présentant un très faible niveau d’expression - non détectable - de ce récepteur et devenant susceptibles à l’infection par le VHC par l’expression ectopique de SR-BI, nous avons clairement démontré que SR-BI est indispensable pour l’entrée du VHC. Afin d’étudier le rôle de SR-BI dans l’entrée cellulaire du VHC, qui présente une singulière hétérogénéité en terme de propriétés biochimiques et de composition en protéines cellulaires intégrées à la surface des particules virales, celles-ci ont été séparées par centrifugation analytique en gradient de densité. Nous avons ainsi défini trois fonctions de SR-BI permettant l’entrée de sous-populations du VHC. D’un part, une fonction d’attachement, correspondant à la capture des particules de densités intermédiaires à la surface cellulaire. Cet attachement résulte de l’interaction entre SR-BI et des composants des lipoprotéines présents à la surface des particules virales, sans mettre en jeu d’interaction avec les GP virales. D’autre part, nous avons défini une fonction d’accès, requise pour toutes les sous-populations du VHC. Cette fonction, elle aussi indépendante de l’interaction directe entre la GP E2 et SR-BI, requiert la fonction physiologique de transfert de lipides de SR-BI. En effet, le blocage de cette fonction de transfert à l’aide de molécules inhibitrices ou par insertion de mutations invalidantes de SR-BI réduit significativement l’entrée du VHC. Enfin, nous avons mis en évidence une troisième fonction, appelée fonction de stimulation, nécessitant l’interaction de la GP E2 et SR-BI, ainsi que la fonction de transfert lipidique deSR-BI et qui, par ailleurs, est régulée par des composants des lipoprotéines. Par l’analyse fonctionnelle de mutants, nous avons défini des déterminants viraux, dans la région HVR1 à l’extrémité N-terminale de la GP E2, et cellulaires, dans la partie N-terminale de SR-BI, critiques pour l’interaction entre E2 et SR-BI et, par conséquent, régulent la fonction destimulation. En conclusion, nous avons démontré que le VHC exploite SR-BI de diverses façons et via des interactions à la fois avec des composants viraux et cellulaires incorporés dans les particules du VHC et ainsi permet l’entrée de particules virales très hétérogènes. / Hepatitis C virus (HCV), which is characterised by its highly heterogeneous biophysical properties, is thought to enter the cell in a slow and multistep manner involving several cell surface molecules. One of these cellular molecules is the Scavenger Receptor B1 (SR-BI), which was identified as an HCV receptor due to its interaction with the HCV glycoprotein E2.Until now, the exact usage of SR-BI by HCV and SR-BI mediated mechanism during cell entry remained unknown. In order to understand SR-BI functions during HCV cell entry, we wanted to study (1) the relevance of HCV E2 binding to SR-BI, (2) the implication of the physiological function of SR-BI itself and (3) how SR-BI mediates cell entry of heterogeneous HCV particles. Owing to the identification of two cell lines that express very low levels of endogenous SRBI, receptor complementation assays revealed, that the ectopic expression of SR-BI is indispensible for HCV entry. Accordingly, we showed for the first time, that SR-BI is an essential HCV entry factor. In order to study HCVcc populations that differ in biophysical properties and host protein composition, we separated them by density gradient analysis and assigned three different SR-BI functions to entry of particular HCV sub-populations. First, an attachment function, that leads to the initial capture of HCV particles of intermediate densities to the cell surface. This attachment function is mediated by an interaction between SR-BI and lipoprotein components on the viral particles but not by the viral glycoproteins. Second, we defined an access function, which is important for all different HCV sub-populations. This access function is also not dependent on the interaction between HCV E2 and SR-BI but involves the physiological function of the receptor. Blocking the lipid transfer function of SRBI upon either mutation or by a specific inhibitor abrogated strongly HCV entry. Finally we defined a third function of SR-BI, that we call enhancement function. This function is triggered upon E2-SR-BI interaction, is dependent on lipoprotein components and involves the lipid transfer function of SR-BI. Upon functional mutagenesis studies, we identified as critical determinants HVR1 (Hypervariable Region 1) residues in E2 and the N-terminus of SR-BI, allowing E2-SR-BI interaction and consequently the implementation of the enhancement function. In conclusion we demonstrate that SR-BI is an unparalled virus entry factor. Its usage by HCV to enter the cell is manifold and intriguingly, owing to the heterogeneous nature of HCV particles, involves different viral components exploiting different aspects of SR-BI.

Nos travaux ont permis d’étudier différents aspects des interactions hôte-pathogènes. L’étude de différents Pattern Recognition Receptors (PRR) autres que les TLR, ainsi que leurs associations a mis en évidence une redondance partielle entre les récepteurs des familles des Scavenger Receptors, lectines de type C et EMR1 in vitro et in vivo dans l’induction de la réponse immunitaire à Mycobacterium tuberculosis. Cette compensation entre les récepteurs contraste avec les rôles indépendants et non redondants des cytokines et de leurs voies associées comme le TNF, l’IL-1R1, L’IFNγR et MyD88, indispensables pour le contrôle de l’infection. Grâce à l’utilisation de nouvelles souris génétiquement modifiées, nous avons pu montrer un rôle minime de la lymphotoxine α dans le contrôle de l’infection par Mycobacterium tuberculosis contrairement au rôle primordial du TNF. Enfin, notre étude s’est poursuivie avec l’analyse de la modulation de la réponse immunitaire de l’hôte par des composants de la paroi des mycobactéries, les PIM. L’élaboration de PIM synthétiques a permis de montrer que ces molécules de faibles poids moléculaires inhibent l’induction des voies TLR4 et TLR2 et possèdent ainsi un potentiel anti-inflammatoire thérapeutique. / In these study, we aimed to investigate different aspects of the host-pathogen interactions. We investigated the involvement of various Pattern Recognition Receptors (PRR) other than TLR, and their associations for the control of M. tuberculosis infection. We highlighted a partial redundancy between members of the Scavenger Receptors family, C-type lectins and EMR1 in response to mycobacteria in vitro and in vivo. This is in sharp contrast with the cytokine pathways like TNF, IL-1R1, IFNγR and MyD88, essentials to control M. tuberculosis infection and which cannot compensate with each other. By using new genetically deficient mice, we showed a limited role for the lymphotoxin α in the control of the infection by Mycobacterium tuberculosis in contrast with the vital role for TNF. Finally, we analysed the modulation of the immune response by mycobacterial cell wall components, PIM. Use of synthetic PIM demonstrated that these small molecules exert an inhibitory activity on TLR4- and TLR2-signaling pathways and may have a therapeutic anti-inflammatory potential.

Hydrogen Sulfide (H2S) is a harmful gas produced during petroleum extraction that leads to corrosion of drilling tools and pipelines. However, a H2S-scavenging liquid compound, when added to pipelines, interacts with liquids that absorbed H2S to create a non-corrosive bi-product. The interaction is associated with the mixing of gases and liquids. This thesis is a study on the effect of superficial gas and liquid velocities on the scavenger's efficiency. This study employs two experimental setups designed to simulate the mixing of gases and liquids within pipelines. A high pressure closed loop was designed and fabricated to determine the influence of gas, liquid velocities and liquid volume on the scavenger's efficiency. All experiments were conducted in this high pressure loop with a thousand feet of coiled tubing to simulate the horizontal section of the pipeline that runs along the ocean floor from the reservoir. This provided practical understanding to petroleum companies to make a better forecast of how the scavenger used in eliminating the H2S, is affected in the process of transporting the liquids and gases from the reservoir to the surface. For an adequate analysis, experiments on four liquid and four gas velocities ranging from 0.2m/s to 0.5m/s and 0.4m/s to 1.1m/s respectively were conducted. Results in this study indicated that increases in superficial gas velocity at low superficial liquid velocity decreases the scavenger efficiency while the opposite is seen at high superficial liquid velocity. In addition, the H2S mass absorption was not a function of liquid volume as would be seen in static reservoirs but more of a function of superficial liquid and gas velocities. With the scavenger interacting with the liquid absorbed H2S, it was expected that the efficiency would increase with the increase in volume but in this study this was not the case. The second experiment is a flow visualization loop which was designed to understand the flow regimes at high pressures. This was done by constructing four 25ft section hoses together with four foot long breaks for visualization. This provided a more fundamental study of the fluid's behavior inside the pipelines allowing for the creation of appropriate flow regime maps in air-water flow. A hundred experiments for two different pressures were conducted at the 25ft location. At high pressures, the flow regime map appeared to shift the transition zones.

Environmental pollution has been a serious concern worldwide. Many degradation methods have been developed to clean sites contaminated with pollutants. More knowledge and better understanding in this field will help to protect our environment. The goal of the research in this thesis is to gain a better understanding of the mechanism of organic pollutant degradation in Fenton reactions and sonochemical reactions. Fenton degradation uses hydroxyl radical to oxidize organic compounds. The radical is produced by catalytic decomposition of hydrogen peroxide with Fe(II). Further research has found that addition of cyclodextrins can enhance degradation efficiency of hydrophobic organic pollutants. To study the mechanism of the enhancement, pollutant-cyclodextrin-Fe(II) aqueous systems were studied by fluorescence and NMR techniques. The results indicated the formation of pollutant/carboxymethyl-â-cyclodextrin/Fe(II) ternary complexes in the solution. With the ternary complex, the catalyst Fe(II) becomes closer to the pollutant, therefore leading to more efficient hydroxyl radical attack on the pollutant. Additional studies showed that hydropropyl-â- cyclodextrin, â-cyclodextrin and á-cyclodextrin bound pollutant well, but bound Fe(II) poorly. Sulfated-â-cyclodextrin did not bind well with pollutant although it bound Fe(II) well. Sonochemical degradation is another important pollutant treatment method in practice. It was found that phenol sonolysis can be enhanced by volatile hydrogen atom scavengers such as carbon tetrachloride and perfluorohexane. The non-volatile hydrogen atom scavenger iodate did not enhance phenol degradation. The first order rate constant for aqueous phenol degradation increased by about 2.2-2.8 times in the presence of 150 ìM carbon tetrachloride. In the presence of less than 1.5 ìM perfluorohexane the first order rate constant increased by about 2.3 times. Hydroquinone was the major observed reaction intermediate both in the presence and absence of hydrogen atom scavengers. Hydroquinone yields were substantially higher in the presence of hydrogen atom scavengers, suggesting that hydroxyl radical pathways for phenol degradation were enhanced by the hydrogen atom scavengers. The additives investigated in this study have potential to improve pollutant degradation efficiency. Other fields may also benefit from the information gained in this study. For example the improvement could be achieved in synthetic processes that rely on hydroxyl radical as a key intermediate.

Introduction Human CD36 is a class B scavenger receptor expressed in a variety of cell types including macrophages and endothelial cells. This heavily glycosylated membrane protein has a large ectodomain (ED) responsible for binding a variety of ligands. These include oxidised LDL and long chain fatty acids, which link CD36 to the development of atherosclerosis and insulin resistance, respectively. CD36 has also been implicated in the phagocyte-uptake of apoptotic cells, anti-angiogenic effects in endothelial cells and development of a variety of fibrotic diseases, through interaction with thrombospondin-1. The main objective of this study was to express, purify and characterise the ligand binding ectodomain of CD36 with a view to better understanding how CD36 binds multiple ligands. Methods A baculovirus expression system was used to express and secrete CD36 ED with a 12-His tag from insect cells using an N-terminal secretion sequence. Subsequent purification was performed using nickel affinity chromatography with functionality of the ED assessed through the ability to bind modified LDL in a solid phase binding assay. The N-glycosylation status of the purified ED was explored through use of the N-glycosidase PNGase F and mass spectrometric analysis. Initial studies to measure binding kinetics involved use of surface plasmon resonance (SPR) and binding of commercially available antibodies, mAb1955, mAb1258 and mAbFA6-152, to purified CD36 ED immobilised on an NTA SPR sensor chip. Results The N-glycosylation status of CD36 ED produced in Sf21 and Hi5 insect cells was different with heterogeneous N-glycosylation being identified at individual glycosylation sites. Solid-phase ligand binding revealed that both glycosylated and deglycosylated ED retain affinity for modified LDL. The purified CD36 ED was found to homo-oligomerise particularly at higher concentrations. MAb1955 and mAb1258 were found to bind to the nickel on the sensor chip precluding microkinetic analysis of binding to CD36. However, mAbFA6-152 was found to bind to the immobilised CD36 ED with high avidity and two-step binding kinetics. Conclusions and future direction This study shows for the first time that N-glycosylation is not important for the binding of modified LDL to CD36. Furthermore it was demonstrated that native CD36 ED produced in Sf21 insect cells can be deglycosylated without denaturation, which may be critical for the success of future crystallisation trials. The characterisation of mAbFA6-152 binding by SPR is proof-of-principle that CD36 ED can be studied using this technique paving the way for future biophysical analysis of ligand binding.