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No description available.

<p> The Scavenger Receptor, Class B, Type I (SR-BI) is an integral membrane protein whose expression in the liver is critical to reverse cholesterol transport by mediating the selective uptake of HDL-derived cholesterol. SR-BI is expressed in a variety of tissues including bone marrow derived macrophages and foam cells in atherosclerotic lesions. We have explored the effect of eliminating SR-BI in leukocytes on advanced stages of atherosclerotic plaque development in apoE KO mice. We observed statistically significant cardiomegaly as a result of the elimination of SR-BI in bone marrow derived cells compared to controls (P=0.02). We report that the elimination of SR-BI in bone marrow derived cells in apoE KO mice induced to undergo atherosclerosis by feeding a high fat diet for four weeks leads to no significant difference in cross-sectional atherosclerotic plaque area at the aortic root (4.9±0.9x10^4 μm^2 when SR-BI-/- apoE-/- --> apoE-/- [n=9] and 5.5±0.9x10^4 μm^2 when SR-BI +/+ apoE-/- --> apoE -/- [n=12], P=0.68) or plaque volume through the aortic sinus (1.8±0.3x 10^7 μm^3 when SR-BI-/- apoE-/- --> apoE-/- [n=9] and 1.9±0.3x10^7 μm^3 when SR-BI +/+ apoE-/- --> apoE -/- [n=12], P=0.69). We demonstrate that macrophage SR-BI protein expression can be decreased by cholesterol associated with lipoproteins. Furthermore, we report that in Raw 264.7 macrophage-like cells the expression of SR-BI can also decrease in response to glucosamine treatment. The expression of SR-BI is decreased significantly in cells overexpressing SR-BI (1d1A[mSR-BI] cells [P=0.003]) due to treatment with glucosamine with increased protein mobility. We support this finding by demonstrating that this difference may be the result of altered glycosylation.</p> / Thesis / Master of Science (MSc)

京都大学 / 新制・課程博士 / 博士(工学) / 甲第25297号 / 工博第5256号 / 京都大学大学院工学研究科材料化学専攻 / (主査)教授 松原 誠二郎, 教授 中尾 佳亮, 教授 杉野目 道紀 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Macrófagos (Mf) produzem PAF e PAF-R e eliminam partículas alteradas via CD36. Uptake de oxLDL requer associação CD36/PAF-R. Avaliamos isto na eferocitose. Bloqueio do PAF-R e de lipid rafts (LR) inibiu eferocitose. Esta induziu associação PAF-R/CD36 e destes com flotilina-1 (marca LR). Eferocitose induziu IL-10 e IL-12p40. Bloqueio do PAF-R inibiu mais IL-10 e inibição da COX-2 teve efeito similar, sugerindo que eferocitose depende da interação PAF-R/CD36 em LR e que isto induz prostanoides e perfil regulador. Mf adquirirem diferentes fenótipos. Estudamos a participação do PAF-R. Bloqueio do PAF-R antes dos estímulos (IFN-g/LPS, IL-4 ou IgG-SRBC/LPS) inibiu marcadores MCP-1, TNF-a, iNOS, receptor manose, arginase-1 e IL-10, mas não IL-12p40, sugerindo que PAF-R modula fenótipo de Mf. PAF e PAF-like são gerados por estressores oxidativos. Ativação do PAF-R induz imunossupressão sistêmica (IS). Mostramos que terapia fotodinâmica (PDT) in vitro gerou ligantes do PAF-R e in vivo inibiu reação de CHS em WT, mas não em PAF-R KO, sugerindo que PDT induz IS via PAF-R. / Macrophages (Mp) produce PAF and PAF-R and scavenge altered particles via CD36. oxLDL uptake requires association CD36/PAF-R. We analyzed that on efferocytosis. PAF-R and lipid rafts (LR) blockage inhibited efferocytosis. Efferocytosis induced association CD36/PAF-R and both with LR marker protein, and induced IL-10 and IL-12p40. PAF-R and COX-2 blockage inhibited more IL-10, suggesting that efferocytosis depends on PAF-R/CD36 interaction in LR and that this induces prostanoids and regulatory profile. Mp acquire different phenotypes. PAF-R participation in that was analyzed. PAF-R blockage before stimuli (IFN-g/LPS, IL-4 or IgG-SRBC/LPS) inhibited markers MCP-1, TNF-a, iNOS, mannose receptor, arginase-1 and IL-10, but not IL-12p40, suggesting that PAF-R modulates Mp phenotype. PAF and PAF-like are generated by oxidative stressors. PAF-R activation induces systemic immunosuppression (SI). We showed that photodynamic therapy (PDT) in vitro generated PAF-R ligands and in vivo inhibited CHS reaction in WT, but not PAF-R KO, suggesting that PDT induces SI via PAF-R.

Der Einsatz von Kohlenstoffmaterialien z.B. in ITER ist damit verbunden, dass es durch physikalische und chemische Zerstäubung zur Bildung von Kohlenwasserstoffen kommt, die im Randschichtplasma zersetzt werden und sich in Form tritiumreicher amorpher Kohlenwasserstoffschichten auf den Wänden ablagern. Deshalb ist ein besseres Verständnis der Bildung, der Zersetzung, des Transports und der Haftung von Kohlenwasserstoffen infolge der Plasma-Wand-Wechselwirkung von großem Interesse. Die genannten Prozesse wurden am linearen Plasmagenerator PSI-2 mit Hilfe verschiedener Diagnostiken für unterschiedliche Plasmen systematisch untersucht. Die Analyse der ablaufenden Reaktionen mittels einfacher Bilanzgleichungen machte es möglich, wichtige Zerfalls- und Bildungskanäle für die verschiedenen Kohlenwasserstoffe und deren Abhängigkeiten von bestimmten Parametern zu ermitteln. Es zeigte sich, dass die starke Zersetzung und Umwandlung von Methan in Wasserstoffplasmen auf die dominierenden Ladungsaustauschreaktionen im Niedertemperaturbereich zurückzuführen ist. Weiterhin wurden Zersetzungslängen für Methan und Ethen gefunden, die im Bereich einiger Zentimeter liegen. Die Untersuchungen der CH-Band-Emission und der Wachstumsprozesse von a-C:H-Schichten ermöglichten die Detektion von globalen und lokalen Zersetzungsprozessen in unterschiedlichen Plasmen, die je nach Plasmadichte (und Gasart) erklärt werden können. Bisher fehlte in allen Modellierungen die atomare Wasserstoffdichte, die hier mit zwei unterschiedlichen Methoden bestimmt wurde. Durch Depositionsexperimente mit und ohne direkten Plasmaeinfluss konnte der durch Stickstoffeinlass verursachte Scavenger Effekt und die damit verbundene Reduktion der Depositionsrate eindeutig nachgewiesen werden. Die Auswertung der QMS-Daten mit Hilfe der Bayesschen Statistik ermöglichte erstmals die Spezifikation der beteiligten Volumenreaktionen. / The materials envisaged for the thermally heavily burdened divertor plates of the international fusion device ITER are CFC materials. As a result of physical and chemical sputtering of these materials many different hydrocarbons are formed which are decomposed at the plasma edge and lead to the deposition of tritium- rich amorphous hydrocarbon films on the vessel walls. Consequently a better understanding of hydrocarbon formation, fragmentation, transport and sticking is an important issue in fusion research. The aforementioned processes are studied systematically at the linear plasma generator PSI-2 using various diagnostics in a range of plasmas. By means of simple balance equations the ongoing reactions could be analysed making it possible to identify important decomposition and formation channels for the various hydrocarbons and their dependence on certain parameters. The strong decomposition and transformation of methane in hydrogen plasmas can be traced back to dominant charge exchange reactions in the low temperature range. In addition, decomposition lengths for methane and ethylene in the range of a few centimetres were found. Spatially resolved measurements of CH band emission and investigations of the growth processes of a-C:H layers enabled the detection of global and local decomposition processes of injected hydrocarbons in different plasmas which can be explained according to the plasma density (and gas type). So far all simulations have lacked information on the density of atomic hydrogen, which in this work has been determined using two different methods. In addition, by means of deposition experiments with and without direct plasma influence, the scavenger effect induced by nitrogen injection and the associated reduction in the deposition rate has been clearly demonstrated. The analysis of the QMS data using Bayesian statistics enabled verification of the volume reactions involved for the first time.

Macrófagos (Mf) produzem PAF e PAF-R e eliminam partículas alteradas via CD36. Uptake de oxLDL requer associação CD36/PAF-R. Avaliamos isto na eferocitose. Bloqueio do PAF-R e de lipid rafts (LR) inibiu eferocitose. Esta induziu associação PAF-R/CD36 e destes com flotilina-1 (marca LR). Eferocitose induziu IL-10 e IL-12p40. Bloqueio do PAF-R inibiu mais IL-10 e inibição da COX-2 teve efeito similar, sugerindo que eferocitose depende da interação PAF-R/CD36 em LR e que isto induz prostanoides e perfil regulador. Mf adquirirem diferentes fenótipos. Estudamos a participação do PAF-R. Bloqueio do PAF-R antes dos estímulos (IFN-g/LPS, IL-4 ou IgG-SRBC/LPS) inibiu marcadores MCP-1, TNF-a, iNOS, receptor manose, arginase-1 e IL-10, mas não IL-12p40, sugerindo que PAF-R modula fenótipo de Mf. PAF e PAF-like são gerados por estressores oxidativos. Ativação do PAF-R induz imunossupressão sistêmica (IS). Mostramos que terapia fotodinâmica (PDT) in vitro gerou ligantes do PAF-R e in vivo inibiu reação de CHS em WT, mas não em PAF-R KO, sugerindo que PDT induz IS via PAF-R. / Macrophages (Mp) produce PAF and PAF-R and scavenge altered particles via CD36. oxLDL uptake requires association CD36/PAF-R. We analyzed that on efferocytosis. PAF-R and lipid rafts (LR) blockage inhibited efferocytosis. Efferocytosis induced association CD36/PAF-R and both with LR marker protein, and induced IL-10 and IL-12p40. PAF-R and COX-2 blockage inhibited more IL-10, suggesting that efferocytosis depends on PAF-R/CD36 interaction in LR and that this induces prostanoids and regulatory profile. Mp acquire different phenotypes. PAF-R participation in that was analyzed. PAF-R blockage before stimuli (IFN-g/LPS, IL-4 or IgG-SRBC/LPS) inhibited markers MCP-1, TNF-a, iNOS, mannose receptor, arginase-1 and IL-10, but not IL-12p40, suggesting that PAF-R modulates Mp phenotype. PAF and PAF-like are generated by oxidative stressors. PAF-R activation induces systemic immunosuppression (SI). We showed that photodynamic therapy (PDT) in vitro generated PAF-R ligands and in vivo inhibited CHS reaction in WT, but not PAF-R KO, suggesting that PDT induces SI via PAF-R.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Schwefellost (SM) ist ein verbotener chemischer Kampfstoff. Nach Aufnahme über die Haut führt SM zu einer Reihe von Symptomen. Auf der molekularen Ebene beruht die Toxizität von SM auf der Reaktion mit DNA und Proteinen. Diese kovalenten Addukte eignen sich zum forensischen Nachweis und können über Flüssigkeitschromatographie gekoppelte Massenspektrometrie (LC-MS) detektiert werden. Ein Hauptziel war es in vitro zu untersuchen ob chemisches Scavenging von N-Acetylcystein (NAC) und Glutathion (GSH) Einfluss in der Behandlung von SM-Vergiftungen hat. Es konnte geklärt werden, dass NAC und GSH a) die Alkylierung von Cys34 in humanen Serum Albumin (HSA) durch SM nicht unterbinden kann und b) den Abbau von SM in gepufferter Lösung nicht beschleunigt. Somit ist Scavenging von SM durch NAC und GSH vernachlässigbar. Trotzdem konnte die Stabilität und die Zuverlässigkeit des Testsystems gezeigt werden. Das zweite Hauptziel war es neue peptidische Biomarker für den Nachweis von SM-Vergiftungen zu finden. Es wurde eine Methode entwickelt, um SM-alkylierte Aminosäurereste in HSA zu finden. Somit konnten 42 SM-alkylierte Peptide gefunden werden. Insgesamt wurden 27 Alkylierungsstellen identifiziert, von denen 24 noch nicht in der Literatur beschrieben wurden. Die vielversprechendste der neue Alkylierungsstellen war das Met329 in HSA. Durch Proteolyse mittels Pepsin wurde das LGM(-HETE)F Tetrapeptid freigeschnitten. Probenvorbereitung und LC-MS Bedingungen wurden optimiert. Allerdings gelang der Nachweis von LGM(-HETE)F in Patientenplasma nicht, was möglicherweise an einer geringeren Stabilität der SM-Alkylierung von Met329 lag. Trotzdem kann sich LGM(-HETE)F als Kurzzeitmarker eignen. Der beobachtete Transfer der HETE-Modifikation vom Met329 zu Glu und Cys Seitenketten stellt einen neuen Einblick in den molekularen Wirkmechanismus von SM dar. Basierend auf diesen Erkenntnissen konnte in Nachfolgestudien die Hemmung von Enzymen durch Alkylierung von Met durch SM gezeigt werden. / Sulfur mustard (SM) is a banned chemical warfare agent. After incorporation, SM may cause tremendous symptoms. On the molecular level, SM reacts with DNA and proteins. These covalent adducts may be useful for forensic or analytical purposes. After adducting SM, proteins can be proteolyzed forming peptides with a SM-modified amino acid residue. These peptide biomarkers can be detected using liquid chromatography-mass spectrometry (LC-MS). One major aim was to develop an in vitro assay to clarify the impact of chemical scavenging of N-acetylcysteine (NAC) and glutathione (GSH) in the treatment of SM. It was found that NAC and GSH had no relevant influence on a) the extent of alkylation of Cys34 in human serum albumin (HSA) and b) the degradation velocity of SM in buffered solution. Accordingly, it was concluded that chemical scavenging of SM by NAC or GSH is negligible. Nevertheless, the robustness and reliability of the developed in vitro assay was shown. The second major aim was to identify novel peptide biomarkers of SM poisoning. An untargeted method to identify SM-alkylated amino acid residues in HSA was developed. Application of this method revealed 42 SM-modified peptides alkylated at 27 different positions. 24 of these positions were not described in the literature so far. A highly interesting target, Met329 in HSA, was investigated in more detail. After pepsin mediated proteolysis, the covalently modified tetrapeptide LGM( HETE)F was generated. Sample preparation and LC-MS conditions were optimized. However, when applying this novel marker to real samples, it could not be detected likely due to its limited stability. Nevertheless, LGM( HETE)F still represents a suitable short term biomarker. The observed transfer of the HETE-moiety from Met329 to Glu and Cys side chains represents a novel insight into the molecular toxicology of SM. Based on these results follow-up studies proved the enzyme inhibitory effect of SM-alkylation of Met residues in keratin kinase.

Red blood cells (RBCs) are vital for oxygen delivery to tissues and constitute the vast majority of all cells in blood. After leaving the red bone marrow as mature cells, RBCs have a lifespan of approximately 120 days before they are removed from the circulation by macrophages, mainly in the spleen and liver. RBC transfusion is a common therapy in modern healthcare. Major surgery, numerous cancer treatments and other, often lifesaving, interventions would be unthinkable without available blood supply. For this reason, hospitals store donated RBCs in blood banks. The metabolic and structural changes that occur during prolonged storage of RBCs (the storage lesion) have been studied in detail in vitro and include oxidative stress, a reduction in glycolysis, increased membrane rigidity and shedding of microparticles from the RBC membrane. Stored RBCs share several features of senescent RBCs, but also with RBCs undergoing an apoptotic-like process called eryptosis. A consequence of the storage lesion is the fact that as much as 25% of stored RBCs could be rapidly removed from the circulation within 24 hours after transfusion. The mechanisms behind this rapid macrophage-mediated recognition and removal of stored RBCs, and its immunological consequences, remain largely unknown. Therefore, the aims of this thesis were to investigate if cryopreserved human RBCs induced an inflammatory response following autologous transfusion into healthy volunteers, and to further understand the mechanisms behind macrophage recognition of stored RBCs in vitro and in vivo. Autologous transfusion of two units of cryopreserved RBCs into healthy human recipients was found to be associated with an increased extravascular RBC elimination already at 2 hours after transfusion. However, there were no signs of an increased production of any of the investigated pro-inflammatory cytokines, indicating that an increase in the destruction of RBCs per se did not induce an inflammatory response. Eryptosis is a form of induced RBC death associated with an increased cytoplasmic Ca2+ uptake. We found that a subset of human RBCs increased their Ca2+ permeability during prolonged storage at +4°C. Using a murine model, to further understand how RBCs with an increased Ca2+ permeability were eliminated by phagocytic cells in the spleen, it was found that such RBCs were taken up by marginal zone macrophages and dendritic cells (DCs) in a manner distinct from that of naturally senescent RBCs. The DC population particularly efficient in this process expressed CD207 and are known for their ability to promote immunological tolerance. Eryptotic cell uptake was not regulated by the phagocytosis-inhibitory protein CD47 on the RBCs. To investigate how RBCs damaged during liquid storage are recognized and taken up by macrophages, a model to store and transfuse murine RBCs was developed. This storage model generated murine RBCs with several characteristics similar to that of stored human RBCs (i.e. loss of ATP, formation of RBC microparticles and rapid clearance of up to 35% of the RBCs during the first 24 h after transfusion). In vitro phagocytosis of human as well as murine stored RBCs was serum dependent and could be inhibited by blocking class A scavenger receptors using fucoidan or dextran sulphate. In conclusion, the findings of this thesis contribute to further understanding how changes inflicted to RBCs during storage direct the fate of these cells in their interaction with cells of the immune system after transfusion. The observation of an increased Ca2+ permeability of stored RBCs, and the possible recognition of such cells by tolerance-promoting DCs, in combination with the findings that class A scavenger receptors and serum factors may mediate recognition of stored RBCs, may result in novel new directions of research within the field of transfusion medicine.