Alexandra Flocke Untersuchung von Stoffwechselparametern und Lipoproteinen im Blutserum von einlings- und zwillingsträchtigen Merino- und Schwarzköpfigen Fleischschafen im peripartalen Zeitraum Medizinische Tierklinik, Veterinärmedizinische Fakultät, Universität Leipzig Eingereicht im Februar 2012 (94 Seiten, 15 Abbildungen, 8 Tabellen, 199 Literaturangaben, 12 Tabellen im Anhang) Schlüsselwörter: Lipoproteine, Schaf, Stoffwechsel, Endotoxin, Ketose Problemstellung: Der Lipoproteinstatus in der kritischen Phase der Hochträchtigkeit wurde beim Schaf bisher nur wenig untersucht. Die Fähigkeit der Lipoproteine, sowohl Lipide zu transportieren und im Körper umzuverteilen, als auch ihre Rolle in der Endotoxinneutralisation machen sie zu einem wichtigen Parameter in stoffwechselbelasteten Situationen. Das Ziel dieser Arbeit ist es, den Lipoproteinstatus zweier verschiedener Leistungsrassen zu analysieren und zu prüfen, ob und inwiefern peripartal Unterschiede bei Ein- und Zwillingsträchtigkeiten bestehen. Zusätzlich werden die Endotoxine der Tiere bestimmt und mögliche Zusammenhänge mit der Stoffwechselsituation und dem Lipoproteinstatus evaluiert. Tiere, Material und Methoden: Untersucht wurden gesunde einlingsträchtige Merinofleischschafe (MFS1), zwillingsträchtige Merinofleischschafe (MFS2) sowie zwillingsträchtige Schwarzköpfige Fleischschafe (SKF2). Im Abstand von je sieben Tagen wurden den Tieren im Zeitraum von der 5. Woche a.p. bis eine Woche p.p. Blutproben aus der V. jugularis externa entnommen. Aus dem Serum wurden die Konzentrationen von β-Hydroxybutyrat (BHB), Glucose, Freie Fettsäuren (FFS), Triacylglycerol (TG), Cholesterol, Bilirubin, Gesamtprotein (alle Hitachi 912), Insulin (RIA-Kit, Firma IBL, Hamburg), α- und β-Lipoproteine (LIPIDOPHOR All In12®, - ohne prä-β-Lipoproteine), freies Endotoxin (LAL-Test) und Anti-Lipid A Antikörper (ALAAK; ELISA) bestimmt. Ergebnisse: Die α-Lipoproteinkonzentration stieg bei den MFS1 vor dem Ablammen signifikant an und erreichte eine Konzentration von = 208,0 ± 40,1 mg/dl (1. Woche a.p.). Die MFS2 wiesen vor dem Lammen einen konstanten Verlauf zwischen = 180,4 ± 31,1 mg/dl (5. Woche a.p.) und = 196,1 ± 42,1 mg/dl (3. Woche a.p.) auf. Die SKF2 zeigten in der 5. Woche a.p. mit = 206,5 ± 39,0 mg/dl eine größtenteils signifikant höhere Konzentration gegenüber den folgenden Untersuchungszeitpunkten. Bei beiden Rassen ließ sich bei den Zwillingsmuttern nach dem Lammen ein signifikanter Abfall der α-Lipoproteinkonzentration auf = 144,3 ± 46,1 mg/dl (MFS2) bzw. = 170,2 ± 48,8 mg/dl (SKF2) feststellen. Es bestand eine gesicherte Korrelation zu Cholesterol. Die β-Lipoproteinkonzentration lag in der 1. Woche p.p. bei den Zwillingsmuttern (MFS2 und SKF2) signifikant niedriger als vor dem Lammen. Die SKF2 wiesen in der 5. und 4. Woche a.p. signifikant niedrigere Konzentrationen als die MFS1 auf. Gesicherte Korrelationen bestanden zu TG, Cholesterol und ALAAK (MFS1). Für die α-Lipoproteinkonzentration wurde ein Referenzbereich von 144,3 – 208,0 mg/dl und für die β-Lipoproteinkonzentration von 13,6 – 28,0 mg/dl bei Muttertieren der beschriebenen Rassen ermittelt. Ausgenommen die Insulinkonzentration in der 1. Woche a.p. bestanden keine signifikanten Differenzen zwischen MFS1 und MFS2. Im Vergleich der Rassen zeigten sich signifikante Differenzen in der Glucosekonzentration in der 2. Woche a.p. und in der Konzentration von Bilirubin in der 3. und 1. Woche a.p.. Die BHB-Konzentration zeigte einen konstanten Verlauf und stieg bei den MFS1 bis auf = 0,96 ± 0,36 mmol/l eine Woche p.p., bei den MFS2 auf = 0,73 ± 0,35 mmol/l und bei den SKF2 auf = 0,63 ± 0,14 mmol/l eine Woche a.p. signifikant an. Gesicherte Korrelationen bestanden zu FFS (MFS2) und Insulin (MFS2; SKF2). Die Glucosekonzentration zeigte sich bei den Zwillingsmuttern nach zunächst abfallenden Konzentrationen in der Hochträchtigkeit eine Woche p.p. signifikant höher ( = 3,63 ± 0,59 mmol/l; MFS2 und = 3,28 ± 0,42 mmol/l; SKF2) als vor dem Lammen. Gesicherte Korrelationen ließen sich zu FFS (MFS1; MFS2), BHB (MFS2) und dem Zeitpunkt der Untersuchung (MFS2; SKF2) feststellen. Die FFS-Konzentration stieg bei den SKF2 zur 1. Woche a.p. auf = 265 ± 131 μmol/l an, fiel eine Woche p.p. signifikant auf = 128 ± 95 μmol/l ab und korrelierte gesichert mit TG. Die TG-Konzentration fiel von der 1. Woche a.p. bis zur 1. Woche p.p. signifikant auf = 0,19 mmol/l (MFS1; SKF2) bzw. = 0,18 ± 0,05 mmol/l (MFS2) ab und korrelierte mit Gesamtprotein (MFS1), Cholesterol (MFS1; MFS2), β-Lipoproteinen (MFS1; MFS2) und freiem Endotoxin (MFS1). Die Cholesterolkonzentration fiel eine Woche p.p. bei den Zwillingsmuttern signifikant ab, bei den MFS1 war der Verlauf konstant. Die Bilirubinkonzentration stieg eine Woche p.p. signifikant an (MFS1; MFS2) und korrelierte mit der Konzentration von freiem Endotoxin. Das Gesamtprotein sank zum Zeitpunkt des Ablammens hin signifikant ab, erreichte jedoch eine Woche p.p. wieder seine Ausgangskonzentration. Eine gesicherte Korrelation konnte mit ALAAK festgestellt werden (MFS2; SKF2). Freies Endotoxin konnte zu jedem Zeitpunkt der Untersuchungsreihe nachgewiesen werden. Dabei lagen die festgestellten Konzentrationen bei x̃ = 0,06 – 1,30 EU/ml. Eine gesicherte Korrelation fand sich mit der Konzentration der ALAAK (MFS2). ALAAK konnten ebenfalls zu jedem Zeitpunkt in Konzentrationen von x̃ = 22,2 - 56,8 (OD-Wert) nachgewiesen werden. Fazit: Die Lipoproteinkonzentrationen in den einzelnen Gruppen zeigten sich a.p. zunächst regulativ den erhöhten peripartalen Belastungen des Lipidstoffwechsels angepasst, p.p. demonstrierte auch die Abnahme der Lipoproteinkonzentrationen das Ende der lipolytischen Aktivität bei den Schafen. Die vorliegenden Ergebnisse der Stoffwechselparameter lassen auf eine stärkere Belastung der Zwillingsmuttern schließen, welche auch die Lipoproteine mit einbezieht. Freies Endotoxin konnte auch bei gesunden Schafen zwar zu jedem Zeitpunkt nachgewiesen, eine gesicherte Korrelation mit der Lipoproteinkonzentration jedoch nicht festgestellt werden.:Inhaltsverzeichnis 1 Einleitung 1 2 Literaturübersicht 4 2.1 Physiologische Gravidität des Schafes 4 2.2 Stoffwechsel von Mutterschafen in der Hochträchtigkeit 5 2.3 Stoffwechselparameter 6 2.3.1 β-Hydroxybutyrat 6 2.3.2 Glucose 7 2.3.3 Insulin 10 2.3.4 Freie Fettsäuren 12 2.3.5 Triacylglycerol 13 2.3.6 Cholesterol 14 2.3.7 Bilirubin 15 2.3.8 Gesamtprotein 17 2.4 Lipoproteine 18 2.4.1 Definition von Lipoproteinen 18 2.4.2 Stoffwechsel der Lipoproteine 21 2.4.2.1 Chylomikronen 21 2.4.2.2 Very-Low-Density-Lipoproteins 22 2.4.2.3 Low-Density-Lipoproteins 23 2.4.2.4 High-Density-Lipoproteins 24 2.4.3 Besonderheiten der Lipoproteine beim Schaf 24 2.5 Endotoxin 27 2.5.1 Definition von Endotoxin 27 2.5.2 Aufbau von Endotoxin 27 2.5.3 Herkunft und Wirkung des Endotoxins 28 2.5.4 Inaktivierung und Elimination des Endotoxins 29 2.5.5 Beziehung zwischen Endotoxin und Fettstoffwechsel 30 3 Tiere, Material und Methoden 32 3.1 Versuchsgut 32 3.2 Versuchsanordnung 33 3.3 Material 34 3.4 Methodik 34 3.4.1 Gesundheitszustand der Versuchsgruppen 34 3.4.2 Bestimmungsmethoden klinisch-chemischer Parameter 34 3.4.3 Bestimmung der Lipoproteine 35 3.4.4 Bestimmung von freiem Endotoxin 37 3.4.5 Bestimmung von Anti-Lipid A Antikörpern 38 3.5 Statistische Auswertung 39 4 Ergebnisse 41 4.1 Tierbestand 41 4.2 Stoffwechselparameter 41 4.2.1 β-Hydroxybutyrat 41 4.2.2 Glucose 44 4.2.3 Insulin 46 4.2.4 Freie Fettsäuren 48 4.2.5 Triacylglycerol 50 4.2.6 Cholesterol 52 4.2.7 Bilirubin 54 4.2.8 Gesamtprotein 56 4.3 Lipoproteine 58 4.3.1 α-Lipoproteine 58 4.3.2 ß-Lipoproteine 60 4.4 Endotoxin 62 4.5 Anti-Lipid A Antikörper 64 5 Diskussion 66 5.1 Versuchsprinzip 66 5.2 Tiere und Versuchsbedingungen 66 5.3 Methodenkritik 67 5.4 Stoffwechselparameter 68 5.4.1 β-Hydroxybutyrat 68 5.4.2 Glucose 71 5.4.3 Insulin 72 5.4.4 Freie Fettsäuren 74 5.4.5 Triacylglycerol 76 5.4.6 Cholesterol 78 5.4.7 Bilirubin 79 5.4.8 Gesamtprotein 81 5.5 Lipoproteine 83 5.5.1 α-Lipoproteine 83 5.5.2 β-Lipoproteine 86 5.6 Endotoxin 87 5.7 Anti-Lipid A Antikörper 89 6 Zusammenfassung 91 7 Summary 93 8 Literaturverzeichnis 95 9 Anhang 116 9.1 Stoffwechselparameter 116 9.1.1 β-Hydroxybutyrat 116 9.1.2 Glucose 117 9.1.3 Insulin 118 9.1.4 Freie Fettsäuren 119 9.1.5 Triacylglycerol 120 9.1.6 Cholesterol 121 9.1.7 Bilirubin 122 9.1.8 Gesamtprotein 123 9.2 Lipoproteine 124 9.2.1 α-Lipoproteine 124 9.2.2 β-Lipoproteine 125 9.3 Endotoxin 126 9.4 Anti-Lipid A Antikörper 127 / Alexandra Flocke Evaluation of metabolic parameters and lipoproteins in the serum of merino and blackhead sheep for meat production with one or two lambs in the peripartal period Large Animal Clinic for Internal Medicine, Faculty of Veterinary Medicine, University of Leipzig Submitted in February 2012 (94 pages, 15 figures, 8 tables, 199 references, 12 tablets in the appendix) Keywords: lipoproteins, sheep, metabolism, endotoxin, ketosis Objective: The lipoprotein status in the critical phase of the peripartal period of sheep has been few reviewed. The ability of lipoproteins to transport and redistribute lipids in the organism and their capacity for endotoxinneutralisation makes them a considerable parameter in metabolic bonded situations. The objective of this research is to show the lipoprotein status of two competitive breeds and to proof if and to what extend differences between ewes with one and two lambs exist. Additionally, the endotoxic situation is assigned and possible correlations with the metabolic system and the lipoprotein status are evaluated. Animals, materials and methods: The research contains healthy merino sheep with one lamb (MFS1), merino sheep with two lambs (MFS2) and blackhead sheep with two lambs (SKF2). At intervals of seven days, blood samples out of the V. jugularis externa were taken from the animals in a period from the 5th week a.p. to one week p.p.. Concentrations in the serum of the following parameters were defined: β-hydroxybutyrat (BHB), glucose, free fatty acids (FFS), triacylglycerol (TG), cholesterol, bilirubin, total protein (all Hitachi 912), insulin (RIA-kit, IBL, Hamburg), α- and β-lipoprotein (LIPIDOPHOR All In12® - without pre-β-lipoprotein), free endotoxin (LAL-test) and anti-lipid A antibodies (ALAAK). Results: The concentration of α-lipoproteins of the MFS1 increased significantly before parturition and finally reached 208,0 ± 40,1 mg/dl (1st Week a.p.). The group of MFS2 showed a constant deviation between = 180,4 ± 31,1 mg/dl (5th Week a.p.) and = 196,1 ± 42,1 mg/dl (3rd week a.p.) before parturition. The SKF2 evidenced with a concentration of = 206,5 ± 39,0 mg/dl in the 5th week a.p. a predominantly significant higher result as during following points in time. Both groups carrying twins showed a significant decrease of the α-lipoprotein concentration down to = 144,3 ± 46,1 mg/dl (MFS2) or = 170,2 ± 48,8 mg/dl (SKF2) after parturition. There were statistically reliable correlations between the α-lipoprotein concentration and cholesterol. The β-lipoprotein concentration from both twin pregnant groups had been significantly lower one week after parturition than before parturition. The results of the SKF2 in week 5 and 4 a.p. were significantly lower than those of the MFS1. Reliable correlations could be determined with TG, cholesterol and ALAAK (MFS1). An α-lipoprotein reference range for ewes of the introduced breeds could be determined from 144,3 - 208,0 mg/dl and for β-lipoproteins from 13,6 - 28,0 mg/dl. There were no significant differences between MFS1 and MFS2 except the concentration of insulin in the 1st week a.p.. In the comparison of the different breeds, significant differences in the concentration of glucose (2nd week a.p.) and bilirubin (3rd week a.p. and 1st week a.p.) could be shown. The concentration of BHB showed a constant deviation and increased significantly to = 0,96 ± 0,36 mmol/l (MFS1; 1st week p.p.), = 0,73 ± 0,35 mmol/l (MFS2, 1st week a.p.) or = 0,63 ± 0,14 mmol/l (SKF2, 1st week a.p.), respectively. Reliable correlations existed with FFS (MFS2) and insulin (MFS2; SKF2). The concentration of glucose of the twin pregnant ewes decreased initially before parturition and increased significantly again in the 1st week p.p. ( = 3,63 ± 0,59 mmol/l; MFS2 and = 3,28 ± 0,42 mmol/l; SKF2). Reliable correlations had been determined with FFS (MFS1; MFS2), BHB (MFS2) and the time of sampling (MFS2; SKF2). Concentrations of FFS increased in the group of SKF2 to = 265 ± 131 μmol/l in the 1st week a.p., decreased again significantly to = 128 ± 95 μmol/l one week p.p. and showed a correlation with TG. The TG concentration decreased significantly from one week a.p. to one week p.p. down to = 0,19 mmol/l (MFS1; SKF2) and = 0,18 ± 0,05 mmol/l (MFS2), respectively. A reliable correlation existed between TG and total protein (MFS1), cholesterol (MFS1; MFS2), β-lipoproteins (MFS1; MFS2) and free endotoxin (MFS1). Both twin pregnant groups had a significant decrease in the cholesterol concentration one week after parturition, in contrast the group of MFS1 showed a constant deviation. The concentration of bilirubin increased significantly one week p.p. (MFS1; MFS2) and showed a reliable correlation with the concentration of free endotoxin. Total protein decreased significantly towards parturition, but reached its initial concentration again one week p.p.. A statistically reliable correlation could be determined with ALAAK (MFS2; SKF2). Free endotoxin could be detected at any point of the exploration. The observed concentrations ranged from x̃ = 0,06 – 1,30 EU/ml. A reliable correlation could be shown with the concentration of ALAAK (MFS2). ALAAK was detected as well in concentrations from x̃ = 22,2 - 56,8 (OD-value) in any taken sample. Conclusion: The lipoprotein concentrations in each group appeared a.p. initially adapted to the peripartal bonds of the increased lipid metabolism. After parturition, the decrease in lipoprotein concentrations, too, demonstrated the end of the lipolytic activity of the ewes. The results of the metabolic parameters lead to the conclusion of a stronger liability of the ewes with twins. This is also valid for the lipoproteins. Free endotoxin could be detected in healthy sheep at any time, but a reliable correlation with the concentration of lipoproteins was not visible.:Inhaltsverzeichnis 1 Einleitung 1 2 Literaturübersicht 4 2.1 Physiologische Gravidität des Schafes 4 2.2 Stoffwechsel von Mutterschafen in der Hochträchtigkeit 5 2.3 Stoffwechselparameter 6 2.3.1 β-Hydroxybutyrat 6 2.3.2 Glucose 7 2.3.3 Insulin 10 2.3.4 Freie Fettsäuren 12 2.3.5 Triacylglycerol 13 2.3.6 Cholesterol 14 2.3.7 Bilirubin 15 2.3.8 Gesamtprotein 17 2.4 Lipoproteine 18 2.4.1 Definition von Lipoproteinen 18 2.4.2 Stoffwechsel der Lipoproteine 21 2.4.2.1 Chylomikronen 21 2.4.2.2 Very-Low-Density-Lipoproteins 22 2.4.2.3 Low-Density-Lipoproteins 23 2.4.2.4 High-Density-Lipoproteins 24 2.4.3 Besonderheiten der Lipoproteine beim Schaf 24 2.5 Endotoxin 27 2.5.1 Definition von Endotoxin 27 2.5.2 Aufbau von Endotoxin 27 2.5.3 Herkunft und Wirkung des Endotoxins 28 2.5.4 Inaktivierung und Elimination des Endotoxins 29 2.5.5 Beziehung zwischen Endotoxin und Fettstoffwechsel 30 3 Tiere, Material und Methoden 32 3.1 Versuchsgut 32 3.2 Versuchsanordnung 33 3.3 Material 34 3.4 Methodik 34 3.4.1 Gesundheitszustand der Versuchsgruppen 34 3.4.2 Bestimmungsmethoden klinisch-chemischer Parameter 34 3.4.3 Bestimmung der Lipoproteine 35 3.4.4 Bestimmung von freiem Endotoxin 37 3.4.5 Bestimmung von Anti-Lipid A Antikörpern 38 3.5 Statistische Auswertung 39 4 Ergebnisse 41 4.1 Tierbestand 41 4.2 Stoffwechselparameter 41 4.2.1 β-Hydroxybutyrat 41 4.2.2 Glucose 44 4.2.3 Insulin 46 4.2.4 Freie Fettsäuren 48 4.2.5 Triacylglycerol 50 4.2.6 Cholesterol 52 4.2.7 Bilirubin 54 4.2.8 Gesamtprotein 56 4.3 Lipoproteine 58 4.3.1 α-Lipoproteine 58 4.3.2 ß-Lipoproteine 60 4.4 Endotoxin 62 4.5 Anti-Lipid A Antikörper 64 5 Diskussion 66 5.1 Versuchsprinzip 66 5.2 Tiere und Versuchsbedingungen 66 5.3 Methodenkritik 67 5.4 Stoffwechselparameter 68 5.4.1 β-Hydroxybutyrat 68 5.4.2 Glucose 71 5.4.3 Insulin 72 5.4.4 Freie Fettsäuren 74 5.4.5 Triacylglycerol 76 5.4.6 Cholesterol 78 5.4.7 Bilirubin 79 5.4.8 Gesamtprotein 81 5.5 Lipoproteine 83 5.5.1 α-Lipoproteine 83 5.5.2 β-Lipoproteine 86 5.6 Endotoxin 87 5.7 Anti-Lipid A Antikörper 89 6 Zusammenfassung 91 7 Summary 93 8 Literaturverzeichnis 95 9 Anhang 116 9.1 Stoffwechselparameter 116 9.1.1 β-Hydroxybutyrat 116 9.1.2 Glucose 117 9.1.3 Insulin 118 9.1.4 Freie Fettsäuren 119 9.1.5 Triacylglycerol 120 9.1.6 Cholesterol 121 9.1.7 Bilirubin 122 9.1.8 Gesamtprotein 123 9.2 Lipoproteine 124 9.2.1 α-Lipoproteine 124 9.2.2 β-Lipoproteine 125 9.3 Endotoxin 126 9.4 Anti-Lipid A Antikörper 127

Hedgehog (Hh) proteins control animal development and tissue homeostasis. They activate gene expression by regulating processing, stability, and activation of Gli/Cubitus interruptus (Ci) transcription factors. Hh proteins are secreted and spread through tissue, despite becoming covalently linked to sterol during processing. Multiple mechanisms have been proposed to release Hh proteins in distinct forms; in Drosophila, lipoproteins facilitate long-range Hh mobilization but also contain lipids that repress the pathway. Here, we show that mammalian lipoproteins have conserved roles in Sonic Hedgehog (Shh) release and pathway repression. We demonstrate that lipoprotein-associated forms of Hh and Shh specifically block lipoprotein-mediated pathway inhibition. We also identify a second conserved release form that is not sterol-modified and can be released independently of lipoproteins (Hh-N*/Shh-N*). Lipoprotein-associated Hh/Shh and Hh-N*/Shh-N* have complementary and synergistic functions. In Drosophila wing imaginal discs, lipoprotein-associated Hh increases the amount of full-length Ci, but is insufficient for target gene activation. However, small amounts of non-sterol-modified Hh synergize with lipoprotein-associated Hh to fully activate the pathway and allow target gene expression. The existence of Hh secretion forms with distinct signaling activities suggests a novel mechanism for generating a diversity of Hh responses.

LPS wird von Gram-negativen Bakterien freigesetzt und führt mit Hilfe von LBP zur Ausschüttung proinflammatorischer Zytokine aus Monozyten und Makrophagen. Diese von LPS ausgelöste Kaskade, ist entscheidend an der Entstehung der Sepsis beteiligt. In dieser Arbeit wurde gezeigt, daß die LPS-induzierte Stimulation von Makrophagen durch murines Serum gehemmt werden kann. Außerdem konnte im Rahmen dieser Arbeit im Mausmodell verdeutlicht werden, welche Rolle Lipoproteine und LBP bei dem protektiven Serumeffekt spielen. Von den in Mausseren verschiedener Mausstämme bestimmten Parametern korrelierte der Phospholipidgehalt relativ gut mit dem inhibitorischen Serumeffekt. Eine Depletion von Lipoproteinen aus den Seren führte zu einer starken Reduktion des inhibitorischen Serumpotentials, während die Verwendung von LBP-defizienten Seren keinen Einfluß auf den Serumhemmeffekt hatte. Lipoproteine sehr geringer, geringer und hoher Dichte verursachten in Gegenwart von LBP eine deutliche Reduktion der LPS-Effekte, die gut mit ihrem Phospholipidgehalt korrelierte. In Abwesenheit von Serum und LBP konnten Lipoproteine LPS-Effekte auch bei hohen Phospholipidkonzentrationen kaum noch inhibieren. In nativen murinen Lipoproteinen hoher Dichte und lipoproteindefizientem Serum ließ sich LBP nachweisen. Die Ergebnisse dieser Arbeit zeigen, daß native Lipoproteine sehr geringer, geringer und hoher Dichten als Akzeptoren für LPS dienen können. Ihr inhibitorisches Potential korreliert am besten mit ihrem Phospholipidgehalt. Der Transport von LPS in diese Lipoproteine wird durch LBP katalysiert, was den protektiven Effekt hoher LBP-Konzentrationen erklärt. Weiterhin konnte gezeigt werden, daß LBP nicht der einzige Bestandteil von murinem Serum ist, der LPS in Lipoproteine transferiert. Vermutlich ist bei den, in dieser Arbeit verwendeten Serumkonzentrationen der PLTP-Gehalt ausreichend, diesen LPS-Transfer in Abwesenheit von LBP zu vollziehen. Bei der Interaktion zwischen Lipoproteinen und LPS handelt es sich um einen physiologischen Weg des Organismus, um auf bakterielle Endotoxine zu reagieren und so der Sepsis entgegenzuwirken. Mit genaueren Kenntnissen über diese Interaktion, bei der Lipidtransferproteine wie LBP und PLTP eine entscheidende Rolle spielen, können eventuell in Zukunft Methoden gefunden werden, diese physiologischen Vorgänge des Körpers zu unterstützen, um so eine Sepsis zu therapieren. / LPS released by gram-negative bacteria is bound by LBP and initiates the release of proinflammatory cytokines in makrophages and monocytes. These cytokines are thought to play a central role in the pathophysiology of sepsis. In these studies I was able to show an inhibitory effect of murine serum on the LPS-induced stimulation of macrophages. Furthermore the role of lipoproteins and LBP in this protective effect of serum was investigated. The inhibitory effect of serum from different mouse strains was best correlated to its phospholipid content. Depletion of serum from lipoproteins strongly reduced its LPS-inhibitory potential while depletion of serum from LBP had no effect. Murine VLDL, LDL and HDL were found to be potent inhibitors of LPS-effects in presence of LBP. In abscence of LBP the inhibitory effect was much weaker. LBP could be detected in murine HDL and murine lipoproteindeficient serum. My data shows that HDL, LDL and VLDL can act as acceptors of LPS. Their inhibitory potential is best correlated to their phospholipid content. LBP catalyses transport of LPS into lipoproteins. This could be an explanation for its protective effect in high doses. Furthermore it could be shown that LBP is not the only serum component that transfers LPS into Lipoproteins. Possibly the used serum contained enough PLTP to perform this transfer in absence of LBP. The interaction between Lipoproteins and LPS is a physiological way of the organism to react on endotoxines and inhibit the development of sepsis. PLTP and LBP play major roles in this interaction. Understanding the pathways of LPS-detoxification may help to support the organism s physiological answer and establish new methods to treat sepsis.

Eukaryotic cells depend on cargo uptake into the endocytic membrane system, which comprises a functionally interconnected network of endosomal compartments. The establishment and maintenance of such diverse compartments in face of the high rates of exchange between them, poses a major challenge for obtaining a molecular understanding of the endocytic system. Rab-GTPases have emerged as architectural key element thereof: Individual family members localize selectively to endosomal compartments, where they recruit a multitude of cytoplasmic effector proteins and coordinate them into membrane sub-domains. Such "Rab-domains" constitute modules of molecular membrane identity, which pattern the endocytic membrane system into a mosaic of Rab-domains. The main objective of this thesis research was to link such "static" mosaic-view with the highly dynamic nature of the endosomal system. The following questions were addressed: How are neighbouring Rab-domains coordinated? Are Rab-domains stable or can they undergo assembly and disassembly? Are the dynamics of Rab-domains utilized in cargo transport? The first part of this thesis research focused on the organization of Rab-domains in the recycling pathway. Utilizing Total Internal Reflection (TIRF) microscopy, Rab11-, but neither Rab4- nor Rab5-positive vesicles were observed to fuse with the plasma membrane. Rab4-positive membranes, however, could be induced to fuse in presence of Brefeldin A. Thus, these experiments complete the view of the recycling pathway by the following steps: a) Rab11-carriers likely mediate the return of recycling cargo to the surface; b) such carriers are presumably generated in an Arf-dependent fission reaction from Rab4-positive compartments. Rab11-chromatography was subsequently carried out in the hope of identifying Rab11-effectors functioning at the Rab4-Rab11 domain interface. An as yet uncharacterized ubiquitin ligase was identified, which selectively interacts with both Rab4 and Rab11. Contrary to expectations, however, the protein (termed RUL for *R*ab interacting *U*biquitin *L*igase) does not function in recycling,but appears to mediate trafficking between Golgi/TGN and endosomes instead.In order to address the dynamics of Rab-domains, fluorescently tagged Rab-GTPases were imaged during cargo transport reactions in living cells. Herefore high-speed/long-term imaging procedures and novel computational image analysis tools were developed. The application of such methodology to the analysis of Rab5-positive early endosomes showed that a) The amount of Rab5 associated with individual endosomes fluctuates strongly over time; b) such fluctuations can lead to the "catastrophic" loss of the Rab5-machinery from membranes; c) Rab5 catastrophe is part of a functional cycle of early endosomes, involving net centripetal motility, continuous growth and increase in Rab5 density. Next, the relevance of Rab5 catastrophe with respect to cargo transfer into either the recycling- or degradative pathway was examined. Recycling cargo (transferrin) could be observed to exit Rab5-positive early endosomes via the frequent budding of tubular exit carriers. Exit of degradative cargo (LDL) from Rab5-positive endosomes did not involve budding, but the rapid loss of Rab5 from the limiting membrane.Rab5-loss was further coordinated with the concomitant acquisition of Rab7, suggesting "Rab conversion" as mechanism of transport between early- and late endosomes.Altogether, this thesis research has shown that first, Rab-machineries can be acquired and lost from membranes. Second, such dynamics provide a molecular mechanism for cargo exchange between endosomal compartments. Jointly, these findings lead to the concept of Rab-domain dynamics modulation in /trans/ between neighbouring domains as mechanistic principle behind the dynamic organization of membrane trafficking pathways.

Hedgehog (Hh) proteins control animal development and tissue homeostasis. They activate gene expression by regulating processing, stability, and activation of Gli/Cubitus interruptus (Ci) transcription factors. Hh proteins are secreted and spread through tissue, despite becoming covalently linked to sterol during processing. Multiple mechanisms have been proposed to release Hh proteins in distinct forms; in Drosophila, lipoproteins facilitate long-range Hh mobilization but also contain lipids that repress the pathway. Here, we show that mammalian lipoproteins have conserved roles in Sonic Hedgehog (Shh) release and pathway repression. We demonstrate that lipoprotein-associated forms of Hh and Shh specifically block lipoprotein-mediated pathway inhibition. We also identify a second conserved release form that is not sterol-modified and can be released independently of lipoproteins (Hh-N*/Shh-N*). Lipoprotein-associated Hh/Shh and Hh-N*/Shh-N* have complementary and synergistic functions. In Drosophila wing imaginal discs, lipoprotein-associated Hh increases the amount of full-length Ci, but is insufficient for target gene activation. However, small amounts of non-sterol-modified Hh synergize with lipoprotein-associated Hh to fully activate the pathway and allow target gene expression. The existence of Hh secretion forms with distinct signaling activities suggests a novel mechanism for generating a diversity of Hh responses.

Eukaryotic cells depend on cargo uptake into the endocytic membrane system, which comprises a functionally interconnected network of endosomal compartments. The establishment and maintenance of such diverse compartments in face of the high rates of exchange between them, poses a major challenge for obtaining a molecular understanding of the endocytic system. Rab-GTPases have emerged as architectural key element thereof: Individual family members localize selectively to endosomal compartments, where they recruit a multitude of cytoplasmic effector proteins and coordinate them into membrane sub-domains. Such "Rab-domains" constitute modules of molecular membrane identity, which pattern the endocytic membrane system into a mosaic of Rab-domains. The main objective of this thesis research was to link such "static" mosaic-view with the highly dynamic nature of the endosomal system. The following questions were addressed: How are neighbouring Rab-domains coordinated? Are Rab-domains stable or can they undergo assembly and disassembly? Are the dynamics of Rab-domains utilized in cargo transport? The first part of this thesis research focused on the organization of Rab-domains in the recycling pathway. Utilizing Total Internal Reflection (TIRF) microscopy, Rab11-, but neither Rab4- nor Rab5-positive vesicles were observed to fuse with the plasma membrane. Rab4-positive membranes, however, could be induced to fuse in presence of Brefeldin A. Thus, these experiments complete the view of the recycling pathway by the following steps: a) Rab11-carriers likely mediate the return of recycling cargo to the surface; b) such carriers are presumably generated in an Arf-dependent fission reaction from Rab4-positive compartments. Rab11-chromatography was subsequently carried out in the hope of identifying Rab11-effectors functioning at the Rab4-Rab11 domain interface. An as yet uncharacterized ubiquitin ligase was identified, which selectively interacts with both Rab4 and Rab11. Contrary to expectations, however, the protein (termed RUL for *R*ab interacting *U*biquitin *L*igase) does not function in recycling,but appears to mediate trafficking between Golgi/TGN and endosomes instead.In order to address the dynamics of Rab-domains, fluorescently tagged Rab-GTPases were imaged during cargo transport reactions in living cells. Herefore high-speed/long-term imaging procedures and novel computational image analysis tools were developed. The application of such methodology to the analysis of Rab5-positive early endosomes showed that a) The amount of Rab5 associated with individual endosomes fluctuates strongly over time; b) such fluctuations can lead to the "catastrophic" loss of the Rab5-machinery from membranes; c) Rab5 catastrophe is part of a functional cycle of early endosomes, involving net centripetal motility, continuous growth and increase in Rab5 density. Next, the relevance of Rab5 catastrophe with respect to cargo transfer into either the recycling- or degradative pathway was examined. Recycling cargo (transferrin) could be observed to exit Rab5-positive early endosomes via the frequent budding of tubular exit carriers. Exit of degradative cargo (LDL) from Rab5-positive endosomes did not involve budding, but the rapid loss of Rab5 from the limiting membrane.Rab5-loss was further coordinated with the concomitant acquisition of Rab7, suggesting "Rab conversion" as mechanism of transport between early- and late endosomes.Altogether, this thesis research has shown that first, Rab-machineries can be acquired and lost from membranes. Second, such dynamics provide a molecular mechanism for cargo exchange between endosomal compartments. Jointly, these findings lead to the concept of Rab-domain dynamics modulation in /trans/ between neighbouring domains as mechanistic principle behind the dynamic organization of membrane trafficking pathways.