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Obwohl Böden unzweifelhaft ein signifikanter Pool von organischem Kohlenstoff sind, ist ihre Bedeutung als potenzielle langfristige Senke für atmosphärischen Kohlenstoff keineswegs klar. Trotz bedeutender wissenschaftlicher Forschritte aus den letzten Jahren zur Klärung der Kohlenstoffdynamik in Böden gibt es nach wie vor offene Fragen insbesondere hinsichtlich der spezifischen geochemischen Mechanismen, die für die Stabilisierung organischen Kohlenstoffs in Böden verantwortlich sind. Vor diesem Hintergrund besteht ein wesentliches Ziel der vorliegenden Dissertation darin, in unterschiedlichen Bodentypen die Konzentration von organischem Kohlenstoff und Stickstoff sowie die mineralogische Zusammensetzung zu untersuchen, um Hinweise auf einen möglichen Einfluss der Tonmineralogie, der spezifischen Oberfläche und der Oxidkonzentration auf die Stabilisierung organischen Materials zu ermitteln. Die Ergebnisse sollen einen Beitrag dazu liefern, die Mechanismen der Fixierung organischer Substanz in Böden besser zu verstehen und das vorhandene Wissen hierüber zu erweitern. Hierzu wurden fünf verschiedene Bodenprofile aus Hessen mit unterschiedlicher mineralogischer Zusammensetzung untersucht. Um die Auswirkungen verschiedener physikalischer und geochemischer Faktoren auf den Gehalt organischer Substanz in den untersuchten Böden festzustellen, wurden folgende Parameter untersucht: -Tonmineralogie, -organische Kohlenstoff- und Stickstoff-Konzentrationen, -%-Kationensättigung, -spezifische Oberfläche, -dithionit- und oxalatlösliche Gehalte an Fe, Al und Mn. Anhand dieser Parameter wurden weiterführende statistische Analysen unter Verwendung der Statistiksoftware SPSS für Windows durchgeführt, um mögliche statistische Zusammenhänge aufzudecken, die für die Stabilisierung von organischem Kohlenstoff in den betrachteten Böden verantwortlich sind. Die im Rahmen der vorliegenden Dissertation ermittelten Ergebnisse zeigen, dass der Tonanteil und die Tonmineralogie der untersuchten Böden nur einen begrenzten Einfluss auf die Stabilisierung organischer Substanz haben. Weiterhin wird gezeigt, dass die in der Literatur propagierte Beziehung zwischen spezifischer Oberfläche und der Konzentration organischen Kohlenstoffs nicht auf alle Böden anwendbar ist. Die Ergebnisse deuten darauf hin, dass die Präsenz von amorphen Eisen- und Aluminiumoxiden der wichtigste Einflussfaktor für die Fixierung von organischem Material in den untersuchten Böden ist. Die größeren Konzentrationen von organischem Kohlenstoff in den kleinsten Fraktionen (Feinschluff und Ton) der Profile sind vor allem darauf zurückzuführen, dass Oxide ebenfalls in diesen Fraktionen aufzufinden sind. Tonminerale haben demnach eine sekundäre Bedeutung, indem sie Komplexe mit den Oxiden bilden, die zur Stabilisierung von organischer Substanz führen können. Insgesamt deuten die Ergebnisse daraufhin, dass Böden keine geeignete Senke für die langfristige Speicherung von organischem Kohlenstoff sind. Obwohl Mechanismen wie die Adsorption von organischer Substanz an Oxide die Stabilisierung organischen Materials unterstützen, scheinen diese nicht stark genug zu sein, um eine permanente Speicherung von organischem Kohlenstoff zu bewirken.
A gene trap strategy was used to identify genes induced in hematopoietic cells undergoing apoptosis by growth factor withdrawal. IL-3 dependent survival of hematopoietic cells relies on a delicate balance between proliferation and apoptosis that is controlled by the availability of cytokines (Thompson, 1995; Iijima et al., 2002). From our previous results of gene trap assay, we postulated that transcriptionally activated antagonistic genes against apoptosis might actually block or delay cell death (Wempe et al., 2001) causing cells to have carcinogenic behavior. The analysis attempted to better understand the outcome of a death program following IL-3 deprivation and to identify those survival genes whose expression is affected by time dependent manner. As described in the chapter 4, there would be two major conclusions evident from the three separate experiments (Genetrap, Atlas cDNA array and Affymetrix chips): Firstly 56% of trapped genes, that are up-regulated by IL-3 withdrawal (28 of 50), are directly related to cell death or survival. Secondly, unlike most array technologies, gene trapping only selects for the transiently induced genes that is independent of pre-existing steady state mRNA levels. In regarding correlations of the genes with potential carcinogenesis, the pre-existing mRNA makes difficult to describe the unique characteristics of deregulated tumor tissue genes. For a joint project with Schering (Schering AG, Berlin), the genes of our GTSTs were examined. The first screen with custom array was used to look for whether the survival genes of our GTSTs are involved in various cancer cell lines, whilst the second screen with Matched Tumor/Normal Array was used to characterize if the selected seven genes (ERK3, Plekha2, KIAA1140, PI4P5Ka/g, KIAA0740, KIAA1036 and PEST domains) are transformation-related genes or not in different tumor tissues. Twenty-six genes were identified as either induced or repressed in one or more cell lines. Genetic information is expressed in complex and ever changing patterns throughout a life span of cells. A description of these patterns and how they relate to the tissue specific cancer is crucial for our understanding of the network of genetic interactions that underlie the processes of normal development, disease and evolution. The development of cancer and its progression is clearly a multiplex phenotype, as a function of time, involving dozens of primary genes and hundreds of secondary modifier genes. There would be a major conclusion evident from the three separate experiments (Genetrap, Affymetrix mouse chip and Matched Tumor/Normal Array): ERK3 could play a significant role in breast, stomach and uterus carcinogenesis with tissue specific regulations. It is clear that ERK3 is obvious putative survival gene in these tumor tissues. Especially, in breast tumors, seven times up-regulation was considerable and the activation of ERK3 could be a feature of breast tumors. My results imply that the unique deregulation of ERK3 is perhaps the major consequence of possible transformation of normal cells into malignant cancer cells, even though further analysis remains to be determined whether an alterated activity of associated survival genes is primarily responsible for a carcinogenesis. However unlike all the other known MAP Kinases, no stimuli and no nuclear substrates of ERK3 is reported. Therefore, it will be necessary first to determine the spectrum of substrates and to identify the proximal effectors for the ERK3 in breast carcinoma cells.
Zahnwale sind die einzige Säugetiergruppe, die umfassend an ein Leben im Wasser angepasst ist und dabei ein aktives Sonarsystem zur Orientierung nutzt. Wahrscheinlich produzieren alle Zahnwalarten sonische oder ultrasonische Klicklaute, deren Echos die Tiere zu einem drei-dimensionalen "akustischen Bild" zusammensetzen. Im Gegensatz zu den meisten anderen Säugetieren produzieren Zahnwale diese Laute im Nasen-Komplex durch einen pneumatisch betriebenen Mechanismus. Jedoch spielt auch der Kehlkopf dabei eine wichtige Rolle, indem er den nötigen Luftdruck in der Nase erzeugt. Die Ergebnisse werden in Bezug auf die physikalischen Voraussetzungen eines Bio-Sonars in einer aquatischen Umwelt interpretiert. Um die morphologischen Eigenschaften (Struktur, Form, Topographie) der Organe im Kopf verschiedener Zahnwalarten vollständig zu erfassen, wurden diese mittels Computertomographie und Magnetresonanztomographie gescannt. Daraufhin wurden die Köpfe makroskopisch präpariert und histologische Schnitte von Gewebeproben angefertigt. Schließlich wurden die Ergebnisse durch digitale dreidimensionale Rekonstruktionen vervollständigt. Diese Studie basiert zum größten Teil auf der Untersuchung von Schweinswalen (Phocoena phocoena) und Pottwalen (Physeter macrocephalus). Zum Vergleich wurden fetale und postnatale Individuen anderer Zahnwalarten herangezogen wie Delphinartige (Delphinus delphis, Stenella attenuata, Tursiops truncatus), Flussdelphinartige (Pontoporia blainvillei, Inia geoffrensis) und der Zwergpottwal (Kogia breviceps). Im Allgemeinen konnte durch die morphologischen Daten dieser Studie die einheitliche "phonic lips-Hypothese der Schallproduktion bei Zahnwalen, wie sie von Cranford, Amundin und Norris [J. Morphol. 228 (1996): 223-285] aufgestellt wurde, bestätigt werden. Diese Hypothese beschreibt eine ventilartige Struktur in der Nasenpassage, den sogenannten "monkey lips/dorsal bursae complex" (MLDB) als Schallgenerator. Der pneumatische Mechanismus lässt die beiden Hälften des MLDB aufeinanderschlagen und erzeugt damit die initiale Schallschwingung im Gewebe ("phonic lips"). Diese Vibration wird über die Melone, einen großen Fettkörper in der vorderen Nasenregion der Zahnwale, fokussiert und in das umgebende Wasser übertragen. Die akzessorischen Nasensäcke und spezielle Schädel- und Bindegewebestrukturen können zu der Fokussierung beitragen. Obwohl die Echolotsignale der Schweinswale sehr spezialisiert zu sein scheinen, weisen die Übereinstimmungen in der Topographie und in der Form der Nasenstrukturen im Vergleich zu Delphinen und Flussdelphinartigen (Pontoporia und Inia) auf eine ganz ähnliche Funktion der Nase bezüglich der Produktion und Emission von Echolotschall hin. Allerdings gibt es einige anatomische Besonderheiten im Nasenkomplex des Schweinswals, welche die besondere Pulsstruktur der Sonarsignale erklären könnte. Diese werden in der Dissertation diskutiert. Bei einem Vergleich der Nasenmorphologie der Pottwale einerseits und der nicht-pottwalartigen Zahnwale andererseits fällt vor allem der Grad der Asymmetrie ins Auge. Im Gegensatz zu dem oben für Delphine und Schweinswale beschrieben Mechanismus betreiben Pottwale die Schallproduktion an den "monkey lips" mit Luft, die im rechten Nasengang unter Druck gesetzt wird (und nicht im nasopharyngealen Raum). Zudem könnte durch Änderung des Luftvolumens im rechten Nasengang die Schalltransmission zwischen den Fettkörpern, und somit die Schallemission, kontrolliert werden. In diesem theoretischen Szenario fungiert der breite rechte Nasengang als eine Art "akustische Schranke", welche zwischen zwei verschiedenen Modi der Klickproduktion wechselt: Der erste Modus mit luftgefülltem Nasengang führt zur Produktion der Kommunikationsklicks ("coda clicks") und der zweite Modus zur Aussendung von Echolotklicks, wenn der Nasengang kollabiert ist. Somit scheinen die zentrale Position und die nahezu horizontale Orientierung des rechten Nasengangs im Kopf der Pottwale als Schnittstelle (Schranke) zwischen den beiden großen Fettkörpern mit dem Mechanismus der Schallproduktion bei veränderten Luftvolumina korreliert zu sein. Die hier beschriebenen und andere Ergebnisse dieser Dissertation deuten darauf hin, dass die Gestalt und das Ausmaß der Nasenasymmetrie nicht mit der systematischen Zugehörigkeit der jeweiligen Art korrelieren, sondern durch den jeweiligen Typus des Sonarsystems als Ausdruck einer bestimmten ökologischen Anpassung bedingt sind. Bei Zahnwalen ist der Kehlkopf charakterisiert durch eine rostrale Verlängerung des Kehldeckels und der beiden Stellknorpel, die ein gänseschnabelartiges Rohr bilden, das von einem starken Sphinktermuskel umrundet und dabei in Position gehalten wird. Auf diese Weise ist das Atemrohr vollständig vom Digestionstrakt getrennt. Aus anatomischer Sicht ist es wahrscheinlich, dass die Schallerzeugung bei Zahnwalen durch eine Kolbenbewegung des Kehlkopfes in Richtung der Choanen zustande kommt, wodurch der Luftdruck im Nasenbereich erzeugt wird. Die Kontraktion des Sphinktermuskels als einem muskulösen Schlauch erzeugt wahrscheinlich die größte Kraft für diese Kolbenbewegung. Jedoch dürften die Muskelgruppen, die den Kehlkopf und das Zungenbein am Unterkiefer und an der Schädelbasis aufhängen, signifikant zur Druckerhöhung beitragen.
The endothelin B receptor belongs to the rhodopsin-like G-protein coupled receptors family. It plays an important role in vasodilatation and is found in the membranes of the endothelial cells enveloping blood vessels. During the course of this work, the production of recombinant human ETB receptor in yeast, insect and mammalian cells was evaluated. A number of different receptor constructs for production in the yeast P. pastoris was prepared. Various affinity tags were appended to the receptor N-and C-termini to enable receptor detection and purification. The clone pPIC9KFlagHisETBBio, with an expression level of 60 pmol/mg, yielded the highest amount of active receptor (1.2 mg of receptor per liter of shaking culture). The expression level of the same clone in fermentor culture was 17 pmol/mg, and from a 10L fermentor it was possible to obtain 3 kg of cells that contained 20-39 mg of the receptor. For receptor production in insect cells, Sf9 (S. frugiperda) suspension cells were infected with the recombinant baculovirus pVlMelFlagHisETBBio. The peak of receptor production was reached at 66 h post infection, and radioligand binding assays on insect cell membranes showed 30 pmoL of active receptor /mg of membrane protein. Subsequently, the efficiency of different detergents in solubilizing the active receptor was evaluated. N-dodecyl-beta-D-maltoside (LM), lauryl-sucrose and digitonine/cholate performed best, and LM was chosen for further work. The ETB receptor was produced in mammalian cells using the Semliki Forest Virus expression system. Radioligand binding assays on membranes from CHO cells infected with the recombinant virus pSFV3CAPETBHis showed 7 pmol of active receptor /mg of membrane protein. Since the receptor yield from mammalian cells was much lower than in yeast and insect cells, this system was not used for further large-scale receptor production. After production in yeast and insect cells, the ETB receptor was saturated with its ligand, endothelin-1, in order to stabilize its native form. The receptor was subsequently solubilized with n-dodecyl-beta-D-maltoside and subjected to purification on various affinity matrices. Two-step affinity purification via Ni2+-NTA and monomeric avidin proved the most efficient way to purify milligram amounts of the receptor. The purity of the receptor preparation after this procedure was over 95%, as judged from silver stained gels. However, the tendency of the ETB receptor produced in yeast to form aggregates was a constant problem. Attempts were made to stabilize the active, monomeric form of the receptor by testing a variety of different buffer conditions, but further efforts in this direction will be necessary in order to solve the aggregation problem. In contrast to preparations from yeast, the purification of the ETB receptor produced in insect cells yielded homogeneous receptor preparations, as shown by gel filtration analysis. This work has demonstrated that the amounts of receptor expressed in yeast and insect cells and the final yield of receptor, isolated by purification, represent a good basis for beginning 3D and continuing 2D crystallization trials.
Hepatitis E virus (HEV) is a positive-stranded RNA virus with a 7.2 kb genome that is capped and polyadenylated. The virus is currently unclassified : the organisation of the genome resembles that of the Caliciviridae but sequence analyses suggest that it is more closely related to the Togaviridae. HEV is an enterically transmitted virus that causes both epidemics and sporadic cases of acute hepatitis in many countries of Asia and Africa but only rarely causes disease in more industrialised countries. Initially the virus was believed to have a limited geographical distribution. However, serological studies suggest that that HEV may be endemic also in the United states and Europe even though it infrequently causes overt disease in these countries. Many different animal species worldwide recently have been shown to have antibodies to HEV suggesting that hepatitis E may be zoonotic. Although two related strains have been experimentally transmitted between species, direct transmission from animal to a human has not been documented. Our main objective in this study is to evaluate the suitability of current available HEV antibody assays for use in low-endemicity areas such as in Germany. Methods: We selected sera on the basis of at least borderline reactivity in the routinely used Abbot EIA. Most were tested as part of routine screening of long-term expatriates in endemic countries. The following assays (recombinant antigens : ORF2 and ORF3) were used: Abbot EIA, Genelabs ELISA, Mikrogen recomBlot and a 'Prototype' DSL-ELISA. We observed a wide range of sensitivity ( average of 56.8%) and specificity ( an average of 61.4%) in these used assays. These results implies that , these assays might be unreliable for detection of HEV infection in areas where hepatitis E is not endemic. However, most anti- HEV assays have not been correlated with the HEV RNA determined by reverse transcription. Many of these unexpected results and discrepancies can be alluded to the following reasons: I. The choice and the size of the HEV antigen. II. Duration of the antibody persistence III. A cross reactivity with different agent IV. Due to geographic species V. A low sensitivity of the available assays. VI. And infection with non-pathogenic HEV strain. (zoonotic strain?). We therefore suggest that, further studies will be required to improve the sensitivity and specificity of the available commercial assays on the market.
In the present study the cryo-immunogold technique was used and optimized for investigating the ultrastructure and immunolabeling of synaptic proteins. It is evidently a suitable method for the localization of membrane proteins since the antigens are not treated with any chemical denaturation before immunolabeling except for the fixation and since the antigens are directly exposed to the surface of the cryo-ultrasections. The v-SNARE VAMP II and the vesicle-associated proteins SV2 and Rab3A were detected extensively at small vesicles in the mossy fiber terminals. The t-SNARE SNAP-25, and N-type and P/Q type Ca2+ channels were allocated to the plasma membrane both at the active zone and outside the active zone. SNAP-25 and N-type Ca2+ channels appeared also at synaptic vesicles. A significantly increased immunolabeling of VAMP II, SV2, Rab3A, SNAP-25 and N-type Ca2+ channels was found at the active zones of fast synapses, indicating a concentration of these proteins at sites of exocytosis. The widespread distribution of the t-SNARE SNAP-25 at the axonal plasma membrane reveals that membrane-targeting specificity cannot be determined solely by v/t-SNARE interactions. Additional control components are required to assure the docking and exocytosis of the synaptic vesicles at active zones. The novel protein Bassoon was only found at active zones of central synapses and showed the highest specific labeling among all proteins investigated. Its labeling pattern implies an association of Bassoon with the presynaptic dense projections, the structural guide for vesicle exocytosis. The involvement of Bassoon in the organization of the neurotransmitter release site suggests that Bassoon may play an important role in determining the specificity of vesicle docking and fusion. In the neurosecretory endings of neurohypophysis the synaptic proteins VAMP II, SNAP- 25, SV2, Rab3A, and the N-type Ca2+ channels showed a preferential labeling over microvesicles. Moreover, the immunolabeling intensity of these proteins over microvesicles corresponded closely to that over synaptic vesicles. This suggests that these synaptic proteins share an identical association with synaptic vesicle and microvesicles. A significant labeling of SNAP-25, the N-type Ca2+ channels and VAMP II was also detected at the plasma membrane near the clustered microvesicles, indicating the competence of microvesicles for docking and exocytosis along the plasma membrane in the absence of active zones. No significant labeling of VAMP II, SNAP-25, SV2 and N-type Ca2+ channel was observed at the membrane of neurosecretory granules. This is in agreement with the notion that synaptic vesicles and microvesicles possess regulatory mechanisms for exocytosis different from those of granules. In contrast, a/ß-SNAP and NSF were found on the granules, and Rab3A and the P/Q-type Ca2+ channels on granules in a subset of terminals. Rab3A is associated specifically with the oxytocin-containing granule population. Interestingly, some plasma membrane proteins, such as SNAP-25 and even N-type Ca2+ channels and P/Q-type Ca2+ channels, were observed not only at the plasma membrane but also at the vesicular organelles. This suggests that these vesicular organelles may be involved in transporting newly synthesized proteins from the soma to the plasma membrane of the terminal. Furthermore, the vesicular pool of the Ca2+ channels may serve in the stimulationinduced translocation into the plasma membrane when required. Using the conventional preembedding method with Epon and the post-embedding method with LR Gold, VAMP II was localized at vesicular organelles of varying size and on horseradish peroxidase filled endocytic organelles in cultured astrocytes, with and without stimulation in the presence of the horseradish peroxidase. This indicates that VAMP II is involved in the cycle of vesicular exocytosis and endocytosis in astrocytes. U373 cells are capable of expressing all three members of the synaptic SNARE complex (v-SNARE VAMP II, t-SNARE syntaxin I and SNAP25). This indicates the competence of U373 to carry out regulated exocytosis by means of the classical SNARE mechanism. In addition, the ubiquitous v-SNARE cellubrevin and the endosome-associated small GTPbinding protein Rab5 could be expressed in U373 cells. All recombinant synaptic proteins investigated in U373 cells revealed a punctuate cellular distribution under the fluorescence microscope, suggesting that they are mainly associated with intracellular compartments. The cryo-electron microscopy provided direct evidence for the association of all expressed proteins with electron-lucent vesicular organelles. It further supports the potential of U373 MG cells to release low molecular weight messengers by a regulated exocytosis mechanism. In addition, myc-VAMP II was found on dispersed granules. Probably, VAMP II also participates in the exocytosis event of granules in U373 cells. Gold labeling for the two presumptive t-SNAREs syntaxin I and SNAP-25 in U373 cells was confined to the vesicular organelles. At the ultrastructural level no significant labeling was identified at the plasma membrane. The high level of colocalization of the two SNARE proteins VAMP II and syntaxin I in the cell body and in cell processes suggests that the two proteins are mostly sorted into identical vesicular organelles. A partial colocalization of VAMP II and cellubrevin as well as of VAMP II and Rab5 was observed under the fluorescence microscope. At the ultrastructural level, a colocalization of VAMP II and cellubrevin as well as of VAMP II and Rab5 was found on some clustered vesicles. The partial colocalization of VAMP II and cellubrevin implies that they similarly function as v-SNAREs. The partial colocalization of Rab5 with VAMP II in U373 cells suggests that the endosomal protein Rab5 is associated with VAMP II-containing organelles during some stages of their life cycle.
The focus of this study were Celtic gold coins excavated from the Martberg, a Celtic oppidium and sanctuary, occupied in the first century B.C. by a Celtic tribe known as the Treveri. These coins and a number of associated coinages, were characterised in terms of their alloy compositions and their geochemical and isotopic signatures so as to answer archaeological and numismatic questions of coinage development and metal sources. This required the development of analytical methods involving; Electron Microprobe (EPMA), Laser Ablation-ICP-MS, solution Multicollector-ICPMS and LA-MC-ICP-MS. The alloy compositions (Au-Ag-Cu-Sn) were determined by EPMA on a small polished area on the edge of the coins. A large beam size, 50µm (diameter), was used to overcome the extreme heterogeneity of these alloys. These analyses were shown to be representative of the bulk composition of the coins. The metallurgical development of the coinages was defined and showed that the earlier coinages followed a debasement trend, which was superceded by a trend of increasing copper at the expense of sliver while gold compositions remained stable. This change occurred with the appearance of the inscribed "POTTINA" coinage, Scheers 30/V. Two typologically different coinages, Scheers 16 and 18 ("Armorican Types") were found to have markedly different compositions which do not fit into the trends described above. A Flan for a gold coin, which may indicate the presence of a mint at the Martberg, was found to have an identicle weight and composition as the Scheers 30/I coins, which preceeded the majority of the coins found at the Martberg in the coin development chronology. The trace element anaylses were made by Laser Ablation-ICPMS using an AridusTM desolvating nebuliser to introduce matrix matched solution standards to calibrate the measurements, which were then normalised to 100%. Quantitative results were obtained for the following elements: Sc, Ti, Cr, Mn, Co, Ni, Cu, Zn, Se, Ru, Rh, Pd, Ag, Sb, Te, W, Ir, Pt, Pb, Bi. The remaining elements remain problematic as they produced incorrect standardisations mainly due to chemical effects in solution such as adsorption onto the beaker walls or oxidation : V, Fe, Ga, Ge, As, Mo, Sn, Re, Os, Hg. Changes in the sources of Au, Ag and Cu were observed during the development of the coinages through the variation of trace elements, which correlate positively with the major components of the coin alloys. Changes in the Pt/Au ratios show that the Scheers 23 coins contain distinctly different gold from the later coinages and that the Scheers 18 gold source was also different. Te/Ag was used to show that the Sch.23 coins also contained different silver and some subgroups were observed in the Sch. 30/V coins. A major change in copper source is indicated by the sudden increase of Sb and Ni with the introduction of the Sch. 30/V coins (POTTINA), which can be linked to a similar change in copper observed in the contemporary silver coinage, Sch. 55 (with a ring). Lead isotopic analyses were made by solution- and Laser Ablation - MC-ICP-MS, The laser technique proved to be in good agreement with the solution analyses with precisions between 1 and 0.1%o (per mil). The development of the laser method opens the way for easy and virtually non-destructive Pb isotopic determinations of ancient gold coins. The results showed that Sch. 23 is very different from the following coinages, Sch. 16 and 18 are also different, forming their own group, and all the later "Eye" staters (Sch. 30/I-VI) lie on a mixing line controlled by the addition of copper from a Mediterranean source, probably Sardinia or Spain. An indication of gold and silver sources should be possible with further analyses of the Sch. 23 and Rainbow Cup gold coins and the Sch. 54 and 55 silver coinages. Copper Isotopic analyses were made by solution- and Laser Ablation - MC-ICP-MS. Both techniques require further development to produce more reproducible results. The results show that there appears to be a trend to more positive d Cu65 values for the later coinages and that the link between the copper used in the Sch. 30/V (POTTINA) coins and the silver Sch. 55 (with a ring) coins is also shown by similarly postive d Cu65 values. The full suite of analyses were also made on samples of gold from the region. They were mostly composed of "placer gold", alluvial gold found in rivers. It was found that when a study is restricted to a limited number of deposits or areas then it is possible to distinguish between deposits based on the concentration of those elements which are least affected by transport related alteration processes. These elements include; the PGE's, due to their refractory nature, and those elements which are usually present in high enough concentrations to remain relatively unaffected, eg: Cu, Pb and Sb. Due to the nature of the coin alloy it is not possible to link the gold used in the coins studied here with gold deposits, as the large amounts of Ag and Cu, added to the coin alloys, have masked the Au signature. However, further Pb isotopic analyses of gold deposits should prove useful in determining from which regions Celtic gold was derived.
Role in routing to the plasma membrane of the L 0 domain of the multidrug resistance protein MRP1
(2003)
Die mehrfache Chemotherapieresistenz (Multidrug Resistance) beruht auf vermehrtem Transport von Xenobiotika aus der Zelle, was zu einer dramatischen Verringerung der intrazellulären Konzentration von chemotherapeutischen Substanzen führt. Dieser Effekt wird von transmembranen Transporter-Proteinen der ABC-Familie verursacht. Zu dieser Familie gehört MRP1, die eine große Vielfalt an Substraten transportieren kann. MRP1 ist ein 190 kDa Glykoprotein mit einer vermuteten Topologie, die zusätzlich zum typischen P-gp ähnlichen Kern (Delta MRP1) eine amino-proximale transmembrane Domäne aufweist, die aus fünf transmembranen Alpha-Helices besteht. Sie ist durch einen cytoplasmatischen Verbindungs-Loop (L0) mit Delta MRP1 verbunden. Wenn MRP1 in polarisierten Zellen exprimiert wird, wird es zu der basolateralen Membran geleitet. In der vorliegenden Arbeit sollte nun die Funktion des amino-terminalen Bereichs von MRP1, der aus der ersten transmembranen Domäne TMD0 und dem cytoplasmischen Verbindungs-Loop L0 besteht, durch Expression und Koexpression von diversen MRP1 Mutanten in polarisierten MDCKII Zellen untersucht werden. Es wurde gezeigt, dass in der L0 Region eine amphipathische Helix vorhanden ist, die für die Funktionalität der MRP1 notwendig ist; dass das isolierte L0-Peptid in der Lage ist, sich mit Delta MRPI zu assoziieren (dadurch erlangt das Protein wieder seine Funktion und lokalisiert sich in der basolateralen Membrane); dass TMD0L0 sich teilweise in der basolaterale Membrane befindet und dass seine Anwesenheit genügt, um die Glycosilierung (Fig. 4.17 in der Dissertation) und die Lokalisierung in der basolateralen Membrane des Delta MRP1 zu ermöglichen (Fig. 4.18 in der Dissertation); dass die Koexpression der zwei komplementären Fragmente eine wild-type-ähnliche Transportaktivität ergibt (Fig. 4.19 in der Dissertation) und dass die beiden Fragmente interagieren (Fig. 4.21 in der Dissertation). Es wurde ausserdem ein chimerisches Protein hergestellt, welches aus TMD0 von MRP1 und L0 von MRP2 besteht und in MDCKII und MDCKII-Delta MRP1 Zellen exprimiert. Es wurde festgestellt, dass das unvollständig glycosiliert ist (Fig. 4.24 in der Dissertation) und dass es sich im endoplasmatischen Reticulum lokalisiert (Fig. 425 in der Dissertation).
In dieser Arbeit wurde der chemische Ozonverlust in der arktischen Stratosphäre über elf Jahre hinweg, zwischen 1991 und 2002, mit Hilfe der so genannten "Ozon-Tracer Korrelationstechnik" (TRAC), untersucht. Bei dieser Methode werden Korrelationen zwischen Ozon und langlebigen Spurenstoffen im Verlauf des Winters im Polarwirbels beobachtet und so der jährliche akkumulierte Ozonverlust berechnet. Die Ergebnisse dieser Arbeit basieren im wesentlichen auf Messdaten der Satelliteninstrumente: HALOE (Halogen Occultation Experiment) auf UARS (Upper Atmosphere Research Satellite) und ILAS (Improved Limb Atmospheric Spectrometer) Instrument auf ADEOS (Advanced Earth Observing Satellite). Das HALOE Instrument misst seit Oktober 1991 kontinuierlich alle zwei bis drei Monate für einige Tage in höheren nördlichen Breiten. ILAS lieferte ausschließlich für den Winter 1996-97 Messungen, die über sieben Monate hinweg in hohen Breiten aufgenommen wurden. Aufgrund der eingeführten Erweiterungen und Verbesserungen der Methode in dieser Arbeit, konnte die Methode anhand einer detaillierten Studie für den Winter 1996-97 validiert werden. Die ILAS Messreihe wurde dazu verwendet, erstmals die Untersuchung der zeitlichen Entwicklung von Ozon-Tracer Korrelationen kontinuierlich für die gesamte Lebensdauer des Polarwirbels durchzuführen. Dabei wurden auch Korrelationen während der Bildung des Wirbels untersucht und im Besonderen mögliche Mischungsvorgänge zwischen Wirbelluft und Luftmassen außerhalb des Wirbels. Ausserdem wurde ein Vergleich der Ergebnisse von ILAS und HALOE Messdaten durchgeführt und Unterschiede in den Ergebnissen tiefgreifend analysiert. Basierend auf HALOE Messungen konnte die erweiterte TRAC Methode über elf Jahren hinweg angewendet werden. Damit war erstmals eine konsistente Analyse von Ozonverlust und Chloraktivierung über diesen Zeitraum möglich. Die Erweiterungen führten zu einer Verringerung und genauen Quantifizierung von Unsicherheiten der Ergebnisse. Ein deutlicher Zusammenhang zwischen meteorologischen Bedingungen, Chloraktivierung und dem chemischen Ozonverlust wurde deutlich. Weiterhin zeigte sich eine Abhängigkeit zwischen den meteorologischen Bedingungen und der Homogenität des Ozonverlustes innerhalb eines Winters, sowie der mögliche Einfluss von horizontaler Mischung auf Luftmassen in einem schwach ausgeprägten Polarwirbel. In dieser Arbeit wurde eine positive Korrelation zwischen den über die gesamte Lebensdauer des Wirbels auftretenden möglichen PSC-Flächen und den akkumulierten Ozonverlusten für die elf untersuchten Jahre deutlich. Es konnte darüber hinaus gezeigt werden, dass der Ozonverlust von deutlich mehr Einflüssen als nur von der Fläche möglichen PSC Auftretens bestimmt wird, sondern zum Beispiel von der Stärke der Sonneneinstrahlung abhängt. Außerdem lassen sich Auswirkungen von Vulkanausbrüchen, wie zum Beispiel im Jahr 1991 der des Mount Pinatubo, identifizieren.
Die Infrarotspektroskopie in Verbindung mit photoaktivierbaren Substraten wurde zur Untersuchung von Substrat-Protein-Wechselwirkungen eingesetzt. Dabei wurden Konformationsänderungen der Ca2+-ATPase des Sarkoplasmatischen Retikulums bei Bindung des Nukleotids, der Phosphorylierung der ATPase und der Hydrolyse des Phosphoenzyms beobachtet. Verwender wurden das native Substrat ATP und seine Analoga ADP, AMPPNP, 2'-deoxyATP, 3'-deoxyATP, ITP, AMP, Pyrophosphat, Ribosetriphosphat und TNP-AMP beobachtet. Diese Analoga waren an spezifischen funktionellen Gruppen des Substrats ATP modifiziert. Modifikation der 2'- und 3'-OH Gruppe des Ribosetriphosphats, der beta- und gamma-Phosphatgruppe und der Aminogruppe des Adenins reduzieren das Ausmaß an bindungsinduzierten Konformationsänderungen. Ein besonders starker Effekt wird für die 3'-OH Gruppe und die Aminogruppe des Adenins beobachtet. Dies zeigt die strukturelle Empfindlichkeit des Nukleotid-ATPase Komplexes auf einzelne Wechselwirkungen zwischen dem Nukleotid und der ATPase. Die Wechselwirkungen einer bestimmten Ligandengruppe mit der ATPase hängen von Wechselwirkungen anderer Ligandengruppen mit die ATPase ab. Die TNP-AMP Bindung verursacht teilweise gegenläufige und kleinere Konformationsänderungen verglichen mit ATP. Die Bindungweise von TNP-AMP ist unterschiedlich zu der von ATP, AMPPNP und anderen Tri- und Diphosphat Nucleotiden. Die Phosphorylierung der ATPase wurde mit ITP und 2'-deoxyATP beobachtet. Ca2E1P wurde in gleichem Ausmaß mit ITP und 2'-deoxyATP wie mit ATP akkumuliert, obwohl das Ausmaß der Konformationsänderungen bei Ca2E1P-Bildung geringer ist. Änderungen der 2'- und 3'-OH des Ribosetriphosphats und der Aminogruppe des Adenins beeinflussen die Reaktionsgeschwindigkeit der Phosphorylierung der ATPase. Es gibt keine direkte Verbindung zwischen dem Ausmaß der Konformationsänderung bei Nukleotid- Bindung und der Rate der Phosphorylierung. Das volle Ausmaß der ATP-induzierten Konformationsänderung ist nicht zwingend für die Phosphorylierung. Die Konformationen von Ca2E1N und Ca2E1P hängen vom Nukleotid ab. Dies weist darauf hin, dass die Struktur von ATPase Zuständen heterogener ist, als bisher erwartet. Die Aussagekraft und der Reichtum an Informationen in den Infrarotspektren zeigen, dass hiermit eine leistungsfähige Methode für die Untersuchung von Enzym-Substrat-Wechsel-Wirkungen und das räumliche Abtasten von Bindungstaschen zur Verfügung steht.
In this study we investigated the regulation of IL-18BPa by IFN-y in the context of colon cancer and human autoimmune diseases. IL-18BPa is a naturally occuring inhibitor that counteracts IL-18 bioactivity. By enhancing IFN-y production IL-18 has been introduced as pivotal mediator of TH1 immune responses. Indeed, many IL-18 effects are mediated by IFN-y. IL-18 bioactivity is connected with the pathogenesis of different inflammatory diseases, for instance, septic shock, colitis, Crohn's disease, myasthenia gravis, multiple sclerosis, rheumatoid arthritis, atherosclerosis, and organ transplant rejection. In addition, IL-18 has tumor-suppressive properties. IFN-y induced IL-18BPa expression was shown on protein and mRNA level in different colon carcinoma cell lines, organ cultures of colonic intestinal biopsy specimens, HaCaT keratinocytes as well as rheumatoid arthritis fibroblastlike synoviocytes (RA-FLS). The IFN-y-mediated induction of IL-18BPa appears to be a more general phenomenom. The capability of IFN-y to induce IL-18BPa also has been confirmed on the promoter level by performing luciferase reporter gene studies with two IL- 18BP promoter fragments. A GAS-site proximal to the transcription start site has been identified to be relevant for IFN-y-mediated induction of these two IL18BP promoter fragments. The induction of IL-18BPa is most likely mediated by STAT-1 in DLD-1 colon carcinoma cells. Sodium butyrate inhibited IFN-y-induced IL-18BPa expression in these cells. On the basis of our observations, we postulate a negative feedback mechanism, by which IFN-y-dependent and -independent IL-18 action might be counterregulated. In this model sodium butyrate is an additional player, that may interrupt the postulated negative feedback loop. A coculture system was performed to simulate an inflammatory TH1 response. This model which is more close to the in vivo situation, confirmed upregulation of IL-18BPa by endogenously produced IFN-y. The role of IL-18BPa is manifold and depends on IL-18 function in each particular case. In autoimmune diseases, for instance, which are often characterized by a TH1 polarized immune response, IL-18BPa might counterregulate IL-18 and/or IL-18-induced IFN-y bioactivity. Important examples are Crohn's disease and rheumatoid arthritis. In CD therapeutic use of IL-18BPa may therefore restore a hypothetically disturbed IL-18/IL-18BP balance. Concerning RA, IL-18BPa expression might contribute to protective functions of IFN-y, observed in different murine models for arthritis and in rheumatoid arthritis patients. Moreover, IL-18BPa might inhibit IL-18-mediated induction of subsequent cardinal inflammatory cytokines responsible for the pathogenesis of these diseases. Indeed, the pharmaceutical industry successfully used IL-18BP as therapeutic agent in a murine model of RA and in phase I clinical trials. On the contrary, in the context of carcinogenesis IFN-y- mediated IL-18BPa expression might be disadvantageous. By counterregulating the IL-18 arm of immune defenses against tumors, IL-18BP may have the potential to promote carcinogenesis. Our hypothesis is underlined by the observation that sodium butyrate, known to be protective in colon cancer, inhibited IFN-y-induced IL-18BPa expression. In parallel, IL-18-induced IFN-y is also responsible for iNOS induction. iNOS-derived NO provides a second possible way for inhibition of IFN-y-dependent and -independent tumor suppressive effects of IL-18. Finally, IFN-y-induced IL-18BPa expression was confirmed on the promoter level. This induction on the promoter level was associated with STAT-1 binding to the GAS element proximal to the start of transcription. It is tempting to speculate that blockage of the cytokine cascade upstream of IL-1 and TNF- a on the level of IL-18 may be of therapeutic benefit. Our data reflect the relationship between inflammation and cancer, in that inflammatory cells and cytokines found in tumors are likely to contribute to tumor growth, progression, and immunosuppression than they are to mount an effective host antitumour response.
In the recent years, high-resolution conditions have been established in solid-state NMR by the combination of magic angle spinning, state-of-the-art r.f. pulse schemes and the introduction of ultra-high magnetic fields. Similar to what is now routine in solution-state NMR, this has opened the way for structure determination by HR-SSNMR methods. Complete structural or dynamical characterization of the biomolecule of interest is most easily achieved if multiple or even uniformly [13C, 15N]-labeled versions are studied. In a first step, experiments that allow the complete assignment of the 13C and 15N resonances have been recently designed. To date, nearly complete chemical shift assignments were reported for two well-ordered proteins, the ±-spectrin SH3 domain and the Crh protein. The SSNMR analysis of the later protein has been presented in Section 4.1. For SSNMR applications, not the molecular size or solubility, but the spectral resolution can be of crucial importance. Experimental parameters and sample inherent conditions such molecular disorder may reduce the overall spectral dispersion. In these circumstances, techniques that allow for spectral simplification without the need of elaborated biochemical procedures (of isotopelabeling) are of special importance. In Section 2, several spectral editing methods have been proposed. These methods not only select resonances due to changesin the physical and chemical environment of the nucleus but they can also directly probe molecular properties such as dynamics and conformational heterogeneity. Once the chemical shifts are available for the biomolecule of interest, methods that permit to obtain structural restraints can be applied. In the case of multiply isotope labeled proteins, such techniques can in principle result in multiple structural parameters. In Section 3.1, we have shown that, similar to solution-state NMR, secondary chemical shifts can be readily employed to study the local backbone conformation. Inaddition, distance constraints between protons may be encoded in high-resolution on rare spins like 13C and 15N and measured. Finally, carbon-carbon constraints may be probed by employing frequency selective r.f. pulse schemes. These dihedral and distance constraints may subsequently lead to the determination of protein secondary to tertiary structure from a single protein sample. In Section 4.2,we have shown that high-affinity ligand binding to membrane proteins can be investigated with solid-state NMR. Here, the neuropeptide neurotensin which binds to the Gprotein coupled receptor NTS1 in sub-nanomolar affinity was investigated.Except for the case of rhodopsin, there is currently no information on the high-resolution structure of any other GPCR or a corresponding high-affinity ligand.Our SSNMR results identify, for the first time, a distinct binding mode of neurotensin that could be of considerable relevance for further pharmacological studies. As exemplified in section 4.3, HR-SSNMR based structural studies can also assist in refining existing (X-ray or solution-state NMR) membrane-protein structures. The presented results provide, for the first time, direct experimental evidence for a double occupancy of the Q0 binding site in the ubiquinone-bc1 complex and may provide the basis for the complete 3D structural determination of the ubiquinone binding pocket. Advancements regarding sample preparation (for example, including modular labeling, in vitro expression and intein technology) and improvements in NMR hardware instrumentation could open up new areas of solid-state NMR research such as the investigation of large protein-protein complexes or the complete 3D characterization of larger membrane proteins. Solid-state NMR studies of multiply-labeled biomolecules will furthermore profit from improved procedures for calculating 3D structures, in particular in the presence of ambiguousor a limited number of structural constraints. Unlike X-ray crystallography, protein motion does not hinder solid-state NMR methods. In fact, complementary to solution-state NMR, it may provide a very efficient means to study protein folding, flexibility and function under biologically relevant conditions. Hand in hand with solution-state techniques and crystallographic methods, solid-state NMR could provide insight into protein function and the chemistry of life with unprecedented accuracy and flexibility.
Im ersten Teil dieser Arbeit sind Protein-Protein Docking-Studien dokumentiert. Bis heute konnten die meisten Protein-Komplex-Strukturen nicht experimentell aufgeklärt werden, so auch die beiden oben genannten Elektrontransfer-Komplexe. Nach einem erfolgreichen Test wurden verschiedene Cytochrom c Oxidase:Cytochrom c Paare mit der gleichen Methode gedockt: COX aus Paracoccus denitrificans mit Pferdeherz Cytochrom c und COX mit dem löslichen Fragment des membrangebundenen Cytochrom C552 (beide aus P. denitrificans). Im zweiten Teil dieser Arbeit wurde die diffusive Annäherung des Cytochrom c an die Cytochrom c Qxidase mit der Brownschen Dynamik Methode simuliert. Die Diffusionsbewegung eines Brownschen Teilchens in wässriger Lösung wird durch die Langevin-Gleichung bestimmt. Der auf dieser Gleichung fußende Ermak-McCammon-Algorithmus ist Grundlage der Simulationsmethode. Die so ermittelten Raten für COX und Pferdeherz, sowie für COX und Cytochrom C552, wurden dann mit experimentell gewonnenen Raten verglichen. Da die Elektrostatik für den Annäherungsprozeß dieser Proteine eine so gewichtige Rolle spielt, wirken sich Mutationen, die mit einer Ladungsänderung einhergehen, merklich aus. Dies ist vor allem dann der Fall, wenn sich die Mutation in der Nähe der Bindungsstelle befindet. Aus dem gleichen Grund ist die Assoziationsrate auch stark von der Ionenstärke der umgebenden Lösung abhängig. Steigt die Ionenkonzentration wird die elektrostatische Komplementarität der Bindingsstellen der beiden Makromoleküle stärker abgeschirmt, und die Rate sinkt. Diese beiden relativen Trends konnten durch die Simulationen gut reproduziert und bestätigt werden. Allerdings liegen die absoluten Resultate merklich über den experimentell gemessenen Raten. Es ist sehr gut möglich, daß post-diffusive Effekte, die nicht in einer Brownschen Dynamik Simulation von starren Körpern berücksichtigt werden können, die Raten erniedrigen. Um den Einfluß der Membranumgebung auf die Wechselwirkung des Elektrontransportsystems zu untersuchen. wurde eine DPPC Doppelschicht um die Oxidase modelliert und energieminimiert. Mit Poisson-Boltzmann Rechnungen wurde das elektrostatische Potential dieses Nanosystems untersucht und mit dem der einzelnen Oxidase verglichen. Durch einen modifizierten Set-up konnten dann auch für dieses Membransystem Brownsche Dynamik Simulationen durchgeführt werden. Der Vergleich mit den vorhergehenden Simulationen ohne Membran erbrachte bemerkenswerte Ergebnisse. Während die Assoziationsraten für Pferdeherz Cytochrom c durch den Membraneinfluß erniedrigt wurden, stiegen sie im Fall des physiologischen Transferpartners c552. Pferdeherz Cytochrom c weist eine positive Nettoladung und einen ausgeprägten bipolaren Charakter auf. Eine große Zahl positiv geladener Seitenketten befindet sich auf der gleichen Hemisphäre wie die Bindungsstelle. Obwohl die DPPC Lipidmoleküle neutral sind, zeigten die Elektrostatikrechnungen, daß die Membranoberfläche abstoßend auf positive Ladungen wirkt. Da sich nun die Bindungsstelle der Oxidase für Cytochrom c nur etwa 10 Å oberhalb der Membran befindet, verringert sich die Wahrscheinlichkeit der Assoziation.
Mitogen activated protein kinases (MAPKs) are found in all eukaryotic cells and represent crucial elements in the signal transduction from the plasma membrane to the nucleus. Although a broad variety of extracellular stimuli activate MAPKs, they evoke very distinct cellular responses. The amplitude and duration of MAPK activation determine signal identity and ultimately cell fate. A tight and finely tuned regulation is therefore critical for a specific cellular response. The role and the regulation of extracellular signal-regulated kinase 5 (ERK5), a MAPK with a large and unique C-terminal tail, were studied in different cellular systems. The study highlights two aspects of ERK5 regulation: control of the phosphorylation state and regulated protein stability. In analogy to other MAPKs ERK5 is activated by dual phosphorylation of threonine and tyrosine residues in its activation motif. A first part of the study concentrates on whether and how the protein tyrosine phosphatase PTP-SL is involved in the downregulation of the ERK5 signal. The direct interaction of both proteins is shown to result in mutual modulation of their enzymatic activities. PTP-SL is a substrate of ERK5 and, independent of its phosphorylation, binding to the kinase enhances its catalytic phosphatase activity. On the other hand, interaction with PTP-SL does not only downregulate enzymatic ERK5 activity but also effectively impedes its translocation to the nucleus. The second part of this study focuses on the interaction of ERK5 with c-Abl and its oncogenic variants Bcr/Abl and v-Abl. In this study these tyrosine kinases are demonstrated to regulate ERK5 by two mechanisms: first, by induction of kinase activity and secondly, by stabilisation of the ERK5 protein. Stabilisation involves the direct interaction of unique ERK5 domains with Abl kinases and is independent of MAPK cascade activation. The level of ERK5 and its intrinsic basal activity – rather than its activation – are essential for v-Abl-induced transformation as well as for survival of Bcr/Abl-positive leukaemia cells. Stabilisation of ERK5 thus contributes to cell survival and should therefore be considered as an additional aspect in therapy of chronic myeloid leukaemia. Taken together, the results obtained in this study demonstrate that diverse pathways regulate ERK5 signalling by affecting kinase activity, localisation and protein stability. While the phosphatase PTP-SL is involved in negative regulation of ERK5, Abl kinases potently activate ERK5 and increase its half-life. Protein stabilisation thus is presented as a novel mechanism in the regulation of MAPKs.
Gegenstand dieser Arbeit sind Eigenschaften angeregter hadronischer Materie sowie physikalische Systeme, in denen diese Materie auftritt bzw. produziert wird. Die Beschreibung der stark wechselwirkenden Materie erfolgt in einem hadronischen, chiral-symmetrischen SU(3)L x SU(3)R Modell, welches die Saturierungseigenschaften von Kernmaterie und die Eigenschaften von Atomkernen reproduziert. Die Untersuchung heißer und dichter unendlicher hadronischor Materie zeigt, dass das vom Modell vorhergesagte Phasendiagramm stark von den Kopplungen der Baryonenresonanzen abhängt. Für kalte hadronische Materie ergibt die Einbeziehung des Baryonendekupletts und die Freiheit in deren Vektorkopplungen eine sehr große Bandbreite an verschiedenen Zustandsgleichungen. Für heiße hadronische Materie mit verschwindendem baryochemischen Potential zeigt sich ebenfalls eine starke Abhängigkeit der Eigenschaften hadronischer Materie von der Ankopplung der baryonischen Resonanzen. Es werden drei verschiedene Parametrisierungen betrachtet. Das resultierende Phasenübergangsverhalten variiert von einem "Crossover" über einen schwachen, zu einem doppelten Phasenübergang erster Ordnung. Es zeigt sich jedoch, dass die beobachteten Eigenschaften von Neutronensternen die Unbestimmtheit bzgl. der Vektorkopplung dieser Freiheitsgrade und damit der Zustandsgleichung deutlich verringern. Das Raum-Zeit Verhalten relativistischer Schwerionenkollisionen bei SPS- und RHIC-Energien wird mittels einer hydrodynamischen Simulation unter Benutzung der chiralen Zustandsgleichungen untersucht. Dabei spiegelt sich das unterschiedliche Phasenübergangsverhalten deutlich im Ausfrierverhalten der hadronischen Materie wider. Die im chiralen Modell berechneten Teilchenzahlverhältnisse werden mit den aus Schwerionenkollisionen von AGS- bis RHIC-Energien erhaltenen experimentellen Daten verglichen. Dabei zeigt sich, dass die verschiedenen Parametersätze des chiralen Modells und die Rechnungen für ein nichtwechselwirkendes, ideales Hadronengas eine ähnlich gute Beschreibung der gemessenen Weite liefern. Die deduzierten Ausfrierwerte für die Temperatur sind sensitiv auf das Phasenübergangsverhalten und liegen unterhalb der jeweiligen kritischen Temperatur. Die vorhergesagten Ausfriermassen sind in allen Parametrisierungen sehr ähnlich mit Abweichungen bis zu 15% von den entsprechenden Vakuumwerten. Die Untersuchung der Eigenschaften von Vektormesonen in dichter Materie erfolgt in der Mittleren-Feld- und in der HartreeNäherung. Hierbei zeigt sich eine signifikante Reduzierung der Teilchenmassen durch Vakuumpolarisationseffekte.
We consider the long-time behaviour of spatially extended random populations with locally dependent branching. We treat two classes of models: 1) Systems of continuous-time random walks on the d-dimensional grid with state dependent branching rate. While there are k particles at a given site, a branching event occurs there at rate s(k), and one of the particles is replaced by a random number of offspring (according to a fixed distribution with mean 1 and finite variance). 2) Discrete-time systems of branching random walks in random environment. Given a space-time i.i.d. field of random offspring distributions, all particles act independently, the offspring law of a given particle depending on its position and generation. The mean number of children per individual, averaged over the random environment, equals one The long-time behaviour is determined by the interplay of the motion and the branching mechanism: In the case of recurrent symmetrised individual motion, systems of the second type become locally extinct. We prove a comparison theorem for convex functionals of systems of type one which implies that these systems also become locally extinct in this case, provided that the branching rate function grows at least linearly. Furthermore, the analysis of a caricature model leads to the conjecture that local extinction prevails generically in this case. In the case of transient symmetrised individual motion the picture is more complex: Branching random walks with state dependent branching rate converge towards a non-trivial equilibrium, which preserves the initial intensity, whenever the branching rate function grows subquadratically. Systems of type 1) and systems of type 2) with quadratic branching rate function show very similar behaviour. They converge towards a non-trivial equilibrium if a conditional exponential moment of the collision time of two random walks of an order that reflects the variability in the branching mechanism is finite almost surely. The equilibrium population has finite variance of the local particle number if the corresponding unconditional exponential moment is finite. These results are proved by means of genealogical representations of the locally size-biased population. Furthermore, we compute the threshold values for existence of conditional exponential moments of the collision time of two random walks in terms of the entropy of the transition functions, using tools from large deviations theory. Our results prove in particular that - in contrast to the classical case of independent branching - there is a regime of equilibria with variance of the local number of particles.
One of the known apoptotic pathways in mammalian cells involves release of mitochondrial Cytochrome c into the cytosol. Cyt c then together with ATP or dATP induces a conformational change in the adaptator protein Apaf-1 (a homologue of the C. elegans CED4 protein) (Zou, Henzel et al. 1997), leading to its oligomerization and the recruitment of several pro-Casp-9 molecules. This protein complex assembly called "apoptosome" leads to the activation of Casp-9 which then initiates or amplifies the caspase cascade. The cell death program can be stalled at several points and we were interested in identifying new proteins inhibiting cell death downstream of Cyt c release. This thesis describes how I have screened a cDNA library derived from a pool of human breast carcinomas in a yeast-based survival screen, using the S. pombe yeast strain HC4 containing an inducible CED4 construct(James, Gschmeissner et al. 1997). The screen resulted in the identification of six proteins displaying cell death-inhibiting activity in S. pombe as well as anti-apoptotic potential in mammalian cells. Those six molecules were RoRet (Ruddy, Kronmal et al. 1997), Aven (Chau, Cheng et al. 2000), Fte-1/S3a (Kho, Wang et al. 1996), PGC2 (Padilla, Kaur et al. 2000; Goetze, Eilers et al. 2002), SAA1-2ß (Moriguchi, Terai et al. 2001) and FBP (Brockstedt, Rickers et al. 1998) of which I selected RoRet, Aven and Fte-1/S3a for further analysis. RoRet is a new anti-apoptotic molecule that can inhibit the mitochondrial pathway via its PRY-SPRY domain. RoRet does not seem to bind to Apaf-1, and does not co-localize with the activated Apaf-1/Caspase-9 complex. Aven was published to act as an anti-apoptotic protein and suggested to function via the recruitment of Bcl-XL to Apaf-1. This work shows that its C-terminal domain can bind to Apaf-1 and has a strong anti-apoptotic activity by itself. Moreover, Aven co-localizes with the activated Apaf-1/Caspase-9 complex suggesting that it is a component of the apoptosome. Furthermore, the expression of Aven is regulated in mammary glands during the pregnancy cycle. Fte-1/S3a has been already implicated in increased transformation capacity of v-Fos in fibroblasts (Kho and Zarbl 1992; Kho, Wang et al. 1996). This work shows that it has anti-apoptotic activity and can protect against Bak- and Apaf-1-induced apoptosis. It can bind directly to activated Apaf-1 at the linker domain between the WD40 repeats and the CED4-like domain, suggesting that it may protect by sequestering the activated Apaf-1 to some organelles whose nature remains to be determined. Moreover, expression studies on mRNA and protein level showed upregulation of Fte-1/S3a in colon, lung and kidney carcinoma. Hmgb1 (Flohr, Rogalla et al. 2001; Pasheva, Ugrinova et al. 2002; Stros, Ozaki et al. 2002) was identified during a survival screen performed with a NIH 3T3 mouse fibroblast cDNA library in a Bak-expressing yeast S. pombe strain. HMGB1 can protect against Bak-, UV-, FasL- and TRAIL-induced apoptosis. Significant overexpression of HMGB1 was found in breast and colon carcinoma, and elevated mRNA amounts were detected in uterus, colon and stomach carcinoma, suggesting that it may be a tumour marker (Brezniceanu et al., 2003).
Resistive Plate Chambers (RPCs) are gaseous parallel plate avalanche detectors that implement electrodes made from a material with a high volume resistivity between 10 high 7 and 10 high 12 omega cm. Large area RPCs with 2mm single gaps operated in avalanche mode provide above 98% efficiency and a time resolution of around 1 ns up to a flux of several kHz/cm high 2. These Trigger RPCs will, as an example, equip the muon detector system of the ATLAS experiment at CERN on an area of 3650 m high 2 and with 355.000 independent read out channels. Timing RPCs with a gas gap of 0.2 to 0.3mm are widely used in multi gap configurations and provide 99% efficiency and time resolution down to 50 ps. While their performance is comparable to existing scintillator-based Time-Of-Flight (TOF) technology, Timing RPCs feature a significantly, up to an order of magnitude, lower price per channel. They will for example equip the 176 m high 2 TOF barrel of the ALICE experiment at CERN with 160.000 independent read out cells. RPCs were originally operated in streamer mode providing large signals which simplifies readout electronics and gap uniformity requirements. However, high rate applications and detector aging issues made the operation in avalanche mode popular. This was also facilitated by the development of new highly quenching C2F4H2-based gas mixtures with small contents of SF6. While the physics of streamers is difficult to study, the avalanche mode opened the possibility for a detailed simulation of the detector physics processes in RPCs. Even though RPCs were introduced in the early eighties and have been (will be) used in experiments, there are still disagreements about the explanation of several aspects of the RPC performance. The high efficiency of single gap RPCs would require a large ionization density of the used gases, which according to some authors contradicts measurements. Even in the case of a large ionization density the gas gain has to be extremely large, in order to arrive at the observed RPC efficiency. This raises other questions: A very strong space charge effect is required to explain the observed small avalanche charges around 1 pC. Doubts have been raised whether an avalanche can progress under such extreme conditions without developing into a streamer. To overcome these difficulties, other processes, like the emission of an electron from the cathode, were suggested. Moreover, the shape of measured charge spectra of single gap RPCs differs largely from what is expected from the statistics of the primary ionization and the avalanche multiplication. In this thesis we discuss the detector physics processes of RPCs, from the primary ionization and the avalanche statistics to the signal induction and the read out electronics. We present Monte-Carlo simulation procedures that implement the described processes. While the fundament of the described model and some results were already published elsewhere [1], the subject of this thesis is the implementation of the space charge effect. We present analytic formulas for the electrostatic potential of a point charge in the gas gap of an RPC. These formulas were developed in collaboration with the University of Graz [2] and were published in [3, 4]. The simulation model presented in [1] is completed by the dynamic calculation of the space charge field using these formulas. Since the gas parameters like drift velocity and the Townsend and attachment coefficients depend on the electric field, they are calculated dynamically as well. The functional dependence of these parameters on the field is obtained with the simulation programs MAGBOLTZ and IMONTE. For the primary ionization parameters, we use the values that are predicted by the program HEED. While the described procedure only simulates the longitudinal avalanche development towards the anode of the RPC, we also present more dimensional models that allow a careful study of the transverse repulsive and attractive forces of the space charge fields, and of the consequences for the avalanche propagation. We shall show that the efficiencies of single gap Timing RPCs is indeed explained by the high primary ionization density (about 9.5 /cm as predicted by HEED) and a large effective Townsend coefficient (around 113 /mm as predicted by IMONTE). We show that the space charge field reaches the same magnitude as the applied electric field in avalanches at large gas gain. This strong space charge effect effectively suppresses large values for the avalanche charges. The shape of the simulated charge spectra is very similar to the measurements. Also the simulated average charges are close to the experimental results. RPCs are operated in a strong space charge regime over a large range of applied voltage, contrary to wire chambers. We apply only standard detector physics simulations to RPCs. The performance of Timing and Trigger RPCs is well reproduced by our simulations. The results concerning the space charge effect were presented and discussed at the 'RPC 2001' workshop [5] and on the '2002 NSS/MIC' conference [6].
In summary, the cooled heavy-ion beams of the ESR storage ring offer excellent experimental conditions for a precise study of the effects of QED in the groundstate of high-Z one- and two-electron ions. This has been demonstrated within the series of experiments conducted at the electron cooler device as well as at the gasjet target. In this work we have used a recently developed experimental approach to obtain the first direct measurement of the two-electron contributions to the ground state binding energy of helium-like uranium. By employing our method, all one-electron contributions to the binding energy such as finite-nuclear size corrections and the one-electron self energy cancel out completely. Note, this is a distinctive feature of this particular kind of QED test and is in contrast to all other tests of bound state QED for high-Z ions such as 1s Lamb shift (in one-electron systems), g-factor of bound electrons, or hyperfine splitting. Compared to former investigations conducted at the superEBIT in Livermore we could already substantially improve the statistical accuracy and extend studies to the higher-Z regime. Moreover, our result has reached a sensitivity on specific two-electron QED contributions. Our value agrees with the theoretical predictions within the experimental uncertainty. Similar to the superEBIT experiment possible sources of systematic errors are essentially eliminated and the final result is limited only by counting statistics. For the case of the 1s Lamb shift in hydrogen-like uranium, the achieved accuracy of +- 4.2 eV is a substantial improvement by a factor of 3 compared to the most precise value up to now [44] (see Fig. 5.6). Our result already provides a test of the first-order QED contributions at the 1.5% level and only a slight improvement is required in order to achieve a sensitivity to QED contributions beyond first-order SE and VP.
The mechanism of peptide transport has been studied on two different ABC transporters of S. cerevisiae. Thereby, the aim of this PhD thesis was to characterise the transporter function on molecular level and shed light on the physiological role of these transporters. The ABC gene YLL048 encodes a novel intracellular transporter translocating peptides from the cytosol to the lumen of the ER. Deletion of the gene resulted in loss of peptide transport activity. The transport activity was fully restored after transformation of the deletion mutant by plasmid-encoded YLL048. Studying the substrate specificity using randomized peptide libraries it was demonstrated that peptides of the size from 6 to 56 amino acids are recognized. So far, no upper limit of the substrate size was obtained. Introduction of D-amino acids in various positions of a nonamer peptide did not impair transport activity. The physiological function of YLL048p is not well understood. The gene product is not essential for cell viability as the deletion mutant did not show any growth phenotype. To examine the possibility that YLL048 encoded protein is part of a quality control of yeast cells involved in the unfolded protein response (UPR), upregulation of YLL048 transcription by heat shock and stress conditions were investigated. We could not observe an influence of stress factors on YLL048 mRNA level. Upregulation of gene expression by the transcription factors Pdr1p and Pdr3p was excluded. The ABC transporter Mdl1p has been identified as peptide transporter of the inner mitochondrial membrane. This protein is required for the export of peptides with the size of 6 to 21 amino acids from the matrix into the intermembrane space. These peptides are generated by m-AAA proteases degrading non-assembled or missfolded membrane proteins. In order to understand the transport mechanism in detail, Mdl1p was expressed in S. cerevisiae and E. coli. Partially enriched protein was reconstituted into liposomes and was active in ATP binding. The association of the NBDs has been described as a central step of the ATPase cycle of ABC transporters, but it is still controversial how both motor domains cooperate and coordinate ATP hydrolysis. To address this question, the Mdl1p-NBD was overexpressed in E. coli and purified to homogeneity. The isolated NBD was active in ATP binding and hydrolysis with a turnover of 0.5 ATP per min and a Km value of 0.2 mM. Isolated NBDs did not show cooperativity in ATPase activity. However, the ATPase activity was observed to be non-linearly dependent on protein concentration suggesting the active form of this enzyme is not a monomer. Very importantly, for the first time an ATP-induced dimer was observed after trapping the NBD by ortho-vanadate or BeFx. The nucleotide composition of the trapped intermediate state was determined and two ADP molecules were simultaneously bound per dimer. An ATP-induced dimer of the ATPase inactive mutant (E559Q) was observed already in the absence of ATPase inhibitor. The E599Q dimer contained two ATP molecules in the absence of Mg2+ at 4°C. Prolonged incubation at 30°C in the presence of Mg2+ induced a stable dimer in which one ATP and ADP molecule were trapped at the same time. Based on these experiments, a new cycle for ATPase activity of ABC transporters was proposed. Binding of ATP to two NBD monomers induces dimerization. Both nucleotides are hydrolysed sequentially. During the hydrolysis cycle the nucleotides cannot be released from the dimer. After hydrolysis of two ATP molecules the domains dissociate and start a new cycle.