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Numorous psychotherapeutic approaches have been put forward to better treat patients with chronic psychological problems or severe personality disorders, among them Schema Therapy (Young, Klosko & Weishaar, 2003). Within Schema Therapy, there are two important theoretical constructs: early maladaptive schemas, which are conceptualized as stable and lasting emotional, behavioral and cognitive patterns regarding ourselves, the world and other people that have their origins in childhood or adolescence and schema modes, which are current states that describe the rapid shifts in emotion, cognition and behaviour that can be seen in patients with severe personality disorders. Schema Therapy has growing popularity with clinicians in the last decade, but only a few studies have tested the empirical validity of the schema mode model. Lobbestael, van Vreeswijk, Spinhoven, Schouten & Arntz (2010) developed and evaluated the Schema Mode Inventory to assess schema modes within the framework of a questionnaire. This instrument has been translated to the German language and the psychometric properties of the instrument indicate an adequate fit to the model established in the original Dutch study (paper 1).
In Schema Therapy, on the one hand mode concepts have been proposed without a focus on one specific disorder (Young et al., 2003), but on the other hand disorder-specific concepts have been established that form the basis for manualization and research. The most popular disorder specific mode concept is that of Borderline Personality Disorder (Arntz & van Genderen, 2009). In randomized controlled trials the effectiveness of Schema Therapy for Borderline Personality Disorder in the individual setting (Giesen-Bloo, van Dyck, Spinhoven, van Tilburg, Dirksen et al., 2006) and the group setting (Farrell, Shaw & Webber, 2009) was evaluated. Whereas Schema Therapy for Borderline Personality Disorder in the individual setting has been manualized (Arntz & van Genderen, 2009), a manual for group Schema Therapy is not yet available, Therefore, particularly since this last evaluation study, there has been a need to better describe, how the Schema Therapy mode model for Borderline Personality Disorder has been applied to the group setting (papers 2 & 3) as well as how both modalities of Schema Therapy treatment, individual and group, are combined in intensive psychotherapy settings such as inpatient or day care (papers 4 & 5). Lastly the question has been put forward as to how effective Schema Therapy for Borderline Personality Disorder is in intensive treatment environments, where it is assumed that the severity of the disorders treated is even higher. It is found that a combined program of individual and group Schema Therapy for Borderline Personality Disorder is effective in the inpatient setting. Subtle variations in treatment programs in three pilot studies leave room for the discussion of possible factors influencing treatment effects (paper 6). The results of all studies are discussed on the background of Schema Therapy’s development as a new psychotherapy approach for the treatment of complex disorders.
Design and optimization of the lattice of the superconducting synchrotron SIS300 for slow extraction
(2011)
The superconducting synchrotron SIS300 is planned to be built at the new Facility for Antiproton and Ion Research (FAIR), at GSI-Darmstadt [1]. SIS300 will be a versatile machine, which by means of a low-energy stretcher-mode or a high-energy ramped-mode will provide slowly extracted heavy ion beams towards the experimental areas. To reach the required maximum field of 4.5 T, cos(θ) magnets are necessary. Thus, SIS300 will become the first superconducting synchrotron worldwide with cos(θ) magnets providing resonant slow extraction.
Since SIS300 will be installed in the same tunnel as the SIS100 synchrotron, the dipole layout of SIS300 cannot be freely chosen. Thus, a standard lattice cannot be applied. A redesign of the SIS300 lattice accepting compromises concerning the positions and phase advances between the optical elements has been proposed. Using the analytical model of the slow extraction, firstly proposed by Kobayashi, and the analytical description of the resonance driving modes, a multiobjective optimization algorithm has been developed for the optimization of the lattice under the given boundary conditions. The final goal of the lattice optimization is a higher efficiency of the slow extraction. The results are evaluated by means of tracking simulations performed with the code Elegant.
The field quality in superconducting cos(θ) magnets is determined by the positions of the superconducting cable and the static and time-dependent effects of the current in the cable. Furthermore, the fast ramp rates of 1 T/s in the dipoles, which are fifty times faster than in any other superconducting cos(θ) magnet, together with the fact that the aperture is smaller than in conventional accelerator magnets, makes it extremely difficult to obtain a high-quality magnetic field. The unavoidable field errors affect the beam dynamics and worsen the slow extraction efficiency. Therefore, the field errors in the SIS300 dipoles have been estimated, and their effects have been taken into account in the optimization algorithm. As a result a compensation scheme has been proposed, in which time-dependent gradients in the sextupoles counteract the decay of the sextupole field errors in the dipole magnets during the slow extraction. For the limits where the compensation was no longer possible, tolerances to the magnet field errors have been determined.
Within the present work, photodissociation reactions on 100Mo, 93Mo and 92Mo isotopes were studied by means of the Coulomb dissociation method at the LAND setup at GSI. Experimental data on these isotopes are important to explain the problem of the underproduction of the lighter p-nuclei - 92; 94Mo - within the models of the p-process nucleosynthesis. The reaction rates used in the nucleosynthesis calculations are usually obtained within the framework of the statistical model. In order to verify the model predictions and reduce the uncertainties, experimental measurements of the reaction cross sections are required. In particular, the data on (γ,n) reactions are of interest, since these reactions were shown to dominate the p-process flow in the molybdenum mass region.
As a result of the analysis of the present experiment, integrated Coulomb excitation cross sections of the 100Mo(γ,n), 100Mo(γ,2n), 93Mo(γ,n) and 92Mo(γ,n) reactions were determined. The measurement of the 93Mo isotope is particularly important, since this nucleus is unstable, and the corresponding cross section has not been measured before.
It should be emphasized that Coulomb dissociation is a unique tool to study photoninduced reactions on unstable nuclei, which is especially relevant in the context of nucleosynthesis network calculations. However, because of to the complexity of the data analysis procedure and a number of model assumptions that are required in order to extract the Coulomb excitation cross section from the data, one of the main aspects of this thesis was to verify the method by comparing the results with the previously published data obtained with real photon beams. Integrated cross sections of the 100Mo(γ,n) and 100Mo(γ,2n) reactions were directly compared to the data by Beil et al., obtained at Saclay with photons from positron annihilation, while an indirect comparison could be performed with a recent photoactivation measurement by Erhard and co-workers. A reasonable agreement was observed for the 1n channel: a scaling factor of 0.8 ± 0.1 between our result and Beil et al. data is consistent with the scaling factor of 0.89±0.09 reported by Erhard et al. between their data and Beil et al. data. Both results are in agreement with the scaling factor of 0.85 ± 0.03 recommended by Berman et al. for the data measured at Saclay on nuclei in the respective mass region. A somewhat lower factor of 0.61 ± 0.09 between the present data and Beil et al. data was obtained for the 2n channel. The discrepancy might be explained by both the substantial efficiency correction that has to be applied to the LAND data in the two-neutron case, as well as by an insufficiently accurate assumption that the Saclay neutron detector efficiency is energy- and multiplicity- independent.
A second important topic of the present thesis is the investigation of the efficiency of the CsI gamma detector. The calorimetric information that it delivers is essential to reconstruct the energy-differential cross section from the present measurement. The data taken with the gamma calibration sources shortly after the experiment were used for the investigation. In addition, a test experiment in refined conditions was conducted within the framework of this thesis. Numerous GEANT3 simulations of the detector were performed in order to understand various aspects of its performance. As a result, the efficiency of the detector was determined to be approximately a factor of 2 lower than the efficiency expected from the simulation. This result is consistent with several independent investigations, which were performed using different methods. At the same time, a remarkable agreement between the simulated and experimental data was achieved under assumption that the inefficiency of the detector is explained by the loss of data from a number of crystals, which are randomly chosen in each event according to their averaged performance ratio (the ”on-off” effect). The reasons for the observed malfunction are yet not fully clear. Regardless of the exact reason, in the present conditions a deconvolution of the measured data from the CsI response is not possible. Consequently, within the framework of this thesis, the results are presented in terms of integrated cross sections. A search for alternative methods of data interpretation, allowing to extract energy-differential information out of the available data, in currently ongoing.
In the more recent experiments at the LAND setup, where the Crystal Ball gamma detector was used as a calorimeter, the reconstruction of the energy-differential cross section with a reasonable resolution was already shown to be feasible. It means that, even considering the uncertainties of the present experiment of the order of 10%, the uncertainties of the statistical model predictions, which are on average estimated to be within a factor of 1.5-2, can already be constrained.
The analysis of the present experiment is still in progress. As a next step, Coulomb excitation cross section for 94Mo will be obtained. The 94Mo(γ,n) reaction cannot be studied by photoactivation, since the life time of the daughter nucleus is too long (4000 y). At the same time, this reaction plays a key role in the p-process nucleosynthesis.
The future of the LAND setup - the R3B setup1 at FAIR2 - will take advantage of a three orders of magnitude higher intensity of the radioactive beams [85], as well as of a completely new detector system. High-resolution measurements of the energy-differential cross sections will be possible for exotic nuclei, which were never accessible in the laboratory before. Such measurements will open great opportunities for nuclear astrophysics, allowing to obtain high-quality experimental data even for regions of the nuclear chart where the statistical model calculations are not applicable.
In Nervensystemen werden zahlreiche Informationen wahrgenommen und verarbeitet um ein adäquates Verhalten hervorzurufen. Für die Untersuchung der funktionellen Zusammenhänge hierbei wurden verschiedene Methoden entwickelt, die eine gezielte Manipulation neuronaler Prozesse ermöglichen. Durch Analyse der resultierenden Effekte können dabei synaptische Proteine, einzelne Neuronen oder neuronale Netzwerke funktionell charakterisiert werden. Bisherige Ansätze verfügen jedoch nur über eine geringe zeitliche und räumliche Auflösung oder erlauben lediglich eine eingeschränkte Anwendung im frei beweglichen Tier.
Diese Nachteile können durch die heterologe Expression von lichtgesteuerten, mikrobiellen Rhodopsinen zur gezielten Manipulation des Membranpotentials umgangen werden. So induziert die Photoaktivierung des Kationenkanals Channelrhodopsin 2 (ChR2; (Nagel et al., Curr Biol 2005)) eine Depolarisation, während die Chloridpumpe Halorhodopsin (NpHR; (Zhang et al., Nature 2007)) für die Hyperpolarisation verwendet werden kann. Dabei ermöglichen die schnellen Kinetiken der Rhodopsine eine zeitlich präzise Steuerung des Membranpotentials. Durch Auswahl geeigneter Promotoren ist zudem oftmals eine zell spezifische Expression möglich. Dieser Ansatz wird daher allgemein als Optogenetik bezeichnet.
In der vorliegenden Arbeit wurden zunächst konventionelle Techniken genutzt, um die Funktion von zwei assoziierten Proteinen eines Acetylcholin Rezeptors in C. elegans zu untersuchen. Des Weiteren wurden verschiedene Methoden für den Fadenwurm entwickelt und angewendet, die die Vorteile optogenetischer Techniken für die funktionelle Charakterisierung synaptischer Proteine und neuronaler Netzwerke nutzbar machen. Hierbei erlaubt die Transparenz von C. elegans die optogenetische Stimulation im lebenden Organismus unter nicht invasiven Bedingungen. Weitere Vorteile von C. elegans als neurobiologischem Modellorganismus liegen in seiner einfachen Handhabung (Hope, 1999) und der stereotypen Entwicklung seines Nervensystems mit bekannten anatomischen Ausprägungen (Sulston and Horvitz, Dev Biol 1977; Varshney et al., PLoS Comput Biol 2011; White et al., Philos Trans R Soc Lond B Biol Sci 1986). Durch ihre Häufigkeit und die experimentelle Zugänglichkeit wird hierbei die neuromuskuläre Synapse oftmals zur Erforschung der synaptischen Reizweiterleitung genutzt (Von Stetina et al., Int Rev Neurobiol 2006). Durch pharmakologische (Lewis et al., Neuroscience 1980; McIntire et al., Nature 1993; Miller et al., Proc Natl Acad Sci U S A 1996; Richmond and Jorgensen, Nat Neurosci 1999) und elektrische Stimulation (Richmond and Jorgensen, Nat Neurosci 1999) können dabei Defekte der Transmission hervorgehoben werden, während Verhaltensexperimente oder elektrophysiologische Messungen der post synaptischen Ströme in Muskelzellen eine quantitative Analyse ermöglichen (Richmond and Jorgensen, Nat Neurosci 1999).
Diese Methoden wurden für die funktionelle Charakterisierung von NRA 2 und NRA 4 verwendet, die beide als akzessorische Proteine zusammen mit dem Levamisol sensitiven Acetylcholin Rezeptor der Körperwandmuskelzellen aufgereinigt wurden (Gottschalk et al., EMBO J 2005). Dabei konnte gezeigt werden, dass NRA 2 und NRA 4 im Endoplasmatischen Retikulum (ER) der Muskelzellen einen Komplex bilden, der die Sensitivität von beiden nikotinischen Acetylcholin Rezeptoren gegenüber verschiedenen cholinergen Agonisten verändert. In diesem Zusammenhang wurde auch nachgewiesen, dass die Oberflächenexpression einzelner Untereinheiten der beiden Rezeptoren durch NRA 2/4 beeinflusst wird. Diese Resultate legen die Vermutung nahe, dass beide Proteine die Zusammensetzung der Rezeptoren und somit ihre pharmakologischen Eigenschaften modulieren. Denkbar ist dabei eine regulatorische Funktion bei der Assemblierung verschiedener Untereinheiten zu einem funktionellen Rezeptor oder bei der Kontrolle des ER Austritts von Rezeptoren mit bestimmter Zusammensetzung. In dieser Hinsicht konnte jedoch keine Interaktion von NRA 2/4 mit der Notch Signalkaskade nachgewiesen werden, wie sie für die homologen Proteine nicalin und NOMO in Vertebraten gezeigt wurde (Haffner et al., J Biol Chem 2007; Haffner et al., EMBO J 2004).
Für die Untersuchung synaptischer Proteine durch optogenetische Techniken wurde ChR2(H134R) selektiv in cholinergen oder GABAergen Motorneuronen exprimiert, um die akute und lichtgesteuerte Freisetzung des jeweiligen Neurotransmitters zu ermöglichen. Die resultierende Stimulation bzw. Inhibition von Muskelzellen wurde hierbei durch elektrophysiologische Messungen der post synaptischen Ströme und durch Analyse von Kontraktionen respektive Relaxationen untersucht. Dabei wurde gezeigt, dass Störungen der synaptischen Reizweiterleitung die Ausprägung und Dynamik dieser lichtinduzierten Effekte beeinflussen und dadurch charakterisiert werden können. So zeigten beispielsweise Mutanten von Synaptojanin und Endophilin nachlassende Effekte bei anhaltender oder wiederholter Stimulation, was durch die gestörte Regeneration synaptischer Vesikel erklärt werden kann (Harris et al., J Cell Biol 2000; Schuske et al., Neuron 2003; Verstreken et al., Neuron 2003).
Die hohe Sensitivität dieser Methode wurde im Nachfolgenden dazu verwendet, die Inhibition cholinerger Motorneuronen durch den metabotropen GABAB Rezeptor zu untersuchen, der in C. elegans aus den beiden Untereinheiten GBB 1 und GBB 2 gebildet wird (Dittman and Kaplan, J Neurosci 2008; Vashlishan et al., Neuron 2008). Dabei konnte zunächst gezeigt werden, dass diese heterosynaptische Inhibition verschiedene lokomotorische Verhaltensweisen der Tiere beeinflusst. Für die mechanistische Untersuchung wurden anschließend cholinerge Motorneuronen durch ChR2(H134R) photoaktiviert, während resultierende Kontraktionseffekte in Abhängigkeit von GBB 1/2 analysiert wurden. Um hierbei die Funktion von GBB 1/2 durch erhöhte GABA Konzentrationen hervorzuheben, wurden zusätzlich GABAerge Motorneuronen optogenetisch stimuliert oder die Wiederaufnahme von GABA aus dem synaptischen Spalt durch Mutation des Membran ständigen GABA Transporters blockiert. So konnte gezeigt werden, dass GBB 1/2 eine akute Inhibition der cholinergen Motorneuronen bewirken, was vermutlich für die Regulation von Bewegungsabläufen eine wichtige Rolle spielt. Die geringe Dynamik der GBB 1/2 induzierten Effekte deutet allerdings darauf hin, dass die synaptische Aktivität durch den metabotropen Rezeptor kaum nachhaltig moduliert wird.
In nachfolgenden Versuchen wurde die optogenetische Stimulation von Motorneuronen außerdem mit der elektronenmikroskopischen Analyse der präsynaptischen Feinstruktur kombiniert. Dadurch konnte die Dynamik der Exozytose und Endozytose synaptischer Vesikel (SV) in Abhängigkeit von neuronaler Aktivität untersucht werden. So wurde gezeigt, dass synaptische Vesikel nahe der aktiven Zone während einer 30 sekündigen Hyperstimulation nahezu komplett aufgebraucht waren. Die vollständige Regeneration der SV Pools benötigte anschließend etwa 12 Sekunden und erfolgte zunächst in der Peripherie der aktiven Zone, was auf eine laterale Heranführung der Vesikel schließen lässt. Nach etwa 20 Sekunden erholte sich ebenfalls die Wirksamkeit der Stimulation von Muskelzellen durch die Motorneuronen, was durch elektrophysiologische Messungen der photo induzierten post synaptischen Ströme gezeigt wurde. Während der Hyperstimulation bildeten sich außerdem große vesikuläre Strukturen, die sich anschließend nach etwa acht Sekunden wieder aufgelöst hatten. In Analogie zu vergleichbaren Experimenten in anderen Organismen liegt die Vermutung nahe, dass es sich dabei um Zwischenprodukte der so genannten Bulk Phase Endozytose handelt, die das Clathrin abhängige Recycling von synaptischen Vesikeln bei starker neuronaler Aktivität ergänzt (Heuser and Reese, J Cell Biol 1973; Miller and Heuser, J Cell Biol 1984; Richards et al., Neuron 2000). Bemerkenswerterweise war der Abbau der vesikulären Strukturen in Synaptojanin und Endophilin defizienten Tieren stark verzögert. Denkbar ist, dass beide Proteine für die Synthese von synaptischen Vesikeln aus den vesikulären Zwischenprodukten der Bulk Phase Endozytose wichtig sind, analog zur ihrer Funktion bei der Clathrin abhängigen Endozytose an der Plasmamembran.
Durch die zielgerichtete Manipulation der Zellaktivität ermöglichen optogenetische Techniken außerdem die funktionelle Charakterisierung von Neuronen und neuronalen Netzwerken. Um die zelluläre Spezifität dieses Ansatzes zu erhöhen, wurde ein Tracking System entwickelt das die Position frei beweglicher Tiere in Echtzeit bestimmt und nachverfolgt. Dadurch konnte die Photoaktivierung optogenetischer Proteine auf definierte Bereiche der Fadenwürmer und somit auf ausgewählte Neuronen innerhalb der Expressionsmuster von verwendeten Promotoren eingeschränkt werden. Des Weiteren ermöglichte hierbei die Auswertung translatorischer Parameter die Analyse verschiedener lokomotorischer Merkmale wie Geschwindigkeit, Bewegungsbahn oder Ausprägung der Körperbiegungen. Dieses System wurde beispielhaft für die konzertierte Photoaktivierung durch ChR2(H134R) bzw. Photoinhibition durch MAC von zwei verschiedenen Gruppen von Neuronen angewendet, um die Integration mechanosensorischer Informationen durch Command Interneuronen zu untersuchen. In diesem Zusammenhang wurde zudem eine Rekombinase basierte Methode für optogenetische Proteine adaptiert, die die Transkription auf die zelluläre Schnittmenge von zwei verschiedenen Promotoren einschränkt und somit die Spezifität der Expression erhöht. Idealerweise kann dieser Ansatz außerdem mit der gezielten Photoaktivierung kombiniert werden, um die zelluläre Selektivität optogenetischer Anwendungen weiter zu verbessern.
Weiterhin ist die Anwendung optogenetischer Techniken bisher durch intrinsische Eigenschaften der verwendeten Rhodopsine auf die relativ kurzzeitige Manipulation des Membranpotentials von Zellen beschränkt. So benötigt ChR2 durch die schnelle Schließung seines offenen Kanals eine kontinuierliche Photoaktivierung, um eine andauernde Depolarisation hervorzurufen. Dies ist jedoch potentiell mit phototoxischen und – besonders bei C. elegans – phototaktischen Nebeneffekten verbunden. Deswegen wurden diverse Mutanten von ChR2 mit stark verlangsamter Inaktivierung (Berndt et al., Nat Neurosci 2009) für ihren Nutzen zur Langzeit Stimulation von erregbaren Zellen im Nematode getestet. Dabei wurde gezeigt, dass ChR2(C128S) durch einen kurzen Photostimulus mit vergleichsweise niedriger Intensität eine anhaltende Depolarisation über mehrere Minuten auslösen kann. Die wiederholte Stimulation in ASJ Neuronen ermöglichte zudem eine langzeitige Depolarisation über mehrere Tage, wodurch die genetisch veranlagte Entwicklung von Tieren manipuliert werden konnte. Durch gezielte Punktmutation konnten außerdem relevante Eigenschaften von ChR2(C128S) für die Langzeit Stimulation weiter verbessert werden.
Als weiteres optogenetisches Werkzeug wurde zudem die Photoaktivierbare Adenylatzyklase alpha (PACa) aus Euglena gracilis (Iseki et al., Nature 2002; Ntefidou et al., Plant Physiol 2003; Schroder-Lang et al., Nat Methods 2007) für die akute und lichtgetriebene Synthese des sekundären Botenstoffs cAMP in C. elegans etabliert. Die Photoaktivierung von PACa in cholinergen Motorneuronen verstärkte dabei die Neurotransmitterfreisetzung und induzierte hyperlokomotorische Phänotypen, vergleichbar zu Mutanten mit erhöhten cAMP Konzentrationen.
Zusammengefasst wurden diverse optogenetische Techniken für C. elegans entwickelt und optimiert, die die zellspezifische und nicht invasive Manipulation des Membranpotentials beziehungsweise die Synthese des sekundären Botenstoffs cAMP durch Licht im frei beweglichen Tier ermöglichen. Diese Methoden können zur gezielten Störung neuronaler Aktivität angewendet werden, um dadurch neurobiologische Fragestellungen im Fadenwurm zu untersuchen. Dies wurde beispielhaft für die Erforschung der synaptischen Reizweiterleitung und die funktionelle Analyse neuronaler Netzwerke demonstriert. Denkbar ist außerdem, diese für C. elegans etablierten Methoden vergleichbar in anderen Modellorganismen anzuwenden. So sind die Fruchtfliege ebenso wie der Zebrafisch Embryo bereits für optogenetische Techniken erprobt (Arrenberg et al., Proc Natl Acad Sci U S A 2009; Schroll et al., Curr Biol 2006). Für Säugetiere wie die Maus, die Ratte und den Makaken wurden zudem bereits Ansätze entwickelt, die die gezielte Photostimulation in lebenden und frei beweglichen Tieren ermöglichen (Han et al., Neuron 2009; Wentz et al., J Neural Eng 2011; Yizhar et al., Nature 2011; Zhang et al., Nat Rev Neurosci 2007).
Statistical machine translation (SMT) should benefit from linguistic information to improve performance but current state-of-the-art models rely purely on data-driven models. There are several reasons why prior efforts to build linguistically annotated models have failed or not even been attempted. Firstly, the practical implementation often requires too much work to be cost effective. Where ad-hoc implementations have been created, they impose too strict constraints to be of general use. Lastly, many linguistically-motivated approaches are language dependent, tackling peculiarities in certain languages that do not apply to other languages. This thesis successfully integrates linguistic information about part-of-speech tags, lemmas and phrase structure to improve MT quality. The major contributions of this thesis are: 1. We enhance the phrase-based model to incorporate linguistic information as additional factors in the word representation. The factored phrase-based model allows us to make use of different types of linguistic information in a systematic way within the predefined framework. We show how this model improves translation by as much as 0.9 BLEU for small German-English training corpora, and 0.2 BLEU for larger corpora. 2. We extend the factored model to the factored template model to focus on improving reordering. We show that by generalising translation with part-of-speech tags, we can improve performance by as much as 1.1 BLEU on a small French- English system. 3. Finally, we switch from the phrase-based model to a syntax-based model with the mixed syntax model. This allows us to transition from the word-level approaches using factors to multiword linguistic information such as syntactic labels and shallow tags. The mixed syntax model uses source language syntactic information to inform translation. We show that the model is able to explain translation better, leading to a 0.8 BLEU improvement over the baseline hierarchical phrase-based model for a small German-English task. Also, the model requires only labels on continuous source spans, it is not dependent on a tree structure, therefore, other types of syntactic information can be integrated into the model. We experimented with a shallow parser and see a gain of 0.5 BLEU for the same dataset. Training with more training data, we improve translation by 0.6 BLEU (1.3 BLEU out-of-domain) over the hierarchical baseline. During the development of these three models, we discover that attempting to rigidly model translation as linguistic transfer process results in degraded performance. However, by combining the advantages of standard SMT models with linguistically-motivated models, we are able to achieve better translation performance. Our work shows the importance of balancing the specificity of linguistic information with the robustness of simpler models.
Forty two samples of the Late Eocene Kiliran oil shale, Central Sumatra Basin, Indonesia were collected from a 102 m long drill core. Palynofacies and geochemical analyses have been carried out to reconstruct the paleoenvironmental conditions and paleoecology during deposition of the oil shale. Amorphous organic matter (AOM) is very abundant (>76%). B. braunii palynomorph is present (3-16%) as the only autochtonous structured organic matter and generally more abundant in middle part of the profile. The stable carbon isotopic composition of organic matter (δ13C) varies from -27.0 to -30.5‰ and is generally more depleted in middle part of the profile. The ratio of total organic carbon to sulfur (TOC/S), used as salinity indicator, ranges from 2.5 to 15.8 and shows variations along the profile. Relatively less saline environments are observed in the middle part profile. Fungal remains are generally present only in middle part of the profile with distinct peak of abundances. The presence of fungal remains is regarded as an indication for a relatively warmer climate during deposition of middle part of the profile. The warmer climate is thought to influence the establishment of a thermocline, limiting the supply of recycled nutrients to the epilimnion. Consequently, the primary productivity in the Kiliran lake decreased during deposition of the middle part of the profile as indicated by the relatively depleted δ13C and the blooming of B. braunii. The chemocline was also shoaling during deposition of the middle part of the profile according to the higher abundance of isorenieratene derivatives of green sulfur bacteria origin. The warmer climate affected also to increase of water supply and thus less saline environments.
Tectonic subsidence is also thought to be a significant factor for the development of the Kiliran lake. The Zr/Rb ratio, an indicator for grain size, ranges from 0.4 to 1.3 and generally increases upwards along the profile. Three sudden decreases of the ratio are observed, indicating rapid change to finer grain size. These decreases are interpreted to indicate rapid deepening events of the lake due to mainly periodic subsidence. During deposition of lower part of the profile, the subsidence rates might have been relatively higher than sediment and water supply rates, resulting in a higher autochtonous fraction in the oil shale. During deposition of middle part of the profile, the sediment and water supply rates were relatively higher promoting distinct progradational sedimentation. Subsequently, the lake became more shallow and smaller during deposition of the upper part of the profile, leading to a relatively higher terrigenous input to the oil shale.
Norneohop-13(18)-ene and neohop-13(18)-ene derived from methanotrophic bacteria are the dominant hopanoid hydrocarbons. The sum of their concentrations varies from 40.6 to 360.0 μg/g TOC. The δ13C of these compounds are extremely depleted (-45.2 to -50.2‰). The occurrence of abundant bacteria including methanotrophic bacteria was responsible for the recycling of carbon below the chemocline of the lake. The effect of the recycling of carbon is observed by the presence of a concomitant depletion (about 7-9‰) in 13C of some specific biomarkers derived from organisms dwelling in the whole phototrophic zone.
4-Methylsterane and 4-methyldiasterene homologues occur in the oil shale as the predominant biomarkers. The sum of the concentrations of all homologues are about 40.3-1,009.2 μg/g TOC with generally higher values in uppermost and lower parts of the profile. Ca accounts as the predominant element in the oil shale, ranging from 5.0 to 16.7%. This element shows generally parallel variation with the 4-methylsterane homologues along the profile. This suggests that the 4-methylsteranes were derived from biological sources favoring more alkaline and more trophic environments. On the other hand, these compounds were less abundant in middle part of the profile which is consistent with less alkaline and less trophic environments promoting B. braunii to bloom.
The 4-methylsterane homologues are considered to originate from Dinoflagellates. Alternation between Dinoflagellates and B. braunii in Paleogene lake systems due to water chemistry changes are known from previous studies. Moreover, freshwater Dinoflagellates have been frequently reported to occur in the basin depocenters. In the present case, distinct alternation between B. braunii abundances and concentrations of 4-methylsterane homologues along the studied oil shale profile suggest that the 4-methylsterane homologues were derived from freshwater Dinoflagellates although dinosterane is not present in the sediment extracts. Water alkalinity and trophic level changes were most likely responsible for the alternation of Dinoflagellates and B. braunii blooming.
We found that the HMTase G9a, that catalyzes H3K9me2 in euchromatin, plays a key modulatory role in type I IFN expression. This finding raises the possibility of targeted intervention with type I IFN expression by using small synthetic inhibitors of G9a. Given the overall minimal negative effect of G9a-deficiency on differentiated cells, the short-term suppression of G9a could be used to potentiate type I IFN expression during chronic viral diseases such as hepatitis C. Accordingly, pharmacological enhancement of methylation, for example by inhibition of the H3K9me2 specific demethylases, could be potentially used to attenuate type I IFN expression and help to control chronic inflammatory and autoimmune conditions. The mechanism responsible for canvassing the epigenetic profile of type I IFN expressing cells are not known. It is plausible, that similar to neurons, where G9a is targeted to specific loci with the help of noncoding RNAs, IFN expressing cells possess similar mechanisms to target H3K9me2 demethylating enzymes to type I IFN loci, thus keeping these loci accessible for IFN-inducing transcription factors. Identification of non-coding RNAs that may contribute to the establishment of the epigenetic state of IFN producing cells will provide a further opportunity for targeted manipulation of IFN expression.
In my thesis, I describe the collaborative experiments that show the ability of synthetic compounds that interfere with the histone readers to suppress inflammation. Our results present a novel concept for the regulation of inflammatory gene expression. The diversity of histone readers and the combinatorial nature of regulation of gene transcription may provide an opportunity for highly selective interference with disease associated transcriptional programs by interfering with specific readers. In the future we plan to address the therapeutic potential of BET antagonists in autoimmune and chronic inflammatory conditions.In summary, the experiments described in my thesis provide an example of how the understanding of the basic mechanisms of chromatin control of gene expression can facilitate novel therapeutic approaches that target chromatin.
Purpose: The aim of this retrospective study is to evaluate the long term implant survival at 5 years, periimplantary conditions and prosthetic maintenance requirements for implant supported mandibular removable dentures retained on only 2 Ankylos® implants placed interforaminally in the mandible and using only conical double crown attachments. Materials and methods: Using the database at the Faculty of Dentistry, University of Frankfurt a selection process was performed to choose patients receiving only 2 Ankylos® implants placed interforaminally in the mandible and using only conical double crown attachments. Implant survival, periimplant condition (periodontal bleeding, plaque index and probing depth), bone loss (from panoramic radiographs) and mobility (using Periotest®) were monitored annually following implant loading. In addition a detailed prosthetic maintenance list was created for each patient based on their yearly checkups and emergency appointments. 37 patients with edentulous mandibles (34 with complete dentures in the upper jaw and 3 with tissue-tooth borne coverdentures) received 2 interforaminal Ankylos® implants (67 in the canine region, 7 in 2nd incisor region). Results: Mean Periotest® values at 5 years (-1.97 ±2.24) were lower than at loading (-1.47 ±2.33). A drop was seen in the Periotest® readings after the first year of loading. The decrease in mean Periotest® values between PTV5 and PTV 1 were not statistically significant (Tukey-Kramer test: p>0.05)
14 patients (37.8%) displayed no resorption at all with an average of 0.801 mm mesially and 0.807mm distally after 5 years. The most increase in bone loss was seen after the first year of loading. There was a gradual increase in bone resorption after the first year of loading. The differences between both distal and mesial bone resorption level at five years and at one year after loading are not significant (Tukey-Kramer test: p<0.05) Plaque and bleeding index values were low at a mean of 0.97 ±0.86 and 0.59 ±0.77 respectively after 5 years of loading. The increase from the first year of loading till the 5th year of loading was significantly higher for plaque measurements but not for bleeding measurements (Tukey-Kramer Test: p<0.05 and p>0.05 respectively). Mean probing depth values were higher after 5 years (2.61 ±0.92 mm) in comparison to the values at loading (2.15 ±0.75 mm). The difference between average values at year 5 and year 1 was statistically significant (Tukey-Kramer test: p<0.05). The most occurring form of maintenance was minor adjustments such as pressure point (15 patient or 40,5%) and relining 11 patients or 29.7%). Teeth breaking off the denture were less common (4 patients or 10.8%). 5 decementations of primary crowns occurred in 4 patients (10.8%) within the 5 year observation time. Other major complications were 4 loose abutments in 3 patients (8.1%), 3 decementations of secondary copings in 3 patients (8.1%) and 1 case (2.7%) in which the prosthetic metal framework fractured. No fracture of abutments or primary crowns occurred during the investigation. Implant survival was 100% percent after 5 years ,1 implants did not fulfil Albrektsson’s success criteria and showed more than 0.2 mm of bone loss per year after the first year of loading with the first year giving a success rate of 98.8%. Conclusion: In conclusion this study has demonstrated that patients have a wider variety of options when it comes to choosing a reliable prosthesis in the lower jaw. Patients with financial limitations can be provided with a reliable prosthetic option using removable dentures retained by conical double crown attachments on 2 implants. The requirements for such a construction are a mechanically stable implant system and a mechanically stable framework. When these prerequirments are fulfilled, the patient can be satisfied with a prosthesis of superior quality to other attachment types and the dentist can rely on the fact that frequent maintenance which costs time and money can be eliminated or at least reduced. Through further innovation this type of construction can also reach patients who are lower down on the economic scale such as elderly patients and retirees.
Recent data indicate that reactive oxygen species (ROS) are produced in the nociceptive system during persistent pain and contribute to pain sensitization. Aim of this study was to investigate potential antinociceptive effects of ROS scavengers in different animal models of pain. Intrathecal injection of ROS scavengers 1-Oxyl-2,2,6,6-tetramethyl -4-hydroxypiperidine (TEMPOL) or Phenyl-N-tert-butylnitrone (PBN) significantly inhibited formalin-induced nociceptive behavior in mice, suggesting that ROS released in the spinal cord are involved in nociceptive processing. Formalin-induced nociceptive behavior was also inhibited by intraperitoneal injection of a combination of vitamin C and vitamin E, but not of vitamin C or vitamin E alone. Moreover, the combination of vitamin C and E dose-dependently attenuated mechanical allodynia in the spared nerve injury (SNI) model of neuropathic pain. The SNI-induced mechanical allodynia was also reduced after intrathecal injection of the combination of vitamin C and E, and western blot analyses revealed that vitamin C and E treatment can ameliorate the activation of p38 MAPK in the spinal cord and in DRGs. These data suggest that a combination of vitamin C and E can inhibit the nociceptive behavior in animal models of pain, and points to a role of the spinal cord as an important area of ROS production during nociceptive processing.
Working memory (WM) contributes to countless activities during everyday live: reading, holding a conversation, making tea and so on. The core processes of WM comprise the phases of encoding, maintenance and retrieval. Successful recognition of stored objects requires several subprocesses such as stimulus encoding and evaluation, memory search and the organisation of a decision and a response. Much research has focused on encoding and maintenance of information but little interest has been directed to the retrieval of information. This is why the present dissertation investigated the neuronal correlates of retrieval of previously stored information and its modulation by load and probe-item similarity.
Here memory load and probe-item similarity were manipulated in order to investigate the neuronal correlates of the recognition process using electroencephalography (EEG). We tested the hypothesis recognition is influenced differently by probe-item similarity and by memory load and that these factors are re Effected by distinct neuronal correlates. Furthermore we tested whether distinct neuronal responses could be related to a summed similarity model.
The analysis of high-density ERP recordings showed both a load effect (load 1>load 3) and a similarity effect In addition, there was an interaction between load and similarity. The load effect was present during the whole epoch and did not change over time, whereas the similarity effect showed two distinct components between 300-600ms. In contrast to the load effect the similarity effect changed its sign over time. For the rest component, match probes elicited the strongest ERP responses, whereas for the second component dissimilar probes yielded the strongest ERP responses. The timing of the similarity effect corresponded well with the early and late P3b complex. The P3b complex is associated with stimulus categorisation and evaluation (early subcomponent) and memory search and criterion testing (late subcomponent).
The results suggest that the difficulty of a task is not only determined by load but also enhanced by probe-item similarity. Since increasing the number of samples (i.e. memory load) can also increase the probe-item similarity (i.e. the probability that one of the samples is perceptually similar to the probe), an independent manipulation of both factors is indispensable to disentangle their particular impact on short-term recognition. Furthermore, I propose that the two distinct neural correlates of the P3b complex reeffects different stages of task processing connected with probe-item similarity. As suggested by summed similarity VI models, these components might reflect the subprocesses of similarity summation (early P3b) and criterion testing (late P3b).
Seit einigen Jahrzehnten ist Lysozym eines der am meisten erforschten Proteine in der Literatur und wird hauptsächlich als Modell Protein zur Aufklärung der Faltungs- und Entfaltungsprozesse genutzt. Da die Frage nach Fehlfaltung und deren Verknüpfung mit neurodegenerativen Krankheiten bis zum heutigen Tag nicht vollständig geklärt ist, besteht hier ein großer Spielraum für weitere Forschungsansätze. In der vorliegenden Arbeit wurden daher zwei Modellsysteme verwendet, Hühereiweiß-Lysozym und menschliches Lysozym, jeweils in ihrem nicht-nativen ungefalteten Zustand. Diese ungefalteten Ensembles wurden mit Hilfe NMR spektroskopischer Methoden untersucht und ergaben sehr detaillierte, zum Teil auch überraschende neue Einblicke in Struktur und Dynamik der beiden Proteine und liefern somit wichtige Erkenntnisse zu Faltungs- und Aggregationsprozessen. ...
Protein translocation across the chloroplast membrane is mediated by molecular machinery composed of protein complexes termed the TOC/TIC (the outer/inner envelope chloroplasts translocases). This translocation process is regulated by metabolic energy in form of GTP and ATP and is influenced by the lipid composition of the membrane. The ability to study the function of a single complex “TOC” in vitro using purified protein or purified chloroplast outer envelope vesicles has been instrumental for our understanding of the mechanism underlying this process.
Indeed, the TOC complex has been purified by previously established procedures. However its functional and structural analyses are impaired by the limited yield of purified protein. Therefore, protocols for native TOC complex purification are described here. The complex isolation is achieved by direct biochemical treatment of biological membrane hosting this complex or by tandem affinity purification of modified protein complex components from generated transgenic plants.
Furthermore, in this thesis, radioactive based in vitro import assays are described, namely those that allow monitoring translocation activity across the outer envelope of chloroplast. Based on the analysis of knock-out plants and isolated complexes it was previously suggested that lipid dependence of protein translocation might exist. Thus, the question was raised whether the lipid composition of the membrane has a direct influence on the behavior and functionality of the TOC translocon, or whether additional components of the chloroplast membrane account for the observed effect in vivo. To answer this question, a technique for vesicle fusion was developed. The principal aim was to explore the effect of an exchange of the lipid environment surrounding the complex translocon. This method helped to demonstrate that the SQDG and PI act stimulatory on the translocation across the outer envelope of chloroplast, whereas DGDG exhibits an inhibitory effect on TOC complex functionality.
Sponges are one of the major components of benthic communities and are considered to be a
key role organism in marine ecosystems. In addition to their importance in terms of
biodiversity, sponges are becoming increasingly attractive to the industry, as they themselves
or associated symbionts, produce various kinds of secondary metabolites of pharmaceutical
properties. Some of them have already been clinically applied.
The taxonomic characters of Porifera are limited to only a few morphological and
histological characters. In addition, sponges of the same species often show a wide
morphological variability, whereas the latter depends on different ecological parameters such
as water depth and current conditions. Thus, the taxonomic classification of sponges often
becomes a scientific challenge.
The fauna of the Yellow Sea rates among the least studied worldwide. At the same time,
according to the UN Atlas of the Ocean, the Yellow Sea is one of the most intensively
exploited marine areas in the world. This is not least due to the dense human population living
in the entire catchment area of the Yellow Sea region. In order to compile medium- and longterm
conclusions about the anthropogenic impact on biota of the Yellow Sea, the knowledge
of species and their distribution is of crucial importance, as these data form the baseline for all
future conservation efforts.
Until now the sponge fauna of the Chinese Yellow Sea is insufficiently investigated.
Thus, there is only one publication on sponges from this region that has been released
hitherto. This paper is dealing with only a view species. However, there is no reference
concerning the present location of the voucher material, on which this publication is based on.
Consequently, no scientific collection on Porifera from the Chinese part of the Yellow Sea
exists to date.
In order to compile a documentation of the recent sponge community of the Chinese
Yellow Sea, 12 study sites along the coast of the Liaoning Peninsula, China, Northeast
Yellow Sea, were investigated with focus on sponge distribution. The corresponding habitats
were characterized in regard to their topographical features, abiotic parameters, and common
composition of benthic megafaunal and macroalgal assemblages.
Due to the lack of comparable studies, a comprehensive literature research on sponges of the
shallow Northwest Pacific Ocean was required. As a result the first compilation of
publications is presented, dealing with sponges from shallow depths of the northwestern
Pacific Ocean.
Abstract
2
In the course of this study, 31 sponge species in total were recorded, which are scientifically
processed. With the exception of four all specimens were determined to species- level.
Twelve out of the total number of species are new to science and are described and classified
according to the recent taxonomic system of the phylum Porifera.
The results of this study indicate considerable differences in species composition between
investigated sites. It is shown that physical factors (particularly current regime, sedimentation,
seasonally related variations in temperatures), as well the availability of suitable substrates are
directly related to the diversity and abundance of investigated sponge communities. In this
context possible adaptation strategies of the corresponding sponges were discussed in detail.
Two sponge species, Clathria (Clathria) asodes and Antho (Acarnia) lithophoenix, formerly
known exclusively from the northeastern Pacific Ocean, are now recorded from the Northwest
Pacific Ocean for the first time. Furthermore, Penares hongdoensis, Clathria (Clathria)
hongdoensis and Celtodoryx girardae were synonymized with Penares cortius, Clathria
(Clathria) acanthostyli, and Celtodoryx ciocalyptoides respectively. Moreover, the occurrence
of eight sponge species, which were known from previous records from the Yellow Sea, could
be confirmed.
As a result of this study the Asian origin of a sponge species that is invasive to the French and
Dutch coasts of the Northeast Atlantic Ocean since the 1990s could be established. Moreover,
it is demonstrated that Celtodoryx girardae from the northeastern Atlantic is in fact
conspecific with Cornulum ciocalyptoides described by Burton (1935) from the Posiet Bay,
Sea of Japan. Apart from taxonomic remarks, variations between populations from both
oceans are examined and discussed thoroughly in regard to possible ecological implications.
The community of documented sponges shows overlapping with the one from the Sea of
Japan. According to the results it is assumed that the endemic degree of the sponges from the
Chinese Yellow Sea is rather low to moderate.
The material obtained in the course of this study was integrated in the collection of the
Senckenbergischen Naturforschenden Sammlungen. Therefore, it is the first scientific
collection of sponges from the Chinese Yellow Sea that can be consulted as a basis for all
further studies on sponges of this region.
The present study is the only investigation of sponges from Dalian and adjacent waters before
the spill occurred in the Dalian harbour in July 2010. Therefore, it provides an essential
baseline needed to assess the impact of the oil spill on benthic communities.
NOSTRIN belongs to the recently defined F-BAR protein family. F-BAR proteins are
multi-domain proteins, which serve as adaptors between plasma membrane and
cytoskeleton components in processes such as membrane protrusion formation,
endocytosis and migration. NOSTRIN encompasses a F-BAR domain at the N-terminus,
which mediates membrane association, followed by a HR1 motif and an intermediate
domain (ID) domain in the middle, and a SH3 domain at the C-terminus. The domain
architecture and ability to form oligomers enable NOSTRIN to coordinate several
interaction partners namely dynamin, caveolin, N-WASP and endothelial nitric oxide
synthase (eNOS) in the process of eNOS trafficking. In this context NOSTRIN was
originally identified and hence termed eNOS traffick inducer. NOSTRIN is expressed in
vascularized tissues (e.g. liver and lung) and in primary endothelial cells.
Aims of the present work were (1) to investigate if NOSTRIN is involved in other
processes besides eNOS trafficking, (2) to analyse the function of NOSTRIN in vivo
through knockdown of NOSTRIN in developing zebrafish and (3) to study the
consequences of the loss of NOSTRIN on signal transduction in a primary cell culture
model derived from NOSTRIN knockout mice.
To study the possible involvement of NOSTRIN in other processes besides eNOS
trafficking a yeast two-hybrid screen was performed in which fibroblast growth factor
receptor 1 (FGFR1) was identified as a putative novel interaction partner of NOSTRIN. In
a series of yeast two-hybrid, pulldown and co-immunoprecipitation experiments the
interaction between NOSTRIN and FGFR1 was confirmed to occur between
endogenously expressed proteins and determined to be direct and to depend on the ID
domain of NOSTRIN and the 130 C-terminal amino acid residues of FGFR1. FGFR1 is
activated by binding of fibroblast growth factors (FGFs) and induces several different
signal transduction pathways (e.g. MAPK and Akt pathway). Overexpression of
NOSTRIN in HeLa cells specifically enhanced FGF2-dependent MAPK activation.
Accordingly, depletion of NOSTRIN attenuated FGF2-dependent MAPK activation and
did not affect FGF2-induced Akt activation.
In summary, NOSTRIN has been identified as a novel interaction partner of FGFR1
involved in FGF2-dependent signal transduction.
The morpholino oligonucleotide-mediated knockdown of NOSTRIN in developing
zebrafish caused vascular leakage and irregular vascular patterning e.g. a loss of the
proper trajectory of intersegmental vessel and interruptions of the dorsal longitudinal
anastomotic vessel. The vascular phenotype was consistent upon use of two different
morpholinos and could be rescued in a dose dependent manner by the injection of
zebrafish NOSTRIN mRNA. Detailed analysis involving confocal and time lapse
microscopy in zebrafish with endothelial specific expression of EGFP revealed that the
knockdown of NOSTRIN impacts in vivo on the migration and morphology of endothelial
tip cells and leads to a reduction of filopodia number and length.
Additionally a NOSTRIN knockout mouse was generated. The analysis of FGFR1 signal
transduction in primary mouse lung endothelial cells (MLECs) from NOSTRIN knockout
and wild type mice revealed that FGF2-dependent MAPK activation was attenuated in
MLECs isolated from NOSTRIN knockout mice when compared to MLECs isolated from
wild type mice. The effect of NOSTRIN on FGF2-dependent signal transduction seems to
be specific, since VEGF-induced MAPK activation was not affected in NOSTRIN
knockout MLECs. The importance of NOSTRIN for FGF2 signal transduction in vivo is
demonstrated by the greatly impaired angiogenic response to FGF2 in NOSTRIN
knockout mice in matrigel plug assay. In a detailed biochemical analysis it was
discovered that NOSTRIN interacts with the activated small GTPase Rac1 and that
overexpression of NOSTRIN enhances Rac1 activation. Furthermore, the interactions of
NOSTRIN with both Rac1 and its GEF Sos1 are required for NOSTRIN-mediated
activation of Rac1. In accordance, activation of Rac1 was not detected upon FGF2
stimulation in NOSTRIN knockout MLECs.
In conclusion, the present work describes a novel function of the F-BAR protein
NOSTRIN in FGFR1 signal transduction. Data presented in this work demonstrate that
NOSTRIN is required for the assembly of a complex consisting of FGFR1, Sos1 and
Rac1 and subsequently for the FGF2-dependent activation of Rac1 in endothelial cells.
The NS5B protein of the hepatitis C virus (HCV) is a RNA-dependent RNA polymerase, which is the key enzyme for viral replication. It is recognized as one of the promising targets for antiviral intervention within the new HCV treatment approach of direct-acting antivirals (DAA). However, several of the known non-nucleoside HCV polymerase inhibitors (NNIs) identified by screening approaches show limitations in the coverage of all six major HCV genotypes (GT). Genotypic profiling therefore has to be implemented early in the screening cascade to discover new broadly active NNIs. This implies knowledge of the specific individual biochemical properties of polymerases from all GTs which is to date limited to GT 1 only. The work submitted here gives a comprehensive overview of the biochemical properties of HCV polymerases derived from all major GTs 1 - 6. Biochemical analysis of polymerases from 38 individual sequences revealed that the optima for monovalent cations, pH and temperature were similar between the GTs, whereas significant differences concerning concentration of the preferred cofactor Mg2+ were identified. Implementing the optimal requirements for the polymerases from each individual GT led to significant improvements in their enzymatic activities. However, the specific activity was distributed unequally across the GTs and could be ranked in the following descending order: 1b, 6a > 2a, 3a, 4a, 5a > 1a. Furthermore, the optimized assay conditions for GT profiling were confirmed by testing the inhibitory activity of four known prototype NNIs, each addressing one of the four NNI binding sites. Additionally, a novel NNI chemotype - identified by screening - is described, the substituted N-phenyl-benzenesulphonamides (SPBS). This inhibitor class showed reversible inhibition of NS5B from HCV 1b Con1 with IC50 values up to 39 nM. Based on the decreased inhibitory activity against a recombinant NS5B protein carrying the mutation L419M, it was assumed that the SPBS inhibitors bound to the thumb site II as it has been described for the carboxy thiophene inhibitors. The postulated binding site was consequently confirmed by analysing a provided co-crystal structure of NS5B in complex with a SPBS analogue. Notably, the two SPBS analogues SPBS-1 and SPBS-2 reported here revealed significant differences in addressing the NH-group of the main chain Y477 by hydrogen-bonds, watermediated or directly, which provoked a shift of the carboxyphenyl group of the inhibitors towards the H475 position for the water-mediated binding mode. Interestingly, the differences observed in the binding mode led to a different cross resistance profile at positions M423 and I482. Using the previously optimized biochemical primer-dependent transcription assay, inhibitory activity of the SPBS could be demonstrated against polymerases from HCV GTs 1a and 1b whereas the inhibitor class failed to inhibit any of the non-GT 1 polymerases. Furthermore, initial antiviral activity for SPBS was demonstrated against the subgenomic replicons of HCV GTs 1a and 1b, respectively, and no considerable cytotoxic potential against a panel of ten different cell types. Finally, concerning a possible future treatment without PEG-IFN α or ribavirin, the SPBS analogues were found to display additive to synergistic effects in combination with the benzothiadiazine, the benzofuran and the indole - representative inhibitors for the binding sites palm I, palm II and thumb I, repectively - in the biochemical assay. Within the same binding site as the SPBS, the reference compound hydroxydihydropyranone displayed additive interactions only with the benzothiadiazine (palm I) in the biochemical assay as well as in cell culture. Hence it could be concluded that, having characterized one individual NNI, no universal predication is possible concerning the combinatory behaviour of NNIs binding to the same binding site. As synergistic, antagonistic or additive interactions are inhibitor-dependent (not binding sitedependent) each novel NNI has to be characterized individually in one-to-one combinations.
The adaptive immune system of jawed vertebrates is based on recognition and elimination of cells that are either invaded by intracellular pathogens or malignantly transformed. One essential component of these processes is the cell surface presentation of antigenic peptides via major histocompatibility complex (MHC) class I molecules to cytotoxic T-cells (CTLs). Cells degrade defective ribosomal products and misfolded or unwanted proteins by the ubiquitin-proteasome pathway. The resulting degradation products are recognized and translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum (ER) lumen, where they are loaded onto MHC I molecules. Assembled peptide-MHC complexes are then shuttled by the secretory pathway to the cell surface for antigen presentation to CTLs, leading in the case of viral infection or malignant transformation to lysis and apoptosis of the target cell. Due to the fact that the TAP complex represents a key control point within the antigen presentation pathway, several viruses have evolved sophisticated strategies to evade immune surveillance by interfering with TAP function.
Detailed studies of the TAP mechanism or its viral inhibition have been severely impeded by difficulties in expressing sufficient amounts of functional heterodimeric TAP complex. Thus, the overexpression of TAP in the methylotrophic yeast Pichia pastoris was established for functional analysis of this important ABC complex. Biomass production was scaled up by fermentation using classical batch and feed methods. Extensive screening of optimal solubilization and purification conditions allowed the isolation of the heterodimeric transport complex. Notably, only the very mild detergent digitonin preserved TAP function. Hereby, the optimal solubilization and purification strategy yielded in 30 mg TAP transporter per liter culture. Remarkably, the protein amount was 50-fold increased compared to previously described expression/purification in cultured insect cells.
The high yield and quality of TAP produced in P. pastoris allowed an extensive analysis of substrate binding and transport kinetics of the transport complex in the membrane, its solubilized and purified state, as well as the reconstituted state. Thereby, a strong and direct effect of the lipid bilayer on ATP hydrolysis and peptide transport was discovered. These important results were extended further by successful functional reconstitution of the antigen translocation machinery in different lipid environments. For the first time, a stimulation of the transport activity by phosphatidylinositol (PI) and phosphatidylethanolamine (PE) was observed, whereas cholesterol was identified as an inhibitor of TAP activity.
Purification of TAP and subsequent thin-layer chromatography (TLC)/liquid chromatography Fourier transform-mass spectrometry (LC FT-MS) fingerprinting of residual lipids exhibited specifically associated glycerophospholipids; mainly PC, PE, and PI species. Strikingly, these lipids not only represent the primary class of phospholipids of the ER but were also shown to be essential for functional reactivation of delipidated, and thus inactive, TAP. The results demonstrate that transport of antigenic peptides by the ABC transporter TAP strictly requires specific glycerophospholipids.
In addition to the biochemical characterization of heterologous produced TAP, the soluble domain of the viral inhibitor US6 from human cytomegalovirus was expressed in E. coli. Optimization of the purification and refolding strategy yielded in functional protein, with a 35-fold increased protein amount compared to previous purification procedures. Protein activity was analyzed by specific inhibition of ATP binding to TAP. Furthermore, high protein yields allowed detailed investigation of TAP-dependent spatial and mechanistic separation of MHC I restricted cross-presentation in professional antigen presenting cells (pAPC).
Stem cells are often referred to as potential candidates for the treatment of different pathologies. Their ability to differentiate into various tissue specific cell types offers the possibility to engineer cell systems or organs for replacement. One of the main questions in stem cell biology is how stemness properties are regulated and to what extend this regulation is intrinsic or conveyed by the direct microenvironment (‘niche’). In order to elucidate such regulatory processes, it is informative to analyze processes or molecules that are shared between different stem cell populations.
One such molecule that is expressed on a wide range of different embryonic and adult as well as tumor stem cells is the ABC transporter Abcg2. ABC transporters in general are transmembrane proteins that actively extrude endo- and exotoxins as well as xenobiotics, thereby protecting cells and organs. Additionally, ABC transporters are responsible for drug resistance in many cancers. A well-described characteristic of stem cells expressing Abcg2 is the formation of the ‘side population’ (SP) phenotype: An active Abcg2 transporter mediates the efflux of a particular fluorescent dye that is taken up by all cells, thus leading to a less brightly stained population. This phenomenon is widely used to characterize and isolate the most primitive stem cell subpopulation from embryonic and adult tissues, including tumors. Besides its role as toxin transporter little is known about the function of Abcg2 in stem cells. This is mainly due to the fact that its physiological substrate in stem cells remains unknown. The identification of such substrates is therefore of high interest because it would directly link the activity of ABC transporters to regulatory mechanisms in stem cell biology.
In the present study we wanted to test the hypothesis that the sphingolipid ceramide is a physiological substrate of the ABC transporter Abcg2. Sphingolipids are potent second messengers and are known to have regulatory functions in stem cells. In particular, the sphingolipid ceramide is described as a mediator of controlled cell death and inducer of differentiation. It is suggested that stem cells need to keep their intracellular ceramide content at low levels in order to prevent apoptosis or differentiation. We propose that Abcg2 and ceramide interact and that this interaction leads to changes in the absolute or relative amounts of ceramide. This in turn influences basic stem cell functions such as self renewal and differentiation.
We show that Abcg2 prevents cells from accumulating fluorescence labeled ceramide. Furthermore, exogenously applied ceramides inhibit the transport activity of Abcg2, measured by a decrease of the side population phenotype. This inhibitory effect is consistent with a competitive inhibition mechanism. Additionally, we show that active Abcg2 can increase the ceramide concentration in cell culture supernatant. Finally we demonstrate that Abcg2 protects from ceramide induced cytotoxicity in human cell lines. In summary, these in vitro results strongly suggest that Abcg2 has the ability to regulate ceramide levels.
Murine hematopoietic stem cells (HSCs) are the best characterized adult stem cell system so far. By using 7-colour fluorescence-activated cell sorting (FACS) we established the purification of the most primitive HSCs, reflected by their high engraftment capability when transplanted to lethally irradiated mice. By using this sorted cell populations it was in addition possible to establish a system to reproducibly manipulate HSCs ex vivo. This experimental system will serve in further elucidating the physiological consequences of Abcg2 mediated changes in ceramide levels on stem cells in vivo.
Taken together, this study shows that Abcg2 has the ability to regulate ceramide levels in cells. This in turn can lead to cellular protection from ceramide induced apoptosis. Additionally, the experimental techniques to further analyze the role of Abcg2 and ceramide in the most primitive hematopoietic stem cells were successfully established, enabling more detailed analysis in the future.
Conclusion: Proteins containing a Jumonji C (JmjC) domain appear in almost all living organisms and catalyze a variety of oxidation reactions. Therefore, they are important regulators in many biological processes such as proliferation and differentiation. They act either as protein hydroxylases, histone demethylases or by regulate mRNA splicing. Given the fact that some of the JmjC domain-containing proteins are shown to be upregulated in response to hypoxia as well as the dependency of JmjC domain catalytic activity on oxygen led to the assumption of an involvement in angiogenesis. For Jmjd6, a member of the JmjC domain-containing protein family, a regulatory involvement in mRNA splicing has been shown. The Jmjd6-/- mouse dies perinatally due to several severe organ malformations, especially in the heart. Despite the pale appearance, the growth retardation and the cardiac defects, it is unclear whether these mice exhibit defects of cells comprising the vasculature. Therefore, the involvement of Jmjd6 in angiogenesis was examined in vitro using angiogenesis assays as well as in vivo using the Jmjd6+/- mouse. An siRNA-mediated knockdown of Jmjd6 in ECs significantly impaired the formation of capillary-like networks in the tube formation assay as well as sprouting in the spheroid assay. Moreover, after siRNA-mediated knockdown of Jmjd6 in ECs cell migration was significantly reduced. These findings were confirmed in the matrigel plug assay in vivo. Implanted matrigel plugs of Jmjd6+/- mice exhibited significantly less perfused vessels compared to wildtype littermates. Furthermore, cultured lung ECs from Jmjd6+/- mice exhibited impaired network forming activity ex vivo compared to cells isolated from wildtype littermates. To elucidate the mechanisms underlying the requirement of Jmjd6 in angiogenesis, an Affymetrix exon-array was performed, which allows detection of changes in gene expression as well as splicing. The siRNA-mediated knockdown of Jmjd6 altered the expression of genes known to play a role in vascular biology. The bioinformatic assessment of alternative splice variants revealed that Jmjd6 silencing affects the splicing of the VEGF receptor 1 (Flt1). Differential splicing of Flt1 was shown to generate a short and soluble form of Flt1 (sFlt1), which sequestrates VEGF and PlGF, and thereby inhibits angiogenesis. In particular, a significant increase in sFlt1 expression was observed. Jmjd6 was recently reported to hydroxylate the splicing factor U2AF65. Therefore, we investigated whether U2AF65 might mediate Flt1 splicing and binds to Flt1 mRNA. Indeed, U2AF65 co-immunoprecipitated with Jmjd6 in ECs, while an interaction of U2AF65 with sFlt1 was demonstrated. Moreover, inhibition of Jmjd6 catalytic function by reduced oxygen concentration altered splicing of Flt1 resulted in an increase of the sFlt1 splice variant. Finally, saturating concentrations of VEGF or PlGF or neutralizing antibodies against sFlt1 significantly reduced the inhibition of sprouting caused by Jmjd6 knockdown in vitro.
Collectively, our results indicate that Jmjd6 has an essential role in the oxygen-dependent regulation of angiogenesis by controlling the splicing of Flt1 mRNA, thereby adjusting the generation of the anti-angiogenic short splice variant sFlt1. Several publications demonstrated a major importance for sFlt1 as a biomarker for many severe human diseases such as preeclampsia, sepsis, cancer, myocardial infarction as well as chronic heart failure. Therefore, the identification of the molecular mechanism behind the generation of sFlt1 might enable the development of new or more precise clinical markers for the diagnosis of the corresponding diseases. Furthermore, the discovery of the enzymes involved in the generation of sFlt1 provides further possibilities to modulate sFlt1 levels and thereby may potentially gives rise to the development of new therapies.
Decorin, a small leucine rich proteoglycan (SLRP) of the extracellular matrix (ECM) is a biologically active molecule with signaling capabilities modulating diverse cellular functions 1. In this report, we explore the role of the matrix proteoglycan decorin in the regulation of inflammation and apoptosis and the resultant biological significance in cancer and diabetic nephropathy. The mechanisms linking immunity and inflammation with tumor development are not well defined. Here we report a novel finding that the soluble form of decorin could autonomously trigger the synthesis of TNFα and IL-12 in macrophages through TLR2 and TLR4 in a p44/42- and p38-dependent manner. In the presence of LPS, decorin enhanced the effects of LPS by signaling additionally via TLR2. Further, decorin could enhance PDCD4 protein expression with subsequent inhibition of LPS-mediated IL-10 protein synthesis by two mechanisms: i) by TLR2/TLR4-dependent stimulation of PDCD4 synthesis and ii) by inhibition of the TGFβ1-induced increase of miR-21, a posttranscriptional suppressor of PDCD4 protein synthesis. Enhanced PDCD4, a translational inhibitor of IL-10, downregulated this anti-inflammatory cytokine, thereby further driving the cytokine profile towards a proinflammatory phenotype.
Importantly, these mechanisms appear to operate in a broad biological context linking pathogen-mediated with sterile inflammation as shown here for sepsis and growth retardation of established tumor xenografts. In sepsis, decorin is an early response gene evoked by inflammation and is markedly elevated in plasma of septic human patients and in plasma and tissues of septic mice. Our findings suggested that in vivo decorin alone mimics the effects of LPS by enhancing the plasma and tissue levels of pro-inflammatory TNFα, IL-12 and PDCD4 but when administered together with LPS, it potentiated the proinflammatory response of this PAMP by inhibiting active TGFβ1, miR-21 and hence the LPS mediated IL-10 production. In vivo, overexpression of decorin in tumor xenografts resulted in decorin/TLR2/4-driven synthesis of PDCD4, TNFα, IL-12 and decorin/TGFβ1/miR-21-mediated inhibition of PDCD4 suppression shifting the immune response to a pro-apoptotic and proinflammatory axis with strong anti-tumorigenic effects resulting in increased apoptosis and growth retardation of solid tumor. Thus, decorin signaling boosts inflammatory activity in sepsis and tumor. In contrast to the proinflammatory and proapoptotic role of decorin in tumor, decorin deficiency in diabetic kidneys led to enhanced apoptosis and increased mononuclear cell infiltration indicating that decorin might give rise to distinct biological outcomes depending on the cell type and biological context. Accordingly, in this study, we used a model of streptozotocin-induced diabetes type 1 in wild-type (Dcn+/+) and decorin-deficient- (Dcn-/-) mice to further elucidate the role of decorin in diabetic nephropathy. In this model, decorin was overexpressed in the mesangial matrix of the glomerulus and in the tubulointerstitium both at the mRNA and protein level in early stages of diabetic nephropathy which declined as the disease further progressed supporting the concept that decorin might act as a part of a natural response to hyperglycemia and to damage caused there from. These observations correlate with the data obtained in renal biopsies from patients at various stages of diabetic nephropathy 15, suggesting clinical relevance of our findings for the human disease. In the diabetic kidney, decorin deficiency was associated with: i) glomerular and tubular overexpression of p27Kip1 and enhanced proteinuria, ii) enhanced expression of TGFβ1 and CTGF resulting in increased accumulation of ECM, iii) overexpression of biglycan and elevated infiltration of mononuclear cells, iv) enhanced apoptosis of tubular epithelial cells despite overexpression of tubular IGF-IR. We further discovered that decorin binds to the IGF-IR in tubular epithelial cells and conveys protection against high glucose-mediated apoptosis providing evidence for a protective role of decorin during diabetic nephropathy development.
Thus, future therapeutic approaches that would either enhance the endogenous production of decorin or deliver exogenous decorin to the diseased solid tumors and/or diabetic kidney might improve the prognosis of these chronic diseases.
The translocation of nuclear-encoded precursor proteins into chloroplasts is a highly ordered process involving the action of several components to regulate this molecular ensemble. Not only GTP hydrolysis and GDP release but also the phosphorylation of TOC GTPases is a widely discussed mechanism to regulate protein import. The receptor component (Toc34) and its isoform of A. thaliana (atToc33) were found to be regulated by phosphorylation. Although the phosphorylation of Toc33 is already known for several years, several questions regarding the molecular components involved in the regulation of the phosphorylation process, precisely what is the protein kinase and where this kinase is initially localized, so far remained unclear.
This thesis aimed at the defining of the phosphorylation status of TOC GTPases in monomeric and/or dimeric states, the identification of the nature of Toc33-PK (protein kinase), and in the same context it aimed at gaining first insights into the physiological significance of Toc33 phosphorylation. To this end, (I) An in vitro and in vivo system for investigating of TOC GTPases Phosphorylation (in monomeric or dimeric state) was developed. Since no information is available about the phosphorylation status of the Toc159 isoforms, the second receptor of the TOC complex, it was interesting to investigate whether these isoforms undergo phosphorylation or not. The results indicated that atToc159 isoforms are able to be phosphorylated by the kinase activity in purified outer envelope membranes (OEMs) of pea, but not atToc132. Moreover, an artificial dimer of psToc34 based on the interaction of a C-terminally fused leucine zipper was not phosphorylated. This result reflected the inability of the OEM kinase to phosphorylate the dimers of TOC GTPases. Also, In vivo labeling of atToc33 was developed and occurred in a dose-dependent manner. Therefore, this results evidenced that in vitro phosphorylation of atToc33 (both endogenous wild type and recombinant expressed proteins) is not artificial labeling but represents a physiological relevance. CD (circular dichroism) measurements revealed that recombinant GTPase domain of atToc33 is preferentially phosphorylated in its folded state. Therefore, it could be suggested that folding of atToc33rec is a prerequisite for its phosphorylation and the phosphorylation event occurs as a posttranslational modification most likely after insertion of Toc33 (Toc34) into the OE of chloroplasts.
Secondly, (II) Isolation and identification of Toc33-PK from OEMs of chloroplasts was performed. Four independent strategies were developed to identify the Toc33-protein kinase: UV-induced and chemically-based crosslinking, different applied chromatographic techniques, identification of PK-Toc33 interaction by means of HDN-PAGE (histidine- and deoxycholate-based native PAGE), and finally mass spectrometric approaches were performed on fractions including the potential kinase activity. UV-induced crosslinking procedure was developed and resulted in covalent bonding of nine proteins to [a-32P] ATP, while chemically-based one was not significant. The applied chromatographic and HDN-PAGE approaches, including mass spectrometry, have revealed the identification of 13 protein kinases. Of these identified kinases, phototropin2 (Phot2, AT5G58140), leucine-rich repeat PK (LRR-PK, AT4G28650.1), and receptor-like transmembrane PK (RLK, AT5G56040.2) were selected as the most promising candidates (ca. kinase type and one transmembrane helix for membrane localization).
(III) The physiological significance of Toc33 phosphoryation was shown to link this process with the environmental changes (especially, the light conditions). Identification of chloroplast OE-located PKs performed by nLC-MALDI-MS/MS resulted in the detection of Phot2. Furthermore, the subcellular localization of Phot2 in OEM of chloroplasts was confirmed by immunoblotting experiments using a-Phot2 antibody. The kinase activity of Phot2 towards TOC GTPases was characterized and revealed that fused GST-KD (kinase domain) protein able to specifically phosphorylate atToc33rec, but not atToc159rec. Also, endogenous atPhot2 was upregulated and heavily detected in the ppi1-S181A plant line (where serine to alanine exchange was performed to abolish the phosphorylation of atToc33). Hence, we suggested that certain signal cascades may directly or indirectly link Toc33 receptor phosphorylation, protein levels of Phot2 (as promising PK candidate), and irradiation conditions (as an inducing signal of the subsequent phosphorylation events). Light-dependent phosphorylation of Toc33 was shown either after de-etiolation conditions or after high light intensities of blue light was performed. Therefore, phosphorylation of Toc33 might be identified as an external regulatory signal to regulate preproteins import into chloroplasts in response to environmental conditions (e.g. light changes) or as a signal of chloroplast biogenesis.
This thesis has light mesons and their vacuum interactions as its topic. In particular, the work examines the question where the scalar antiquark-quark states are found in the physical spectrum -- in the energy region below or above 1 GeV. Contrary to the naive expectation, the mentioned states are found in the region above 1 GeV. This has consequences for the building of order parameters for the chiral symmetry breaking of Quantum Chromodynamics (QCD).
Proteomic analysis is the large-scale identification and characterization of proteins including post translational modifications. Proteomics encompasses a number of approaches including bottom-up and top-down workflows which are widely used independently and complementary as tools for the successful study of protein species. However, up to the present day these techniques have not been able to overcome every analytical limitation. Mass spectrometry has played a vital role alongside proteomics in providing the required analytical means of detecting protein amounts down to the atomole range. Soft ionization methods such as matrix assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) have permitted the transfer of peptides and intact proteins into the gas phase without extensive degradation. The introduction of recent developments in MALDI technology such as the highly sensitive 4-chloro-alpha-cyanocinnamic acid matrix (Cl-CCA) as well as the commercial availability of a MALDI-LTQ-Orbitrap which boosts peptide mass accuracy below 3 parts per million (ppm), have offered new prospective in protein analysis. The aim of the current study is to incorporate these new aspects and provide further advancements in gel-based as well as gel-free proteomic workflows.
Peptides of proteolytically digested proteins are routinely analyzed by means of peptide mass fingerprinting (PMF) often combined with MS/MS analyses to complement and substantiate PMF results by peptide sequence information. The most widely used protease for enzymatic digestion is trypsin, since it exhibits a very specific cleavage behavior limited to C-terminal hydrolyses after basic amino acids. However, less specific enzymes such as chymotrypsin, elastase and pepsin have emerged as useful tools in the analysis of particular protein classes e.g. membrane, cereal, and phosphorylated proteins. In this work a comprehensive bottom-up proteomic investigation including in-solution and in-gel protein digestions of analytes covering small to large, acidic to basic, and hydrophobic to hydrophilic proteins in combination with a series of less specific enzymes are presented in order to show the superiority of the novel MALDI matrix Cl-CCA. The Cl-CCA matrix proved to be highly superior compared to standard α-cyano-4-hydroxycinnamic acid (CHCA) since an average detection of more than 2- to 3-fold peptide amount was possible depending on the used protease and, therefore, resulting in strongly increased sequence coverage. Additionally, protein identification of chymotrypsin and elastase in-gel digested protein standards was evaluated. The MALDI-LTQ-Orbitrap providing peptide mass accuracy below and up to 3 ppm in combination with Cl-CCA as matrix and newly optimized digestion conditions led to unambiguous protein identifications of all chymotryptic digests outperforming its tryptic counterparts in the case of hydrophobic bacteriorhodopsin and α-globin from hemoglobin A (α-HgbA). In addition, significantly higher sequence coverage and increased number of detected peptides was acquired. Moreover, a proposed workaround for elastase digestions was capable of providing a solution for successful identification results.
Apart from digestions of singly separated proteins, solution isoelectic focusing (sIEF) was evaluated. OFFGEL fractionation is an efficient means of fractionating peptides and proteins according to their isoelectric point (pI) values through immobilized pH gel (IPG) strips after which samples are recovered in solution. Consequently, an issue of peptide recovery arises as a category of peptides relatively insoluble to the recovery solution should be present. A method was developed including the scraping of gel matrix from the IPG strips and peptide extraction using acetonitrile as organic solvent in combination with analytical techniques such as nLC-MALDI-MS/MS for peptide identification. The nature of the peptide species remaining in-gel was analysed and attributed to peptide solubility. A general trend in which a high percentage of neutral and hydrophobic peptides remaining entrapped in the IPG gel strip was observed.
The present work also examines a new top-down proteomic workflow involving protein elution from cleavable gels containing the labile crosslinker ethylene-glycol-diacrylate (EDA). Protein amounts of as low as 100 ng loaded onto EDA gels were detected using MALDI-TOF MS in the linear acquisition mode. Proteins from 8.5 up to 78 kDa were successfully measured including a hydrophobic 15 kDa core protein attaining a GRAVY score of +0.079. Additionally, the method was compatible with one dimensional protein separation as well as for 2-D IEF/SDS-PAGE. Lastly, two methods for protein identification were tested and found to be compatible to the proposed technique.
Clathrin-mediated endocytosis (CME) involves spatially and temporally restricted molecular dynamics.
Although protein kinases and the actin cytoskeleton contribute to the process, whether and how
functions of kinases and actin are integrated remains unknown. Here, we demonstrate that neural
Wiskott-Aldrich syndrome protein (N-WASP) and protein kinase CK2 form a complex and localize on
clathrin-coated vesicles (CCVs). N-WASP binds to and is phosphorylated by CK2, thereby reducing the
kinase activity of CK2. By contrast, N-WASP-promoted actin polymerization is decreased upon both
phosphorylation and binding of CK2. Knockdown of N-WASP and CK2, alone or in combination, results
in impaired endocytosis of epidermal growth factor (EGF) and increased cell-surface levels of EGF
receptor (EGFR). In order to rescue the phenotype of N-WASP-CK2 knockdown cells, both N-WASP and
CK2 activities and abilities to assemble in a complex are required. In summary, this study shows that the
N-WASP-CK2 complex integrates in a single circuit different activities contributing to CME of EGFR and
that the interplay between the two proteins optimizes this process.
Towards a THz Bloch laser
(2011)
The realisation of tunable THz laser sources working at room temperature would give
rise to further applications in this range of the electromagnetic spectrum. The THz
Bloch laser could therefore become the basis for a technological breakthrough. Beside
this practical relevance, the physics of the gain mechanism has been investigated
theoretically for a long time and the experimental implementation of a self-starting
laser still has not been achieved.
At the beginning of this thesis the basic principles of Bloch oscillations and the
related Bloch gain are described. The need of a superlattice structure to make Bloch
oscillations possible in a semiconductor material is discussed. In this context, the effect
of negative differential resistance and its influence on the field distribution due to Gunn
domains is explained. The latter lead to an inhomogeneous field which may suppress
the Bloch gain mechanism. The Krömer criterion is introduced and the concept of
field-pinning layers to improve the field homogeneity is deduced. Finally, the design of
the laser material is shown and different types of laser waveguides are compared.
In chapter 3 detailed recipes for the processing of samples are given. Different types of
contacts (ohmic and Schottky), the wafer bonding process required for double-metal
lasers and the application of different photoresists for different purposes are described.
An explanation of the formation of waveguides due to dry etching, wet etching
and ion implantation follows. Dry etching is an established technique in the field
of microstructure processing but the challenge of etching about 20 μm has led to
problems. The high etching depth also makes wet etching difficult but this method
could be improved due to a hard bake of the photoresist. The protection of critical
areas on the surface of the samples with photoresist during ion implantation was
increased by optimising the spin coating process. However, a full implantation of the
active layer between the waveguides was not achieved which was the reason for the
development of the hybrid technology. Here a prior wet etching of about 10 μm is
performed and the rest of the material is implanted.
The experimental setup is shown in chapter 4. An alternative method for the electrical
contacting with the help of a copper bar is introduced. This improves the current
distribution and the risk of an electrical breakdown during the measurements could
therefore be lowered. Devices for THz beam guidance and spectroscopic measurements
are shown and the method of biasing the samples with pulses below 100 ns and
determining the effective voltage applied to the sample is depicted. These short pulses
are required to prevent the samples heating up drastically due to high power.
Chapter 5 contains the current-voltage characterisation of several structures including
I-V-samples, Bloch laser samples and a quantum cascade laser. Different contacts
(ohmic and Schottky) and different techniques for the formation of the ridges have
been used in the processing of these samples (performed at the University of Frankfurt
in all cases) and their influence on the I-V-dependence is discussed. The properties of
the THz emission of the quantum cascade laser are in good agreement with published
results from lasers processed with the same material. Another important result of
this chapter is that the Bloch laser samples show unstable behaviour compared to the
quantum cascade structure even with short pulses (of about 10 ns) where the risk of an
electrical breakdown or the building of filaments is low. THz radiation emitted from
one of the Bloch laser samples could not be observed.
Two aspects that may have prevented the Bloch laser to emit are discussed in
chapter 6. The saturation of the gain for higher amplitudes of the THz wave is
investigated in single mode and multiple mode operation (the latter could occur due
to the Bloch gain being expected to be broadband). In both cases it is shown that
the saturation effect would limit the output power only to values clearly above the
detection limit. In the subsequent section the distribution of the electric field is
simulated with SILVACO software. Structures with transit layer lengths above the
Krömer criterion are compared with structures which include field-pinning layers. It is
shown that the latter are useful to avoid propagating Gunn domains as they build up
in similar structures without field-pinning layers. Nevertheless, the electric field inside
the superlattice regions is not stable. Beside spatial inhomogeneities also temporal
variations of the field magnitude are observed. The lack of a suitable field distribution
is expected to be the main reason for the samples not to work.
The miniaturization of electronics is reaching its limits. Structures necessary to build integrated circuits from semiconductors are shrinking and could reach the size of only a few atoms within the next few years. It will be at the latest at this point in time that the physics of nanostructures gains importance in our every day life. This thesis deals with the physics of quantum impurity models. All models of this class exhibit an identical structure: the simple and small impurity only has few degrees of freedom. It can be built out of a small number of atoms or a single molecule, for example. In the simplest case it can be described by a single spin degree of freedom, in many quantum impurity models, it can be treated exactly. The complexity of the description arises from its coupling to a large number of fermionic or bosonic degrees of freedom (large meaning that we have to deal with particle numbers of the order of 10^{23}). An exact treatment thus remains impossible. At the same time, physical effects which arise in quantum impurity systems often cannot be described within a perturbative theory, since multiple energy scales may play an important role. One example for such an effect is the Kondo effect, where the free magnetic moment of the impurity is screened by a "cloud" of fermionic particles of the quantum bath.
The Kondo effect is only one example for the rich physics stemming from correlation effects in many body systems. Quantum impurity models, and the oftentimes related Kondo effect, have regained the attention of experimental and theoretical physicists since the advent of quantum dots, which are sometimes also referred to as as artificial atoms. Quantum dots offer a unprecedented control and tunability of many system parameters. Hence, they constitute a nice "playground" for fundamental research, while being promising candidates for building blocks of future technological devices as well.
Recently Loss' and DiVincenzo's p roposal of a quantum computing scheme based on spins in quantum dots, increased the efforts of experimentalists to coherently manipulate and read out the spins of quantum dots one by one. In this context two topics are of paramount importance for future quantum information processing: since decoherence times have to be large enough to allow for good error correction schemes, understanding the loss of phase coherence in quantum impurity systems is a prerequisite for quantum computation in these systems. Nonequilibrium phenomena in quantum impurity systems also have to be understood, before one may gain control of manipulating quantum bits.
As a first step towards more complicated nonequilibrium situations, the reaction of a system to a quantum quench, i.e. a sudden change of external fields or other parameters of the system can be investigated. We give an introduction to a powerful numerical method used in this field of research, the numerical renormalization group method, and apply this method and its recent enhancements to various quantum impurity systems.
The main part of this thesis may be structured in the following way:
- Ferromagnetic Kondo Model,
- Spin-Dynamics in the Anisotropic Kondo and the Spin-Boson Model,
- Two Ising-coupled Spins in a Bosonic Bath,
- Decoherence in an Aharanov-Bohm Interferometer.
The canonical Wnt/β-catenin and the Shh pathway as well as the Notch signaling cascade
are key regulators in stem cell biology and are independently associated with the development
of cancer. Despite the knowledge of a balanced signaling for cellular maintenance, the
fundamental biochemical mechanisms of crosstalk are still poorly understood. This study
demonstrates that the outcome of interaction between Wnt and Shh is cell type specific. A
combined inhibitory mechanism of the Shh and Notch2/Jagged2 pathways on dominant
active β-catenin signaling in the adult tongue epithelium keeps Wnt/β-catenin signaling
restricted to physiological tolerable levels. In the opposite crosstalk the activation of
Wnt/β-catenin signaling in medulloblastoma (MB) of the Shh subtype, in turn inhibits the Hh
pathway.
The inhibitory mechanism of Shh and Notch2/Jagged2 on Wnt/β-catenin signaling is
independent of the degradation complex of β-catenin and takes place inside the nucleus.
Furthermore, the negative feedback on Wnt/β-catenin signaling by the Shh pathway relies
on transcriptional activity of Gli1/2A. Inhibition of Gli1/2A with the specific inhibitor GANT61
abrogated the negative impact of Shh on β-catenin signaling in vitro. Although the negative
feedback loop of Shh is still functional in human SCC25 cells, the inhibitory effect of
Notch2/Jagged2 is lost and contributes to the cancerogenic phenotype of these cells. In the
inverse situation, the activation of β−catenin signaling has a negative feedback on
constantly active Shh signaling and significantly inhibits the Hh pathway. This was shown in
Ptch+/- and Math1-Cre:SmoM2Fl/+ MB tumor spheres in vitro, in which inhibition of sphere
formation and growth was observed and Hh target gene transcription was down-regulated.
This demonstrates for the first time that the activation of canonical Wnt/β-catenin signaling
in primary MB cells with a Hh pathway over-activation has a negative effect on the growth of
these cells in vitro.
In summary the results show that crosstalk of Wnt/β-catenin and Shh signaling has context
specific outcome on pathway activity. Elucidation of the molecular interactions will improve
our understanding of Wnt and Hh associated tumors and contribute to the development of
new therapeutic strategies.
The study of meson production in proton-proton collisions in the energy range
up to one GeV above the production threshold provides valuable information about
the nature of the nucleon-nucleon interaction. Theoretical models describe the interaction
between nucleons via the exchange of mesons. In such models, different
mechanisms contribute to the production of the mesons in nucleon-nucleon collisions.
The measurement of total and differential production cross sections provide information
which can help in determining the magnitude of the various mechanisms.
Moreover, such cross section information serves as an input to the transport calculations
which describe e.g. the production of e+e− pairs in proton- and pion-induced
reactions as well as in heavy ion collisions.
In this thesis, the production of ω and η mesons in proton-proton collisions at 3.5
GeV beam energy was studied using the High Acceptance DiElectron Spectrometer
(HADES) installed at the Schwerionensynchrotron (SIS 18) at the Helmholtzzenturm
f¨ur Schwerionenforschung in Darmstadt.
About 80 000 ω mesons and 35 000 η mesons were reconstructed. Total production
cross sections of both mesons were determined. Furthermore, the collected statistics
allowed for extracting angular distributions of both mesons as well as performing
Dalitz plot studies.
The ω and η mesons were reconstructed via their decay into three pions (π+π−π0)
in the exclusive reaction pp −→ ppπ+π−π0. The charged particles were identified
via their characteristic energy loss, via the measurement of their time of flight and
momentum, or using kinematics.
The neutral pion was reconstructed using the missing mass method. A kinematic
fit was applied to improve the resolution and to select events in which a π0 was
produced.
The correction of measured yields for the effects of spectrometer acceptance was done
as a function of four variables (two invariant masses and two angles). Systematic
studies of the acceptance for different input distributions were performed.
The measured yields were normalized to the number of measured events of elastic
scattering. Systematic errors due to the methods of the data analysis and the
background subtraction were investigated.
Production angular distributions of ω and η mesons were measured. Both mesons
exhibit a slightly anisotropic angular distribution.
The Dalitz plot of ω meson production shows indications of resonant production.
However, the deviation of the distribution from the one expected by phase space
simulations is not large.
The Dalitz plot of η meson production shows a signal of the production via the
N(1535) resonance, The contribution of N(1535) to the production was quantified
to be about 47%. The angular distribution of η mesons does not show significant
differences between resonant and non resonant production.
The total production cross section of ω mesons in the reaction pp −→ ppω was
determined to be 106.5 ± 0.9 (stat) ± 7.9 (sys) [μb] where stat indicates statistical
error and sys indicates systematic error, while that of η mesons was determined to
be 136.9 ± 0.9 (stat) ± 10.1 (sys) [μb] in the reaction pp −→ ppη
Occurrence and sources of 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) in the aquatic environment
(2011)
The aim of the present study was to identify the sources of 2,4,7,9-tetramethyl-5-decyne-4,7-
diol (TMDD) into the aquatic environment and to investigate its occurrence in rivers and
wastewater treatment plants (WWTPs). Therefore, TMDD was analyzed in 441 wastewater
samples from influents and effluents of 27 municipal WWTPs, in 6 sludge samples, in 52
wastewater samples from 3 sewage systems of municipal WWTPs, in 489 surface samples
from 24 rivers, in 9 wastewater samples of 3 paper-recycling industries and in 65 groundwater
samples. TMDD was also analyzed in household paper products, in 23 samples of toilet
papers, in 5 types of paper towels and in 12 types of paper tissues. The samples were collected
between 2007 and 2011. The water samples were extracted with solid phase extraction (SPE)
and the household paper samples with Soxhlet extraction. Gas chromatography-mass
spectrometry (GC-MS) was used for quantification purposes.
Between November 2007 and January 2008, TMDD was detected in the river Rhine at Worms
with permanent high concentrations (up to 1330 ng/L). The results showed that TMDD is
uniformly distributed across the river at Worms. An increase of the mean TMDD
concentration from approximately 500 ng/L to 1000 ng/L was registered in January 2008. Due
to the minor fluctuations of the TMDD concentration during the sampling period it is
expected that the input of TMDD into the river is continuous. Therefore, TMDD might rather
originate from effluents of municipal WWTPs than from temporal sources. The mean TMDD
load based on the analysis of 147 water samples collected in the River Rhine was 62.8 kg/d
which is equivalent to 23 t/a suggesting that TMDD must be used and/or produced in high
quantities in order to be found in those high concentrations. To determine if TMDD is
discharged by effluents of municipal WWTPs into the rivers, 24 hours influent and effluent
samples of four municipal WWTPs in the Frankfurt/Rhine-Main metropolitan region were
collected during November 2008 and February 2010 and analyzed for TMDD. The TMDD
influent concentrations varied between 134 ng/L and 5846 ng/L and the effluent
concentrations between <LOQ (limit of quantitation) and 3539 ng/L. The TMDD elimination
rates in the four WWTPs varied between 33% and 68%. The results showed that effluents of
municipal WWTPs are an important source of TMDD in the aquatic environment because
TMDD is not completely removed from the sewage during the wastewater treatment. Weekly
and daily variations of the TMDD concentration in the influents of two municipal WWTPs
indicated that both private households and indirect industrial dischargers contribute to the
introduction of TMDD into the municipal sewage systems. A more detailed study of the
TMDD elimination rate in the different wastewater treatment stages was carried out in the
WWTP Niederrad/Griesheim in Frankfurt am Main. The results showed that the removal of
TMDD is mainly carried out during the aerobic biological treatments, where the elimination
rate was 46%. In contrast, during the anoxic treatment the removal efficiency was only 1.4%
and during the mechanical treatment the elimination rate was 19%.
To determine the sources of TMDD in the sewage, household paper products (paper tissues,
toilet papers and paper towels) were analyzed for TMDD using Soxhlet extraction. TMDD
was detected in 83% of the samples (n=40). The highest mean TMDD concentrations were
found in recycled toilet paper (0.20 μg/g) and in paper towels (0.11 μg/g). In paper tissues and
non-recycled toilet paper the mean TMDD concentrations were lower 0.080 μg/g and
0.025 μg/g respectively. According to these results the high TMDD influent concentrations
found previously in municipal WWTPs (mean 1.20 μg/L) cannot be explained due to
migration of TMDD from the household paper products into the sewage. Thus indirect
industrial dischargers are the cause of the high influent TMDD concentrations. Effluents of
municipal WWTPs with different indirect industrial dischargers (textile-, metal processing-,
food processing-, electroplating-, paper-recycling- and printing ink factories) were analyzed.
The highest mean TMDD concentrations were found in the effluents of municipal WWTPs
that have paper-recycling (71.3 μg/L) and printing ink factories (138 μg/L) as indirect
industrial dischargers. These results were confirmed by analyzing process wastewater of three
paper-recycling factories located in Germany. High TMDD concentrations were detected and
fluctuated between 1.83 μg/L and 113 μg/L. TMDD was also analyzed in the wastewater of a
non-recycling-paper factory but its concentration was much lower (0.066 μg/L) indicating that
TMDD is introduced into the processing water during the papermaking process due to the use
of waste paper. Analyses of wastewater samples from different parts of the sewage pipes of a
municipal WWTP in Hesse, which receives the wastewater from a printing ink factory, were
carried out. The TMDD concentration in the wastewater sample from the sewage pipe of the
printing ink factory was much higher (3,300 μg/L) than the TMDD concentration detected in
the other wastewater samples from the sewage system (0.030 μg/L – 0.89 g/L). These results
confirm the printing ink production as one of the principal sources of TMDD in the sewage.
Analysis of surface water samples of the River Modau downstream from the effluent of the
WWTP Nieder-Ramstadt showed TMDD concentrations of up to 28.0 μg/L. These high
TMDD concentrations might be caused by the indirect wastewater discharges of a paint
factory connected to the municipal sewage system. These results indicate that TMDD is
introduced into the municipal WWTPs principally by indirect industrial dischargers and they
are mainly paint and printing ink factories. The paper-recycling factories also represent an
important source of TMDD in municipal WWTPs but indirectly. According to statements
given by the representatives of two paper recycling factories neither TMDD or any other
TMDD containing product is used or added during the papermaking process. Therefore,
TMDD is washed out from the printing inks of the coloured waste paper and concentrated in
the process wastewater in the closed water circuits of paper-recycling factories reaching rivers
and municipal WWTPs.
The occurrence and distribution of TMDD in surface waters in Germany was also studied.
The results showed that TMDD is widely distributed across different rivers systems in the
federal states of Hesse, North-Rhine-Westphalia, Bavaria, Baden-Wuerttemberg and
Rhineland-Palatinate. In Hesse, TMDD was detected in the some of main rivers with mean
concentrations of 812 ng/L (Schwarzbach, Hessian Ried), 374 ng/L (Kinzig), 393 ng/L (Main,
at Frankfurt), 539 ng/L (Werra), 326 ng/L (Fulda), 151 ng/L (Emsbach) and 161 ng/L
(Nidda). In small rivers (creeks) the mean TMDD concentrations varied between <LOQ
(Diemel, Urselbach) and 1890 ng/L (Darmbach). The results showed that the TMDD
concentrations in creeks are highly influenced by both effluents of WWTPs and by the
distance between the sampling point and the nearest WWTP. Surface samples from sampling
locations downstream from WWTPs dischargers showed higher TMDD concentrations (mean
518 ng/L) than sampling locations upstream from WWTPs dischargers (mean 35.1 ng/L).
The behavior of TMDD during bank filtration was investigated at two locations, at a water
utility company at the Lower River Rhine (urban area) and at the Oderbruch polder (rural
area). The results indicated that TMDD is removed from the surface water by bank filtration
at both sampling locations. The removal process is probably carried out in the first meters of
the aquifer (hyporheic zone) by biodegradation processes, since TMDD does not tend to be
absorbed by sediments and it was not found in the groundwater of monitoring wells. In
groundwater samples from the Hessian Ried (n=23) TMDD was found only in five samples
and the highest TMDD concentration was 135 ng/L. According to these results, TMDD does
not represent a concern for drinking water in Germany, since it does not reach the
groundwater with high concentrations and it has a low toxicity potential.
The input of TMDD into the North Sea was estimated to be 60.7 t/a by considering the mean
transported loads of TMDD by the River Rhine at Wesel (58.3 t/a) and Meuse in the
Netherlands (2.40 t/a). The estimated discharge of TMDD by German municipal WWTPs
(8.19 t/a) and paper-recycling factories (9.24 t/a) into rivers seems to be too low considering
that the mean TMDD load in the River Rhine downstream from Wesel is 58.3 t/a. However,
due to the high density of population and industries at the Lower Rhine it is expected that
more relevant sources of TMDD are located along the Rhine River increasing the transported
load.
According to the results of this PhD project TMDD is a non-ionic surfactant contained in
products, which are applied on surfaces (printing inks and paints) and has the potential to
reach the aquatic environment. Therefore, TMDD should fulfill the requirement of a
biodegradability of 80% established by the “Law on the Environmental Impact of Detergents
and Cleaning Products” in Germany. However, due to the partial elimination rates of TMDD
obtained in municipal WWTPs (between 33% and 68%) and to the absence of information
about the execution of the biodegradation test on TMDD, it is unknown if TMDD is in
accordance with this law. Otherwise, its use as surfactant in such products is questionable.
During the last years, chemopreventive activity of NSAIDs against a great variety of tumors was highly investigated. COX-2 seemingly plays a major part in tumorigensis and tumor development, underlined by several studies in animals and humans. At first, NSAIDs were thought to accomplish chemoprevention by inhibition of COX-2 as their so far known mode of action comprises unselective inhbition of COX-enzymes. However, further studies revealed COX-independent mechanisms. Sulindac is known as a well established drug used to treat inflammation and pain exerting the most prominent chemopreventive action, mainly in colorectal cancer or FAP and can be classified into the group of NSAIDs inhibting both COX-isoformes. As interference with the AA metabolism is evident, it was speculated whether Ssi has targets other than COX-enzymes providing evidence and explanation of its beneficial side effect profile and its ability to reduce tumor growth. 5-LO is another master enzyme in the AA cascade which produces inflammatory lipid mediators (LTs) upon stimulation in inflamed tissues. The present work should answer the question if Ssi targets the 5-LO pathway and should examine the molecular mechanisms behind Ssi-mediated 5-LO inhibiton. As COX-2 is upregulated during carcinogenesis and is inhibited by Ssi, further investigations should show regulatory effects of Ssi on 5-LO gene expression in MM6-cells and whether Sp1 as a common transcriptional factor is involved in such a regulation. As the use of NO-NSAIDs seem to be a promising strategy concerning their chemopreventive and gastroprotective effects compared to the parent NSAIDs, a possible interaction with the 5-LO pathway as a second, potent target should additionally be elucidated. In the first section it was demonstrated that the pharmacologically active metabolite of sulindac, Ssi, targets 5-LO. Ssi inhibited 5-LO in ionophore A23187- and LPS/fMLP-stimulated human PMNL (IC50 ≈ 8 -10 μM). Importantly, Ssi efficiently suppressed 5-LO in human whole blood at clinically relevant plasma levels (IC50 = 18.7 μM). Ssi was 5-LO-selective as no inhibition of related lipoxygenases (12-LO, 15-LO) was observed. The sulindac prodrug and the other metabolite, sulindac sulfone, failed to inhibit 5-LO. Mechanistic analysis demonstrated that Ssi directly suppresses 5-LO with an IC50 of 20 μM. Together, these findings may provide a novel molecular basis to explain the COX-independent pharmacological effects of sulindac under therapy. In the second part of the work dealing with the analysis of Ssi’s inhibitory mechanism on 5-LO it was presented that Ssi shows a lack of potency in cellular systems where membrane constituents are existent. The addition of microsomal fractions of PMNLto crude 5-LO enzyme were able to recover enzyme activity to ~ 100 %. Selectively 5-LO activity stimulating lipids like PC, participating in 5-LO membrane interactions within the regulatory C2-like domain of 5-LO, counteracted the Ssimediated inhibition on 5-LO-wt in a concentration-dependent manner. Lastly, a protein mutant lacking three trp resudies essential for linking the enzyme to nuclear membranes and deploying catalytic activity was not influenced by Ssi and shows enzyme activity in a cell-free assay. Ssi displays the first 5-LO inhibitor on the market interacting with the C2-like domain of the enzyme and therfore can stand for a novel lead structure of 5-LO inhibitors. An influence on 5-LO gene expression by Ssi could be detected in differentiated MM6-cells, described in the results chapter 3 (4.3). Ssi downregulated the 5-LO mRNA level after 72 hrs of incubation in differentiated MM6-cells to ~ 20 % of output control at concentrations of 10 μM. Concomitantly, mRNA levels of Sp1 were suppressed. Reporter gene studies revealed Sp1 most probably as a regulating agent involved in the Ssi-mediated 5-LO mRNA downregulation as co-transfection of increasing amounts of Sp1 could abrogate the effect. A ChIP assay could identify Sp1 as a critical transcriptional factor as Sp1 binding to the 5-LO promoter decreased in presence of Ssi. Lastly, three NO-NSADIs (NO-sulindac, NOnaproxen, NO-aspirin) were tested for the ability of 5-LO product inhibition. In intact PMNL, all compounds showed effective inhibition of 5-LO activity and NO-sulindac was most potent with an IC50 value of ~ 3 μM. NO-ASA inhibited 5-LO with IC50 values of ~ 30 μM and showed a non-competitive mode of action in cell-based assays. On human recombinant 5-LO all compounds again showed inhibitory potency whereas NO-sulindac again suppressed LT biosynthesis with an IC50 vaue comparable to intact cellular systems. Unfortunately, all inhibitors showed a loss of potency when tested for inhibition of 5-LO product synthesis in human whole blood as higher concentrations up to 100 μM were needed to reach at least 55 % enzyme inhibition. However, this strategy of 5-LO inhibition seems promising and needs further experimental approaches to gain more insight into the mechanism of 5-LO inhibition by NONSAIDs.
5-lipoxygenase (5-LO) catalyzes the first two steps in leukotriene (LT) biosynthesis. In a two step reaction the enzyme oxygenates arachidonic acid (AA) to form the highly unstable epoxide leukotriene A4 (LTA4) in dehydrating a hydroperoxide intermediate (20). LTA4 can then be further metabolized by two terminal synthases yielding either the potent chemoattractant leukotriene B4 (LTB4) or the cysteinyl leukotrienes (CysLTs). 5-LO enzyme expression is primarily found in mature leukocytes (22) where it can either reside in the cytoplasm or in the nucleus associated with euchromatin (29). Its enzymatic activity is embedded in a complicated network in intact cells regulating LT synthesis by various factors dependent on the cell type and nature of stimulus. Factors such as the amount of free AA released by phospholipase A2 enzymes, levels of enzymes involved, catalytic activity per enzyme molecule and availability of different small molecules influence 5-LO activity (36).
The 5-LO derived LTs are lipid mediators which were shown to primarily mediate inflammatory and allergic reactions and their role in the pathogenesis of asthma is well defined. CysLTs are among the most potent bronchoconstrictors yet studied in man and play an important role in airway remodeling. LTB4 has no bronchoconstrictory effects in healthy and asthmatic humans but displays potent chemoattractant properties on neutrophils and increases leukocyte adhesion to the vessel wall endothelium (22). Therefore, LTB4 enhances the capacity of macrophages and neutrophils to ingest and kill microbes. In concert with LTB4, histamine and prostaglandin E2 (PGE2) CysLTs are thought to maintain the tone of the human airways (82).
Besides their well studied role in asthma, 5-LO derived LTs have also been implicated to play a role in cardiovascular diseases and cancer. In contrast to healthy tissues, LT pathway enzymes and receptors were found to be abundantly expressed in cancer tissues, atherosclerotic lesions in the aorta, heart and carotid artery (86). Pharmacological inhibition of 5-LO potently suppressed tumour cell growth by inducing cell cycle arrest and triggering cell death via the intrinsic apoptotic pathway (92, 93). In several studies LTs were found to exhibit cardiovascular actions by promotion of plasma leakage in postcapillary venules, coronary artery vasoconstriction and impaired ventricular contraction leading to reduced coronary blood flow and cardiac output (24). Unfortunately, the precise molecular mechanisms through which LTs influence carcinogenesis and cardiovascular diseases are still incompletely understood.
In contrast, an increasing number of studies questions the correlation between 5-LO and cancer (95-97) since extreme LT concentrations were applied to induce proliferative effects in the majority of the publications. A few studies exist which show susceptibility towards 5-LO products in physiological concentrations or achieve anti-proliferation by applying low concentrations of 5-LO inhibitors (98) ...
The aim of this study is a better understanding of radiation processes in regional climate models (RCMs) in order to quantify their impact and to reduce possible errors. A first important task in finding an answer to this question was to examine the accuracy of the components of the radiation budget in regional climate simulations. To this end, the simulated radiation budgets of two regional climate simulations for Europe were compared with a satellite-based reference. In the simulations with the RCM COSMO-CLM there were some serious under- and overestimations of short- and long-wave net radiation in Europe. However, taking into account the differences in the reference datasets, the results of the COSMO-CLM were quite satisfactory.
Using statistical methods, the influence of potential sources of uncertainties was estimated. Uncertainties in the cloud cover and surface albedo had a significant impact on uncertainties in short-wave net radiation, the explained variance of uncertainties in cloud cover was two to three times higher than that of uncertainties in surface albedo. Uncertainties in the cloud cover resulted in significant errors in the net long-wave radiation. However, the influence of uncertainties in soil temperature on errors in the long-wave radiation budget was low or even negligible. These results were confirmed in a comparison with simulations of the REMO and ALADIN regional climate models. It is reasonable to expect that a better parameterization of relatively simple parameters such as cloud cover and surface albedo is a means of significantly improving the simulation of radiation budget components in the COSMO-CLM.
An important question for the application of RCMs is to examine whether the results of radiation uncertainties and their impact factors are comparable if the model is applied in a region that is not the one for which it was originally created. Comparisons of the simulated radiation budgets of different RCMs for West Africa showed that problems in the simulation of short- and long-wave radiation fluxes were a widespread problem. Most of the tested models showed some considerable under- or overestimation of the short- and long-wave radiation fluxes.
Similar to Europe uncertainties in cloud cover were also in the simulations for Africa a significant factor affecting uncertainties in the simulated radiation fluxes. However, for the African simulations uncertainties in the parameterization of surface albedo were much more important than in Europe. On average, overland uncertainties in the cloud cover and surface albedo were of similar importance. Uncertainties in soil temperature simulations were of higher importance in Africa, and reached overland similar values of the mean explained variance (R2 ≈ 0.2) such as uncertainties in the cloud cover. This indicates a geographical dependence of the model error. This study confirmed the assumption that an improved parameterization of relatively simple parameters such as the surface albedo in RCMs leads to a significant improvement in the modeled radiation budget, particularly in Africa.
The influence of errors in the simulated radiation budget components on the simulation of climate processes, such as the West-African monsoon (WAM), was investigated in a next step. The evaluation of ERA-Interim and ECHAM5 driven COSMO-CLM simulations for Africa showed that the main features of the WAM were well reproduced by the model, but there were only slight improvements compared to the driving data. The index of convective activity in the model simulations was much too high and precipitation was underestimated in large parts of tropical Africa. The partly considerable differences between the ERA-Interim and ECHAM5 driven simulations demonstrated the sensitivity of the RCM to the boundary conditions and in particular to the sea surface temperature. An excessive northwards shift of the monsoon in the model was influenced by the land-sea temperature gradient and the strength of the Saharan heat low. Consequently, a part of the error was due to the driving data and the model itself produced another part.
By modifying the parameterization of the bare soil albedo the errors in the radiation budget and 2 m temperature in the Sahara region were significantly reduced. Similarly, the overesti-mation of precipitation and convection has been reduced in the Sahel. The effect of this modifi-cation on the examined WAM area was low. This confirmed that especially in desert regions, errors in the surface albedo were a driving factor for errors in the radiation budget. However, there are other important factors not yet sufficiently understood that have a strong influence on the quality of the simulation of the WAM.
The analysis of the actual state, the quantification of error sources and the highlighting of connections made it possible to find means to reduce uncertainties in the simulated radiation in RCMs and to have a better understanding of radiation processes. However, the magnitude of the errors found, the number of possible influencing factors, and the complexity of interactions, indicate that there is still a need for further research in this area.
According to the World Health Organization (WHO) bacterial resistance to antibiotic drug therapy is emerging as a major public health problem around the world. Infectious diseases seriously threaten the health and economy of all countries. Hence, the preservation of the effectiveness of antibiotics is a world wide priority. The key to preserving the power of antibiotics lies in maintaining their diversity. Many microorganisms are capable of producing these bioactive products, the so called antibiotics. Specifically in microorganisms, polyketide synthases (PKS) and non-ribosomal peptide synthases (NRPS) produce these natural bioactive compounds. Besides being used as antibiotics these non-ribosomal peptides and polyketides display an even broader spectrum of biological activities, e.g. as antivirals, immunosuppressants or in antitumor therapy. The wide functional spectrum of the peptides and ketides is due to their structural diversity. Mostly they are cyclic or branched cyclic compounds, containing non-proteinogenic amino acids, small heterocyclic rings and other unusual modifications such as epimerization, methylation, N‐formylation or heterocyclization. It is has been shown that these modifications are important for biological activity, but little is known about their biosynthetic origin.
PKS and NRPS are multidomain protein assembly lines which function by sequentially elongating a growing polyketide or peptide chain by incorporating acyl units or amino acids, respectively. The growing product is attached via a thioester linkage to the 4’-phosphopantetheine (4’-Ppant) arm of a holo acyl carrier protein (ACP) in PKSs or holo peptidyl carrier protein (PCP) in NRPSs and is passed from one module to another along the chain of reaction centers. The modular arrangement makes PKS and NRPS systems an interesting target for protein engineering. More than 200 novel polyketide compounds have already been created by module swapping, gene deletion or other specific manipulations. Unfortunately, however, engineered PKS often fail to produce significant amounts of the desired products. Structural studies may faciliate yield improvement from engineered systems by providing a more complete understanding of the interface between the different domains. While some information about domain-domain interactions, involving the most common enzymatic modules, ketosynthase and acyltransferase, is starting to emerge, little is known about the interaction of ACP domains with other modifying enzymes such as methyltransferases, epimerases or halogenases.
To further improve the understanding of domain-domain interactions this work focuses on the curacin A assembly line. Curacin A, which exhibits anti-mitotic activity, is from the marine cyanobacterium Lyngbya majuscula. This outstanding natural product contains a cyclopropane ring, a thiazoline ring, an internal cis double bond and a terminal alkene. The biosynthesis of curacin A is performed by a 2.2 Mega Dalton (MDa) hybrid PKS-NRPS cluster. A 10-enzyme assembly catalyzes the formation of the cyclopropane moiety as the first building block of the final product. Interestingly, for these enzymes the substrate is presented by an unusual cluster of three consecutive ACPs (ACPI,II,III). Little is known about the function of multiple ACPs which are supposed to increase the overall flux for enhanced production of secondary metabolites.
The first task in this work was to elucidate the structural effect of the triplet ACP repetition by nuclear magnetic resonance (NMR). The initial data show that the excised ACPI, ACPII or ACPIII proteins resulted in [15N, 1H]-TROSY spectra with strong chemical shift perturbations (CSPs), suggesting an effect on the structure. The triplet ACP domains display a high sequence identity (93- 100%) making structural investigation using usual NMR techniques due to high peak overlap impossible. To enable the investigation of the triplet ACP in its native composition we developed a powerful method, the three fragment ligation. Segmental labeling allows incorporating isotopes into one single domain in its multidomain context. As a result we could prepare the triplet ACP with only one domain isotopically labeled and therefore assign the full length protein. In this way our method paved the way to study the structural effects of the triplet ACP repetition. We could show unexpectedly, that, despite the fact that the triplet repeat of CurA ACPI,II,III has a synergistic effect in the biosynthesis of CurA, the domains are structurally independent.
In the second part of this work, we studied the structure of the isolated ACPI domain. Our results show that the CurA ACPI undergoes no major conformational changes upon activation via phosphopantetheinylation and therefore contradicts the conformational switching model which has been proposed for PCPs. Further we report the NMR solution structures of holo-ACPI and 3-hydroxyl-3-methylglutaryl (HMG)-ACPI. Data obtained from filtered nuclear overhauser effect (NOE) experiments indicate that the substrate HMG is not sequestered but presented on the ACP surface.
In the third part of this work we focussed on the protein-protein interactions of the isolated ACPI with its cognate interaction partners. We were especially interested in the interaction with the halogenase (Cur Hal), the first enzyme within the curacin A sub-cluster, acting on the initial hydroxyl-methyl-glutaryl (HMG) attached to ACPI. Primarily we studied the interaction using NMR titration and fluorescence anisotropy measurements. Surprisingly no complex between ACPI and Cur Hal could be detected. The combination of an activity assay using matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy and mutational analysis revealed several amino acids of ACPI that strongly decrease the activity of CurA Hal. Mapping these mutations according to their effect on the Cur Hal activity onto the structure of HMG-ACPI displays that these amino acids surround the substrate and form a consecutive surface. These results suggest that this surface is important for Cur Hal recognition and selectivity. Our research presented herein is an excellent example for protein-protein interactions in PKS systems underlying a specific recognition process.
Membrane proteins (MPs) constitute about 30% of the genome and are essential in many cellular processes. In particular structural characterisation of MPs is challenged by their hydrophobic nature resulting in expression difficulties and structural instability upon extraction from the membrane. Despite these challenges, progress in sample preparation and the techniques to solve MP structures has led to 281 unique MP structures as of January 2011. Through the combination of a cell-free expression system and selective labelling strategies, this thesis aimed to advance the structure determination of α-helical MPs by NMR spectroscopy and resulted in the structure determination of a seven-ransmembrane-helix protein. Results were obtained for the 5-lipoxygenase-activating protein (FLAP) and proteorhodopsin (PR). The detergent-based cell-free expression mode proved most efficient for production of both targets, but optimisation of FLAP and PR followed different routes. The presence of a retinal cofactor in PR greatly facilitated the search for an appropriate hydrophobic environment. For structural studies, NMR spectra of FLAP indicated favourable properties of the lysolipid LPPG. In contrast, PR was stable and homogenous in the short-chain lipid diC7PC. As NMR spectra of α-helical MPs are generally characterised by broad lines and signal overlap, selective labelling strategies were essential in the assignment process of both targets. For the backbone assignment of FLAP the transmembrane segment-enhanced (TMS) labelling was developed, employing the six amino acids AFGILV. These residues cluster predominantly in transmembrane helices and form long stretches allowing a large extent of backbone assignment. Besides that, the combinatorial labelling enables identification of unique pairs in the sequence based on a mixture of 15N and 1-13C-labelled amino acids. To find the optimal labelling pattern for a given primary structure, the UPLABEL algorithm has been made available and successfully applied in the backbone assignment of PR. Both selective labelling approaches greatly benefitted from the use of a cell-free expression system to reduce isotope scrambling. Additionally, the de novo structure of PR was determined with an average backbone rmsd of 1.2 Å based on TALOS-derived backbone torsion angles, intrahelical hydrogen bond restraints and distance restraints from the NOE and paramagnetic relaxation enhancement (PRE). A major bottleneck in the NMR structure determination of MPs concerns the number of long-range distances which are often limited. In PR, side chain assignment was enabled by stereo-array isotope labelling as well as selective labelling which provided 33 long-range NOEs. These NOEs stabilised the symmetry of the seven helix bundle. With a total number of 1031, the majority of long-range distances were derived from PREs. The structure of PR reveals differences to its homologues such as the absence of an anti-parallel β-sheet between helices B and C and allows conclusions towards the mechanism of colour tuning.
The role of small leucine-rich proteoglycans, biglycan and decorin, in podocytopathy and albuminuria
(2011)
Biglycan is a member of the small leucine-rich proteoglycan (SLRP) family and is involved in the assembly of extracellular matrix components. In macrophages soluble biglycan acts as an endogenous ligand of the innate immunity receptors TLR2 and TLR4. Data addressing the role of biglycan in renal pathology are surprisingly limited. In a normal kidney, biglycan is expressed mainly in the tubulointerstitium; however, in the course of various renal diseases its expression may be altered. The biological role and mechanisms of biglycan action in the pathology of renal diseases, especially those affecting glomeruli, remain poorly understood.
Albuminuria is the first detectable clinical abnormality in diabetic nephropathy. In this study we detected increased biglycan mRNA expression in glomeruli of renal biopsies of patients with incipient diabetic nephropathy, with predominant localization in podocytes. This novel finding raised the question about the role and mechanisms of biglycan action in diabetic podocyte injury and whether the mechanisms of biglycan signaling causing podocyte injury and albuminuria could be extrapolated to other glomerular diseases.
To investigate the role of biglycan in the cause of diabetic podocyte injury and albuminuria we used the murine model of STZ-induced diabetic nephropathy and wild type (Bgn+/0) and biglycan deficient (Bgn-/0) mice. We observed that biglycan was expressed on mRNA and protein levels in podocytes of diabetic Bgn+/0 mice and that diabetic Bgn+/0 mice also had significantly higher albuminuria compared to non-diabetic mice 6 and 12 weeks after disease induction. Biglycan deficiency was shown to be an important factor in albuminuria development. Namely, we observed that diabetic Bgn-/0 mice had significantly lower levels of urinary albumin compared to diabetic Bgn+/0 mice. We showed that less severe podocyte loss in the urine of diabetic Bgn-/0 mice was associated with significantly higher nephrin and podocin glomerular expression compared to diabetic Bgn+/0 mice. Our data suggested that biglycan deficiency was protective against podocyte loss into urine and might be beneficial against development of albuminuria in diabetes.
Biglycan contributed to podocyte actin rearrangement due to increased phosphorylation of Rac1 in vitro. Furthermore, biglycan induced caspase-3 activity and production of reactive oxygen species (ROS), thus enhancing apoptosis in cultured podocytes. Biglycan-induced ROS generation was TLR2/TLR4-dependent. Overexpression of soluble biglycan in wild type mice induced albuminuria under normal conditions and significantly increased albuminuria under pathological conditions (murine model of LPS-induced albuminuria). Inhibition of Rac1 activity in vivo decreased the albuminuria induced by biglycan overexpression. In patients with glomerular diseases, biglycan was detected in urine and was associated with nephrin appearance in the urine of these patients and with increased albuminuria. Collectively, our results elucidate a novel mechanism for biglycan-induced TLR2- and TLR4-dependent, Rac1- and ROS-mediated podocytopathy leading to podocyturia, albuminuria development and progression of glomerular diseases. Interfering with biglycan actions and blocking its signaling via TLR2 and TLR4 might be a potential therapeutic strategy against these diseases. To achieve this goal, the specific mechanisms for binding of biglycan to TLR2 and TLR4 must be elucidated and effective ways of preventing this binding must be developed. Nevertheless, biglycan remains the “danger signal” that activates innate immune receptors in non-immune cells and triggers the deleterious mechanisms leading to aggravation of renal injury.
Which factors determine whether a stimulus is consciously perceived or unconsciously processed? Here, I investigate how previous experience on two different time scales – long term experience over the course of several days, and short term experience based on the previous trial – impact conscious perception. Regarding long term experience, I investigate how perceptual learning does not only change the capacity to process stimuli, but also the capacity to consciously perceive them. To this end, subjects are trained extensively to discriminate between masked stimuli, and concurrently rate their subjective experience. Both the ability to discriminate the stimuli as well as subjective awareness of the stimuli increase as a function of training. However, these two effects are not simple byproducts of each other. On the contrary, they display different time courses, with above chance discrimination performance emerging before subjective experience; importantly, the two learning effects also rely on different circuits in the brain: Moving the stimuli outside the trained receptive field size abolishes the learning effects on discrimination ability, but preserves the learning effects on subjective awareness.
This indicates that the receptive fields serving subjective experience are larger than the ones serving objective performance, and that the channels through which they receive their information are arranged in parallel. Regarding short term experience, I investigate how memory based predictions arising from information acquired on the trial before affect visibility and the neural correlates of consciousness. To this end, I vary stimulus evidence as well as predictability and acquire electroencephalographic data.
A comparison of the neural processes distinguishing consciously perceived from unperceived trials with and without predictions reveals that predictions speed up processing, thus shifting the neural correlates forward in time. Thus, the neural correlates of consciousness display a previously unappreciated flexibility in time and do not arise invariably late as had been predicted by some theorists.
Admittedly, however, previous experience does not always stabilize perception. Instead, previous experience can have the reverse effect: Seeing the opposite of what was there, as in so-called repulsive aftereffects. Here, I investigate what determines the direction of previous experience using multistable stimuli. In a functional magnetic resonance imaging experiment, I find that a widespread network of frontal, parietal, and ventral occipital brain areas is involved in perceptual stabilization, whereas the reverse effect is only evident in extrastriate cortex. This areal separation possibly endows the brain with the flexibility to switch between exploiting already available information and emphasizing the new.
Taken together, my data show that conscious perception and its neuronal correlates display a remarkable degree of flexibility and plasticity, which should be taken into account in future theories of consciousness.
Ubiquitin is a highly conserved protein involved in several cellular processes like protein degradation, endocytosis, signal transduction and DNA repair. The discovery of ubiquitin-like proteins (UBL) and ubiquitin-like domains (ULD) increases the number of regulation pathways where the property of the ubiquitin-fold is profitable.
Autophagy is the catabolic pathway used in cells to deliver cytosolic components and dysfunctional organelles to the lysosome for degradation. MAP1LC3 proteins are ubiquitin-like proteins involved in one hand for the expansion of the autophagosome, which sequesters cytosolic substrates. In the other hand, these proteins (LC3- and GABARAP- subfamilies) bind to autophagic receptors linked to polyubiquitinated proteins aggregates. For this project, the 3D structure of the GABARAPL-1/NBR1-LIR complex was determined and confirmed that GABARAPL-1 belongs to the MAP1LC3 proteins family, structurally characterized by an ubiquitin-fold, consisting of a central beta-sheet formed by four beta-strands and two alpha-helices on one side of the beta-sheet, preceded N terminally by two alpha-helices, resulting in the formation of two hydrophobic pockets, hp1 and hp2. The autophagic receptor NBR1 interacts with GABARAPL-1 through the hp1 and hp2 with its LIR motif taking an extended beta conformation upon binding, forming an intermolecular beta-sheet with the second beta-strand of GABARAPL 1. This LC3- interacting region (LIR) consists of an Theta XX Gamma sequence preceded by acidic amino acids, with Theta and Gamma represented by any aromatic and hydrophobic residues, respectively. Interaction studies of the LIR domains of p62, Nix and NBR1 with different members of the MAP1LC3 proteins family indicate that the presence of a tryptophan in the LIR motif increases the binding affinity. Substitution to other aromatic amino acids or increasing the number of negatively charged residues at the N-terminus of the LIR motif, however, has little effect on the binding affinity due to enthalpy-entropy compensation, suggesting that effector proteins can interact with a wide variety of different sequences with similar and moderate binding affinities.
Additionally to be present in proteins dealing with protein folding and degradation, ubiquitin-like domain were found protein involved in the regulation of signal transduction like TBK1, a serine/threonine kinase responsible for induction of immune response. In this second project, based on the NMR chemical shifts of the TBK1 domain contained between amino acids 302 and 383, secondary structure prediction programs (TALOS and CSI) confirmed the presence of an Ubiquitin-like domain in TBK1 by identifying one alpha-helix and four beta-strands sequentially aligned like following beta-beta-alpha-beta-beta. This alignment corresponds perfectly with the secondary structure elements of Ubiquitin and proved that TBK1_ULD belongs to the UBL protein superfamily. The similarity to ubiquitin was even bigger by the presence in addition of a small beta-strand and a short helix, which are observed as the beta 5-strand and a 310-helix in Ubiquitin, respectively. The first attempts on the 3D structure determination confirmed the Ub-fold but due to the lack of assignment in TBK1_ULD, only a structure based on ubiquitin as a model was determined. Interaction studies of TBK1_ULD with the IAD-SRR domain of IRF3 showed that both side of the molecule seems involved and that the TBK1/IRF3 interaction is more complex than a one to one binding process. Unfortunately, the instability of TBK1_ULD associated to the difficulty in the purification of IAD-SRR did not allow to further study this interaction more precisely.
Finally, to overcome the difficulty encountered in NMR experiments because of low expression and/or poor solubility, an expression vector using the intrinsic property of ubiquitin was designed. Fused to proteins or peptides targets, this construct produced proteins and peptides in a larger amount than with traditional expression vectors and also with a less cost than chemical synthesis for pure labeled peptides for NMR structural studies. The presence of a hexa histidine tag was useful for the isolation and the purification of the constructs. The existence of a TEV cleavage site was created to keep the possibility of releasing the ubiquitin moiety from the expressed protein or peptide. Moreover, the ubiquitin-tag could also still be attached to the protein/peptide of interest when biophysical methods like NMR, ITC or CD spectroscopy are applied, providing the same results than for the protein/peptide moiety alone.
Agriculture of crops provides more than 85% of the energy in human diet, while also securing income of more than 2.6 billion people. To investigate past, present and future changes in the domain of food security, water resources and water use, nutrient cycles, and land management it is required to know the agricultural land use, in particular which crop grows where and when. The current global land use or land cover data sets are based on remote sensing and agricultural census statistics. In general, these only contain one or very few classes of agricultural land use. When crop-specific areas are given, no distinction of irrigated and rainfed areas is made, whereas it is necessary to distinguish rainfed and irrigated crops, because crop productivity and water use differ significantly between them.
To support global-scale assessments that are sensitive to agricultural land use, the global data set of Monthly Irrigated and Rainfed Crop Areas around the year 2000 (MIRCA2000) was developed by the author. With a spatial resolution of 5 arc-minutes (approximately 9.2 km at the equator), MIRCA2000 provides for the first time, spatially explicit irrigated and rainfed crop areas separately for each of the 26 crop classes for each month of the year, and includes multi-cropping. The data set covers all major food crops as well as cotton, while the remaining crops are grouped into three categories (perennial, annual and fodder grasses). Also for the first time, crop calendars on national or sub-national level were consistently linked to annual values of harvested area at the 5 arc-minutes grid cell level, such that monthly growing areas could be computed that are representative for the time period 1998 to 2002.
The downscaling algorithm maximizes the consistency to the grid-based input data of cropland extent [Ramankutty et al., 2008], crop-specific total annual harvested area [Monfreda et al., 2008], and area equipped for irrigation [Siebert et al., 2007]. In addition to the methodology, this dissertation describes differences to other datasets and standard scaling methods, as well as some applications. For quality assessment independent datasets and newly developed quality parameters are used, and scale effects are discussed.
Supplementary Appendices document crop calendars for irrigated and rainfed crops for each of the 402 spatial units (Appendix I), data sources of harvested area and of cropping periods for irrigated crops, country by country (Appendix K), as well as data quality parameters (Appendix L, including spreadsheet files).
It has been estimated that about 1% of live births carry severe congenital heart defects and 20-30% among them have valve malformations. Despite its medical importance the underlying cause of many valvular diseases remains undiscovered. Thus, it is important to identify genes that play a crucial role in cardiac valve formation and maturation.
A temporal RNA expression analysis of heart development suggested that the extracellular matrix protein Nephronectin might be a novel regulator of valve development and/or trabeculation. Nephronectin is transiently expressed during rat heart development at the time of heart valve morphogenesis and trabeculation. Moreover, the extracellular matrix is known to be crucial for organogenesis. It is a complex, dynamic and critical component that regulates cell behavior by modulating the activity, bioavailability, or presentation of growth factors to cell surface receptors.
In order to verify the hypothesis that Nephronectin is a novel regulator of valve formation and/or trabeculation the zebrafish was chosen as model system. Females are able to spawn at intervals of 5 days laying hundreds of eggs in each clutch. Development progresses rapidly with precursors to all major organs appearing within 36 hours post fertilization. Zebrafish embryos develop externally, are translucent and continue to grow for several days despite developing severely malformed, non functional hearts. In addition, gene expression can be easily modulated. During the present study it has been shown that Nephronectin expression is correlated to valve development and trabeculation. Morpholinomediated knockdown of Nephronectin in zebrafish caused failure of valve formation and trabeculation resulting in > 85% lethality at 7 days post fertilization.
Cardiac valve formation is initiated at the junction of atrium and ventricle and is characterized by extracellular matrix deposition and endocardial cell differentiation. In accordance with the above-described phenotype the earliest observed abnormality in Nephronectin morphants was an extended tube like structure at the atrio-ventricular boundary. In addition, the expression of myocardial genes involved in cardiac valve formation (cspg2, fibulin1, tbx2b, bmp4) was expanded and endocardial cells along the extended tube like structure exhibited characteristics of atrio-ventricular cells (has2, notch1b and Alcam expression, cuboidal cell shape). Inhibition of has2 in Nephronectin morphants rescued the endocardial but not the myocardial expansion. In contrast, diminishment of BMP signaling in npnt morphants resulted in reduced ectopic expression of myocardial and endocardial atrio-ventricular markers. Taken together, these results identify Nephronectin as a novel upstream regulator of BMP4-HAS2 signaling playing a crucial role in atrio-ventricular canal differentiation.
This thesis combines behavioral and cognitive approaches regarding the Web for analyzing users' behavior and supposed interests.
The work is placed in a new field of research called Web Science, which includes, but is not restricted to, the analysis of the World Wide Web. The term Web Science is affected by Tim Berners-Lee et al., who invited the researchers to "create a science of the web" [BLHH+06a]. The thesis is structured in two parts, reflecting the intersection of disciplines that is required for Web Science.
The first part is related to computer science and information systems. This part defines the Gugubarra concepts and algorithms for web user profiling and builds upon the results by Mushtaq et al. [MWTZ04]. This profiling aims at understanding the behavior and supposed interests of users. Based on these concepts, a framework was implemented to support the needs of web site owners. The core technologies used are Java, Spring, Hibernate, and content management systems. The design principles, architecture, implementation, and tests of the prototype are reported.
The second part is directly related to behavioral economics and is connected to the areas of economics, mathematics, and psychology. This part contributes to behavior models, as was claimed by Tim Berners-Lee et al.: "Though individual users may or may not be rational, it has long been noted that en masse people behave as utility maximisers. In that case, understanding the incentives that are available to web users should provide methods for generating models of behaviour..."[BLHH+06b]. The focus here is on studies that investigate the user's choice of online information services in a multi-attribute context. The introduced research framework takes into account background and local context effects and builds upon theoretical foundations by Tversky and Kahneman [TK86]. The findings provide useful insights to behavioral scientists and to practitioners on how to use framing strategies to alter the user's choice.
The role of peroxisome proliferator-activated receptor gamma during sepsis-induced lymphopenia
(2011)
Sepsis is one of the most common diseases on intensive care units all over the world and accounts there for the highest mortality rate. One of the hallmarks of sepsis is an accelerated T-cell apoptosis, resulting in a compromised immune state with the inability to eradicate pathogens. This promotes organ damage or even organ failure. A multiple organ dysfunction evolves, which often ends up in septic shock and death. Recently, it was shown that severe T-cell depletion correlates with sepsis mortality. When inhibiting T-cell apoptosis, an increased mouse survival was observed in experimental sepsis. ...
This dissertation consists of three essays, which study the implication of financial frictions in business cycles and monetary policy making. The first essay develops a Dynamic Stochastic General Equilibrium (DSGE) model to study how the instability of the banking sector can amplify and propagate business cycles. Model simulations show that in an economic down turn, in addition to credit demand contraction induced by low firm net worth, low bank capital
position can create strong credit supply contraction, and have a quantitatively significant effect on business cycle dynamics. The second essay studies the optimal Taylor-type monetary policy rules based on the model developed in the first chapter and find that with interest rate smoothing, 'leaning against the wind' can significantly dampen the procyclicality of financial distortions, and increase the welfare of the economy. The third chapter examines the role of households frugality in a financial crisis and finds that higher savings by more frugal households provide an important cushion for the fall in private investment funding.
Owing to long-term similarities with regard to orbital climate forcing (i.e., low eccentricity and a dampened influence of precession), Marine Isotope Stage (MIS) 11 represents one of the closest astronomical analogues for present and future climate. Hence, insights into the climate variability of MIS 11 can contribute to a better understanding of the climatic evolution of the present (Holocene) interglacial as it would occur without human interference. In order to elucidate the natural climate variability during MIS 11, this study examines predominantly annually laminated lake sediments of Holsteinian age from Dethlingen, northern Germany. The Holsteinian interglacial is widely accepted to be the terrestrial equivalent of MIS 11c in central Europe and can be biostratigraphically correlated with the Hoxnian, Mazovian and Praclaux interglacials on the British Isles, in Poland and in France, respectively. These correlations yield the potential to cross-check the results from individual sites on a regional scale. This study is based on a multi-proxy approach including palynological, micropaleontological, sedimentological, geochemical and time series analyses within a wellconstrained chronological framework that has been established through varve counting and regional bio-stratigraphic correlations with other annually laminated archives of Holsteinian age. In particular, the here-presented study aims at (i) fingerprinting the long-term (centennial- to millennial-scale) and short-term (sub-decadal- to decadal-scale) climate variability during the Holsteinian interglacial, (ii) deciphering the nature, tempo and trigger mechanisms of abrupt climate change under interglacial boundary conditions, and (iii) assessing its impact on terrestrial ecosystems. With regard to long-term climate variability, the vegetation succession at Dethlingen as inferred from pollen data provides insights into the mesocratic to telocratic forest phases of a glacial-interglacial cycle spanning ~11500 (± 1000) years of the 15-16-ka-long Holsteinian interglacial. The development of temperate mixed forests suggests a general prevalence of mild climatic conditions during the Holsteinian. The older parts of the interglacial are characterised by the strong presence of boreal tree taxa (e.g., Picea), whereas the younger parts of the interglacial are marked by the expansion of sub-Atlantic to Atlantic forest elements (e.g., Abies, Buxus, Ilex, Quercus) and the decline of boreal tree taxa. This vegetation succession suggests a general warming trend and decreasing seasonality over the course of the Holsteinian interglacial. Based on the maximum pollen abundances of indicator tree taxa (e.g., Buxus and Quercus), peak warmth was reached during the later stages of the interglacial; it was accompanied by high humidity. The forest succession of the Holsteinian interglacial was punctuated by abrupt and gradual changes in the abundances of temperate plant taxa. These vegetation changes indicate considerable intra-interglacial climate variability. In particular, two marked declines of temperate taxa leading to the transient development of boreal and sub-boreal forests were triggered by centennial-scale climate oscillations, here termed Older and Younger Holsteinian Oscillations (OHO and YHO). These oscillations occurred ~6000 and ~9000 years after the onset of the interglacial pioneer forestation in central Europe, respectively. To assess the impact of abrupt climate change on terrestrial ecosystems during the Holsteinian and to investigate the underlying driving mechanisms, the intervals spanning the OHO and the YHO at Dethlingen were subjected to decadal-scale palynological and sedimentological analyses. Based on these data, the OHO comprises a 90-year-long decline of temperate taxa associated with expansion of Pinus and non-arboreal pollen, and a subsequent 130-year-long recovery of temperate taxa marked by the pioneer expansion of Betula and Alnus. Owing to its highly characteristic imprint on vegetation dynamics, the OHO can be identified in pollen records from the central European lowlands north of 50º latitude, from the British Isles to Poland. A close inspection of individual pollen records from that region reveals the prevalence of colder winters during the OHO, with a gradient of decreasing temperature and moisture availability, and increased continentality towards eastern Europe. This climate pattern points to a weakened influence of the westerlies and/or stronger influence of the Siberian High connected to the OHO. The vegetation dynamics during the YHO are characterised by a decline of temperate taxa (particularly of Carpinus) and the expansion of pioneer trees (mainly Betula). In contrast to the OHO, frost-sensitive taxa (e.g., Ilex, Buxus and Hedera) continued to thrive. This suggests that mean winter temperatures remained relatively high (>0 ºC) during the YHO pointing to a decrease of summer warmth related to the climatic deterioration. The YHO, which has a duration on the order of 300 years, is centered within a long-term (~1500-year) decline and subsequent, millennial-scale recovery of temperate taxa. Because the impact of the OHO and the YHO on the vegetation at Dethlingen was markedly different, both short-term climate oscillations may have been caused by different trigger mechanisms. For the OHO, the inferred regional-scale winter cooling over central Europe lasting for several decades points to a decrease in ocean heat transport, most likely related to a transient slowdown in North Atlantic Deep Water formation. This view is supported by the strong resemblance of the OHO to the 8.2 ka event of the Holocene with regard to the duration, imprint on terrestrial ecosystems, spatial pattern of the climatic impact, timing within the respective interglacial, and prevailing interglacial boundary conditions. In contrast, the presence of frost-sensitive taxa during the YHO appears to exclude a reduction in oceanic heat transport as postulated for the OHO. Instead, the long-lasting, gradual changes in the abundances of temperate taxa suggest a connection to orbital forcing, with the triggering mechanism causing the centennial-scale vegetation setback itself remaining unclear. The characteristics of short-term climate variability were investigated based on microfacies and time series analyses of a ~3200-year-long, annually laminated window of the Dethlingen record. The annual laminations at Dethlingen comprise biogenic varves consisting of two discrete sub-layers. The light layers, which are controlled by the intensity of diatoms blooms during spring/summer, reflect changes in the productivity of the Dethlingen palaeolake. In contrast, the dark layers, which consist predominantly of amorphous organic matter and fragmented diatom frustules, represent sediment deposition during autumn/winter. Spectral analyses of the thicknesses of the light and dark layers have revealed several peaks exceeding the 95% and 99% confidence levels that are near-identical to those known from modern instrumental data and Holocene records. Decadal-scale signals at periods of 90, 25, and 10.5 years are likely associated with the 88-, 22- and 11-year solar cycles; hence, solar activity appears to have been a forcing agent in productivity changes of the Dethlingen palaeolake. Sub-decadal-scale signals at periods between 3 and 5 years and ~6 years may reflect an influence of the El Niño-Southern Oscillation (ENSO) and the North Atlantic Oscillation (NAO) on varve formation during winter.
Blood vessel formation is a well orchestrated process where multiple components including different cells types, growth factors as well as extracellular matrix proteins act in synergistic and highly regulated manner to support the growth of new blood vessels. During embryonic development this process is marked as vasculogenesis and entails the differentiation of mesodermal cells into angioblasts and their subsequent fusion into a primitive vascular plexus. Angiogenesis, in contrast, describes the formation of new vessels from the pre-existing vasculature and it occurs in the embryo during remodeling of the primitive plexus into a mature vascular network. Furthermore, in the adult, angiogenic processes play a role in various physiological and pathological conditions. Angiogenesis is governed by a set of factors and molecular mechanisms whose identification has been a major focus of cardiovascular research for the past several decades. Most recently, Epidermal growth factor-like domain 7 (EGFL7) has been described as a novel molecular player in this context. This secreted protein is produced by endothelial cells and has been implicated in vessel development. Studies performed in zebrafish revealed an important role for EGFL7 in lumen formation during vasculogenesis although the underlying molecular mechanism has not been elucidated yet. In contrast, the investigation of EGFL7’s functions during angiogenic sprouting has faced several challenges and the role of EGFL7 in angiogenesis remained elusive. The purpose of this thesis was to identify the functions of EGFL7 during angiogenic mode of vessel formation in a systematic fashion using numerous in vitro as well as in vivo approaches.
Previously it has been suggested that EGFL7 might associate with the extracellular matrix from where it could exert its effects. Indeed, we could show that EGFL7 accumulates on the outer surface of endothelial cells in vivo by demonstrating its co-localization with collagen IV, a major constituent of the basal lamina. Furthermore, after its secretion to the extracellular matrix (ECM), EGFL7 seemed to interact with some components of the extracellular matrix including fibronectin and vitronectin, but not collagens and laminin.
A major group of receptors that mediate the interaction between the cells and the ECM are integrin receptors. Our co-immunoprecipitation studies revealed that EGFL7 associated with integrin αvβ3 which is highly expressed in endothelial cells and known to be important for vessel growth. Importantly, this EGFL7-αvβ3 integrin interaction was dependent on Arg-Gly-Asp (RGD) motif present within the second EGF-like domain of EGFL7 protein. Adhesion assays performed with human umbilical vein endothelial cells (HUVEC) revealed that EGFL7 promoted endothelial cell adhesion compared to BSA used as a negative control, however, adhesion seemed to be less efficient as compared to bona fide ECM proteins such as fibronectin and vitronectin. In addition, cultivation of endothelial cells on EGFL7 was characterized by the absence of mature focal adhesions and stress fibers, but was paralleled by increased phosphorylation of kinases typical for integrin activation signaling cascade such as FAK, Src and Akt. This led us to the hypothesis that EGFL7 creates an environment that supports a motile phenotype of endothelial cells by serving as a modulator of existing interactions between the cells and the surrounding matrix. Indeed, EGFL7 increased random migration of HUVEC on fibronectin in an αvβ3 integrin dependent manner as shown using a live cell imaging platform. Most importantly, this was paralleled by a decrease in endothelial cell adhesion to fibronectin which is consistent with previous reports on secreted proteins that support a medium strength of adhesion and such promote cellular migration. To assess the overall effect of EGFL7 on the process of blood formation several in vitro and in vivo approaches were employed. First, the addition of EGFL7 to Matrigel injected subcutaneously into mice significantly increased the invasion of endothelial cells into the plugs. Second, a spheroid-based sprouting assay in three-dimensional collagen matrix clearly demonstrated the ability of EGFL7 to support angiogenic sprouting in an integrin dependent manner. This is consistent with the observed effects of EGFL7 on endothelial cell migration. Third, using in vivo assays such as the chick chorioallantoic membrane (CAM) assay as well as a zebrafish model system we were able to validate the importance of the EGFL7-integrin interaction for the process of angiogenesis in vivo. Taken together, I identified some of the major cellular functions EGFL7 modulates during angiogenesis. In addition, with integrin αvβ3 I unraveled a novel interaction partner of EGFL7 that delivers a mechanistical explanation for EGFL7’s effects on blood vessel formation. Most importantly, data presented in this PhD thesis contribute substantially to the existing literature on EGFL7 unambiguously assigning a role for this protein in the process of angiogenesis.
Nanotechnology is a rapidly developing branch of science, which is focused on the study of phenomena at the nanometer scale, in particular related to the possibilities of matter manipulation. One of the main goals of nanotechnology is the development of controlled, reproducible, and industrially transposable nanostructured materials.
The conventional technique of thin-film growth by deposition of atoms, small atomic clusters and molecules on surfaces is the general method, which is often used in nanotechnology for production of new materials. Recent experiments show, that patterns with different morphology can be formed in the course of nanoparticles deposition process on a surface. In this context, predicting of the final architecture of the growing materials is a fundamental problem worth studying.
Another factor, which plays an important role in industrial applications of new materials, is the question of post-growth stability of deposited structures. The understanding of the post-growth relaxation processes would give a possibility to estimate the lifetime of the deposited material depending on the conditions at which the material was fabricated. Controllable post-growth manipulations with the architecture of deposited structures opens new path for engineering of nanostructured materials.
The task of this thesis is to advance understanding mechanisms of formation and post-growth evolution of nanostructured materials fabricated by atomic clusters deposition on a surface. In order to achieve this goal the following main problems were addressed:
1. The properties of isolated clusters can significantly differ from those of analogous clusters occurring on a solid surface. The difference is caused by the interaction between the cluster and the solid. Therefore, the understanding of structural and dynamical properties of an atomic cluster on a surface is a topic of intense interest from the scientific and technological point of view. In the thesis, stability, energy, and geometry of an atomic cluster on a solid surface were studied using a liquid drop approach which takes into account the cluster-solid interaction. Geometries of the deposited clusters are compared with those of isolated clusters and the differences are discussed.
2. The formation scenarios of patterns on a surface in the course of the process of cluster deposition depend strongly on the dynamics of deposited clusters. Therefore, an important step towards predicting pattern morphology is to study dynamics of a single cluster on a surface. The process of cluster diffusion on a surface was modeled with the use of classical molecular dynamics technique, and the diffusion coefficients for the silver nanoclusters were obtained from the analysis of trajectories of the clusters. The dependence of the diffusion coefficient on the system’s temperature and cluster-surface interaction was established. The results of the calculations are compared with the available experimental results for the diffusion coefficient of silver clusters on graphite surface.
3. The methods of classical molecular dynamics cannot be used for modeling the self-assembly processes of atomic clusters on a surface, because these processes occur on the minutes timescale, what would require an unachievable computer resource for the simulation. Based on the results of molecular dynamics simulations for a single cluster on a surface a Monte-Carlo based approach has been developed to describe the dynamics of the self-assembly of nanoparticles on a surface. This method accounts for the free particle diffusion on a surface, aggregation into islands and detachment from these islands. The developed method is allowed to study pattern formation of structures up to thousands nm, as well as the stability of these structures. Developed method was implemented in MBN Explorer computer package.
4. The process of the pattern formation on a surface was modeled for several different scenarios. Based on the analysis of results of simulations was suggested a criterion, which can be used to distinguish between different patterns formed on a surface, for example: between fractals or compact islands.This criteria can be used to predict the final morphology of a growing structure.
5. The post-growth evolution of patterns on a surface was also analyzed. In particular, attention in the thesis is payed to a systematical theoretical analysis of the post-growth processes occurring in nanofractals on a surface. The time evolution of fractal morphology in the course of the post-growth relaxation was analyzed, the results of these calculations were compared with experimental data available for the post-growth relaxation of silver cluster fractals on graphite substrate.
All the aforementioned problems are discussed in details in the thesis.
Savannas are the most important timber and non-timber forest products (NTFPs) providing ecosystems in West Africa. They have been shaped by traditional human land-use (i.e. agriculture, grazing, and harvesting) for thousands of years. In the last decades, land-use has drastically changed due to the rapid population growth and the growing production of cash-crop in West Africa and this process is still continuing. The percentage of land intensively used for agriculture has increased, while the length of fallow periods has decreased. Such changes have enormous ecological, economic, and social consequences. In the context of land-use changes, there is an urgent need to better understand and evaluate the impact of land-use on savannas. Such an understanding provides insights on appropriate management activities that ensure the maintenance of savannas and guarantee the availability of savanna products for subsistence and commercial use of rural West African people.
The major objective of the present thesis was to study the impact of land-use on savanna vegetation and diversity as well as on populations of two important NTFP-providing tree species in a semi-arid area in West Africa. The study area was located in the south-eastern part of Burkina Faso and comprised the protected W National Park and its adjacent communal area.
In the first study (chapter 2), I investigated in cooperation with a colleague from Burkina Faso (Blandine Nacoulma) the impact of land-use on the savanna vegetation. We analyzed which environmental factors determine the occurrence of the vegetation types and investigated the effect of land-use on vegetation structure and the occurrence of life forms and highly valued tree species. Furthermore, we tested whether land-use has an impact on plant diversity pattern and if this impact differed between the vegetation types and layers (woody and herb layer). Vegetation relevés were performed and the vegetation and plant diversity of the protected W National Park were compared with those of its surrounding communal area. Our results reveal five vegetation types occurring in both areas. Elevation and physical soil characteristics and thus soil water availability for plants played the most important role for the occurrence of the vegetation types. The influence of land-use on plant diversity differed between the five vegetation types and the two layers. The impact was highest on the vegetation types with the most favorable soil conditions for cultivation and lowest on rocky habitats with poor soils. While the diversity of the woody layer was increased under human land-use, the diversity of the herb layer was diminished. Overall, as land-use effects were not only negative, our findings suggest that land-use does not automatically lead to a loss of plant species and to a degradation of savanna habitats. We conclude that both protected and communal areas are of great importance for the conservation of savanna vegetation and diversity. Our study highlights furthermore the importance of different management strategies for each vegetation type.
In the following two studies (chapter 3 and 4), the impact of land-use - and in particular of harvesting - on populations of Adansonia digitata L., the baobab tree, and Anogeissus leiocarpa (DC.) Guill. & Perr. was examined. These two tree species were chosen as they provide several NTFPs for the local population and as they show different levels of human protection and opposed life histories. Thus, they may react differently to land-use. Stands of the protected W National Park were compared with those of its surrounding communal area (in fallows, croplands, and villages). I applied dendrometric methods to study the population structures and combined it with rates and patterns of NTFP-harvesting (debarking and chopping/pruning). Furthermore, the impact of land-use and harvesting on the fruit production of A. digitata and on the sprouting ability of A. leiocarpa were studied. The inverse J-shaped size class distribution curve indicates that the stands of A. digitata were in a healthy state in the park, while the low number of smaller size classes in fallows, croplands, and villages may give evidence of an ageing population. However, a high number of seedlings were recorded in villages. The stands of A. leiocarpa were also in healthy states in the park and likewise in fallows. In contrast, the absence of saplings gives evidence of a declining population in croplands. Both species were strongly harvested by local people and harvesting was tree size-specific. Pruning in interaction with tree-size had a significant impact on fruit production of A. digitata. While smaller trees were more vulnerable to pruning, bigger trees benefited from slight-pruning. A. leiocarpa had a great ability to respond to chopping by sprouting. The sprouting ability increased even with higher chopping intensity. Results suggest that despite the intense harvesting and the land-use impact, populations of both species are still well preserved. While A. digitata can withstand the harvesting and land-use pressure by its longevity, extremely low adult mortality rates, and particularly due to positive human influences, A. leiocarpa is able to withstand the use pressure by its fast growing, high recruitment, and high sprouting ability. I conclude that a none protected tree species (A. leiocarpa) might not necessarily be at higher risk to the harvesting and land-use impact than a protected tree species (A. digitata) as the adverse impact of harvesting and land-use can be compensated by its specific life history.
Important additional information to such ecological findings can be provided by local people. Learning from traditional knowledge and management systems of local people will help to produce culturally and ecologically reasonable conservation and management strategies. Thus, I investigated local uses and management strategies of A. digitata and A. leiocarpa in the last two studies (chapter 5 and 6). Quantitative ethnobotanical surveys among the Gulimanceba people were conducted in the communal area in order to document uses of the different plant parts, harvesting modes, perceptions about the population status, and conservation status of both species. Hereby, differences in knowledge between gender, generations, and people from different villages were tested. Interviews reveal that both species are harvested for multipurpose and emphasize the high importance of both species for local people. Especially the leaves and fruits of A. digitata add valuable minerals and vitamins to the otherwise micronutrient-“poor” staple crops of the Gulimanceba people. In comparison with other studies in West Africa, it has turned out that people in this area could benefit even more from A. leiocarpa, e.g. for dyeing of clothes, for treatment of malaria and skin problems. Local knowledge did not differ between genders and generations, while it slightly differed between people from different villages. The lack of age differences suggests that the traditional knowledge about these two species is passed on from one generation to another. Differences between people from different villages might be explained by influences from the neighboring countries Niger and Benin. Current local harvesting modes and management strategies of both species resulted in sustainable use. However, ongoing land-use intensifications require adapted harvesting and management techniques to guarantee the persistence of these economically important species. These results provide, in combination with the ecological findings (chapter 3 and 4), appropriate management recommendations for A. digitata and A. leiocarpa that are reliable under currently practiced management strategies.
The present work comprises different projects within the scope of public health. In detail, they all aim at combating the high-burden diseases HIV/AIDS, malaria and tuberculosis more effectively. Since there was, and still is, no harmonization between the existing biowaiver guidelines, the biowaiver dissolution test conditions by WHO and FDA were compared against each other using drug products, which had already demonstrated BE to the comparator in vivo. Thereby it could be shown that the dissolution conditions proposed by the WHO are more appropriate for granting biowaivers than those of the FDA. Further, the applicability of the WHO dissolution test conditions was investigated using the APIs ethambutol, isoniazid and pyrazinamide (all BCS Class III) as model compounds. These investigations demonstrated that the concept of the biowaiver proved to work properly, i.e. leading to no false positive BE decision and an acceptable incidence of false negative BE decisions. In addition, four new biowaiver monographs were published addressing important APIs in the treatment of HIV/AIDS and malaria. Before these efforts, there were only a very few biowaiver monographs available for antiviral or antimalarial APIs, i.e. the database of biowaiver monographs has been clearly improved. The last part of the present work dealt with the extension of the biowaiver concept to related areas such as the WHO Prequalification of Medicines Programme. Investigations revealed that the biowaiver tools are generally eligible for prequalification of drug products containing ethambutol, isoniazid, pyrazinamide, or lamivudine to prove BE between an appropriate comparator and the test candidate. By contrast, some APIs are excluded from the biowaiver procedure. In conclusion, the implementation of the biowaiver tools for prequalification of biowaivable APIs is, along with BCS-based biowaiver approval of new generics, an important step towards making essential, high-quality drug products more cost-effective and, as a consequence, more accessible for a larger percentage of the population. In that way, the treatment conditions for those in need living in the developing countries can be improved enormously, so that those who are poor do not have to receive poor treatment. The quality standard of essential medicines will increase worldwide, thereby helping to combat the high-burden diseases better and, in turn, lead to an improvement of the global health status.
The current work investigated the association of trait anxiety and the neural efficiency of cognitive processing for affectively neutral (not threat-related) information. In a sample of 46 healthy volunteers, three fMRI experiments were conducted to test the prediction derived from attentional control theory (Eysenck et al., 2007) that high as compared to low trait-anxious individuals expend more neural effort on tasks requiring the top-down control of attention to reach a given level of performance. In a colour-word Stroop task requiring the inhibition of irrelevant stimulus information and associated responses as well as in a working-memorymanipulation task requiring the shifting of attention between items in working memory, trait anxiety (as measured with the State-Trait Anxiety Inventory; Spielberger et al., 1970) was positively associated with task-related increases in the activation of two adjacent regions in the right dorsolateral prefrontal cortex (DLPFC). The finding that along with a stronger activation of this brain region commonly implicated in top-down control processes, the high-anxious subjects showed equal (working memory manipulation) or worse (Stroop) performance when compared to low-anxious subjects, does support the assumption that processing is less efficient in the high anxious. However, in contrast to the predictions, trait anxiety did not show a significant association with task-related brain activation in a task-switching paradigm requiring shifting between task sets. It is discussed how different attentional control demands of the task may account for differences in the effects of trait anxiety on overt behavioural performance and underlying neural processes. In addition to DLPFC activation, trait anxiety modulated the functional connectivity of distributed regions involved in processing of the Stroop and the working-memory-manipulation task. It is discussed how the observed differences in regional DLPFC activation and network connectivity relate to each other. A possible interpretation suggests that activation increases in the DLPFC reflect an attempt to compensate for suboptimal connectivity by investing more effort in prefrontally supported control processes. Overall, the current work shows an association of trait anxiety with the neural efficiency of cognitive processing in affectively neutral tasks involving attentional control. Furthermore, it suggests that investigations of neural efficiency should take into account difference in functional integration in addition to regional activation.
Time-resolved spectroscopic analysis of fucoxanthin-chlorophyll proteins and isolated carotenoids
(2011)
The aim of this thesis was to elucidate the excitation energy transfer in the fucoxanthin-chlorophyll proteins (FCPs) isolated from the diatom Cyclotella meneghiniana in detail and to clarify the role of the different pigments contained. In a first step the excited state dynamics of the free pigments were studied by means of time-resolved absorption spectroscopy. The FCPs contain three different carotenoid species. Besides the main light-harvesting carotenoid fucoxanthin (fx) the xanthophyll cycle pigments diadinoxanthin (ddx) and diatoxanthin (dtx) are found in substoichiometric amounts. Fx is contained in an unusual carotenoid-to-chlorophyll ratio of about one. In case of ddx and dtx, changing the solvent polarity showed no significant effects on the absorption spectrum and the excited state dynamics were hardly influenced. In contrast, a solvent dependence is observed in the absorption spectrum and excited state dynamics of fx. The S1 lifetime depends strongly on the solvent polarity and an additional broad excited state absorption band red shifted compared to the S1 excited state absorption appears. The occurrence of the described features can be explained with an intramolecular charge transfer state, which is stabilized in a polar environment and appears only in carotenoids with a conjugated carbonyl group. Despite its rather short excited state lifetimes of less than 200 fs (S2) and 30-60 ps (S1), fx acts as a very efficient energy donor in the FCPs. The ultrafast energy transfer dynamics of the isolated proteins FCPa and FCPb were investigated in a comprehensive study using transient absorption in the visible and NIR spectral region complemented with polarized transient absorption spectroscopy. The excitation energy transfer was not influenced significantly by changing the light conditions during the growth, which yields an altered amount of ddx and dtx. It can be concluded that the contribution of the xanthophyll cycle pigments to the energy transfer is not significant. The altered oligomerization state results in a more efficient energy transfer for the trimeric FCPa, which is also reflected in different Chl a fluorescence quantum yields. Thus, an increased quenching in the higher oligomers of FCPb can be assumed. The observed dynamics change drastically for two different excitation wavelengths λ = 500 nm and λ = 550 nm, which both lead to the population of the S2 excited state of individual carotenoids, namely blue and red absorbing fx molecules. The differing absorption maxima result from distinct microenvironments within the protein. For FCPa an additional slow time constant of 25 ps was found after excitation at 500 nm. By means of polarized transient absorption spectroscopy applied to FCPa different transition dipole moments for the S1 and the ICT state of fx could be identified. Based on the presented studies a detailed model explaining the excitation energy transfer pathways could be developed. In agreement with the faster overall transfer rate which is also evident in the anisotropy data in case of 550 nm excitation, upon excitation at 500 nm one slow transfer channel is active. It can be attributed to a blue absorbing fx not strongly associated with a Chl a molecule. Most likely excitation energy transfer takes place between the S1/ICT states of two different fx molecules before the energy is transferred to Chl a. Additional transient absorption experiments with an improved time resolution were performed to investigate the oscillations observed. These coherent effects superimposed the kinetics of isolated carotenoids as well as FCPs within the first 500 fs. The oscillations showed a very unusual damping behavior and vanished already after two oscillation periods. In case of fx, the solvent environment as well as the excitation wavelengths had an influence on the oscillations. The frequencies of the oscillations were 70-100 cm^-1 for fx in solvents with varying polarity and 50-80 cm^-1 for the FCPs. These results could further confirm the assumption that the red absorbing fx molecules are located in a more polar environment within the protein compared to the blue absorbing fx. To clarify the origin of the oscillations in more detail, further experiments with a controlled chirp of the applied pulses and comparison between different carotenoids in various solvents are required. This approach promises to give further insight in the excited state dynamics and to answer the question whether dark states are involved. Right now, the coherent excitation of the strongly coupled excited states 1Bu+ (S2) and 1Bu- resulting in electronic quantum beats and the existence of an additional short lived excited state absorption (S2-SN2) in the visible spectral region are the most reasonable explanations for the occurrence of the coherent effects in the transient absorption spectra of carotenoids.
Since combinatorial chemistry and high throughput screening have become a common technique in the drug discovery phase the number of compounds being considered has increased frequently. These structures are often characterized by high molecular weight, high lipophilicity and low solubility in aqueous and physiological media. Due to the generally poor bioavailability, new in vitro techniques were needed for screening of pharmacokinetic properties. An important parameter for these screening methods is the implementation at an early state of drug discovery phase, to find potential lead structures, before investment costs become significant. The established in vitro methods for the prediction of membrane interaction are not reliable especially for poorly soluble compounds. A new method that is fast and easy to use, requires only small amounts of NCE and which can provide more reliable predictions is needed. In this study, a new screening technique based on surface activity profiling for the prediction of oral drug absorption was evaluated with special emphasis on the predictability of biological membrane interaction of poorly soluble drug compounds. It was demonstrated that drug absorption through a bilayer membrane can be modeled by the orientation of compounds at the air/water interface. Thus amphilicity of a drug is generally related to both oral absorption and blood brain barrier penetration. In turn, amphiphilicity is influenced by the lipophilicity, size and charge distribution of a drug. Surface activity profiling was determined by analysis of surface pressure profiles using the Gibbs adsorption isotherm. The surface activity measurements were carried out using a multichannel tensiometer Delta 8, which was developed by Kibron to be utilized in conjugation high throughput screening in early drug discovery processes. For this study two test sets were analyzed, one for the prediction of gastrointestinal wall interaction and the second for the prediction of the penetration behavior at the blood brain barrier. Both test sets consist of drug compounds with a wide range of absorption properties and consist mainly of compounds with poor water solubility. Since the drugs characteristics varied, they were classified according to water solubility and surface activity and a sample preparation method for each group was established. For the prediction of oral drug absorption, three different methods were established to model the interaction of compound and gastrointestinal wall. For drug compounds with solubility above 1mmol/L the traditional shake-flask method enabled the determination of the amphiphilic properties of drug compounds in pure aqueous media. Compounds with solubility below 1mmol/L tend to not to exhibit any increase in surface activity. Thus surface tension measurements of compounds, which exhibited a limited surface activity due to poor aqueous solubility, were conducted from stock solutions prepared with various organic solvents. Mainly polar organic solvents were used. A mixture of DMSO and DMF resulted in the best combination of properties: the intensive solubility enhancing effect of DMF and the lower intrinsic surface activity of DMSO. The polar solvent ruptured the water clusters, so that highly lipophilic structures had a higher affinity to the solvent and higher concentrations could be obtained. For these compounds higher maximum surface pressure were generated than was possible in pure aqueous media. The surface pressure data were correlated with the fraction absorbed values in vivo. However it was found that poor water solubility is not the only limiting step to exhibiting any surface activity. Some compounds were showed no surface activity in either solvent system. Therefore a micelle vehicle method was established using short chain phospholipids to mimic the gastrointestinal wall. It could be concluded from the results, that non surface active drugs can interact with the phospholipids micelle vehicle in a way analogous to their interaction with the membrane bilayer. The relative critical micelle concentration was correlated with the fraction absorbed of this test set. A sample preparation schema based on the three types of drugs was established. This schema enabled us to predict the absorbance of slightly soluble and poorly soluble drugs with acceptable reliability for early compound screening. For the prediction of blood brain barrier penetration using surface activity profiling as analyzing method, a test set with very poorly soluble characteristics was chosen. The sample preparation method was based on a strictly aqueous approach using the ‘shake flask’ method. The surface tension measurements enabled correlation of the amphiphilic properties of the very poorly soluble drug compounds with BBB uptake. From the aqueous surface pressure profiles and the determination of physicochemical parameters, it was found that blood brain barrier is more likely when a drug provides a small cross-sectional area, As, at the interface. The cross-sectional area is the only parameter which is independent from the maximal concentration in aqueous media and it is particularly suitable for lower solubility compounds. In summary, it was shown that amphilicity is related to biological membrane interaction in the human body and that surface activity profiling with appropriate sample preparation can be used as a reliable screening tool for the prediction of oral drug absorption of poorly soluble drugs. Furthermore an in vitro screening method of blood-brain-barrier penetration was established.
Table of Contents – Cumulative Dissertation Thesis German Summary Summary of Analysis Results for German-Speaking Readers Introductory Paper Introduction and Overview of the Dissertation Paper 1 Vykoukal, Jens / Wolf, Martin / Beck, Roman (2009) Services Grids in Industry: On-Demand Provisioning and Allocation of Grid-based Business Services In: Business & Information Systems Engineering (BISE), 1(2), 177-184 Paper 2 Vykoukal, Jens / Setzer, Michael / Beck, Roman (2008) Grid Architecture for Risk Management: A Case Study in a Financial Institution In: Proceedings of the 12th Pacific Asia Conference on Information Systems (PACIS), Suzhou, China Paper 3 Vykoukal, Jens / Pahlke, Immanuel / Beck, Roman (2011) Impact of Grid Assimilation on Operational Agility in Turbulent Environments: An Empirical Investigation in the Financial Services Industry In: Proceedings of the 19th European Conference on Information Systems (ECIS), Helsinki, Finland Paper 4 Vykoukal, Jens / Wolf, Martin / Beck, Roman (2009) Does Green IT Matter? Analysis of the Relationship between Green IT and Grid Technology from a Resource-based View Perspective In: Proceedings of the 13th Pacific Asia Conference on Information Systems (PACIS), Hyderabad, India Paper 5 Vykoukal, Jens (2010) Grid Technology as Green IT Strategy? Empirical Results from the Financial Services Industry In: Proceedings of the 18th European Conference on Information Systems (ECIS), Pretoria, South Africa Paper 6 Vykoukal, Jens / Beck, Roman / Wolf, Martin (2010) Impact of Pressure for Environmental Sustainability on Grid Assimilation: Empirical Results from the Financial Services Industry In: Australasian Journal of Information Systems (AJIS), 17(1), 83-106 Appendix: Publications, Curriculum Vitae
Almost two decades ago, microRNAs were discovered as novel posttranscriptional regulators of gene expression. Since then, research efforts have uncovered their involvement in the control of various cellular processes including migration, proliferation and cell survival. Even more complex events, such as the formation of new blood vessels or organ development, have been shown to be tightly regulated and orchestrated by microRNAs. Due to their crucial regulatory role in tissue homeostasis in vertebrates, it does not come as a big surprise that dysregulated microRNA ex-pression is associated with pathology of diverse diseases. In this regard, the miR-17-92 cluster is a prime example since it has become famous for its amplified expression in tumours and its on-cogenic potential. Our lab demonstrated the expression of the members of the miR-17-92 cluster, namely miR-17, -18a, -19a, -20a, -19b and -92a, in endothelial cells and provided evidence for the anti-angiogenic activity of miR-92a in ECs as well as its important regulatory role in tissue re-covery after ischemia. In this work we addressed the function of the remaining members of the miR-17-92 cluster, i.e. miR-17, miR-18a, miR-19a and miR-20a, in endothelial cells and angiogenesis. Surprisingly, the individual members all displayed anti-angiogenic properties in endothelial cells in vitro, although overexpression of the whole cluster in transformed colonocytes was shown to promote tumour angiogenesis in a mouse model. In this context, we provide evidence that the individual miRs differentially affect the paracrine angiogenic activity of endothelial and tumour cells. Moreover, Antagomir-mediated inhibition of miR-17/20 in a mouse tumour model did not affect tumour angi-ogenesis, although miR-17/20 inhibition profoundly increased vascularization of Matrigel plugs. Thus, our research efforts suggest a differential involvement of the members of the miR-17-92 cluster in physiological and tumour angiogenesis. Additionally, we identified Janus kinase (JAK) 1 as a novel miR-17 target in endothelial cells and demonstrated the involvement of JAK1 in angio-genesis and in the phosphorylation of STAT3 in response to different cytokines in vitro. Overall, inhibition of specific members of the miR-17-92 cluster might represent an attractive therapeutic strategy to enhance angiogenesis in ischemic diseases. In the second part of the present work we investigated the therapeutic value of Antagomir-mediated microRNA inhibition in animal models of pulmonary arterial hypertension. Collectively, inhibition of miR-17 by the respective Antagomir revealed a significant improvement of pulmonary hemodynamics and cardiac function in both the chronic hypoxia mouse model and the mono-crotaline-induced lung injury rat model. Histomorphometric analysis of the lungs of the pulmonary hypertensive mice and rats uncovered a significant reduction of disease associated musculariza-tion of pulmonary arteries in Antagomir-17 treated animals compared to the control animals indicating interference with smooth muscle cell proliferation or survival. Probing of lung tissue of the pulmonary hypertensive rats for selected miR-17 targets uncovered a profound increase in the expression of the cyclin dependent kinase inhibitor p21 in the Antagomir-17 treated rats suggest-ing that inhibition of miR-17 impairs proliferation by impeding cell cycle progression. Analysis of miR-17 function in human smooth muscle cells in vitro corroborated the results from the animal experiments by demonstrating pro-proliferative activity of miR-17 and decreased levels of p21 in these cells. Collectively, our results indicate that Antagomir-17 improves pulmonary hemodyna-mics and cardiac function by interfering with vascular remodelling within the lung. Hence, inhibi-tion of miR-17 might be of therapeutic value to ameliorate the disease pattern in pulmonary arte-rial hypertension. In summary, the present work provides insights into the regulatory functions of members of the miR-17-92 cluster, especially miR-17, in blood vessels and suggests that specific inhibition of members of the miR-17-92 cluster might be a novel option to treat vascular diseases.
Visual perception has increasingly grown important during the last decades in the robotics domain. Mobile robots have to localize themselves in known environments and carry out complex navigation tasks. This thesis presents an appearance-based or view-based approach to robot self-localization and robot navigation using holistic, spherical views obtained by cameras with large fields of view. For view-based methods, it is crucial to have a compressed image representation where different views can be stored and compared efficiently. Our approach relies on the spherical Fourier transform, which transforms a signal defined on the sphere to a small set of coefficients, approximating the original signal by a weighted sum of orthonormal basis functions, the so-called spherical harmonics. The truncated low order expansion of the image signal allows to compare input images efficiently, and the mathematical properties of spherical harmonics also allow for estimating rotation between two views, even in 3D. Since no geometrical measurements need to be done, modest quality of the vision system is sufficient. All experiments shown in this thesis are purely based on visual information to show the applicability of the approach. The research presented on robot self localization was focused on demonstrating the usability of the compressed spherical harmonics representation to solve the well-known kidnapped robot problem. To address this problem, the basic idea is to compare the current view to a set of images from a known environment to obtain a likelihood of robot positions. To localize the robot, one could choose the most probable position from the likelihood map; however, it is more beneficial to apply standard methods to integrate information over time while the robot moves, that is, particle or Kalman filters. The first step was to design a fast expansion method to obtain coefficient vectors directly in image space. This was achieved by back-projecting basis functions on the input image. The next steps were to develop a dissimilarity measure, an estimator for rotations between coefficient vectors, and a rotation-invariant dissimilarity measure, all of them purely based on the compact signal representation. With all these techniques at hand, generating likelihood maps is straightforward, but first experiments indicated strong dependence on illumination conditions. This is obviously a challenge for all holistic methods, in particular for a spherical harmonics approach, since local changes usually affect each single element of the coefficient vector. To cope with illumination changes, we investigated preprocessing steps leading to feature images (e.g. edge images, depth images), which bring together our holistic approach and classical feature-based methods. Furthermore, we concentrated on building a statistical model for typical changes of the coefficient vectors in presence of changes in illumination. This task is more demanding but leads to even better results. The second major topic of this thesis is appearance-based robot navigation. I present a view-based approach called Optical Rails (ORails), which leads a robot along a prerecorded track. The robot navigates in a network of known locations which are denoted as waypoints. At each waypoint, we store a compressed view representation. A visual servoing method is used to reach a current target waypoint based on the appearance and the current camera image. Navigating in a network of views is achieved by reaching a sequence of stopover locations, one after another. The main contribution of this work is a model which allows to deduce the best driving direction of the robot based purely on the coefficient vectors of the current and the target image. It is based on image registration as the classical method by Lucas-Kanade, but has been transferred to the spectral domain, which allows for great speedup. ORails also includes a waypoint selection strategy and a module for steering our nonholonomic robot. As for our self-localization algorithm, dependance on illumination changes is also problematic in ORails. Furthermore, occlusions have to be handled for ORails to work properly. I present a solution based on the optimal expansion, which is able to deal with incomplete image signals. To handle dynamic occlusions, i.e. objects appearing in an arbitrary region of the image, we use the linearity of the expansion process and cut the image into segments. These segments can be treated separately, and finally we merge the results. At this point, we can decide to disregard certain segments. Slicing the view allows for local illumination compensation, which is inherently non-robust if applied to the whole view. In conclusion, this approach allows to handle the most important criticism to holistic view-based approaches, that is, occlusions and illumination changes, and consequently improves the performance of Optical Rails.
The construct diversity describes the collective amount of differences among members within a social unit. The present dissertation is based on the assumption that, through engagement with diversity, people acquire an understanding of what role diversity plays in the societies, organizations, work groups, or other social units they are part of. This understanding of the role diversity plays in a given social unit provides a vantage point from which people will engage with diversity in the future. These vantage points from which people engage with diversity are the general subject matter of the present dissertation. Two main research questions are addressed in this regard: First, whether the role diversity is given in a particular context does have effects on groups and the individual members therein. Second, if such effects exist, it seeks to explore the processes and mechanisms they are based on. Both questions are addressed from different perspectives in the three main chapters of this dissertation. Chapter 5 contains two meta-analyses on the effects of diversity beliefs and diversity climates. Diversity beliefs are individual attitudes that describe the degree to which diversity is ascribed an instrumental value for achieving beneficial outcomes or avoiding detrimental ones. Diversity climates depict such a value of diversity on the group-level. Building on the social identity approach, I explain how diversity beliefs and climates can obviate diversity’s detrimental effects and foster beneficial ones. As both diversity beliefs and climates can cause such effects, they are considered together in the main analyses in the chapter. In the first part of the chapter, a meta-analysis on these moderator effects of diversity beliefs/climates is presented (k = 23). The majority of studies that addressed such effects reported significant results. The patterns of these results showed that, in general, diversity will be more positively related to beneficial outcomes the more it is valued. However, the analysis also revealed that there are at least two types of patterns of this moderation. So far, it cannot be explained which pattern will occur under what circumstances. In the second part of the chapter, a meta-analysis on the main effects of diversity beliefs/climates on beneficial outcomes is presented (k = 71). These effects did not receive much attention in the primary studies. Based on the social identity approach and the fact that diversity is a ubiquitous feature of modern organizations, I argue that they are important nonetheless. The meta-analysis revealed a significant positive main effect of diversity beliefs on beneficial outcomes (r = .25; p < .0001). However, the effect sizes varied considerably across studies. Both moderator and main effects were found across a broad array of outcomes, study designs, levels of analysis, and operationalizations of the constructs involved. They were found irrespective of whether diversity beliefs or diversity climates were considered. The heterogeneity of results in the meta-analyses suggests that there is still much to be learned about when differences in vantage points from which people engage with diversity will have an effect and about the processes that underlie these effects. Chapter 6 is, therefore, predominantly concerned with these underlying processes. Most of the previous research has treated pro-diversity beliefs and pro-similarity beliefs as opposite poles of one underlying continuum. There is, however, evidence that people can hold both types of beliefs simultaneously. Therefore, I propose that both diversity in certain aspects and similarity in other aspects can simultaneously constitute valid and valued parts of an organization’s identity, and that, hence, identifying with the organization can create two forms of solidarity among the employees: organic solidarity – based on meaningfully and synergistically interrelated differences, and mechanic solidarity – based on the common ground that all employees share. Furthermore, I propose that both forms of solidarity can coexist and that both are positively related to the quality of collaboration within the organization. Thus, organizational identification is proposed to influence quality of collaboration indirectly through both organic and mechanic solidarity. These propositions were tested with regard to the collaboration of different teams within two organizations: a German university (Study 1, N = 699) and a Taiwanese hospital (Study 2, N = 591). The results from both studies confirm the predictions. However, the relative importance of each form of solidarity varied across study contexts and across different facets of the quality of collaboration. Chapter 7 also builds on the findings from the meta-analyses and is again predominantly focussed on the processes underlying the effects of diversity beliefs and diversity climates, yet from a different angle. Previously, diversity beliefs and climates have often been discussed with regard to their potential to influence whether diversity will lead to more and deeper elaboration of information within the group. In chapter 7 a theoretical model is developed that complements these cognitive processes by addressing the emotional side of diverse groups. Central to the model is the assumption that group diversity can stimulate group members to engage with each other emotionally, resulting in higher levels of state affective empathy: an emotional state which arises from the comprehension and apprehension of fellow group members’ emotional state. State affective empathy, in turn, is known to lead to a variety of beneficial team processes that can ultimately enhance individual and group-level performance. Thus, the central proposition of the model is that the relationship between diversity and performance is mediated through state affective empathy. The other propositions in the model specify moderators that determine when diversity will indeed have this empathy-stimulating effect. Diversity beliefs and climates are considered second-order moderators that shape the relationship between diversity and empathy through their influence on the first-order moderators. In general, it is proposed that diversity is related to empathy more positively if it is valued by the group or its members. In summary, the results from the meta-analyses in chapter 5, the results from the field studies in chapter 6, and the theoretical arguments presented in chapter 7 can be interpreted such that differences in vantage points from which people engage with diversity can indeed affect groups and their members. Therefore, the first research question of the present dissertation can be answered affirmatively from three different perspectives. However, it also became clear that there is still much uncertainty about the mechanisms underlying these effects. In line with the second research question of the present dissertation, these mechanisms were examined more closely in chapter 6 and 7. The field studies in chapter 6 highlighted the role of identification as the driving force behind the effects of different vantage points on diversity. Furthermore, they also corroborate the proposition that valuing diversity and valuing similarity can be co-occurring phenomena that both influence the collaboration within the group positively. The theoretical model presented in chapter 7 opens up a new emotional way in which diversity beliefs and climates can influence whether diversity will lead to better or worse performance. In sum, therefore, also with regard to the second research question of the present dissertation, progress has been made.
The phenomenon of magnetism is a pure quantum effect and has been studied since the beginning of civilization. The practical use of magnetic materials for technical purposes was well established in the 19th century; still nowadays there is no lack of new high-tech applications based on magnetism for example in information technology to store and process data. This thesis does not focus on the development of new applications of magnetism in technology, nor enhancement of known fields of application. Instead, the intention is to use a quantum theory of magnetism for obtaining new insights on physical effects that accompany the phenomenon of magnetism. Therefore three different model systems, each of which are believed to describe a class of real compounds, are considered. Starting from the idea that magnetism can be understood by use of the so-called Heisenberg model that microscopically characterizes the interaction between localized magnetic moments, we restrict ourselves to the case where a long-range magnetic order is present. In order to deduce consequences resulting from this microscopic picture we use the spin-wave theory that is introduced in the first chapter. Central objects of this theory are the magnons which are elementary quantum excitations in ordered magnets. An application of these mathematical techniques to a model that describes an antiferromagnet in an external magnetic field is presented in the second chapter. Quantities like the spin-wave velocity and the damping of magnons are calculated using a Hermitian operator approach in the framework of spin-wave theory. A strong renormalization of the magnetic excitations arises because the symmetry of the system is reduced due to the external magnetic field. In the second model system, that describes thin films of a ferromagnet, concepts of classical physics meet quantum physics: The magnetic dipole-dipole interaction that is also known in everyday life from the magnetic forces between magnets and was initially formulated in the theory of electromagnetism, is included in the microscopic model. Having a special compound in mind where the magnetic excitations are directly accessible in experiments, the energy dispersions of magnon modes in thin-film ferromagnets are deduced. Our approach is essentially a basis for further investigations beyond this thesis to describe strong correlations and condensation of magnons. A recent realization of data processing devices with spin waves puts the understanding of physical processes in these ferromagnetic films in the focus of upcoming research. The third model system brings in the so-called frustration where the interactions between the spins are such that the total energy cannot be minimized by an appropriate alignment of the magnetic moments in the classical picture. In the simplest case this appears because the antiferromagnetically coupled spins are located on a triangular lattice. This situation will lead to strong quantum fluctuations which make this model system interesting. Finally the overall symmetry is reduced by inclusion of spin anisotropies and an external magnetic field. Instead of focusing on the properties of the magnetic excitations, the effect of the magnetic field on the properties of the lattice vibrations is subject to the investigation. This is interesting because the characteristics of lattice vibrations can be measured experimentally using the supersonic technique.
The enzyme quinol:fumarate reductase (QFR) from the anaerobic epsilon-proteobacterium Wolinella succinogenes is a membrane protein complex that couples the catalysis of the oxidation of menaquinol to menaquinone to that of the reduction of fumarate to succinate. This is the terminal step in fumarate respiration, a form of anaerobic respiration in which oxygen is replaced by fumarate as the terminal electron acceptor in many anaerobic microorganisms. In QFR, both the heme groups (low-potential distal and high-potential proximal heme b group in transmembrane subunit C) are part of the electron transport chain between the two catalytic sites of the redox enzyme. Although the reduction of fumarate by menaquinol is exergonic, it is not exergonic enough to support the generation of a transmembrane electrochemical proton potential delta p. Evidence has previously shown that this reaction is catalysed by a novel mechanism, involving the facilitation of transmembrane electron transfer by transmembrane proton transfer via an essential compensatory transmembrane proton transfer pathway ("E-pathway") which is inactive in the oxidized state of the enzyme. The two key constitutents of the the pathway are the amino acid residue Glu C180 of the transmembrane helix V (located in subunit C) and the ring C propionate of the distal heme bD. The aim of the project was to obtain, by employing a combination of time-resolved as well as static spectroscopic approaches, a detailed insight of the transmembrane electron coupled proton transfer mechanism. Minute changes in both the oxidized and reduced states of a redox protein system can be selectively and sensitively monitored by static Fourier Transformed Infrared (FTIR) difference spectroscopy. The technique employed in this context, electrochemically induced FTIR difference spectroscopy, is complemented by computer-based electrostatic calculations. In order to elucidate the catalytic mechanism of the important reactions in QFR, it is necessary to investigate these in a time-resolved manner. Rapid scan FTIR difference spectroscopy is a suitable technique that allows the course of the reaction to be monitored in a time dependent fashion. The techniques employed in this context are time-resolved (tr-FTIR) and transient absorption spectroscopy. In the following, the details of individual sub-projects are discussed in brief. ...
The ubiquinol:cytochrome c oxidoreductase is a key component of several aerobic respiratory chains in different organisms. It is an integral membrane protein complex, made up of three catalytic subunits (cytochrome b, cytochrome c1 and Rieske iron sulphur protein) and up to eight additional subunits in mitochondria. The complex oxidizes one quinol molecules and reduces two cytochrome c during the Q cycle, originally described by Peter Mitchell. Electrons are split between the low and the high potential chain and protons are released on the positive side of the membrane, increasing the protonmotive force needed by the ATP-synthase for energy transduction. The cytochrome bc1 complex from P. denitrificans is a perfect model for structural and functional studies. Bacteria are easy to grow and the genetic material is readily accessible for genetic manipulation. Moreover, the P. denitrificans aerobic respiratory chain is very close to the mitochondrial one: the complexes involved in electron transfer resemble the ones found in mitochondria, but lack most of the additional subunits. As a unique feature, P. denitrificans has a strongly acidic domain at the N-terminal region of the cytochrome c1, a sequence of 150 aminoacids which does not correlate with any known protein. An analogous composition can be found in the eukaryotic cytochrome bc1 complex as a part of an accessory subunit, proposed to be involved in facilitating electron transfer between the complex and the electron acceptor cytochrome c. In order to study the function of this domain in the P. denitrificans cytochrome bc1 complex, a deletion mutant has been previously cloned and modified with an affinity tag as a C-terminal extension of cytochrome b. The complex is purified by affinity chromatography and characterized by steady-state kinetics using not only horse heart cytochrome c but also the endogenous electron acceptor, the membrane bound cytochrome c552, employed here as a soluble fragment. Steady–state kinetics indicate that the deletion of the long acidic domain had effects neither on the turnover rate nor on the apparent affinity for the substrate. To understand wether the deletion affects the reaction between the cytochrome bc1 complex and the substrate, laser flash photolysis experiments are performed, showing that the interaction observed was not changed in the complex missing the acidic domain. The results presented in this work confirm the ones previously obtained by Julia Janzon using soluble fragments of the same interaction partners. The deletion, however, affected the oligomerization state of the complex, as shown by LILBID (Laser Induced Liquid Bead Ion Desorption) analysis. The wild type complex has a tetrameric structure, better described as a “dimer of dimers”. The deletion of the acidic domain on the cytochrome c1 results in the separation of the two dimers, yielding the canonical dimer. Therefore, the complex deleted in the acidic domain is used for cloning and expression of a heterodimeric complex, containing an inactivating mutation in the quinol oxidation site in only one monomer, thus allowing a selective switch-off for half the complex. Such a complex is needed for the verification of an internal regulation mechanism, the half-of-the-sites reactivity. According to it, the dimeric structure of the cytochrome bc1 complex has functional implications, since the two monomers can communicate and work in a coordinated manner. This approach confirms that substrate oxidation does effectively take place only in one of the two monomers constituting the dimer, and that the binding of substrate at the Qo and Qi site regulates the switch between active and inactive monomer. Moreover, this mechanism works also as an effective protection against the reaction of quinone intermediates with oxygen and the formation of reactive oxygen species (ROS), responsable for cellular aging. The motion of the ISP head domain is also addressed in this work; in particular the mechanism which regulates the movements towards the cytochrome c1 and the electron bifurcation at the quinol oxidation site. Laser flash kinetics in presence of several inhibitors and the substrate allow studying the response of the ISP to the binding of different species at the quinol oxidation site. The binding of ligand at the Qo site in the complex triggers the conformational switch in the ISP head domain, supporting the mechanism proposed in the literature according to which the Qo site is able to “sense” the presence of substrate and transfer the information to the ISP, regulating its mobility. The internal electron pathway between the ISP and the cytochrome c1 has been analyzed also by stopped-flow kinetics, in presence and absence of inhibitors. The results indicate that two kinetic phases describe the reduction of cytochrome c1 by the ISP, and a model for the simulation of the data is proposed.
Dynamics of chaotic strings
(2011)
The main topic of this thesis is the investigation of dynamical properties of coupled Tchebycheff map networks. At every node of the network the dynamics is given by the iteration of a Tchebycheff map, which shows strongest possible chaotic behaviour. By applying a coupling between the various individual dynamics along the links of the network, a rich structure of complex dynamical patterns emerges. Accordingly, coupled chaotic map networks provide prototypical models for studying the interplay between local dynamics, network structure, and the emergent global dynamics. An exciting application of coupled Tchebycheff map lattices in quantum field theory has been proposed Beck in Spatio-temporal chaos and vacuum fluctuations of quantized fields' (2002). In this so-called chaotic string model, the coupled map lattice dynamics generates the noise needed for the Parisi-Wu approach of stochastic quantization. The remarkable obversation is that the respective dynamics seems to reproduce distinguished numerical values of coupling constants that coincide with those observed in the standard model of particle physic. The results of this thesis give insights into the chaotic string model and its network generalization from a dynamical point of view. This leads to a deeper understanding of the dynamics, which is essential for a critical discussion of possible physical embeddings. Apart from this specific application to particle physics, the investigated concepts like synchronization or a most random behaviour of the dynamics are of general interest for dynamical system theory and the science of complex networks. As a first approach, discrete symmetry transformations of the model are studied. These transformations are formulated in a general way in order to be also applicable to similar dynamics on bipartite network structures. An observable of main interest in the chaotic string model is the interaction energy. In Spatio-temporal chaos and vacuum fluctuations of quantized fields' (2002) it has been observed that certain chaotic string couplings, corresponding to a vanishing interaction energy, coincide with coupling constants of the standard model of elementary particle physics. Since the interaction energy is basically a spatial correlation measure, an interpretation of the respective dynamical states in terms of a most random behaviour is tempting. In order to distinguish certain states as most random', or evoke another dynamical principle, a deeper understanding of the dynamics essential. In the present thesis the dynamics is studied numerically via Lyapunov measures, spatial correlations, and ergodic properties. It is shown that the zeros of the interaction energy are distinguished only with respect to this specific observable, but not by a more general dynamical principle. The original chaotic string model is defined on a one-dimensional lattice (ring-network) as the underlying network topology. This thesis studies a modification of the model based on the introduction of tunable disorder. The effects of inhomogeneous coupling weights as well as small-world perturbations of the ring-network structure on the interaction energy are discussed. Synchronization properties of the chaotic string model and its network generalization are studied in later chapters of this thesis. The analysis is based on the master stability formalism, which relates the stability of the synchronized state to the spectral properties of the network. Apart from complete synchronization, where the dynamics at all nodes of the network coincide, also two-cluster synchronization on bipartite networks is studied. For both types of synchronization it is shown that depending on the type of coupling the synchronized dynamics can display chaotic as well as periodic or quasi-periodic behaviour. The semi-analytical calculations reveal that the respective synchronized states are often stable for a wide range of coupling values even for the ring-network, although the respective basins of attraction may inhabit only a small fraction of the phase space. To provide analytical results in closed form, for complete synchronization the stability of all fixed points and period-2 orbits of all chaotic string networks are determined analytically. The master stability formalism allows to treat the ring-network of the chaotic string model as a special case, but the results are valid for coupled Tchebycheff maps on arbitrary networks. For two-cluster synchronization on bipartite networks, selected fixed points and period-2 orbits are analyzed.
Pulsed electron-electron double resonance (PELDOR) is a pulsed EPR method that can reliably and precisely provide structural information regarding duplex RNAs and DNAs by measuring long-range distances (1.5-7 nm) utilizing distance-dependent magnetic dipole-dipole interaction between two nitroxide spin labels. In this thesis the application field of PELDOR spectroscopy has been expanded. For the first time the global architecture of tertiary folded RNA has been mapped in vitro. Moreover, the first application of PELDOR for determining structural aspects of RNA and DNA molecules inside cells has been presented. RNA has the central role in cellular processes and gene regulation. It can adopt complex three dimensional structures, which in combination with its conformational dynamics is essential for its function as biological catalyst, structural scaffold and regulator of gene expression. Riboswitches are cis-acting RNA segments that modulate gene expression by direct binding of small molecules with high affinity and specificity. Neomycin-responsive riboswitch is an engineered riboswitch developed by combination of in vitro selection and in vivo screening. Upon insertion into the 5‟ untranslated region of mRNA and binding the cognate ligand it is able to inhibit translational initiation in yeast. Using enzymatic probing the secondary structure had been postulated comprising global stem-loop architecture with a terminal and an internal loop. In the first part of this thesis, the global conformational arrangement of this 27 nucleotides long RNA element has been studied by means of site-directed spin labeling and PELDOR spectroscopy. Spin-labeled neomycin-responsive riboswitch mutants were synthesized via a Sonogashira cross-coupling reaction between 5-membered pyrroline ring based nitroxide radical (TPA) and 5-iodo-uridine. The labeling positions were chosen outside of the binding pocket and UV melting curves revealed that spin-labeling neither disturbs the secondary structure nor interferes with ligand binding. Efficient ligand binding was proven by thermal stabilization of 20.3±3.3 oC upon addition of neomycin, as well as by cw EPR spectra. PELDOR time traces with long observation time windows and with good signal to noise ratio and modulation depth were recorded for all double-labeled samples allowing a reliable data analysis. The fact that there were no shifts in the measured distances upon addition of neomycin implied the existence of a prearranged tertiary structure of the neomycin-sensing riboswitch without a significant global conformational change induced by ligand binding. Measured distances were in very good agreement with the NMR structure of the ligand-bound state of the riboswitch indicating the intrinsic propensity of the global RNA architecture toward its energetically favored ligand-bound form at low temperature. The results harvested in this work represent the first application of PELDOR for mapping the global structure of a tertiary folded RNA. In the second part of this thesis the possibility of applying PELDOR on nucleic acids (NAs) in cellular environment has been investigated. It was shown before that global NA structure depends on matrix conditions, such as concentration of ions and small molecules, molecular crowding, viscosity and interactions with proteins. Therefore, PELDOR spectroscopy on a double-labeled 12-base pair DNA duplex, the 14-mer cUUCGg tetraloop hairpin RNA and the 27-mer neomycin-sensing riboswitch has been used to obtain long-range distance constraints on such systems in Xenopus laevis oocytes and to compare them with in vitro measurements. The reduced lifetime of nitroxide spin labels under cellular conditions has been a major challenge in these measurements. Investigation of nitroxide reduction kinetics in-cell has revealed that the 5-membered pyrrolidine and pyrroline rings are significantly slower reduced compared to 6-membered piperidine ring based nitroxides. Due to prolonged lifetime of the TPA nitroxides covalently attached to NA molecules PELDOR signals could be measured with good signal-to-noise ratios up to 70 minutes of incubation time. The partial loss of coupled spin labels due to nitroxide reduction only led to a decrease in the modulation depth upon increasing the incubation time. No alterations in the measured distances between in vitro and in-cell experiments implies the existence of stable overall conformations of the 14-mer cUUCGg tetraloop hairpin RNA and the 27-mer neomycin-sensing riboswitch, whereas the 12-bp duplex DNA experiences stacking in-cell but retaining the secondary structure. Thus, for the first time nanometer distance measurements were performed inside cells, clearly laying a foundation for the application of PELDOR spectroscopy to study biological processes in cells, such as diffusion, interaction with proteins and other factors or chemical reactions.
Nonequilibrium phase transitions in chiral fluid dynamics including dissipation and fluctuation
(2011)
Chiral fluid dynamics combines the fluid dynamic expansion of a hot and dense plasma created in a heavy-ion collision with the explicit propagation of fluctuations at the chiral phase transition of quantum chromodynamics. From systems in equilibrium long-range fluctuations are expected at a conjectured critical point. Heavy-ion collisions are, however, finite in size and time and very dynamic. It is thus likely that nonequilibrium effects diminish the signal of a critical point. They can, however, stimulate phenomena at a first order phase transitions, like nucleation and spinodal decomposition. Both of phase transition scenarios are investigated in this work. Based on the linear sigma model with constituent quarks a consistent quantum field theoretical approach using the two-particle irreducible effective action is developed to derive both, the local equilibrium properties of the expanding quark fluid and the damping and noise terms in the Langevin equation of the order parameter of the phase transition, the sigma field. Within this formalism it is possible to obtain a conserved energy-momentum tensor of the coupled system. It describes the energy dissipation from the sigma field to the heat bath during relaxation. Within this model we investigate nonequilibrium phenomena in a scenario with a critical point and a first order phase transition. We observe long relaxation times at the phase transition, phase coexistence at the first order phase transition and critical slowing down at the critical point. We find a substantial supercooling in a first order phase transition in our model and due to the energy-momentum exchange also reheating is present. While at the critical point the correlation length increases slightly we find an enhanced intensity of nonequilibrium fluctuations at the first order phase transition, which leads to an increased production of sigma mesons.
In recent years using symmetry has proven to be a very useful tool to simplify computations in semidefinite programming. This dissertation examines the possibilities of exploiting discrete symmetries in three contexts: In SDP-based relaxations for polynomial optimization, in testing positivity of symmetric polynomials, and combinatorial optimization. In these contexts the thesis provides new ways for exploiting symmetries and thus deeper insight in the paradigms behind the techniques and studies a concrete combinatorial optimization question.
A pattern is a word that consists of variables and terminal symbols. The pattern language that is generated by a pattern A is the set of all terminal words that can be obtained from A by uniform replacement of variables with terminal words. For example, the pattern A = a x y a x (where x and y are variables, and the letter a is a terminal symbol) generates the set of all words that have some word a x both as prefix and suffix (where these two occurrences of a x do not overlap). Due to their simple definition, pattern languages have various connections to a wide range of other areas in theoretical computer science and mathematics. Among these areas are combinatorics on words, logic, and the theory of free semigroups. On the other hand, many of the canonical questions in formal language theory are surprisingly difficult. The present thesis discusses various aspects of the inclusion problem of pattern languages. It can be divide in two parts. The first one examines the decidability of pattern languages with a limited number of variables and fixed terminal alphabets. In addition to this, the minimizability of regular expressions with repetition operators is studied. The second part deals with descriptive patterns, the smallest generalizations of arbitrary languages through pattern languages ("smallest" with respect to the inclusion relation). Main questions are the existence and the discoverability of descriptive patterns for arbitrary languages.
This thesis consist of three chapters of which each investigates a topic from financial and monetary economics. In the first chapter a novel method to analyze the monetary policy of central banks is presented. In the second chapter (joint work with Professor Michael Binder, Goethe-University Frankfurt) the effects of conditional loan programs of the International Monetary Fund (IMF) on participating countries' output growth are investigated. In the third chapter (joint work with Professor Jan Pieter Krahnen, Goethe-University Frankfurt) a network model of interconnected bank balance sheets which gives rise to systemic risk is developed and used to analyze the implications of a bank levy related to banks' contribution to systemic risk. All three chapters give important insights to the policy design of macroeconomic institutions such as central banks, the IMF, and agencies charged with macroprudential supervision.
This study comprises a survey on ecology, morphology and taxonomy of parasitic fungi infecting Pteridophytes and Orchidaceae found by the author on several field trips to Western Panama as part of the project plant parasitic micro-fungi of Western Panama (ppMP). In Panama, approximately 9500 species of vascular plants are found. Of these, Orchidaceae are with ca. 1150 (ca. 12%) species by far the most speciose family. The Pteridophytes in Panama comprise ca. 940 species in 31 families. Most fungal pathogens on Orchidaceae in tropical regions were described from plants in culture or from material intercepted at borders by plant quarantine services and not from their natural habitats. Therefore, little is known about distribution and ecology of these pathogens in their natural range. The author determined and classified several hundred Orchidaceae-species and Pteridophytes at the sites selected in the context of the project. This work facilitated the identification of many host plants (at least to genus-level) even in sterile condition in the field. About 65 species of Pucciniales are known to infest Orchidaceae and ca. 38% of them are described from tropical America. All available types of Pucciniales on Orchidaceae in tropical America were studied and compared with 91 specimens of rust fungi on orchids collected by the author in Panama. Several hundred additional specimens housed in the BPI, almost all intercepted from plant quarantine services, were used for comparison. As result of this work, it is suggested to combine Uromyces stenorrhynchi Henn. to Sphenospora and, as this is the oldest epithet, to synonymize S. kevorkianii Linder, S. mera Cumm. and S. saphena Cumm. with it. Further, it could be demonstrated that Uredo aurantiaca Montemartini, U. cyrtopodii Syd. & P. Syd., U. epidendri Henn., U. guacae Mayor, U. gynandrearum Corda, U. lynchii (Berk.) Plowr., U. neopustulata Cumm. (≡U. pustulata Henn.), U. nigropuncta Henn., U. oncidii Henn., U. ornithidii F. Kern., Cif. & Thurst., and presumably U. scabies Cke., are anamorphs of this variable species. U. gynandrearum is the oldest anamorph-name for all these taxa. Therefore, it can be established that this rust infects more than 80 species of Orchidaceae in three subfamilies. In total, the anamorph of this species was collected by the author on 17 different species of Orchidaceae in Panama which, apart from one species, are all new hosts to science. The molecular data obtained by the author confirm this view, although more data, especially from material from the whole range of distribution of U. gynandrearum, are necessary. Puccinia spiranthicola Cumm. was found to be a synonym of P. cinnamomea Diet. & Holw. and was found by the author on three different Orchidaceae in two subfamilies. Uredo pleurothallidis Keissl. is now considered a synonym of U. wittmackiana Henn. and the latter as the anamorph of Puccinia oncidii Cumm. In the anamorph genus Uredo, a new species was found infecting at least five different species of Sobralia and Elleanthus (Sobraliinae) at different localities. Molecular data indicate it to be related to the currently polyphyletic Phakopsoraceae. For the rusts with suprastomatal sori on Orchidaceae, now separated from Hemileia and placed in the genus Desmosorus (nom. inval.), the current concept with only one taxon is rejected and the establishment of three subspecies is suggested. The complicated taxonomy is discussed and makes it necessary to validate the genus-name and make a new combination. Another Hemileia-anamorph species was found by the author and is considered to be new to science. This is the first species of this alliance in America on Orchidaceae. Molecular data obtained by the author confirm the separation of Desmosorus from Hemileia and the position of the new species. For rusts on Pteridophytes, a new species of Milesia, (teleomorph: Milesina) and a new anamorphic species of Uredinopsis was found, both on hosts hitherto not known. In Calidion, the presumable anamorph-genus of Uncol, the species C. cf. cenicafeae Salazar & Buriticá was found on several new hosts. Further, the teleomorph was found. Morphologically, this teleomorph did not agree with the description of Uncol by the author of the genus, although the anamorph characteristics left no doubt that it is Calidion. Apparently, the description of Uncol is inadequate, but cannot be improved, as the type is unavailable. Molecular data obtained by the author show this species to be closest to Desmosorus. For Uredo superficialis Speg., the anamorph of Desmella, nine new hosts in eight different fern families were found by the author and the collaborators of the ppMP-project. Ecological data indicate that this species includes different host specific races, which, however could not be distinguished morphologically. For all these rusts, a thorough discussion of the ecology in their habitats is given. In total, 21 LSU rDNA sequences from 6 different rust species on Orchidaceae and Pteridophytes were obtained and analyzed with the Maximum Parsimony and Minimum Evolution method. Here, the position of several groups could be confirmed, and some anamorphs could be assigned to different teleomorphic relationships. Within the Ascomycota and their anamorphs, several hitherto unknown species and species not known from these hosts or not known from Panama were found and analyzed. On Orchidaceae, the following fungi belonging to the Ascomycota are described, illustrated and discussed: In the Phyllachorales, a hitherto not known Phyllachora sp. was found on Oncidium warszewiczii Rchb. f. and was compared with the other species of this order currently known from Orchidaceae. In the Asterinaceae s. l. Lembosia cf. epidendri Meir. Silva & O. R. Pereia was found on Maxillaria crassifolia (Lindl.) Rchb. f., which is a new host and new host alliance for this fungus hitherto only known from Brazil. The fungus is described and compared with all species of Asterinaceae currently known on Orchidaceae. In the Meliolaceae, Meliola orchidacearum Cif. was found on Camaridium biolleyi (Schltr.) Schltr. and an Epidendrum sp. which are new hosts and new host alliances of this fungus which was hitherto only known from the Caribbean Islands. It is described, illustrated and compared with the type. In the Glomerellaceae, Glomerella cingulata and its anamorph Colletotrichum gloeosporioides were found on several hosts. The species is illustrated, described and compared with data from literature. In the anamorphic Mycosphaerellaceae, Pseudocercospora odontoglossii (Prill. & Delacr.) U. Braun, a species currently only known from culture, was found on the new host Pleurothallis imraei Lindl. It is illustrated, described and compared with data from literature. On ferns, the following other fungi are described, illustrated and discussed: A conspicuous undescribed form of Polycyclus was found by the author on Elaphoglossum ciliatum (C. Presl.) T. Moore (Dryopteridaceae) and Serpocaulon loriceum (L.) A. R. Sm. (Polypodiaceae). A conspectus of Parmulariaceae infecting ferns is given and demonstrated that Polycyclina should be synonymized under Polycyclus. Summing up, it can be assessed, especially for the Pucciniales, that the most speciose plant family in Panama carries remarkable few species of specific parasites, and that many of them seem to be distributed over a wide range of species which often are not closely related. One reason amongst others seems to be that parasites need a minimum density of host plants in a habitat to survive. As orchid species often occur with only few (and often small) individual plants at a given locality, the probability for a specific pathogen to infect a plant gets too low, hence high diversity by low abundance of hosts might be an impediment for specific pathogens. In this case, unspecific parasites, or such which are infecting larger alliances, are in advantage. Other reasons could be specific traits of orchids, like succulence and mycotrophy which might hamper fungal infections.
Forty two samples of the Late Eocene Kiliran oil shale, Central Sumatra Basin, Indonesia were collected from a 102 m long drill core. The oil shale core represents the deposition time of about 240.000 years. Palynofacies and geochemical analyses have been carried out to reconstruct the paleoenvironmental conditions and paleoecology during deposition of the oil shale. Amorphous organic matter (AOM) is very abundant (>76%). B. braunii palynomorphs are present (3-16%) as the only autochtonous structured organic matter and generally more abundant in the middle part of the profile. The stable carbon isotopic composition of bulk organic matter (13C) varies from -27.0 to -30.5‰ and is generally more depleted in the middle part of the profile. The ratio of total organic carbon to sulfur (TOC/S), used as salinity indicator, ranges from 2.5 to 15.8 and shows variations along the profile. Slightly less saline environments are observed in the middle part of the profile. Fungal remains are generally present only in this part with a distinct peak of abundance. The presence of fungal remains is regarded as an indication for a relatively warmer climate during deposition of the middle part of the profile. The warmer climate is thought to influence the establishment of a thermocline, limiting the supply of recycled nutrients to epilimnion. Consequently, the primary productivity in the Kiliran lake decreased during deposition of the middle part of the profile as indicated by the relatively depleted 13C values and the blooming of B. braunii. The chemocline was also shoaling during the deposition according to the higher abundance of total isorenieratane and its derivatives originated from green sulfur bacteria dwelling in the photic zone euxinia. The warmer climate is also thought to influence the slightly decrease of water salinity during deposition of the middle part of the profile. The occurrence of B. braunii in Kiliran lake is also recognized from organic geochemical data. The distribution of n-alkanes is characterized by the unusual high amount of C27 n-alkane relative to the other long-chain n-alkanes. The concentrations of C27 n-alkane vary from 30.1 to 393.7 μg/g TOC and are generally in parallel with the abundances of B. braunii palynomorphs along the profile. The 13C values of this compound are about -31‰ and up to 2‰ enriched relative to those of the adjacent long-chain n-alkanes. B. braunii race A can thus be regarded as the significant biological source of the C27 n-alkane. Lower amounts of lycopane are observed in many oil shale samples (0 to 54.7 μg/g TOC). The 13C value of this compound is 17.2‰. This strong enrichment of 13C suggests that the lycopane was derived from B. braunii race L. The concentrations of lycopane develop generally in opposite with those of C27 μalkane. It is likely that both B. braunii races bloomed in alternation in the lake, probably due to changes on specific water chemistry. Norneohop-13(18)-ene and neohop-13(18)-ene derived from methanotrophic bacteria are the dominant hopanoid hydrocarbons. The sum of their concentrations varies from 40.6 to 360.0 μg/g TOC. The 13C of these compounds are extremely depleted (-45.2 to -50.2‰). The occurrence of abundant bacteria including methanotrophic bacteria was responsible for the recycling of carbon below the chemocline of the lake. The effect of the recycling of carbon is observed by the presence of a concomitant depletion (about 7-9‰) in 13C of some specific biomarkers derived from organisms dwelling in the whole phototrophic zone. 4-Methylsterane and 4-methyldiasterene homologues occur in the oil shale as the predominant biomarkers. The sum of the concentrations of all homologues are about 40.3-1,009.2 μg/g TOC with generally higher values in the uppermost and lower parts of the profile. Calcium (Ca) accounts as the predominant element in the oil shale, ranging from 5.0 to 16.7%. This element shows generally parallel variation with the 4-methylsterane and 4-methyldiasterene homologues along the profile. This suggests that these compounds were derived from biological sources favoring more alkaline and more trophic environments. On the other hand, these compounds were less abundant in the middle part of the profile which is consistent with less alkaline and less trophic environments promoting B. braunii to bloom. Alternation between Dinoflagellates and B. braunii in ancient lacustrine environments due to water chemistry changes have been known from previous studies. In the present case, distinct alternation between B. braunii abundances and concentrations of 4-methylsterane and 4-methyldiasterene homologues along the studied oil shale profile suggest a hypothesis that these compounds were derived from freshwater Dinoflagellates although dinosterane is not present in the sediment extracts. Water alkalinity and trophic level changes were most likely responsible for the alternation of Dinoflagellates and B. braunii blooming.
Subject of this thesis is the non-perturbative investigation of the thermal transition in Quantum Chromodynamics by means of lattice gauge theory and a particular type of lattice fermions, the so-called twisted mass fermions. These fermions offer the possibility of improvement as compared to the standard Wilson-type formulation. We investigate the properties of these fermions at finite temperature, i.e. the structure of the bare parameter space as well as leading order cutoff effects in the weak coupling limit. Then we focus on two-flavour simulations at finite pion mass. We identify the (pseudo-)critical temperatures for our set of pion masses (300 to 500 MeV) and discuss the extrapolation to the chiral limit for which the nature of the transition is still an open question. Besides pseudo-critical temperatures we consider the magnetic equation of state and screening observables. We find that the assumption of a second order transition (in the 3d O(4) universality class) agrees with our data without being able to exclude alternatives. Finally, we discuss the future inclusion of strange and charm quarks in dynamical twisted mass simulations and look at the corresponding cutoff effects in the free limit.
Die Familie der Proteorhodopsine (PR) besteht aus Hunderten von PR Molekülen, die unter Lichteinwirkung Protonen pumpen und somit eine bedeutende Rolle für die Energiegewinnung spielen könnten. Da der pKa Wert des Proton Akzeptors der Schiff‘schen Base (SB) (~7.2) dem pH Wertes der Ozeane (~7.9) ähnelt, wird auch über eine regulatorische Funktion spekuliert. Wird in Erwägung gezogen, dass 24 000 PR Moleküle pro SAR86 Zelle vorhanden sind (Beja et al. 2001) und dass 13% der Bakterien der Meeresoberfläche PR besitzen (Sabehi et al. 2005) liefert dieses Protein wahrscheinlich einen bedeutenden Energiebeitrag neben der Photosynthese. Einblicke in den Mechanismus der Energieumwandlung erfordern sowohl die Untersuchung des Chromophores, welches die Lichtenergie absorbiert als auch der Struktur des Apoproteins, das durch die Generierung eines Protonengradienten zur Energiegewinnung beiträgt. Der Fokus der Doktorarbeit liegt auf dem Chromophor und seiner Umgebung. Eine erste Charakterisierung der SB und des Retinals erfolgt durch UV/VIS und NMR Messungen (Pfleger et al. 2008). Die 13C chemische Verschiebungen von 10,11-13C2 Retinal und die 15N chemische Verschiebung der protonierten SB, gebildet durch K231, zeigt eindeutig, dass im Grundzustand nur eine Konformation der Retinals, all-trans, vorliegt. Die 15N chemische Verschiebung weist außerdem auf eine starke Wechselwirkung der SB mit ihren Gegenionen hin. Desweiteren kann durch Messungen der 15N chemischen Verschiebung der SB bei verschiedenen pH Werten der pKa Wert der SB abgeschätzt werden, auf ~12. Diese Stabilisierung der positiv geladenen protonierten Form der SB weist auf die Existenz eines Wasserclusters hin, das durch die hohe Dielektrizitätskonstante die protonierte Form der SB stabilisieren könnte. Um zu überprüfen, ob Wasser an der SB gebunden ist, wird ein sogenanntes 15N-1H HETCOR Experiment durchgeführt. Der Bereich der 15N chemischen Verschiebung der SB korreliert mit einer Protonenresonanz bei ~5 ppm, welche im Bereich einer Wasserresonanz liegt und die durch D2O austauschbar ist. Dies indiziert eine wichtige Bedeutung von Wasser in der Nähe der SB für die Funktion von PR. Der Einfluss von Mutationen des Histidins H75 und des Aspartats D97 auf die 15N chemische Verschiebung der SB sowie die Auswirkung von Histidinmutationen auf das Chromophor deuten eine direkte Wechselwirkung von Aspartat 97 und der SB an, nicht aber eine direkte Wechselwirkung von H75 und der SB. Neben dem Chromophor ist außerdem das Signalpeptid Gegenstand der Untersuchung der Doktorarbeit. Motivation für die Untersuchung war die Inhomogenität der Proben, die im Zusammenhang mit ungleich prozessiertem PR stehen könnten. Ein zweiter Teil beschäftigt sich mit neuen Konzepten der Datenaufnahme, da das S/R in der Festkörper NMR ein limitierender Faktor darstellt. Diese beinhalten Verstärkung der Relaxation (RELOAD) sowie die Refokussierung von T2 bei Verwendung eines Prozessierungsschrittes, der „half echo alternating transformation“ (HEAT).
This thesis is based on the following publications (in chronological order): 1. Biegel, E., S. Schmidt & V. Müller (2009) Genetic, immunological and biochemical evidence for a Rnf complex in the acetogen Acetobacterium woodii. Environ. Microbiol. 11: 1438-1443. My contribution: Amplification, sequence determination and analysis of Rnf homologues, enrichment of the Rnf complex 2. Biegel, E. & V. Müller (2010) Bacterial Na+-translocating ferredoxin:NAD+ oxidoreductase. Proc. Nat. Acad. Sci. U. S. A. 107: 18138-18142. My contribution: I designed and performed all experiments shown and interpreted the data. 3. Biegel, E., S. Schmidt, J. Gonzáles & V. Müller (2010) Biochemistry, evolution and physiological function of the Rnf complex, a novel ion-motive electron transport complex in prokaryotes. Cell. Mol. Life Sci., in press. DOI: 10.1007/s00018-010-0555-8. My contribution: I was involved in writing all chapters except chapters: „phylogenetic analyses of rnf genes“ and „distribution of rnf genes“. 4. Biegel, E. & V. Müller (2010) A Na+-translocating pyrophosphatase in the acetogenic bacterium Acetobacterium woodii. J. Biol. Chem., in press. DOI: 10.1074/jbc.M110.192823. My contribution: I designed and performed all experiments shown and interpreted the data.
Effort estimates are of utmost economic importance in software development projects. Estimates bridge the gap between managers and the invisible and almost artistic domain of developers. They give a means to managers to track and control projects. Consequently, numerous estimation approaches have been developed over the past decades, starting with Allan Albrecht's Function Point Analysis in the late 1970s. However, this work neither tries to develop just another estimation approach, nor focuses on improving accuracy of existing techniques. Instead of characterizing software development as a technological problem, this work understands software development as a sociological challenge. Consequently, this work focuses on the question, what happens when developers are confronted with estimates representing the major instrument of management control? Do estimates influence developers, or are they unaffected? Is it irrational to expect that developers start to communicate and discuss estimates, conform to them, work strategically, hide progress or delay? This study shows that it is inappropriate to assume an independency of estimated and actual development effort. A theory is developed and tested, that explains how developers and managers influence the relationship between estimated and actual development effort. The theory therefore elaborates the phenomenon of estimation fulfillment.
In der vorliegenden Arbeit konzentrierte ich mich auf mediterrane wirbellose Tierarten, welche sich als Konsequenz ihrer Lebensweise nur schlecht ausbreiten können. Nichtsdestotrotz haben es Süßwasserkrabben der Gattung Potamon und Landschnecken der Gattung Tudorella geschafft, große Gebiete zu besiedeln, die heute durch das Mittelmeer getrennt sind. Für beide Gruppen wurde spekuliert, dass Menschen an ihrer Ausbreitung beteiligt waren. Es war mein Ziel die biogeographischen Muster dieser beiden Gattungen zu analysieren und abzuschätzen, ob Menschen tatsächlich Vektoren ihrer Ausbreitung waren. Meine Analysen fanden auf drei Ebenen statt: Taxonomie, Gattung und Art.
In human neuroscientific research, there has been an increasing interest in how the brain computes the value of an anticipated outcome. However, evidence is still missing about which valuation related brain regions are modulated by the proximity to an expected goal and the previously invested effort to reach a goal. The aim of this dissertation is to investigate the effects of goal proximity and invested effort on valuation related regions in the human brain. We addressed this question in two fMRI studies by integrating a commonly used reward anticipation task in differential versions of a Multitrial Reward Schedule Paradigm. In both experiments, subjects had to perform consecutive reward anticipation tasks under two different reward contingencies: in the delayed condition, participants received a monetary reward only after successful completion of multiple consecutive trials. In the immediate condition, money was earned after every successful trial. In the first study, we could demonstrate that the rostral cingulate zone of the posterior medial frontal cortex signals action value contingent to goal proximity, thereby replicating neurophysiological findings about goal proximity signals in a homologous region in non-human primates. The findings of the second study imply that brain regions associated with general cognitive control processes are modulated by previous effort investment. Furthermore, we found the posterior lateral prefrontal cortex and the orbitofrontal cortex to be involved in coding for the effort-based context of a situation. In sum, these results extend the role of the human rostral cingulate zone in outcome evaluation to the continuous updating of action values over a course of action steps based on the proximity to the expected reward. Furthermore, we tentatively suggest that previous effort investment invokes processes under the control of the executive system, and that posterior lateral prefrontal cortex and the orbitofrontal cortex are involved in an effort-based context representation that can be used for outcome evaluation that is dependent on the characteristics of the current situation.
In the work presented herein the microscopic transport model BAMPS (Boltzmann Approach to Multi-Parton Scatterings) is applied to simulate the time evolution of the hot partonic medium that is created in Au+Au collisions at the Relativistic Heavy Ion Collider (RHIC) and in Pb+Pb collisions at the recently started Large Hadron Collider (LHC). The study is especially focused on the investigation of the nuclear modification factor R_{AA}, that quantifies the suppression of particle yields at large transverse momentum with respect to a scaled proton+proton reference, and the simultaneous description of the collective properties of the medium in terms of the elliptic flow v_{2} within a common framework.
This dissertation contains three essays on monetary policy, dynamics of the interest rates and spillovers across economies. In the first essay I examine the effects of monetary policy and its interaction with financial regulation within a micro-founded macroeconometric framework for a closed economy with a heterogeneous banking system, facing a period of low interest rates. I analyse the interplay between monetary policy and banking regulation and study the role of agents’ expectations for the effectiveness of unconventional monetary policy tools. In the next essay, I argue that openness is crucial for understanding the dynamics of the term structure. In an empirical application, I show that my model of the term structure fits well the yield curve in-sample and has a sound ability to forecast interest rates out-of-sample. The model accounts for the expectations hypothesis, replicates the forward premium anomaly and reconciles the uncovered interest rate parity implications. The last essay is concerned with the dynamics of co-movement among macroeconomic aggregates and the degree of convergence or decoupling amongst economies. The model includes measures of financial and trade-based interdependencies and incorporates feedback between macroeconomic variables and time-varying weights. The findings point at the importance of asset price movements and financial linkages.
Organische Materialien haben bis zur Mitte des 20. Jahrhunderts hinsichtlich ihrer elektronischen Eigenschaften keine besondere Aufmerksamkeit auf sich gezogen. Größeres Interesse an diesen Materialien entstand erst durch die Entdeckung einer ungewöhnlich hohen elektrischen Leitfähigkeit des organischen Perylen-Bromin Ladungstransfer-Komplexes durch Inokuchi et al. im Jahr 1954. Diese neue Klasse von Materialien besteht typischerweise aus Donor- und Akzeptor-Molekülen, die in einer bestimmten Stöchiometrie aneinander gebunden sind. Elektrische Ladung wird zwischen den Donor- und Akzeptor-Molekülen transferiert. Um diesen Prozess zu beschreiben, entwickelte Robert Mulliken in den 60er Jahren ein theoretisches Gerüst. Abhängig von der Anordnung der Moleküle und transferierten elektrischen Ladung kann der Ladungstransfer-Komplex (oder Salz) ein Isolator, ein Halbleiter, ein Metall oder sogar ein Supraleiter sein. Noch mehr Aufmerksamkeit erhielten Ladungstransfer-Materialien mit der Entdeckung des ersten quasi-eindimensionalen organischen Metalls TTF-TCNQ (tetrathiafulvalene-tetracyanoquinodimethane) im Jahr 1973. ...
In this thesis, laboratory investigations have been conducted to investigate several processes occurring during the melt segregation (crystal settling and compaction processes), as well as during emplacement of plutons. With the help of three different sets of centrifuge experiments rates of these three magmatic processes have been evaluated. In the first series of the centrifuge experiments, the diapiric ascent of buoyant material from two source layers at different depths was studied. Through five models, the hypothesis of ascending diapirs was tested and it was demonstrated whether a rising diapir ascends straight upward or if its ascent might be deviated by another buoyant, softer – and consequently easier to travel through – layer which is located within the overburden strata. We were interested under which conditions they can be formed. For this purpose we placed perturbations on top of both the buoyant layers; either with a set-off of both the protrusions (for three of these experiments), or with both protrusion sitting directly on top of each other (for one of the experiments). In the first experiment, we omitted the perturbations, to test which pathways diapirs take which grow from natural Rayleigh-Taylor instabilities. Three others experiments differed in the viscosity contrast between the overburden and the buoyant material. Through the experimental runs, the effects of different overburden viscosities and perturbation positions on the number of the diapirs were observed. The modeling results show that two diapirs rising from the offset perturbations do not take the same pathway through the overburden layer. Rather, each diapir takes a different pathway, with the deeper diapir piercing through its overburden while rising, regardless if it was a buoyant layer or denser overburden layers. However, when the two perturbations were situated directly above each other in the different PDMS layers, this resulted in the formation of one big diapir rather than several smaller ones, and the overburden layer was less deformed than with offset perturbations. Diapiric structures as those derived from the models without perturbation and where the perturbation are offset occur within Great Kavir Basin (Iran), where numerous salt diapirs grew from several salt horizons, which show a similar spatial distribution. The resulting structure observed in the model where the two perturbations situated directly above each other, is close to what is observed in composite batholiths such as the Flasergranitoid Zone within the Bergsträßer Odenwald Crystalline Complex (Germany). The second series of models were aimed to study crystal settling within a magma. For this purpose experiments with an artificial magma of 30 vol% olivine in 70 vol% basaltic melt were conducted to elucidate the formation mechanisms and time scales of gravitational cumulates. Through the experiments, two physical processes have been observed: (i) purely mechanical compaction, and (ii) chemical compaction induced by dissolution and re-precipitation of settled crystals. The results reveals that the mechanical settling of the dense olivine suspension occurs at about 1/6 the speed of simple Stokes settling, and a sedimentation exponent n of 4.1 is found. Evidences of chemical compaction induced by dissolution and re-precipitation of settled crystals have been highlighted by a detailed analysis of the fine structure of olivine grain boundaries. This last has revealed (1) the presence of Ca, which is characteristic only for MORB-melt, at the interface of two adjacent Ol-grains even when no melt is present; (2) a not fully crystallized boundary layer between two adjacent olivine grains. The crystal size distribution curves and the grain size growth exponent n ~3.6 indicate that diffusion controlled Ostwald ripening is the dominant crystal growth mechanism in concentrated magmatic suspensions. Finally, the formation times in natural olivine adcumulates have been calculated. The last series of centrifuge experiments deals with the crystal-melt settling-floating mechanism in a system composed of natural two pyroxene gabbro. The results have revealed a vertical evolution of the major and trace elements in the melt phase. Then, a numerical modelling of the sedimentation process of the crystals has been made in order to describe the compaction evolution with time. In comparing the numerical simulation with the centrifuge modelling, the stratification of the compacted layer in the runs is reproduced in numerical models. Moreover, on the base of the numerical and centrifuge modelling, a sedimentation exponent describing a deviation of settling in concentrated suspensions from Stokes sedimentation has been evaluated. Finally, the numerical simulation is applied to the Muskox intrusion to estimate the formation time and the melt fraction evolution in using the hindered sedimentation model calculations.
This work deals with the use of dielectrics with high permeability, so-called high-k dielectrics in organic thin-film field-effect transistors (FETs). The central part was the preparation of the high-k dielectric and its implementation in transistors, in which organic semiconductors were used as active layer. A field-effect transistor can be used to measure the charge carrier mobility. Employing high-k dielectrics the carrier concentration in the active layer can be greatly increased. In this way, high charge carrier concentrations in organic layers can be achieved without chemical doping. As high-k dielectric strontium titanate (STO) was selected. It is also available as a niobium-doped and therefore conducting substrate material. Thus, one has an ideal substrate for the growth of the dielectric layer in conjunction with a substrate which acts as gate electrode. As the organic semiconductor the small molecules pentacene and copper phthalocyanine (CuPc) were sublimated, as electrical contacts gold was used. As a key part of this work an ultra high vacuum chamber system was constructed for in situ preparation of field effect transistors. For the deposition of the organic thin films a molecular beam deposition chamber was built, including a manipulator and effusion cells as evaporation sources. For the preparation of the dielectric a sputtering chamber was set-up. Another chamber was used in conjunction with an effusion cell for the deposition of the gold contacts. For the structured deposition of the different layers in the devices a shadow mask system was implemented. Movable masks could be positioned by means of a wobble stick onto the sample carriers. The system thus allowed for the use of masks in all chambers. The different thin films required in the transistor structure were first individually prepared and characterized. For the characterization primarily X-ray diffraction and optical microscopy were used. The growth of pentacene was analyzed on aplha-AlO substrates. With X-ray diffraction the (00l) reflections of the thin film phase were observed. In growth studies of CuPc aplha-AlO and STO substrates were used. With X-ray diffraction the aplha-phase was detected. With increasing substrate temperature an increase in crystallinity, but also an increase in surface roughness was observed. The sputtering of STO as a high-k dielectric was studied and optimized. Simultaneously, a high deposition rate, a smooth film surface and good crystallinity of the layer were required. As the most important parameters the substrate temperature, pressure and sputtering power were identified. Argon and oxygen were employed as sputtering gases, as substrate MgO was used. The films showed in comparison to crystalline STO a distortion to larger lattice constants. The degree of distortion decreased with increasing chamber pressure, on the other hand, deposition rate decreased with increasing chamber pressure as well. By combining the individual deposition processes FETs in bottom-gate geometry were prepared. The first step was always sputtering of the STO dielectric on niobium-doped STO substrates. Subsequently, the electrodes and the organic layer were deposited. For comparison transistors on silicon substrates with silicon dioxide (SiO2) as the dielectric were prepared. To study the transistor properties a measurement setup was build. A dielectric constant of about 190 for the STO in the transistors was achieved. The transistors with CuPc as active layer showed p-type conduction behavior. The transistors with STO as dielectric had a much stronger response than those with SiO2. They reached mobilities of 2E-4 cm2/Vs at very low applied voltages of 3V. It could thus be demonstrated that STO is suitable as a dielectric for organic FETs, and that through the use of high-k dielectrics high charge carrier densities can be achieved.
NK cells are part of the innate immune system, and are important players in the body’s first defence line against virus-infected and malignantly transformed cells. While T cells recognize neoplastic cells in an MHC-restricted fashion, NK cells do not require prior sensitization and education about the target. In leukemia and lymphoma patients undergoing allogeneic hematopoietic stem cell transplantation not only T cells but also NK cells have been found to mediate potent graft-versus-tumor effects. Hence, autologous or donor-derived NK cells hold great promise for cancer immunotherapy. Since the generation of highly purified NK cell products for clinical applications is labor-intensive and time consuming, established human NK cell lines such as NK-92 are also being considered for clinical protocols. NK-92 cells display phenotypic and functional characteristics similar to activated primary NK cells. While NK-92 cells are highly cytotoxic towards malignant cells of hematologic origin, they do not affect healthy human tissues. NK-92 cells can be expanded under GMP-compliant conditions, and can therefore be provided in sufficient numbers with defined phenotypic characteristics for clinical applications. Safety of NK-92 cells for adoptive immunotherapy was already shown in two phase I/II clinical trials...
Capoeta damascina (Teleostei: Cyprinidae) is one of the most common freshwater fish species, found throughout the Levant, Mesopotamia, Turkey and Iran. According to the state of knowledge prior to this study, C. damascina, which is distributed over a wide range of isolated water bodies, was not a well-defined species. It was questionable whether it represents a single species or a complex of closely related species with high intraspecific and comparatively low interspecific variability. The goal of this study was to investigate the taxonomy, systematic position of the C. damascina species complex and the phylogenetic relationships among its members, based on morphological features as well as molecular phylogeny. Samples obtained from throughout the geographic range of this species complex were subjected to comparative morphological analyses in order to define, properly diagnose and separate species within the C. damascina complex. To elucidate phylogenetic relationships among members of the C. damascina species complex, samples were subjected to genetic analyses, using two molecular markers targeting the mitochondrial cytochrome oxidase I (COI, n = 103) and the two adjacent divergence regions (D1-D2) of the nuclear 28S rRNA genes (LSU, n = 65). Based on morphological and molecular genetic data, six closely related species were recognized within the C. damascina complex: C. buhsei, C. caelestis, C. damascina, C. saadii, C. umbla and an undescribed species, Capoeta sp.1. Analyses of the morphometric and meristic data obtained in this study revealed phenotypic variability among the various populations within a species and among the different species. Such differences in morphological characters reflect genetic differences, environmentally induced phenotypic variation or both, as the meristic phenotype of fish is sometimes a consequence of environmental parameters acting on the genotype. Based on phylogenetic analyses, two main lineages were identified within the C. damascina species complex: a western lineage represented by C. caelestis, C. damascina and C. umbla and an eastern lineage represented by C. buhsei, C. saadii and Capoeta sp.1. The close phylogenetic relationships between C. damascina and C. umbla and the sharing of same haplotypes between one specimen of C. damascina from Euphrates and another of C. umbla from Tigris reflect one of three possibilites: recent speciation, mitochondrial introgression or a combination of both. The results obtained in this study indicate that speciation of the above-mentioned six taxa is quite recent and that their dispersal and present-day distribution can be related to Pleistocene events. The drying out of the Persian Gulf, probably during one of the first glacials of the Pleistocene, led the ancestor of the C. damascina species complex in Mesopotamia to reach the rivers of the Gulf and of Hormuz basins and differentiate there, giving rise to the eastern lineage (ancestor of C. buhsei, C. saadii and Capoeta sp.1). As connections presumably existed among the different river drainages and basins in Iran during the wet periods of the Pleistocene, the ancestor of C. buhsei, C. saadii and Capoeta sp.1 was subsequently able to colonize the various Iranian drainages and differentiate there, giving rise to C. buhsei, C. saadii and Capoeta sp.1. After the separation from the eastern lineage, the western lineage, represented by the ancestor of C. damascina, C. umbla and C. caelestis, most likely reached the Levant from the Tigris-Euphrates system during the Pleistocene glacials, when river connections existed in the regions of the upper courses of Ceyhan Nehri (southern Turkey) and some western affluents to the Euphrates. From Ceyhan Nehri, it dispersed into other rivers in southern Turkey during Pleistocene periods of low sea levels until it reached Göksu Nehri and evolved into C. caelestis. The sister population differentiated into C. damascina and C. umbla. Based on the results obtained in this study, it is likely that C. damascina colonized the Levant and southern Turkey during the Pleistocene glacials. This is well supported by the low genetic variability among the C. damascina populations. Direct connections existed among the river drainages in the Levant during the Pleistocene periods of low sea level, thus serving as a pathway for the dispersal of C. damascina. The results of this study provide a coherent picture of the taxonomic position, phylogenetic relationships and evolutionary history of the C. damascina species complex and explain present patterns of distribution considering paleogeographic events.
The objective of this thesis is to develop new methodologies for formal verification of nonlinear analog circuits. Therefore, new approaches to discrete modeling of analog circuits, specification of analog circuit properties and formal verification algorithms are introduced. Formal approaches to verification of analog circuits are not yet introduced into industrial design flows and still subject to research. Formal verification proves specification conformance for all possible input conditions and all possible internal states of a circuit. Automatically proving that a model of the circuit satisfies a declarative machine-readable property specification is referred to as model checking. Equivalence checking proves the equivalence of two circuit implementations. Starting from the state of the art in modeling analog circuits for simulation-based verification, discrete modeling of analog circuits for state space-based formal verification methodologies is motivated in this thesis. In order to improve the discrete modeling of analog circuits, a new trajectory-directed partitioning algorithm was developed in the scope of this thesis. This new approach determines the partitioning of the state space parallel or orthogonal to the trajectories of the state space dynamics. Therewith, a high accuracy of the successor relation is achieved in combination with a lower number of states necessary for a discrete model of equal accuracy compared to the state-of-the-art hyperbox-approach. The mapping of the partitioning to a discrete analog transition structure (DATS) enables the application of formal verification algorithms. By analyzing digital specification concepts and the existing approaches to analog property specification, the requirements for a new specification language for analog properties have been discussed in this thesis. On the one hand, it shall meet the requirements for formal specification of verification approaches applied to DATS models. On the other hand, the language syntax shall be oriented on natural language phrases. By synthesis of these requirements, the analog specification language (ASL) was developed in the scope of this thesis. The verification algorithms for model checking, that were developed in combination with ASL for application to DATS models generated with the new trajectory-directed approach, offer a significant enhancement compared to the state of the art. In order to prepare a transition of signal-based to state space-based verification methodologies, an approach to transfer transient simulation results from non-formal test bench simulation flows into a partial state space representation in form of a DATS has been developed in the scope of this thesis. As has been demonstrated by examples, the same ASL specification that was developed for formal model checking on complete discrete models could be evaluated without modifications on transient simulation waveforms. An approach to counterexample generation for the formal ASL model checking methodology offers to generate transition sequences from a defined starting state to a specification-violating state for inspection in transient simulation environments. Based on this counterexample generation, a new formal verification methodology using complete state space-covering input stimuli was developed. By conducting a transient simulation with these complete state space-covering input stimuli, the circuit adopts every state and transition that were visited during stimulus generation. An alternative formal verification methodology is given by retransferring the transient simulation responses to a DATS model and by applying the ASL verification algorithms in combination with an ASL property specification. Moreover, the complete state space-covering input stimuli can be applied to develop a formal equivalence checking methodology. Therewith, the equivalence of two implementations can be proven for every inner state of both systems by comparing the transient simulation responses to the complete-coverage stimuli of both circuits. In order to visually inspect the results of the newly introduced verification methodologies, an approach to dynamic state space visualization using multi-parallel particle simulation was developed. Due to the particles being randomly distributed over the complete state space and moving corresponding to the state space dynamics, another perspective to the system's behavior is provided that covers the state space and hence offers formal results. The prototypic implementations of the formal verification methodologies developed in the scope of this thesis have been applied to several example circuits. The acquired results for the new approaches to discrete modeling, specification and verification algorithms all demonstrate the capability of the new verification methodologies to be applied to complex circuit blocks and their properties.
This dissertation investigated the development of the complementiser that from the demonstrative pronoun in the Germanic languages; each chapter dealt with a different aspect. In the introduction, the terms ‘reanalysis’ and ‘analogy’ and their relevance for grammaticalisation were explained, and the issues of the chapters were presented. The second chapter introduced some information about the Germanic language family and the languages which were relevant for this investigation, namely Gothic, Old English, Old Icelandic, Old Saxon and Old High German. Previous assumptions about the diachrony of that were presented and discussed. One of these proposals which mainly draws on evidence from West Germanic involves the idea that the source construction contained two independent main clauses with a demonstrative pronoun (that) at the end of the first clause (cf. e.g. Paul 1962, § 248). In contrast to this, the Gothic evidence showed that the source construction of the reanalysis of ϸatei was not a proper paratactic construction (at least in Gothic) but already a complex construction which contained a complementiser (ei) in the appositional subordinate clause (cf. also e.g. Longobardi 1994 for the diachrony of ϸatei). This contradiction raised the question whether the analysis of the Gothic that-complementiser also applies to the diachrony of that in West Germanic. This issue was taken up in the third chapter which presented an overview of subordination and complementisers in Northwest Germanic. The aim was to show that the Northwest Germanic languages also show a subordinating particle, which functions like the Gothic ei, namely ϸe (OE), er/es (OI), the (OHG, OS). As a result, the subordinating particle could be observed in relative and adverbial clauses in all Northwest Germanic languages. In complement clauses, which are most crucial for the argumentation, the subordinating particle is found in Old English and Old Icelandic but not in Old Saxon. In Old High German, there are only combinations of the with a following pronoun, theih and theiz, in ‘Otfrids Evangelienbuch’ (see Wunder 1965). Consequently, the presence of a subordinating particle is confirmed in North and West Germanic. The fact that the patterns of subordination are quite similar in all Germanic languages suggested a unitary analysis of the development of that in Germanic was appropriate. In chapter four, the similarities and differences between the Germanic languages with respect to the development of that were explained. It was argued that the preconditions of the reanalysis were the same, whereas the consequences of the reanalysis are realised differently in each language. The most important precondition was that the appositional source construction (explained in more detail below) was generally available in Germanic. Since the demonstrative pronoun at the end of the matrix clause and the subordinating particle of the subordinate clause were adjacent, phonological combination might have been crucial for the subsequent reanalysis to take place. After reanalysis, however, different changes can be observed in the different languages. For instance, it appears that during the Old English period the final syllable of the form ϸætte was deleted (see chapter 4 for references), whereas the final –ei is still present in the Gothic ϸatei, and completely absent in Old High German and Old Saxon. The source structure of the reanalysis was discussed in detail in a separate subsection. The appositional source construction, which was already assumed for the reanalysis of Gothic ϸatei, was compared with analyses of clitic left dislocation which propose that two constituents with the same theta-role derive from a Big DP (see e.g. Grewendorf 2009, Belletti 2005). Based on the Big DP analysis of Grewendorf (2009), it was claimed that the appositional clause, introduced by the subordinating particle, is generated in the Spec of a DP, and adjoined to this DP on the surface. It was argued that this whole complement DP-node occurred in an extraposed position in OV-languages so that the verb, when it stays in-situ, does not appear between the demonstrative pronoun and the subordinating particle. The structure in (1) illustrates the syntactic source structure which is assumed to apply to the development of the complementiser that in Germanic. ...
In total, this dissertation comprises three research papers. Objective of all of these papers are to detect mistakes of private investors when conducting mutual funds investments and to analyze the implications. Moreover, the question is addressed whether financial advisors help private investors to avoid these investment mistakes. All three research papers use the same data base which has been provided by a German online brokerage house. The detailed data set allows contributing to existing literature on mutual fund investments, smart decision making, household finance as well as financial advice on an investor- and transaction-specific level. The first paper addresses the question which particular decision criteria private investors use when purchasing mutual funds. It can be shown that funds volume is the dominating decision criterion, whereas historical performance is only of minor importance. As performance persistence exists in the underlying data set, it can be concluded that the majority of investors make investment mistakes. In the second paper it is shown that smart investors, i.e. investors who purchase mutual funds by chasing historical performance, are older, wealthier, more experienced and less likely to be overconfident. In addition, it can be verified that there exists a positive impact of the ability to select mutual funds by chasing historical performance on the overall investment success. Hence, the quality of mutual fund selection ability is an ex-ante measure for investment success. Finally, the third paper analyzes the influence of financial advice on mutual fund decision making of private investments. Evidence can be provided that financial advisors do not help their customers to purchase mutual funds by chasing historical performance. In fact, advisors recommend high-volume mutual funds from well-known fund families. Apparently, financial advisors are much more salesmen than real advisors. These results hold when controlling for potential endogeneity issues.