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Gridded maps of meteorological variables are needed for the evaluation of weather and climate models and for climate change monitoring. In order to produce them, values at locations where no observing stations are available need to be estimated from point-wise observations. For the interpolation of meteorological observations deterministic and stochastic methods are often combined. Deterministic methods can account for ancillary information such as elevation, continentality or satellite observations. Stochastic methods such as kriging reproduce observed values at the station locations and also account for spatial variability. In the first two studies of this thesis, a flexible interpolation method for the gridding of locally observed daily extreme temperatures is developed that also provides an optimal estimate of the interpolation ncertainty. In the third study, an observational dataset is created using this interpolation method and then applied to evaluate a climate simulation for Africa.
In the first study, the Regression-Kriging-Kriging (RKK) method is tested for the interpolation of daily minimum and maximum temperatures (Tmin and Tmax) in different regions in Europe. RKK accounts for elevation, continentality index and zonal mean temperature and is applicable in regions of differing station density and climate. The accuracy of RKK is compared to Inverse Distance Weighting, a common deterministic interpolation method, and to Ordinary Kriging, a common stochastic interpolation method. The first step in RKK is to use regression kriging, in which multiple linear regression accounts for topographical effects on the temperature field and kriging minimizes the regression error, to interpolate climatological means. In the second step daily deviations from the monthly climatology are interpolated using simple kriging. Owing to the large climatological differences across the investigation area the interpolation is performed in homogeneous subregions defined according to the Köppen-Geiger climate classification. Cross validation demonstrates the superiority of RKK over the simpler algorithms in terms of accuracy and preservation of spatial variability. The interpolation performance however strongly varies across Europe, being considerably higher over Central Europe (highest station density) than over Greenland (few stations along the coast line). This illustrates the strong impact of the station density on the accuracy of the interpolation result. Satellites provide comprehensive observations of climate variables such as land surface temperature (LST) and cloud cover (CC). However, LST is associated with high uncertainty (standard error ~ 1-2°C), preventing its direct application in meteorology and climatology. The second study investigates the usefulness of LST and CC as predictors for the gridding of daily Tmin and Tmax. The RKK algorithm is compared with similar interpolation methods that apply LST and CC in addition to the predictors used with the RKK algorithm. The investigation is conducted in two regions, Central Europe and the Iberian Peninsula, which differ strongly in average cloud cover (Central Europe is approximately 30% cloud free and the Iberian Peninsula approximately 60 % cloud free). RKKLST (in which monthly mean LST is used as an additional predictor) yields for Central Europe no clear improvement over RKK, yet it reduces the interpolation error over the Iberian Peninsula. This finding can be explained by the higher percentage of cloud free pixels over that region in summer which enables a more robust determination of monthly mean LST. Adding a regression step for daily anomalies (using the predictor CC) yields the RKRK method and improves the preservation of spatial variability over the Iberian Peninsula. Moreover, a successive reduction of the station number (from 140 to 10 stations) reveals an increasing superiority of RKKLST and RKRK over RKK in both regions.
The application of a gridded observational dataset for climate monitoring or climate model validation requires knowledge of the uncertainties associated with the dataset. The estimation of the interpolation uncertainty, here the inter quartile range is the used uncertainty measure, is therefore an important issue within the frame of this thesis. By means of cross validation it is shown that the largest uncertainties occur in regions of low station density (e.g. Greenland), in mountainous regions and along coastlines (in these regions model evaluation results should be interpreted carefully). The magnitude of the interpolation error mainly depends on the station density, while the complexity of terrain has substantially less influence. On average over all regions and investigation days the target precision of the uncertainty estimate is reached. However, on local scales and for single days it can be clearly over- or underestimated. The application of satellite-derived predictors (LST and CC) yields no noteworthy improvement of the uncertainty estimate.
In the last study two regional climate simulations for Africa using the ERA-Interim driven COSMO-CLM (CCLM) model at two different horizontal resolutions (0.22° and 0.44°) are validated. It is assessed whether observed patterns and statistical properties of daily Tmin and Tmax are correctly represented in the model. The ERA-Interim reanalysis and a specially created observational dataset are used as reference. The observational dataset is generated by applying the RKRK algorithm (developed within the second study). The investigations show an occasionally large bias in Tmin and Tmax. The hemispheric summers are generally too warm and the temporal variability in temperature is too high, particularly over extra tropical Africa. The diurnal temperature range is overestimated by about 2°C in the northern subtropics but underestimated by about 2°C over large parts of the African tropics. CCLM reproduces the observed frequency distribution of daily Tmin and Tmax in all African climate regions, and the extreme values in the lower percentiles (5, 10, 20%) for Tmin are well simulated. The higher percentiles (80, 90, 95%) for Tmax are however overestimated by 2-5°C. For both Tmin and Tmax the 0.22° simulation is on average 0.5°C warmer than the 0.44° simulation. Additionally, the higher percentiles are about 1°C warmer for both Tmin and Tmax in the higher resolution run, while the lower percentiles in both runs match very well. Although the temperature pattern is represented in more detail along the coastlines and in topographically complex regions, the higher resolution simulation yields no qualitative improvement.
To summarize, the choice of the appropriate algorithm mainly depends on the interpolation conditions. In cases where the station density is high across the target region and the predictor space is adequately covered by observing stations, the computationally less demanding RK algorithm should be preferred. In regions where the station density is low the more robust RKRK algorithm should be the first choice. Due to the strong physical relation of both CC and LST to Tmin and Tmax the missing information is at least partially compensated for. The estimation of the interpolation uncertainty could be improved by applying a normal score transformation to the data prior to a kriging step. This is because the kriging assumption that the increments of the variable of interest are second order stationary can be approximately met by a normal score transformation.
Der Begriff psychologische Akkulturation beschreibt jene Veränderungen, die infolge des dauerhaften Aufeinandertreffens verschiedener kultureller Gruppen auf individueller Ebene zu beobachten sind (Berry, 1997). Die vorliegende Arbeit umfasst drei Publikationen, die sich mit Akkulturationsprozessen von Kindern und Jugendlichen mit Migrationshintergrund in Deutschland befassen. Zunächst wird ein Überblick über den aktuellen Stand der Forschung zur Situation junger Migranten in Deutschland vorgelegt. An zentraler Stelle steht dabei die Frage, wie die Migrationsgeschichte und Immigrationspolitik Deutschlands sowie die öffentliche Einstellung gegenüber Migranten die transkulturelle Adaptation von Kindern und Jugendlichen nicht-deutscher ethno-kultureller Herkunft beeinflussen. Bereits bestehende wissenschaftliche Erkenntnisse werden verknüpft mit den Ergebnissen neuerer empirischer Studien um zu einem tieferen Verständnis der Ursachen für die vielfach berichteten problematischen Verläufe psychologischer und soziokultureller Adaptation von Migranten beizutragen. Neben anderen Risiken und protektiven Faktoren wird diskutiert, wie sich Besonderheiten Deutschlands als Aufnahmeland, wie z.B. die Eigenarten des Schulsystems, auf Adaptationsverläufe auswirken können. Unsere eigenen Studien tragen zum Verständnis der Anpassungsprozesse junger Migranten bei, indem sie aufzeigen, dass nicht die Akkulturationsstrategie der Integration, sondern speziell die Orientierung an der deutschen Kultur bei Individuen zu den günstigsten psychologischen und soziokulturellen Ergebnissen zu führen scheint. Im Rahmen dieser Arbeit wird weiterhin ein empirischer und methodologischer Beitrag zur Akkulturationsforschung geleistet, indem ein Messinstrument zur Erfassung psychologischer Akkulturation bei Kindern im deutschen Sprachraum – die Frankfurter Akkulturationsskala für Kinder (FRAKK-K)– entwickelt, validiert und schließlich anhand einer Fragestellung praktisch angewandt wird. Die Skalenentwicklung und –optimierung erfolgte auf der Grundlage von zwei Studien, welche Daten von 387 Grundschülern aus zwei städtischen Regionen in Deutschland umfassen (Frankenberg & Bongard, 2013). Die Ergebnisse konfirmatorischer Faktorenanalysen sprechen für zwei Faktoren, Orientierung an der Aufnahmekultur und Orientierung an der Herkunftskultur, die jeweils mittels 6 Items erfasst werden. Beide Subskalen weisen eine zufriedenstellende interne Reliabilität und Kriteriumsvalidität auf und lassen sich zwecks Erfassung der Akkulturationsstrategie kombinieren (i.e. Assimilation, Integration, Separation und Marginalisierung). In einer ersten praktischen Anwendung der Skala wird der Frage nachgegangen, inwiefern erweiterter Musikunterricht und Orchesterspiel in der Grundschule über verstärkte Gruppenkohäsion zur Förderung kultureller Integration beitragen können.
Grundschüler, die in einem Orchester gespielt haben, zeigen über einen Zeitraum von 1,5 Jahren einen stärkeren Anstieg der Orientierung an der deutschen Kultur als Schüler, die keinen erweiterten Musikunterricht erhielten. Musikschüler fühlen sich außerdem stärker in die Klassengemeinschaft integriert. Dies deutet darauf hin, dass die Erfahrung der Zusammenarbeit und des Musizierens innerhalb einer Gruppengemeinschaft zu einer stärkeren Orientierung an der deutschen Kultur geführt hat. Die Orientierung an der Herkunftskultur blieb unbeeinflusst. Somit können Programme, die jungen Migranten die Gelegenheit bieten Musik innerhalb einer größeren, kulturell heterogenen Gruppe aufzuführen, als eine effektive Intervention zur Förderung der kulturellen Anpassung an die Mehrheitskultur und der Integration innerhalb – und außerhalb – des Klassenzimmers führen.
Abschließend werden die Ergebnisse der empirischen Untersuchungen vor dem Hintergrund des aktuellen Forschungsstandes zu neueren Akkulturationsmodellen sowie zu der Terminologie und den methodischen Herausforderungen des Forschungsfeldes in Beziehung gesetzt und kritisch reflektiert. Daraus abgeleitet werden Implikationen für zukünftige Interventionen und Forschung diskutiert.
Studies on the focusing performance of a Gabor lens depending on nonneutral plasma properties
(2013)
The concept of the Gabor lens goes back to an idea by Dennis Gabor, who proposed a magnetron-type trap as an effective diverging lens for electron beams (collecting lens for positive ion beams).
Electrons confined inside the lens volume by orthogonal magnetic and electric fields, create an electric space charge field that causes a radial symmetric focusing force on an ion beam passing through the lens volume.
Since the beginning of the 1990s, a new design of this lens type as well as numerical models to describe the confined plasma cloud have been developed at the Institute for Applied Physics (IAP, Johann Wolfgang Goethe-University Frankfurt).
Thanks to an improved understanding of the plasma confinement as a function of the external fields, two lenses have successfully been tested for low beam currents and remain in operation.
In the scope of this work, the performance of a prototype Gabor lens for the transport of intense, i.e. space charge dominated ion beams, was investigated at the High Current Test Injector (HOSTI) of GSI Helmholtzzentrum für Schwerionenforschung GmbH for the first time.
To ensure an optimal focusing performance of the Gabor lens a homogeneous and stable electron confinement is required. Therefore, new non-interceptive diagnostic methods were developed to investigate the parameters and state of the confined nonneutral plasma column as a function of the external fields.
An essential part of the studies was the time-resolved diagnostic of an occurring plasma instability and the determination of the electron temperature via optical spectroscopy. The latter necessitated the detailed investigation of atomic excitation as well as the measurement of optical-emission cross sections.
A comparison of the results from both experiments i.e. the beam transport measurements at GSI and the diagnostic experiments performed at IAP concerning the plasma state, gave first indications of possible interaction processes between the nonneutral plasma and the ion beam.
Transylvanian Saxons' migration from Romania to Germany: the formation of a 'return' diaspora?
(2013)
Processes and patterns of migration on a global scale have changed in profound ways during the last two decades (Smith and King, 2012). In the European context, this is exemplified by transformations to the traditional mobility patterns from East to West Europe (Koser and Lutz, 1998), with migrants more likely to be involved in temporary circular and transnational mobility (Favell, 2008). Since the end of the Second World War, historical and political events in Europe have facilitated the mobility of ethnic Germans from Eastern Europe to Germany. Subsequently, the fall of the Iron Curtain has permitted unrestrained East-West movements, which resulted in mass migrations towards the West and diaspora fragments in the East. However, after settlement in the West, ethnic Germans have also been absorbed within wider temporary and transnational movements (Koser, 2007). Within this context, this thesis examines the post-migratory lives of three generations of Transylvanian Saxons in Germany by exploring the cultural, social, economic and political dimensions of this community. This thesis aims to contribute to on-going academic debates about diasporas by explicitly responding to Hoerder s (2002) call for more studies on ethnic German diasporas. It shows that Transylvanian Saxons, who relocated to the ancestral homeland, do not disrupt identities and lives forged in diaspora, but rather, they negotiate complex identities and belongings in relation to both home and homeland . It reveals a double diaspora and the necessity to perceive identity and diaspora as dynamic processes and constantly evolving in relation to time, space and place. This double diasporic allegiance in the case of the Transylvanian Saxons suggests interrogating the formation of a return diaspora and its importance for processes of international migration.
The human brain is an unparalleled system: Through millions of years of evolution and during a lifespan of learning, our brains have developed remarkable abilities for dealing with incoming sensory data, extracting structure and useful information, and finally drawing the conclusions that result in the actions we take. Understanding the principles behind this machinery and building artificial systems that mimic at least some of these capabilities is a long standing goal in both the scientific and the engineering communities. While this goal still seems unreachable, we have seen tremendous progress when it comes to training data-driven algorithms on vast amounts of training data, e.g. to learn an optimal data model and its parameters in order to accomplish some task. Such algorithms are now omnipresent: they are part of recommender systems, they perform speech recognition and generally build the foundation for many semi-autonomous systems. They start to be integral part of many technical systems modern technical societies rely on for their everyday functioning. Many of these algorithms were originally inspired by biological systems or act as models for sensory data processing in mammalian brains. The response properties of a certain population of neurons in the first stages of the mammalian visual pathway, for example, can be modeled by algorithms such as Sparse Coding (SC), Independent Component Analysis (ICA) or Factor Analysis (FA). These well established learning algorithms typically assume linear interactions between the variables of the model. Most often these relationships are expressed in the form of a matrix-vector products between a matrix with learned dictionary-elements (basis vectors as column vectors) and the latent variables of these models. While on the one hand this linear interaction can sometimes be justified by the physical process for which the machine learning model is proposed, it is on the other hand often chosen just because of its mathematical and practical convenience. From an optimal coding point of view though, one would generally expect that the ideal model closely reflect the core interactions of the system it is modeling. In vision for example, one of the dominant processes giving rise to our sensory percepts are occlusions. Occluding objects are omnipresent in visual scenes and it would not be surprising if the mammalian visual system would be optimized to process occluding structures in the visual data stream. Yet, the established mathematical models of the first stages of the visual processing path (like, e.g., SC, ICA or FA) all assume linear interactions between the active image components. In this thesis we will discuss new models that aim to approximate the effects of occluding components by assuming nonlinear interactions between their activated dictionary elements. We will present learning algorithms that infer optimal parameters for these models given data. In the experiments, we will validate the algorithms on artificial ground truth data and demonstrate their ability to recover the correct model parameters. We will show that the predictions made by these nonlinear models correspond better to the experimental data measured in-vivo than the predictions made by the established linear models. Furthermore, we systematically explore and compare a large space of plausible combinations of hyperparameters and preprocessing schemes in order to eliminate any effects of artefacts on the observed results. Training nonlinear sparse coding models is computationally more demanding than training linear models. In order to perform the numerical experiments described in this thesis we developed a software framework that facilitates the implementation of massive parallel expectation maximization (EM) based learning algorithms. This infrastructure was used for all experiments described in here, as well as by collaborators in projects we will not discuss. Some of the experiments required more than 1017 floating point operations and were run on a computer cluster running on up to 5000 CPU Cores in parallel. Our parallel framework enabled these experiments to be performed.
Due to recent technical developments, it became evident that the mammalian transcriptome is much more complex than originally expected. Alternative splicing(AS) and the transcription of long non-coding RNAs (lncRNAs) are two phenomenas which have been greatly underestimated in their frequency. Nowadays it is accepted that almost every gene has at least one alternative isoform and the number of lncRNAs exceeds the one of protein-coding genes.
We built user-friendly web interfaces which can process Affymetrix GeneChip Exon 1.0 ST Arrays (exon arrays) and GeneChip Gene 1.0 ST Arrays (gene arrays)for the analysis of alternative splicing events. Results are presented with detailed annotation information and graphics to identify splice events and to facilitate biological validations. Based on two studies using exon arrays, we show how our tools were used to profile genome-wide splicing changes under silencing of Jmjd6 and under hypoxic conditions. Since gene arrays are not intended for AS analysis originally, we demonstrated their applicability by profiling alternative splicing events during embryonic heart development.
To measure lncRNAs expressions with exon arrays, we completely re-annotation all probes and built a lncRNA specific annotation. To demonstrate the applicability of exon arrays in combination with our annotation, we profiled the expression of tens of thousands of lncRNAs. Further, our custom annotation allows for a detailed inspection of lncRNAs and to distinguish between isoforms, as we validated by RTPCR.
To allow for a general usage to the research community, we integrated the annotation in an easy-to-use web interface, which provides various helpful features for the analysis of lncRNAs.
In our daily life, we carry out lots of tasks like typing, playing tennis, and playing the piano, without even noticing there is sequence learning involved. No matter how simple or complex they are, these tasks require the sequential planning and execution of a series of movements. As an ability of primary importance in one’s life, and an ability that everyone manages to learn, action-sequence learning has been studied by researchers from different fields: psychologists, neurophysiologists as well as roboticists. In the concept of sequence learning, perceptual learning and motor learning, implicit and explicit learning have been studied and discussed independently.
We are interested in infancy research, because infants, with underdeveloped brain functions and with limited motor ability, have little experience with the world and not yet built internal models as presumption of how to interpret the world. A series of infant experiments in the 1980s provided evidence that infants can rapidly develop anticipatory eye movements for visual events. Even when infants have no control of those spatial-temporal patterns, they can respond actually prior to the onset of the visual event, referred as "Anticipation".
In this work, we applied a gaze-contingent paradigm using real-time eye tracking to put 6- and 8-month-old infants in direct control of their visual surroundings. This paradigm allows the infant to change an image on a screen by looking at a peripheral red disc, which functions as a switch. We found that infants quickly learn to perform eye movements to trigger the appearance of new stimuli and that they anticipate the consequences of their actions in an early stage of the experiment.
Attention-shift from learning one stimulus to the next novel stimulus is important in sequence learning. In the test phase of infant visual habituation with two objects, we propose a new theory of explaining the familiarity-to-novelty shift. In our opinion an infant’s interest in a stimulus is related to its learning progress, the improvement of performance. As a consequence, infants prefer the stimulus which their current learning progress is maximal for, naturally giving rise to a familiarity-to-novelty shift in certain situations. Our network model predicts that the familiarity-to-novelty-shift only emerges for complex stimuli that produce bell-shaped learning curves after brief familiarization, but does not emerge for simple stimuli that produce exponentially decreasing learning curves or for long familiarization time, which is consistent with experimental results. This research suggests the infant's interest in a stimulus may be related to its current learning progress. This can give rise to a dynamic familiarity-to-novelty shift depending on both the infant's learning efficiency and the task complexity.
We know that for both infants and adults, the performance on certain motor-sequence tasks can be improved through practice. However, adults usually have to perform complex tasks in complicated environments; for example, learning multiple tasks is unavoidable in our daily life. In existing research, learning multiple tasks showed puzzling and seemingly contradictory results. On the one hand, a wide variety of proactive and retroactive interference effects have been observed when multiple tasks have to be learned. On the other hand, some studies have reported facilitation and transfer of learning between different tasks.
In order to find out the interaction between multiple-task learning, and to find an optimal training schedule, we use a recurrent neural network to model a series of experiments on movement sequence learning. The network model learns to carry out the correct movement sequences through training and reproduces differences between training schedules such as blocked training vs. random training in psychophysics experiments. The network model also shows striking similarity to human performance, and makes prediction for tasks similarity and different training schedules.
In conclusion, the thesis presents learning sequences of actions in infants and recurrent neural networks. We carried out a gaze-contingent experiment to study infants’ rapid anticipation of their own action outcomes, and we also constructed two recurrent neural network models, with one model explaining infant attention shift in visual habituation, and the other model directing to task similarity and training schedule in motor sequence control in adults.
Life-attenuated measles virus (MV) vaccines have revealed their capacity to routinely induce life-long immunity against MV after just a single or two low-dose injections. Moreover, MV vaccines have been shown to be extensively safe and well tolerated, in general. Thus, MV is a prime candidate for a recombinant vaccine platform to protect also against other pathogens after vaccination. For this purpose, foreign genes can be inserted into additional transcription units (ATU) in recombinant MV genomes so that the encoded foreign proteins are co-expressed with MV proteins in infected cells. These so-called bivalent MV should protect against infection by MV or the pathogen, which the encoded foreign protein had been derived from. Bivalent MVs have already been shown to be effective vaccines against e.g. dengue virus or hepatitis B virus infections by inducing humoral and sometimes also cellular immune responses. In most of these studies, soluble or soluble versions of the pathogens' antigens were used for generation of bivalent MVs.
We hypothesized that the form of the antigen expressed by bivalent MVs is crucial for the potency and constitution of the induced immune responses. Therefore, three different forms of an antigen expressed by bivalent MVs were analyzed, here. The model antigen chosen for this purpose has been the envelope protein (Env) of SIVsmmPBj1.9. In its natural mature form, Env is composed of the surface unit gp120 and the transmembrane unit gp41, which stay non-covalently linked after proteolytic processing of the common precursor protein gp160. However, gp120 can be shed by infected cells or virus particles. Therefore, natural gp160 antigen was used as shedding form. Furthermore, stabilized covalently-linked gp160 variants and soluble gp140 variants were used in this thesis. These different antigen forms were inserted either behind the P or behind the H expression cassettes into the MV genome. The respective bivalent MVs were rescued and characterized. Expression of SIVsmmPBj1.9 Env variants by the bivalent MVs was confirmed by immuno blot and in situ immunoperoxidase assays. Replication curves of bivalent MV showed that growth of MVs expressing the different Env variants was slightly delayed by approximately 24 h compared to control viruses.
For immunization of transgenic, MV-susceptible IFNAR-/--CD46Ge mice, which are the current standard to analyze MV vaccines in a small animal model, an optimal dose of 1x105 TCID50 was determined. For the evaluation of humoral immune responses in transgenic mice, two ELISA systems for the detection of total α-MV and α-SIV antibodies and neutralization assays for detection of neutralizing antibodies against MV and SIV in sera of immunized mice were established. Mice immunized with any of the bivalent MVs showed significant humoral immune responses against MV comparable to those elicited by the parental MV vaccine strain without further genetic modifications. Mice immunized with MVvac2-gp140(P) expressing the soluble gp140 variant revealed highest α-SIV titers with a maximal OD of up to 0.4. Second highest levels of α-SIV antibodies were detected in mice that were immunized with the shedding variants or soluble Env in other positions. MVs expressing the stabilized variants induced only very low α-SIV antibody titers. Neutralizing antibodies directed against SIV could be detected in sera of mice immunized with MVs expressing the soluble or shedding variants, but not in sera of mice immunized with MVs expressing the stabilized variants. In sera of control mice immunized with PBS no antibodies could be detected, as expected. Thus, soluble and shedding antigens induced humoral immune responses, whereas stabilized antigens induced only weak humoral immune responses but no neutralizing antibodies. Analysis of cellular immune responses is still ongoing.
Besides Env, further SIV antigens could be tested for their potency to induce humoral as well as cellular immune responses.
Besides being used as a vaccine platform, recombinant MVs are evaluated as future agent for cancer therapy due to their significant inherent tumor-lytic, so-called oncolytic activity. Currently, the anti-tumoral activity of MV is analyzed in clinical phase I trials. MV strains with high fusion activity are used as oncolytic agents. The fusion protein F of MV strain NSe is highly fusogenic, in contrast to e.g. F of MVwt323, a clone of the pathogenic strain IC-B. Sequence analysis of these two proteins identified one coding nucleotide difference at aa 94 in the F2 domain: a valine (V) in FNSe and a methionine (M) in Fwt323. To evaluate impact of this difference, residues at aa 94 were exchanged. After transient-transfection of MV F and H expression plasmids in receptor-positive cells, V94 in the F2 subunit of FNSe or Fwt323 led to about 6-fold higher fusion activity compared to F proteins with M94. The co-expressed H protein (HNSe or Hwt323) did not influence fusion activity, indicating that the receptor (CD46 or SLAM) bound by H does not quantitatively affect the F proteins' activation. Analysis of F and H showed that formation and transport of MV glycoprotein complexes are not altered by substitution in aa 94 of FNSe or Fwt323.
Furthermore, recombinant MVNSe, MVNSe-F-M94, MVwt323, or MVwt323-F-V94 were rescued. Viral replication revealed slightly higher titers for recombinant MVs expressing M94 in F after 96 h of replication, compared to MVs expressing V94. MVs expressing V94 in F2 showed 2.5-fold higher fusion activity on CD46- and SLAM-positive Vero-hSLAM cells and 2-fold higher fusion activity on B95a cells expressing only SLAM compared to MVs expressing F with M94. Fusion activity of recombinant MVs can thus be modulated by substituting a single aa. V94 in the F protein results in highly fusion active MVs with possibly increased direct cytotoxicity in infected tumors, whereas M94 in F could be associated with decreased fusion activity for therapies, where higher virus titers are required.
Angiogenesis, the formation of new blood vessels from existing ones, is a fundamental biological process required for embryonic development; it also plays an important role during postnatal organ development and various physiological and pathological remodeling processes in the adult organism. Vascular endothelial growth factor (VEGF) and its main receptor, VEGF receptor-2 (VEGFR-2), play a central role in angiogenesis. VEGFR-2 expression is strongly upregulated in angiogenic vessels, but the mechanisms regulating VEGFR-2 expression are not well understood. We found in this study that the G-protein α subunit Gα13 plays an important role in the regulation of VEGFR-2 expression. In vitro, we found that knockdown of Gα13 reduced VEGFR-2 expression in human umbilical vein endothelial cells and impaired responsiveness to VEGF-A. This phenotype was rescued by adenoviral normalization of VEGFR-2 expression. Gα13-dependent VEGFR-2 expression involved activation of the small GTPase RhoA and transcription factor NF-κB; it was abrogated by deletion of the NF-κB binding site at position -84 of the VEGFR-2 promoter. In vivo, endothelial cell-specific loss of Gα13 resulted in reduced VEGFR-2 expression, impaired responsiveness towards VEGF-A in Matrigel assays, and reduced retinal angiogenesis. Importantly, also tumor vascularization was diminished in the absence of endothelial Gα13, resulting in reduced tumor growth. Taken together, we identified Gα13-dependent NF-κB activation as a new pathway underlying the transcriptional regulation of VEGFR-2 during retinal and tumor angiogenesis.
Cheating and Cheaters in Pfaffe Amis and Reinhart Fuchs An Alsatian poet named Heinrich, writing around 1180, composed a beast epic, based on French sources, about a trickster fox named Reinhart. Some sixty years later, a poet known to us only as Der Stricker composed a work of similar length and structure, about a trickster priest named Amis, and his diligent efforts to cheat various anonymous individuals out of their money. Other works by this poet bear out the Stricker's consistent emphasis on strategy over brute force, prudence and intelligence over unconsidered actions. These stories both illustrate that power, when not directed by intelligence, is useless or dangerous, even to the one who wields it. Tricksters and cheating also appear in a surprising range of works contemporary to the Stricker's Pfaffe Amis and Heinrich's Reinhart Fuchs. Romances have their own trickster characters, conducting their cheats using methods and structures that recall those of these two Schwank-type epics. Cheaters like Amis, and Tristan's Isolde generate twin situations. One of them is true/hidden, and can influence the characters, and one is false/apparent, to which the victim characters are forced to respond. This artificial, apparent reality persists even after the cheater has left the scene, occasionally taking on a truth of its own. Both Reinhart and Amis, whatever their motivations, work evil everywhere they go; and yet the audience is expected to treat them as sympathetic characters. Because the trickster universe functions to turn systems upside-down, it also rejects the concepts of good and evil, forming a universe in which all that matters is who wins and who loses. The place of the villain belongs now to the fool; any character who becomes deceived deserves to be, and is treated with indignation by the narrator, just as the traditional villain might be.
Goethe and the Sublime
(2013)
The dissertation situates the Goethean sublime in an obscured countermovement of resistance to the aestheticization the concept underwent in the 18th century. Before the encounter with the English aesthetic concept of the sublime, the German notion of das Erhabene (the sublime) named not a category of aesthetic experience, but a social affect. In contrast to the Sublime of Edmund Burke's theory, which explicitly excludes melancholy from the sources of the Sublime, das Erhabene is an affect related to the self-overcoming of melancholic subjectivity. As the aestheticized notion of the sublime displaced das Erhabene, Goethe became one of the most radical innovators of the aesthetics of the sublime. But as is demonstrated in chapters on The Sorrows of Young Werther, Elective Affinities, Faust and Wilhelm Meister, he did so with the aim of recovering the displaced meaning of das Erhabene as social affect. Goethe's sublime aims to show at every turn that the so-called "aesthetic experience" of the sublime is really displaced social affect. His treatment of the sublime therefore constitutes a radical critique of the establishment of aesthetics as an independent sphere of inquiry. There is for Goethe no way to understand aesthetic experience independently of its social context. By reconnecting the sublime it to the original social meaning of das Erhabene, Goethe recovers the aesthetics of the sublime as a means of mediating and facilitating the movement of subjectivity from frustrated stasis to divine creativity; i.e., from exclusion to participation in the material creation of reality.
Long-distance seed dispersal is a crucial process allowing the dispersal of fleshy-fruited tree species among forest fragments. In particular, large frugivorous bird species have a high potential to provide inter-patch and long-distance seed transport, both important for maintaining fundamental genetic and demographic processes of plant populations in isolated forest fragments. In the face of increasing worldwide forest fragmentation, the investigation of long-distance seed dispersal and the factors influencing seed dispersal processes has recently become a central issue in ecology. In my thesis, I studied the movement behaviour and the seed dispersal patterns of the trumpeter hornbill (Bycanistes bucinator), a large obligate frugivorous bird, in KwaZulu-Natal, South Africa. I investigated (i) the potential of trumpeter hornbills to provide long-distance seed dispersal within different landscape structures, (ii) seasonal variations in ranging behaviour of this species, and (iii) the potential of this species to enhance the functional connectivity of a fragmented landscape. I used highresolution GPS-data loggers to record temporally and spatially fine-scaled movement data of trumpeter hornbills within both continuous forests and fragmented agricultural landscapes during the breeding- and the non-breeding season. First, combining these data with data on seed-retention times, I calculated seed dispersal kernels, able to distinguish between seed dispersal kernels from the continuous forests and those from the fragmented agricultural landscapes. The seed dispersal distributions showed a generally high ability of trumpeter hornbills to generate seed transport over a distance of more than 100 m and for potential dispersal distances of up to 14.5 km. Seed dispersal distributions were considerably different between the two landscape types, with a bimodal distribution showing larger dispersal distances for fragmented agricultural landscapes and a unimodal one for continuous forests. My results showed that the landscape structure strongly influenced the movement behaviour of trumpeter hornbills, and this variation in behaviour is likely reflected in the shape of the seed dispersal distributions. Second, for each individual bird I calculated daily ranges and investigated differences in daily ranging behaviour and in the process of range expansion comparatively between the breeding- and the non-breeding season. I considered differences in habitat use and possible consequences resulting for seed dispersal function during different seasons. I found that within the breeding season multi-day ranges were built from strongly overlapping and nearly stationary daily ranges which were almost completely restricted to continuous forest. In the non-breeding season, however, birds assembled multi-day ranges by shifting their range site to a generally different area, frequently utilizing the fragmented agricultural landscape. Thereby, several small daily ranges and few large daily ranges composed larger multi-day ranges within the non-breeding season. Seasonal differences in ranging behaviour and range assembly processes resulted in important consequences for seed dispersal function, with short distances and less spatial variation during the breeding season and more inter-patch dispersal across the fragmented landscape during the non-breeding season. Last, I used a projection of simulated seed dispersal events on a high-resolution habitat map to assess the extent to which trumpeter hornbills potentially facilitate functional connectivity between plant populations of isolated forest fragments. About 7% of dispersal events resulted in potential between-patch dispersal and trumpeter hornbills connected a network of about 100 forest patches with an overall extent of about 50 km. Trumpeter hornbills increased the potential of functional connectivity of the landscape more than twofold and seed dispersal pathways revealed certain forest patches as important stepping-stones for seed dispersal among forest fragments. Overall, my study highlights the overriding role that large frugivorous bird species, like trumpeter hornbills, play in seed dispersal in fragmented landscapes. In addition, it shows the importance of fine-scaled movement data combined with high-resolution habitat data and consideration of different landscape structures and seasonality for a comprehensive understanding of seed dispersal function.
Lipid mediators have been referred as bioactive lipids, whose change in lipid levels resulted in functional or pathophysiological consequences. They are in the focus of biological research, nevertheless this is a late recognition due to the many difficulties of working with bioactive lipids due to their properties: hydrophobic, unstable and they occur in only in small quantities. Liquid chromatography and mass spectrometry have facilitated the work with them. Especially in this field, cardiovascular diseases and inflammatory mediated diseases and cancer are pathophysiological events where LMs are deregulated. Additionally, if the modulation of one LM pathway is not sufficient to overcome a disease, the combination of targeting two or more pathways could be effective. Needless to say, lipid signaling cascades are complicated pathways and possible shunting into other pathways when inhibiting or genetically deleting enzymes should be taken into consideration.
The first part of this work has focused on enzymes that metabolize eicosanoids, like mPGES-1 and 5-LO. mPGES-1 is an important enzyme metabolizing PGH2 and one of the key players of the AA cascade. Its product, PGE2 plays an important role in different inflammatory processes. Inhibition of the mPGES-1 might be a promising step to circumvent COX dependent side effects of NSAIDs. The class of quinazoline compounds around the lead structure FR20 has been investigated on isolated human and murine enzyme, in HeLa cells and in different human whole blood (HWB) settings to establish the possible effects of these compounds on eicosanoid profiling. Novel compounds with inhibitory activities in the submicromolar range (IC50: 0.13 µM - 0.37 µM on isolated enzyme) were obtained which were also effective in cells and HWB. Furthermore, pharmacological profiling of toxicity and lipid screening with LC/MS-MS revealed that compounds also reduce PGE2 levels in intact cells and whole blood; they do not impair cell viability but lack the ability to inhibit the murine mPGES-1 enzyme. This problem could be overcome by means of chemical synthesis varying the scaffold (quinoline, quinazoline) or introducing biosteric replacement in the phenyl moieties.
5-LO is a relevant enzyme that plays an important role in eicosanoid signaling in particular in leukotriene biosynthesis. Leukotrienes are involved in asthma, allergic rhinitis, glomerulonephritis, rheumatoid arthritis, sepsis, cancer and atherosclerosis. Moreover, genetic variants in the genes of the 5-LO pathway have been associated with the risk of development of acute myocardial infarction and stroke. Eicosanoids are increased in infectious exacerbations of chronic obstructive pulmonary disease (COPD). They are also elevated in the airways of stable COPD patients compared to healthy subjects. Therefore, 5-LO has attired the scientific community as a possible therapeutic target to treat the several disease conditions listed before. In this study an extensive evaluation of imidazo[1,2-a]pyridines as a suitable lead structure for novel 5-LO targeting compounds was presented Within the three publications, 5-LO inhibitory activity of synthesized compounds was investigated in intact PMNL, a cell-free assay, in human whole blood and rodent cells to both elucidate structure-activity relationships and compounds were in vitro pharmacological evaluated. Chemical modifications for lead optimization via straight forward synthesis were used to combine small polar groups (hydroxy, and methoxy groups) which led to a suitable candidate with desired in vitro pharmacokinetic profile in terms of solubility and intrinsic clearance without showing any cytotoxicity. More than 70 imidazo[1,2-a]pyridine derivatives have been synthesized, resulting in more than 50 active compounds. Although it was not possible to introduce a solubility group without impairing the 5-LO inhibitory activity, combination of small polar groups lead to a more favorable solubility and in vitro metabolic stability. Overall, the development of 5-LO inhibitors with high efficacy and selectivity in vivo will provide a possible treatment for patients having one of the diseases where leukotriene biosynthesis plays an important role.
Other types of 5-LO inhibitors have been synthesized during this work, NO-NSAIDs can be postulated as novel 5-LO inhibitors that could circumvent the undesired side-effects of inhibiting COX isoforms (ulcer perforation, gastrointestinal bleeding and in some cases death). It is suggested that NO group is released in situ or after compounds are metabolized. NO-NSAIDs maintain the same anti-inflammatory properties by inhibiting 5-LO in clinical relevant concentrations. NO-NSAIDs are currently under clinical trial for the treatment of diseases where inflammation plays an important role. Synthesis of NO-NSAIDs is straightforward and can be applied for most NSAIDs recently published. Among them, the most promising candidate is NO-sulindac that was able to inhibit 5-LO product formation in intact PMNL, purified 5-LO and HWB in micromolar concentration. Additional experiments regarding their mechanism are currently being performed.
The present study could show that dual inhibitors are an interesting approach that is practicable. It has been used in the recent years to overcome side-effects and diseases concerning more pathophysiological conditions. MetS is an example of a conjunction of symptoms: hyperglycemia, hypertriglyceridemia, hypertension and obesity. Due to its complex nature, the current treatment strategies of MetS require multiple pharmacological compounds regulating lipid and glucose homeostasis as well as blood pressure and coagulation. This study describes the first synthesis of dual sEH/PPAR modulators as potential agents for treatment of MetS. Following a combinatorial approach, an acidic head group known as a pharmacophore important for PPARα/γ dual agonistic activity was combined with different hydrophobic urea derivatives in order to introduce an epoxide mimetic (sEH pharmacophore). The resulting compounds yielded high inhibition of sEH and different patterns of PPAR agonistic activity. This study demonstrates that the pharmacophores of PPAR agonists and sEH inhibitors can be easily combined, resulting in a simplified blueprint of a dual sEH/PPAR modulator. Further in vivo pharmacological evaluation studies are needed in order to evaluate, which pattern of PPAR activation shows the most promising profile for treatment of metabolic syndrome.
Another example of dual pharmacology has been presented in this work. Natural products derived compounds were able to target sEH and exhibit promising antiproliferative properties. The principle of addressing multiple targets by natural products can be transferred to synthetic multi-target ligands. In conclusion, several (E)-styryl-1H-benzo[d]imidazoles were synthesized and evaluated on recombinant sEH after an initial hit (IPS) that lead to potent sEH inhibitors exhibiting antiproliferative activities. Following the natural product-inspired design, the desired biological activity from a bacterial secondary metabolite has been enhanced and transferred to a synthetic compound series. The resulting compounds were accessible via an easy synthetic route and offered a possibility to investigate the structure-activity relationships. The natural product inspired drug design extends the valuable role of natural products as drugs and drug precursors to templates for fully synthetic bioactive molecules. Simplification of natural products by means of chemical synthesis could lead to an interesting field in the treatment of cancer.
Affinity chromatography has been used to unravel unknown- and off-target effects which either contribute to the biological effect of the inhibitor or that counteract or lead to undesired side-effects. During this PhD work, two main projects related to this technique have been established. In the first one, related to an imidazo[1,2-a]pyridine inhibitor (EP6), it has been shown that epoxide-sepharose is a reliable material in order to couple compounds bearing an alcohol. Coupling of an analogue of EP6 to the sepharose has been accomplished and affinity towards 5-LO was demonstrated. The challenging step is to discern from unspecific protein binders and analysis via SDS-PAGE separation and mass spectrometry. Further experiments using other cell types or improving SDS-PAGE analysis (e.g. 2D gel analysis) should be useful to unravel EP6 off-target effect. During the second project related to off-target effects of celecoxib and DMC, the main problem was the coupling of the functional group to the sepharose. Affinity towards COX-2 could not be demonstrated pointing out the inefficient coupling method. Higher pH values during coupling reaction should be tested in further experiments. Nevertheless, affinity chromatography is a useful technique to unravel cellular mechanisms.
Sphingolipid metabolism is also a recent area that attired the attention of cancer researchers, due to their important roles in cell proliferation and apoptosis. Ceramide metabolism inhibitors were synthesized and evaluated on different assay systems in order to assess their efficacy on several cancer lines. Remarkably, 2,2-dimethyl-1,3-dioxolan-4-yl)methanamine (32) was a useful scaffold to mimic the sphingoid base. This key intermediate was used to produce ceramide analogues that could enter the cell and target apoptosis machinery. EB143 (38) increased ceramide levels in an in vitro ceramide synthase assay in a dose-response manner meaning that ceramide synthase was not inhibited but the ceramide de novo synthesis was activated. This effect was due to the fact that EB143 is a cytotoxic compound with an interesting antiproliferative profile. Further chemical modifications should be carried out to modulate this effect.
COX and LO inhibitors are cancer-preventive not only by inhibiting specific antiapoptotic AA metabolites but also by facilitating accumulation of AA which promotes neutral SMase activity and increases the proapoptotic ceramide. Several 5-LO inhibitors have been evaluated on several cancer lines and sphingolipid levels were measured in order to obtain a relationship. A549, Capan-2 and MCF-7 cells line were incubated with synthetic 5-LO inhibitors and zileuton. Compounds were cytotoxic to all cancer cell lines except from A549. Needless to say, zileuton did not exhibit a cytotoxic profile. Synthetic 5-LO inhibitors were able to modify ceramide levels but were useless when coincubating with sphingolipid metabolism inhibitors (myoricin, amitryptiline etc.) and inconsistent results were obtained. On the contrary, zileuton selectively increased Cer-C16 levels and in less extend Cer-C24:1. When using a SPT inhibitor (myoricin) alone was able to reduce C24:1 and Cer-C16:0 levels below the control, a similar effect occurred when incubation the cells with zileuton and myriocin. Interestingly, treatment of zileuton together with either amitryptiline or desipramine led to a decrease in Cer-C24:1 and levels Cer-C16:0 but the inhibition was not complete indicating that probably the de novo pathway has an important role. Further investigations on mRNA level should be carried out in order to discern which CerS is activated.
The main objective of the present thesis was the synthesis of lipid signaling modulators and their evaluation in vitro as therapeutic strategy to overcome pathophysiological conditions (cancer, metabolic syndrome, etc). It has been accomplished on many relevant targets like 5-LO, mPGES-1, sEH and PPAR and these lipid signaling modulators could be used in the treatment of diseases conditions where lipid mediators play an important role.
We study the price-setting problem of market makers under perfect competition in continuous time. Thereby we follow the classic Glosten-Milgrom model that defines bid and ask prices as the expectation of a true value of the asset given the market makers partial information that includes the customers trading decisions. The true value is modeled as a Markov process that can be observed by the customers with some noise at Poisson times.
We analyze the price-setting problem by solving a non-standard filtering problem with an endogenous filtration that depends on the bid and ask price process quoted by the market maker. Under some conditions we show existence and uniqueness of the price processes. In a different setting we construct a counterexample to uniqueness. Further, we discuss the behavior of the spread by a convergence result and simulations.
In the past century, scientists have realized that venoms are a source of a number of natural substances presenting a wide range of pharmacological properties and often displaying a high specificity for their targets. Thus, the field of toxinology came into being, which is defined as the study of toxic substances of biological origin. Toxins are found in a wide variety of animals, including fish, cone snails, scorpions, snakes, and even some mammals. To be classified as venom, these must contain substances, i.e. toxins, which disturb physiological processes and must be deliberately delivered to the target animal. Snakes have evolved one of the most sophisticated mechanisms for venom delivery. Envenomation by snakebite can induce and inhibit aggregation/agglutination of platelets as well as inhibit/activate hemostasis, but also disrupt other physiological functions via neurotoxins and angioneurin growth factors. Snake venoms contain a substantial amount of C-type lectin-related proteins (CLRPs) which are known to function, notably, as integrin inhibitors. CLRPs are heterodimers composed of homologous α and β subunits which can assemble either covalently or noncovalently to oligomers, resulting in αβ, (αβ)2 and (αβ)4 structures. Some of the main targets of CLRPs are membrane receptors, coagulation factors, and proteins essential to hemostasis. The platelet collagen receptors GPVI and α2β1 integrin as well as the von Willebrand factor receptor GPIb play important roles in platelet activation and aggregation and are considered main targets of antithrombotic drugs. In this thesis, the integrin α2β1 is particularly considered as it is the sole collagen-binding integrin on platelets. Reduced expression of this platelet receptor results in dysfunction of platelet responses. Equivalently, overexpression of α2β1 integrin results in an increased risk of thrombosis. As a result, selective inhibitors of the collagen-α2β1 interaction could give rise to effective antithrombotic drugs. Integrins are large receptors which mediate cell-cell contacts and the binding of cells to the extracellular matrix (ECM). Therefore, they play a role in physiological processes, e.g. hemostasis and immunity, as well as in pathological processes, e.g. tumor angiogenesis and atherosclerosis. 18 α and 8 β integrin subunits, with nine α subunits containing an additional A domain, associate non-covalently to form 24 heterodimers with distinct binding specificities. Integrin collagen receptors are a subclass of four receptors which all utilize the β1 subunit. The α2β1 integrin is a collagen-binding receptor expressed not only on platelets, but also on endothelial and epithelial cells. Consequently, this integrin is also essential for cell adhesion and migration playing a role in angiogenesis as well as tumor metastasis. To date, there are five known antagonists of α2β1 integrin: EMS16, rhodocetin, vixapatin, and most recently rhinocetin and flavocetin-A. The first four have been shown to be specific for the integrin α2A domain, the major collagen-binding domain. All these antagonists are CLRPs and present new leads for drug design. In the past few years, many insights into the structure and function of rhodocetin were obtained. Monoclonal antibodies proved to be advantageous in disclosing this information, making them not only useful as therapeutic agents, but also as tools for protein characterization. The venom of the Vipera palaestinae snake was recently shown to contain an α2β1 integrin inhibitor, which prevented the integrin from binding collagen. This inhibitor, called vixapatin, was the initial focus of this dissertation. Vixapatin’s interaction with the α2β1 integrin needed further characterization on a molecular and cellular level to assess its medical potential and monoclonal antibodies were to be used as a tool. Originally, vixapatin had been isolated by reversed-phase high-performance liquid chromatography. To avoid the stringency of this method, for this study, it was replaced with gentler chromatographic methods. First, the α2β1 integrin inhibitor was isolated from the crude snake venom with affinity chromatography using the α2A domain as bait, establishing a method to quickly screen venoms for α2β1-binding proteins which affect the collagenintegrin interaction. The applicability of this method to other snake venoms was shown by isolating an α2A domain-specific toxin from the venom of Trimeresurus flavoviridis. To allow further characterization of both these toxins, gel filtration and ion exchange chromatography were employed to purify the protein without the α2A domain. These classical protein purification methods resulted in similar separation patterns of both the V. palaestinae and T. flavoviridis venom proteins. Purified proteins exhibiting the potential of inhibiting integrinbinding to collagen were analyzed by two-dimensional gel electrophoresis. Both VP-i and flavocetin-A, the integrin inhibitors from V. palaestinae and T. flavoviridis, respectively, were shown to have more complex structures than was evident from the purification. Each consisted of four low-molecular-weight proteins which assembled into two bands (for VP-i) or one single band (for flavocetin-A) under non-reducing conditions. Mass spectrometry analyses revealed VP-i to belong to the family of CLRPs, just like vixapatin does. However, these two proteins differed in their primary sequences and only showed homology to one another. The toxin purified from T. flavoviridis revealed this toxin to be flavocetin-A, a heterodimeric CLRP which had so far only been shown to have GPIb-binding activity. At the time of flavocetin-A’s purification, flavocetin-B was co-purified; flavocetin-B consists of the same two α and β subunits, plus an additional γ subunit. As no sequence information is known to date for the γ subunit, it may be one of the additional proteins purified here, along with an additional δ subunit. Therefore, the toxin isolated here may actually consist of four different subunits forming a tetramer of two different heterodimers, generating an (αβ)2(γδ)2 structure. This proposed (αβ)2(γδ)2 flavocetin-A structure has binding sites for both α2β1 integrin and GPIb, with no sterical overlap, as shown by affinity chromatography using the α2A domain and the extracellular domain of the GPIb receptor. The potential of VP-i and flavocetin-A to inhibit integrin-binding to type I collagen was shown during purification: Both toxins efficiently bind to the integrin α2A domain; also, VP-i and vixapatin bind to the A domain with the same affinity. Surface plasmon resonance showed the interaction of flavocetin-A with the α2β1 integrin to be extremely strong and association to be very fast. Furthermore, both toxins were shown to inhibit binding of the wildtype integrin to collagen: VP-i and flavocetin-A acted antagonistically on cell adhesion and cell migration. Initially, the interaction between VP-i and α2β1 integrin was to be further characterized with the help of monoclonal antibodies. However, this proved problematic, the procedure requiring various optimizations. Although, after expert consultation, some monoclonal antibodies could be obtained, the cells were extremely sensitive and gave unsatisfactory results when tested as detection tools in Western blot and immunoassays. Concluding, two novel α2β1 integrin inhibitors were discovered: VP-i and flavocetin-A, which were purified using the same procedure and which have similar functions. Both are Ctype lectin-related proteins which effectively inhibit cell adhesion and migration. This underlines that nature has instrumentalized CLRPs to specifically inhibit α2β1 integrin. Further characterization of VP-i and flavocetin-A will be able to provide leads for future drug development.
During the height of the Second World War pressure from Great Britain resulted in the transfer of thousands of German prisoners of war (PoWs) from British to Canadian control. To house them, Canada built a system of PoW camps, including Riding Mountain Camp in southwestern Manitoba. The PoWs sent there soon realized their good fortune: they lived in warm barracks, ate abundant food, and were able to purchase goods from a mail order catalog. But while the PoWs were well treated, they were at the same time subjected to a concerted reeducation campaign organized by the Allies. This reveals that these Canadian camps were not merely warehouses for the PoWs, but in fact, classic reforming institutions.
Initially subjected to ideological training under Nazism, the PoWs were next subjected to another kind of education under the Canadians. Evidence collected from oral history interviewing, archival research, and three seasons of field archaeology combine to reveal that material culture was a key nexus in this competition for the minds of the PoWs. In addition to providing books and teaching courses on history and political science, the Canadians introduced the PoWs to a democratic, capitalistic way of life by familiarizing them with North American consumer goods and by allowing them to fraternize with Canadian civilians. The Nazi bureaucracy, in turn, used material things to try to keep the PoWs from turning to the other side. For example, by sending them crisp new Wehrmacht uniforms from Germany, heartening Christmas cards, and packages filled with German goods adorned with Nazi symbolism.
Plants absorb sunlight via photosynthetic pigments and convert light energy intochemical energy in the process of photosynthesis. These pigments are mainly bound to antenna protein complexes that funnel the excitation energy to the photosynthetic reaction centres. The peripheral antenna of plant photosystem II (PSII) consists of the major light-harvesting complex of PSII (LHC-II) and the minor LHCs CP29, CP26 and CP24. Light intensity can change frequently and plants need to adapt to high-light conditions in order to avoid photodamage. When more photons are absorbed than can be utilised by the photosynthetic machinery, excessive excitation energy is dissipated as heat by short-term adaptation processes collectively known as non-photochemical quenching (NPQ). A decrease in PSII antenna chlorophyll (Chl) fluorescence yield and a reduction in the average Chl fluorescence lifetime are associated with NPQ. The main component of NPQ is the so-called energy-dependent quenching (qE), and it is triggered by the rapid drop in thylakoid lumenal pH resulting from the plant’s photosynthetic activity. This process is thought to take place at the PSII antenna complexes, which therefore not only capture and transfer light energy but are also involved in balancing the energy flow. The decrease in lumenal pH acivates the enzyme violaxanthin de-epoxidase (VDE), which converts the xanthophyll violaxanthin (Vio) into zeaxanthin (Zea) in the xanthophyll cycle. In addition, the PSII subunit PsbS was discovered to be essential for qE by screening qE-deficient Arabidopsis thaliana mutants. This membrane protein is considered a member of the LHC superfamily, which also includes LHC-II and the minor LHCs. Previous studies on PsbS isolated either from native source or refolded in vitro have produced inconsistent results on its pigment binding capacity. Interestingly, a pH-dependent change in the quaternary structure of PsbS under high light conditions has been reported. This observed dimer-tomonomer transition very likely follows the protonation of lumenal glutamates upon the drop in pH and is accompanied by a change in PSII supercomplex localisation. PsbS dimers are preferentially found in association with the PSII core, whereas PsbS monomers co-localise with LHC-II.Despite the identification of !pH, Zea and PsbS as key players in qE, both the nature of the quencher(s) as well as the underlying molecular mechanism leading to excess energy dissipation still remain unknown. Several models have been put forward to explain the reversible switch in the antenna from an energy-transmitting to a quenched state. Proposals include a simple pigment exchange of Vio for Zea, and aggregation or an internal conformational change of LHC-II. Charge transfer (CT)quenching in the minor LHCs or quenching by carotenoid dark state (Car S1)-Chl interactions have also been suggested. However, none of these qE models has so far been capable of accommodating all the physiological observations and available experimental data. Most importantly, the function of PsbS remains an enigma. A recent qE model suggested that monomerisation of PsbS enables the protein to transiently bind a carotenoid and form a quenching unit with a Chl of a PSII LHC. In view of the various proposed qE mechanisms, this thesis aimed at understanding the interplay of the different qE components and the contribution of the PSII subunits LHC-II, the minor LHCs and PsbS to qE. The initial approach was to investigate the properties of the PSII subunits in the most simple in vitro model system, namely in detergent solution. For this purpose, LHC-II was isolated either from native source or refolded from recombinantly produced protein. Investigation of the minor LHCs and PsbS required heterologous expression and refolding. In addition, experiments were performed on aggregated LHC-II. Aggregates of LHC-II have been used as a popular model system for qE because they exhibit highly quenched Chl fluorescence. At the final stage of this doctoral work, a more sophisticated model system to approximate the thylakoid membrane was developed by reconstitution of the PSII subunits LHC-II and PsbS into liposomes. This system not only allowed for investigation of these membrane proteins in their native environment, but also for mimicking the xanthophyll cycle by distribution of Zea within the membrane as well as !pH by outside buffer exchange. The role of Zea in qE was first investigated with detergent solubilised antenna proteins. The requirement of this xanthophyll for qE is well-known, but the specific contribution to the molecular quenching mechansim is unclear. Previous work had shown that replacement of Vio for Zea in LHC-II was not sufficient to induce Chl fluorescence quenching in Zea-LHC-II, as suggested by the so-called molecular gearshift mechanism. However, by means of selective two-photon excitation spectroscopy, an increase in electronic interactions between Car S1 and Chls was observed for LHC-II upon lowering the pH of the detergent buffer. Electronic Car S1-Chl coupling became even stronger when Zea-LHC-II was probed. The extent of Car S1-Chl coupling correlated directly with the extent of Chl fluorescence quenching, in a similar way as observed previously in live plants under high-light conditions. However, very similar results were obtained with LHC-II aggregates. This implied that the increase in electronic interactions and fluorescence quenching was independent of Zea and low pH. Further experiments on aggregates of LHC-II Chl mutants indicated that the targeted pigments were also not essential for the observed effects. It is proposed that the same molecular mechanism causes an increase in electronic Car S1-Chl interactions and Chl fluorescence quenching in Zea-LHC-II at low pH as well as in aggregated LHC-II. Most likely, surface exposed pigments form random quenching centres in both cases. On the other hand, it was possible that Zea could act as a direct quencher of excess excitation energy in the minor LHCs. However, enrichment of refolded CP29, CP26 and CP24 with Zea did not lead to a change in the Chl excited state lifetime. Formation of a carotenoid radical cation, previously implied in CT quenching, was also not observed, although artificial generation of such a radical cation was principally possible as shown for CP29. During the course of this work, a study reporting the formation of Zea radical cations in minor LHCs was published. Therefore, Zea-enriched minor LHCs were again investigated on the experimental apparatus used in the reported study. Indeed, the presence of at least one carotenoid radical cation for each minor complex was detected. It is suggested that either the preparation method of incubating the refolded minor LHCs with Zea in contrast to refolding the complexes with only Zea and lutein causes the observed differences or that the observed spectral radical cation signatures are due to experimental artifacts. While the experiments with LHC-II and the minor LHCs gave useful insights into the putative qE mechanism, the quencher site and the mode of action of Zea could still not be unambiguously identified. Most importantly, these studies could not explain the function of the qE keyplayer PsbS. Therefore, the focus of the work was shifted to PsbS protein production, purification and characterisation. In view of inconsistent reports on the pigment binding capacity of this PSII subunit, refolding trials with and without photosynthetic pigments were conducted. The formation of a specific pigmentprotein complex typical for other LHCs was not observed and neither was the earlier reported “activation” of Zea for qE by binding to this protein. Nevertheless, PsbS refolded without pigments displayed secondary structure content in agreement with previous studies, indicating pigment-independent folding. Reconstitution of pigmentfree, refolded PsbS into liposomes confirmed that the protein is stable in the absence of pigments. Zea distributed in PsbS-containing liposomes also showed no spectral alteration that would indicate its “activation”. With the ability to reconstitute PsbS, it was then possible to proceed to modelling qE in a proteoliposome system. For this purpose, PsbS was co-reconstituted with LHC-II, which has been reported to interact with PsbS. One-photon excitation (OPE) and two-photon excitation (TPE) spectroscopy measurements were performed on LHC-II- and LHC-II/PsbS-containing liposomes. This enabled both quantification of Chl fluorescence quenching as well as determination of the extent of electronic Car S1-Chl interactions. The effect of Zea was investigated by incorporating it in the proteoliposome membrane. It was shown that Zea alone was not able to induce significant Chl fluorescence quenching when only LHC-II was present. However, when LHC-II and PsbS were co-reconstituted, pronounced Chl fluorescence quenching and an increase in electronic Car S1-Chl interactions were observed and both effects were enhanced when Zea was present. Western blot analysis indicated the presence of a LHC-II/PsbS-heterodimer in these proteoliposomes. In addition to the OPE and TPE measurements, the average Chl fluorescence lifetime was determined in detergent-free buffer at neutral pH and directly after buffer exchange to low pH. No significant changes in the average lifetime were observed for LHC-II proteoliposomes when either Zea was present or after exchange for low pH buffer. This indicated that Zea alone cannot act as a direct quencher, which concurs with the OPE measurements. Moreover, the complex was also properly reconstituted as no aggregation or significant Chl fluorescence quenching were observed. The average lifetime was not significantly affected in LHC-II/PsbS-proteoliposomes, independent of Zea or pH. However, a shortlived component in the presence of a long-lived component was not resolvable with the time resolution of the fluorescence lifetime apparatus.
Implications for qE model systems and the in vivo quenching mechanism are discussed based on the experiments in detergent solution, on LHC-II aggregates and with the proteoliposome model system.
Introduction: Postural control is a prerequisite to many everyday and sporting activities which requires the interaction of multiple sensorimotor processes. As long as we have no balance disorders, the maintenance of an erect standing position is taken for granted with automatic running control processes. It is well known that with increasing age or disease balance problems occur which often cause fall-related injuries. To assess balance performance, posturography is widely applied in which body sway is traditionally viewed as a manifestation of random fluctuations. Thus, the amount of sway is solely used as an index of postural stability, that is, less sway is an indication of better control. But, traditional measures of variability fail to account for the temporal organisation of postural sway. The concept of nonlinear dynamics suggests that variability in the motor output is not random but structured. It provides the stimulus to reveal the functionality of postural sway. This thesis evaluates nonlinear analysis tools in addition to classic linear methods in terms of age-related modifications of postural control and under different standing conditions in order to broaden the existing knowledge of postural control processes.
Methods: Static posturographic analyses were conducted which included the recording of centre of pressure (COP) time series by means of a force plate. Linear and nonlinear methods were used to quantify postural sway variability in order to evaluate both the amount and structure of sway. Classic time and frequency domain COP parameters were computed. In addition, wavelet transform (WT), multiscale entropy, detrended fluctuation analysis, and scaled windowed variance method were applied to COP signals in order to derive structural COP parameters. Two experiments were performed. 1) 16 young (26.1 ± 6.7 years), healthy subjects were asked to adopt a bipedal stance under single- and dual-task conditions. Three trials were conduced each with a different sampling duration: 30, 60, and 300 seconds [s]. 2) 26 young (28.15 ± 5.86 years) and 13 elderly (72 ± 7 years) subjects stood quietly for 60 s on five different surfaces which imposed different biomechanical constraints: level ground (LG), one foot on a step (ST), uphill (UH), downhill (DH), and slope (SL). Additional to COP recordings, limb load symmetry was assessed via foot pressure insoles.
Results: We found a higher sensitivity of structural COP parameters to modulations of postural control and partly an improved evaluation of sway dynamics in longer COP recordings. WT revealed a reweighing of frequency bands in response to altered standing conditions. Scaling exponents and entropy values of COP signals were task-dependent. Higher entropy values were found under the dual-task and condition ST. The time scales affected under the altered standing positions differed between groups and sway directions. Mainly larger posturograms were found in the elderly. Age effects were especially revealed in position ST and concerning medial-lateral COP signals. Load asymmetry was stronger in elderly subjects for LG, UH, and DH positions.
Discussion: Modifications of multiple time scales corresponds to an interplay of control subsystems to cope with the altered task demands. The affected time scales are age-dependent suggesting a change of control processes. Higher irregularity under the dual-task indicates a more complex motor output which is interpreted as less attentional investment into postural control. Larger complexity is evident for ST in contrast to LG position. ST obviously challenges lateral sway which is counteracted differently between groups. Load asymmetry suggests that especially elderly subjects adopt a step-initiation strategy.
Conclusion: A continued application of nonlinear methods is necessary to broaden the understanding of postural control mechanisms and to identify classifiers for balance dysfunctions. Structural COP parameters provide a more comprehensive indication of postural control system properties between groups and task demands. COP recordings of at least 60 s are recommended to adequately quantify COP signal structure. The analysis of postural strategies in everyday activities increases the ecological validity of postural control studies and can provide valuable information regarding the development of effective rehabilitation programs.
The spider genus Eusparassus Simon, 1903 (Araneae: Sparassidae: Eusparassinae; stone huntsman spider) is revised worldwide to include 30 valid species distributed exclusively in Africa and Eurasia. The type species E. dufouri Simon, 1932 is redescribed and a neotype is designated from Portugal. An extended diagnosis for the genus is presented. Eight new species are described: Eusparassus arabicus Moradmand, 2013 (male, female) from Arabian Peninsula, E. educatus Moradmand, 2013 (male, female) from Namibia, E. reverentia Moradmand, 2013 (male, female) from Burkina Faso and Nigeria, E. jaegeri Moradmand, 2013 (male, female) from South Africa and Botswana, E. jocquei Moradmand, 2013 (male, female) from Zimbabwe, E. borakalalo Moradmand, 2013 (female) from South Africa, E. schoemanae Moradmand, 2013 (male, female) from South Africa and Namibia and E. mesopotamicus Moradmand and Jäger, 2012 (male and female) from Iraq, Iran and Turkey. 22 species are re-described six of them are transferred from the genus Olios Walckenaer, 1837. Six species-groups are proposed: the dufouri-group [8 species: E. dufouri, E. levantinus Urones, 2006, E. barbarus (Lucas, 1846), E. atlanticus Simon, 1909, E. syrticus Simon, 1909, E. oraniensis (Lucas, 1846), E. letourneuxi (Simon, 1874), E. fritschi (Koch, 1873); Iberian Peninsula to parts of north-western Africa], walckenaeri-group [3 species: E. walckenaeri (Audouin, 1826), E. laevatus (Simon, 1897), E. arabicus; eastern Mediterranean to Arabia and parts of north-eastern Africa], doriae-group [7 species: E. doriae (Simon, 1874), E. kronebergi Denis, 1958, E. maynardi (Pocock, 1901), E. potanini (Simon, 1895), E. fuscimanus Denis, 1958, E. oculatus (Kroneberg, 1846) and E. mesopotamicus; Middle East to Central and South Asia], vestigator-group (3 species: E. vestigator (Simon, 1897), E. reverentia, E. pearsoni (Pocock, 1901); central to eastern Africa and an isolated area in NW India], jaegeri-group [4 species: E. jaegeri, E. jocquei, E. borakalalo, E. schoemanae; southern and south-eastern Africa], tuckeri-group [2 species: E. tuckeri (Lawrence, 1927), E. educatus; south-western Africa). Two species, E. pontii Caporiacco, 1935 and E. xerxes (Pocock, 1901) cannot be placed in any of the above groups. Two species are transferred from Eusparassus to Olios: O. flavovittatus (Caporiacco, 1935) and O. quesitio Moradmand, 2013. 14 species are recognized as misplaced in Eusparassus, thus nearly half of the described species prior to this revision were placed mistakenly in this genus. Neotypes are designated for E. walckenaeri from Egypt, E. barbarus, E. oraniensis and E. letourneuxi (all three from Algeria) to establish their identity. The male and female of Cercetius perezi Simon, 1902, which was known only from the immature holotype, are described for the first time. It is recognized that the monotypic and little used generic name Cercetius Simon, 1902 — a species, which had been known only from the immature holotype — as a synonym of the widely used name Eusparassus. The case proposal 3596 (conservation of name Eusparassus) is under consideration by ICZN.
The first comprehensive molecular phylogeny of the family Sparassidae with focus on the genus Eusparassus is investigated using four molecular markers (mitochondrial COI and 16S; nuclear H3 and 28S). The monophyly of Eusparassus and the dufouri, walckenaeri and doriae species-groups are recovered with the latter two groups more closely related. The monophyly of the tuckeri-group is not supported and the position of E. jaegeri as the only available member of the jaegeri-group is not resolved within the Eusparassus clade. DNA samples of the vestigator-group were not accessible for this study. The origination of the genus Eusparassus around 70 million years ago (MA) is estimated according to molecular clock analyses. Using this recent result in combination with some biogeographic and geological data, the Namib Desert is proposed as the place of ancestral origin for Eusparassus and putative Eusparassinae genera.
Further analyses are done on the phylogenetic relationships of Sparassidae and its subfamilies. The Eusparassinae are not confirmed as monophyletic, with the two original genera Eusparassus and Pseudomicrommata in separate clades and only the latter clusters with most other assumed Eusparassinae, here termed the "African clade". Monophyly of the subfamilies Sparianthinae, Heteropodinae sensu stricto, Palystinae and Deleninae is recovered. The Sparianthinae are supported as the most basal clade, diverging considerably early (143 MA) from all other Sparassidae. The Sparassinae and genus Olios are found to be polyphyletic. The Sparassidae are confirmed as monophyletic and as most basal group within the RTA-clade. The divergence time of Sparassidae from the RTA-clade is estimated with 186 MA in the Jurassic. No affiliation of Sparassidae to other members of the "Laterigradae" (Philodromidae, Selenopidae and Thomisidae) is observed, thus the crab-like posture of this group was proposed a result of convergent evolution. Only the families Philodromidae and Selenopidae are found members of a supported clade. Including a considerable amount of RTA-clade representatives, the higher-level clade Dionycha is not but monophyly of the RTA-clade itself is supported.
This thesis aims to analyse in a first step the physical and chemical properties of soil profiles along pedomorphological transects in different land used conditions (protected, partly protected as well as cultivated and pastured areas) in North West Benin and in South East Burkina Faso. The information about soils, which are carried out in consideration of the pedogenesis processes like weathering types, saprolitisation, formation of laterite crusts and denudation within the planation surfaces are therefore correlated in a second step with the structure and dynamic of woody plant around individual soil profiles. The relationship soil properties and woody plant is investigated in order to assess the reciprocal influence between the diversity of woody plants and soil characteristics within a small scale study and under different land use conditions.
A common vertical and lateral differentiation of physical and chemical properties regardless of the partly protected, protected and cultivated status of the sites can be noticed. Thus, in the cultivated site of Kikideni and in the partly protected zone of Natiabouani (South East Burkina Faso) sandy loam and sandy clay loam soil surfaces are widespread because of the occurrence of similar erosion processes like sheet wash, rill and gully erosion while in the central part of the Pendjari National Park loamy soil textures are prevailing. In fact, the steepness of the relief and the length of the slopes in the Pendjari Park seem to limit the development of some erosion forms as gully. Furthermore, the classification of soils reflects the variation of pedological processes along the transects and thus the occurrence of different soil types. The status of the sites may play an insignificant role in the differentiation of soil properties within the scale of small pedomorphological transects. A direct comparison of the vegetation type in the land use respectively partly protected and in the total protected sites (National Park of Pendjari) reveals a transition from the shrub savanna to the tree savanna. In conclusion it is important to insist on the fact that the variations of soil parameters within small slopes and the different sites are more conditioned by varying erosion processes and drainage conditions than the status protected or land use sites while the composition and diversity of plants is influenced by the status of the sites, the prevailing management tools, the pedogenetic conditions as well as the presence of wild animals like elephants. The ordination diagram shows that the organic matter is better correlated to the subgroup representing principally the sites of the hunting zone of the Pendjari Park and might be an explaining factor to the distribution of these sample sites groups. CEC ratios in the partly protected site of Natiabouani represent the highest measured in all sites. Nevertheless, statistical analysis of the CCA (canonical correspondence analysis) indicates generally a low correlation. This tendency is consolidated by the Monte Carlo test (p=0.14) which is a good indicator of species and environmental conditions. The detailed analysis of soil properties and the vegetation dynamic as well as their relationship within small pedomorphological transects represent an important pedological and botanical data collection involving different compartments. This thesis contributes to the better understanding of the savanna landscapes of West Africa and may provide essential scientific background for each development project directed towards interdisciplinary and integrative researches.
Biological membranes separate the cell interior from the outside and have diverse functions from signal transduction, apoptosis to transportations of ions and small molecules in and out of the cell. Most of these functions are fulfilled by proteins incorporated in the membrane. However, lipids as the main component of membrane not only serve as structural element for bilayer formation but they are also directly involved e.g. signalling processes and bilayer properties are important to mediate protein interactions. To fully understand the role of lipids, it is necessary to develop a molecular understanding of how certain membrane components modify bulk bilayer structure and dynamics. Membranes are known to have many different motions in different conditions and time scales. Temperature, pH, water content and many other conditions change membrane dynamics in a high degree. In addition to this, time scales of motions in membranes vary from ns to ms range corresponding to fast motion and slow motion, respectively. Therefore, membranes are needed to be studied systematically by varying the conditions and using methods to investigate motions in various time scales separately. The aim of this study was therefore perform a combined solid-state NMR / molecular dynamics study on model membranes. Different substrates, such as potential drugs, polarizing agents and signaling lipids were incorporated into bilayers and their location within the membrane and their effect onto the membrane was probed. NSAIDs (non-steroidal anti-inflammatory drugs), pirinixic acid derivatives, ceramides and polarizing agents were the substrates for membranes in this study. There were several experimental methods that were applied in order to investigate effects of these substrates on membrane dynamics. Different kind of phospholipids including POPC, DMPC and DPPC were used. In addition to experimental work, with the information gathered from solid state NMR experiments molecular dynamics simulations were performed to obtain more information about the membranes at the molecular level. As a result, combination of solid-state NMR with molecular dynamics simulations provides very systematic way of investigating membrane dynamics in a large range of time scales.
Pirinixic acid derivatives were special interest of this study because of their activity on peroxisome proliferator-activated receptor (PPAR) as an agonist as well as on enzymes of microsomal prostaglandin E2 synthase-1 (PGE2s) -1 and 5-lipoxygenase (5-LO) as dual inhibitor. Two potent pirinixic acid derivatives, 2-(4-chloro-6-(quinolin-6-ylamino)pyrimidin-2-ylthio)octanoic acid (compound 2) and 2-(4-chloro-6-(quinolin-6-ylamino)pyrimidin-2-ylthio)octanoate (compound 3), have been worked and their insertion depts were investigated by combining of solid state NMR and molecular dynamics simulations. Both experimental and theoretical results pointed out that compound 3 was inserted the phospholipid bilayer more deeply than 2. NSAIDs – lipid mixtures have been also studied here. It is known that consumption of NSAIDs as in mixture with lipids results much fewer side effects than consumption of the drugs alone. Thus, it is crucial to understand interactions of NSAIDs with lipids and investigate the possible complex formation of drugs with lipids. In this study, interactions of three widely used NSAIDs, ibuprofen, diclofenac and piroxicam, with DPPC were investigated by solid-state NMR. 1H and 31P NMR results depicted that ibuprofen and diclofenac had interactions with lipids, which is an indication of drug-lipid complex formation whereas piroxicam didn’t show any interactions with lipids suggesting that no complex formation occurred in the case of piroxicam. Ceramides are known to play key roles in many cell processes and many studies showed that the functions of ceramides are related with the ceramide effects on biological membranes. Therefore, in this study, influences of ceramides on biophysics of lipid bilayers were investigated by using various solid state NMR techniques and molecular dynamics simulations. Results from molecular dynamics simulations clearly showed that ceramide and lipids have strong interactions. More evidences about ceramide-lipid interactions were provided from 1H and 14N NMR results. In addition, it was indicated by both simulation and experimental methods that ceramide increased the rigidity of DMPC by increasing chain order parameters. BTbk is a biradical, which is used as polarizing agent for dynamic nuclear polarization (DNP) experiments and found to be more efficient than other widely used polarizing agents such as TOTAPOL. Since it is a hydrophobic compound, which prefers to stay inside lipid bilayer it is important to investigate the location and orientation of bTbk along the bilayer in order to understand its enhancement profile in DNP measurements. In this study, both NMR relaxation time measurements and molecular dynamics simulations revealed that bTbk tends to stay more close to hydrophobic chain of lipids than the interfacial part of lipids at bilayer surface.
In the first part of this work, a brief introduction on lipid membranes as well as a theoretical summary on both methods of solid-state NMR and molecular dynamics simulations is given. Then, in the second part methodology is introduced for both solid-state NMR spectrometer and theoretical calculations. Afterwards, results of different membrane systems are discussed in the following parts for both solid state NMR and MD. Finally, in the last part, a summary and the conclusion of the overall results together with some future plans are explained.
In this thesis the integral membrane protein diacylglycerol kinase (DAGK) from E.coli is investigated with solid-state NMR. The aim is to gain an insight into the enzyme’s mechanism through integration of kinetic, structural and dynamic data. The biological function of DAGK is the transfer of the γ-phosphate group from Mg*ATP to diacylglycerol (DAG) building phosphatidic acid (PA)[6] as port of the membrane-derived oligosaccharide cycle[31,34]. Surprisingly, DAGK does not share structural or sequential similarities with other kinases[12]. Typical sequence motives found in other kinases, which catalyze phosphoryl transfer reactions, are not found[13]. In its physiological form DAGK is a homo-trimer with nine transmembrane helices, three catalytic centers and a size of 39.6 kDa.
First, the set-up of a real-time 31P MAS NMR experiment is shown. This experiment allows measuring in real-time the simultaneous ATP hydrolysis in the aqueous phase and lipid substrate phos-phorylation in the membrane phase with atomic resolution under magic angle spinning[56]. After fast transfer of the sample into the NMR spectrometer the enzymatic reaction is started with a temperature jump. This approach of real-time MAS NMR in a dual-phase system was demonstrated for the lipid substrate analogs dioleoyl- (DOG) and dibutyrylglycerol (DBG), with a C8 and C4 aliphatic chain, respectively. The combination of 31P direct and cross polarization functions as a dynamic filter. In the 31P direct polarized experiment nuclei in both phases are detected, while in the 31P cross polar-ized experiment, only nuclei in the membrane phase are detected. Rates for substrate turnover, i.e. degradation of γP-, βP, αP-ATP and build-up of βP-, αP-ADP, free phosphate as side reaction, and PA are obtained, which reveal a Michaelis-Menten behavior with regard to Mg*ATP and DBG. Here Mg*ATP and DBG follow a random-equilibrium model, where every substrate can bind indepen-dently from the other substrate. Analyses of the peak integrals from educts and products of the enzymatic reaction, revealed the stoichiometry of the reaction: 1.5 ATP molecules are used to phos-phorylate one DBG molecule. The excess of ATP is attributed to the basal ATPase activity. Further-more, experiments with ATPγS, usually regarded as a non-hydrolysable ATP-analog, where carried out. Surprisingly, DAGK hydrolyzes ATPγS and also transfers the thio-phosphate group to the lipid acceptor DBG, which points to a certain degree of plasticity in the active center. A phosphorylated enzyme intermediate was not detected. These results suggest the building of a ternary complex of Mg*ATP, DBG and DAGK performing a direct-phosphoryl transfer reaction, without passing through a phosphorylated enzyme intermediate. Experiments with the transition state analog ortho-vanadate (Vi) showed a decoupling of the ATP hydrolysis activity from lipid substrate phosphorylation. This indicates a specific transfer site for the γ-phosphate group from ATP to DAG, which can be blocked by Vi.
A general disadvantage of NMR spectroscopy compared to other spectroscopic methods is its inherent low sensitivity. One possible starting point for the improvement of signal-to-noise per unit time is the reduction of the spin-lattice relaxation time of protons[209]. Usually 95 % of the experi-mental time is required for the relaxation of the 1H to equilibrium. The addition of paramagnetic species can be used to reduce the 1H T1[233]. In a comprehensive study four different paramagnetic agents were tested: Cu2+-EDTA, Cu2+-EDTA-tag, Gd3+-TTAHA and Gd3+-DOTA. The titration of these paramagnetic complexes showed the principle feasibility of this approach, but differences between the tested species exist. The most promising complex is Gd3+-DOTA which, at a concentration of 2 mM, causes a 10-time improvement of signal-to-noise ratio per unit time. This allowed measuring 2D 13C-13C correlation spectra of proteoliposomes in one tenth of the usual required experimental time (i.e. 10 hours vs. 4 days) with good signal-to-noise.
For the investigation of structural or dynamic changes in the protein upon substrate interaction with MAS NMR, the spectral properties CP efficiency and resolution of the DAGK in liposomes needed to be improved. The most critical step during sample preparation is the reconstitution of the membrane protein from detergent micelles into a membrane of synthetic lipids under detergent removal. For this procedure the important criteria are enzymatic activity, measured in a coupled ATPase assay[55], and homogeneity of the proteoliposomes, which was tested e.g. on a discontinuous sucrose step gradient. Therefore an extensive study was carried out, in which different detergents, lipids and lipid mixtures, techniques for detergent removal and different protein-to-lipid ratios were tested. A direct correlation between high ATPase activity and good resolution was not found. Moreover, active DAGK in a mixture of DMPC and cholesterol, which emulates the membrane features of a membrane containing DAG, showed the best CP efficiency and resolution.
The assignment of the protein backbone and amino acid side chains the first mandatory step towards the investigation of structural and dynamical features influencing and defining the enzymatic mechanism by MAS NMR. As the assignment procedure is very time consuming for a total protein, a special labeling scheme for DAGK was developed, which allows assigning most of the protein areas presumably involved in enzyme catalysis. The assignment of DAGK with solution NMR[132] was not transferable to the MAS NMR spectra. Most important for the assignment process were the unique pairs[335], two consecutive amino acids which only appear once in the amino acid sequence. These unique pairs served as anchor points. Five different multinuclear MAS NMR experiments (DARR, NCO, NCA, NCACX, NCOCX) were required for the sequential assignment. It was possible to assign 35 % of the total amino acid sequence with one sample and 8 experiments acquired at 850 MHz. The secondary structure analysis showed subtle differences to the DAGK assignment with solution NMR[132], which can be attributed to the different environment in lipid bilayers and detergent micelles.
Data about structural and dynamical changes under substrate interaction can reveal details about the enzymatic mechanism. Therefore changes in chemical shift in 2D heteronuclear correlation experiments in the apo-state and under substrate saturated conditions with the substrates Mg*AMP-PNP, a non-hydrolysable ATP-analog, DOG, a mixture of Mg*AMP-PNP and DOG as well as inhibited by Vi were recorded. The most significant peak changes were observed at the interface membrane-cytoplasm as well as the the N-terminal amphipathic helix. The residues revealing chemical shift perturbations correlate with conserved residues or such residues, for which importance for catalysis and/or folding could be shown in mutation studies[8]. Especially noticeable were the changes at the amino acids Asn 72, Lys 64, His 87, Tyr 86 and Asp 95.
Beside changes of the chemical shift, changes of line width or signal doubling were observable. These changes can point to a correlation with dynamic reorientations in the μs-ms time regime, which are most relevant for enzymatic processes. The protein backbone dynamics in the apo-state as well as saturated with the substrates or inhibited with Vi were investigated with a 15N-CODEX experiment, which is based on the reorientation of the CSA tensor upon dynamical changes[350]. Specific effects of the different substrates or analogs on the protein backbone dynamic were revealed complementing the structural data and the chemical shift perturbation experiments.
Im Zentralen Nervensystem (ZNS) kommunizieren neuronale Synapsen über eine Kombination von chemischen und elektrischen Signalen, die in ihrer Umgebung eine spezifische Komposition von Ionen benötigen. Um eine strenge Kontrolle des ZNS-Milieus zu gewährleisten, hat sich in Säugetieren eine endotheliale Blut-Hirn-Schranke (BHS) entwickelt. Die BHS limitiert den parazellulären Molekül Transport und wird von den Kapillargefässen des Gehirns gebildet, wobei die physische Barrier von den Tight Junctions (TJs) des vaskulären Endothels generiert wird. Das Gehirnendothel ist Teil einer neurovaskulären Einheit (NVE), zu der auch Perizyten (PZ), Astrozyten (AZ), Mikroglia und Interneurone zählen. Fehlkommunikation oder defekte zelluläre Komponenten in der NVE führen in der Regel zu Störungen in der BHS Funktion und können schwerwiegende neuronale Erkrankungen zur Folge haben.
Vor einigen Jahren haben wir und andere Forschungsgruppen herausgefunden, dass der Wnt/β-Catenin Signalweg essentiell für die Vaskularisierung des Gehirns während der Embryonalentwicklung ist und darüber hinaus auch eine bedeutende Rolle in der Induktion der BHS spielt. Des Weiteren konnte im Zebrafischmodell eine Aktivierung des kanonischen Wnt Signalweges auch im adulten Organismus nachgewiesen werden. Allerdings ist die Quelle der Wnt Wachstumsfaktoren bis dato unbekannt. Der Wnt Signalweg ist eine hoch konservierte und komplexe zelluläre Signalkaskade, die in allen mehrzelligen Organismen vorkommt. Wnt Wachstumsfaktoren sind sekretierte, hydrophobe Signalmoleküle, die sowohl über lange als auch kurze Strecken entweder den β-Catenin-abhängingen („kanonischen“) oder β-Catenin-unabhängingen („nicht-kanonischen“) Wnt Signalweg aktivieren können.
Da die meisten ZNS Erkrankungen mit einem Zusammenbruch der BHS-Funktion assoziiert sind, ist die Forschung bestrebt die Mechanismen, die der Entstehung und Aufrechterhaltung der BHS zugrunde liegen, zu ermitteln und zu verstehen. Das Ziel meiner Doktorarbeit war es herauszufinden, ob AZ Wnts produzieren und ob deren Wirkung auf das Gehirnendothel an der Aufrechterhaltung der BHS beteiligt ist. Zu diesem Zweck, habe ich ein in vitro BHS Kokultivierungs-Modellsystem etabliert das erstmalig ausschliesslich auf der Verwendung von murinen AZ und Gehirnendothelzellen basiert. Zu Beginn der Studie wurden sowohl primäre AZ als auch eine murine Gehirnendothel-zelllinie (MBE) bezüglich ihrer zell-spezifischen Eigenschaften charakterisiert. Dabei konnte belegt werden, dass sowohl die primären AZ als auch die MBE Zelllinie, aufgrund ihrer Proteinexpressionsprofile als repräsentative Vertreter ihres Zelltyps eingestuft werden können. Die darauffolgenden Untersuchungen konnten zeigen, dass primäre AZ über mehrere Passagen hinweg fast alle 19 Wnt Liganden auf mRNA Ebene exprimierten. Ferner konnte in primären Gehirnendothelzellen und zwei Gehirnendothelzelllinien die korrespondierenden Frizzled (FZD) Rezeptoren und low density lipoprotein receptor-related protein (LRP) Korezeptoren nachgewiesen werden. Dieser Befund legte Nahe, dass AZ und Gehirnendothelzellen die basalen Eigenschaften besitzen, um über den Wnt Signalweg miteinander zu kommunizieren. Die Stimulation von pMBEs mit Astrozyten konditioniertem Medium (AKM) induzierte die Hochregulation von Claudin-3 einem bekannten kanonischen Wnt Zielgens. Interessanterweise konnte diese Regulation teilweise durch die Zugabe von dickkopf 1 (Dkk1), einem Wnt/β-Catenin Antagonisten, inhibiert werden.
Um die physiologische Rolle der Wnt Liganden zu bestimmen, habe ich mir die Eigenschaft des universellen Sekretionsmechanismus der Wachstumsfaktoren, welcher von dem Transmembranprotein evenness interrupted (Evi) abhängig ist, zu Nutze gemacht. Die Verpaarung von Evifl/fl mit hGFAP-Cre Mäusen erlaubt die AZ-spezifische Deletion des Evi Proteins (Evi KO), was zur Folge hat, dass die Astrozyten der Nachkommen keine Wnt Wachstumsfaktoren sekretieren können.
In vitro führte der Verlust von Wnts in AKM zu einer teilweisen Delokalisierung von Junction Proteinen. Während die Kokultivierung mit Evi WT AZ einen straken Anstieg im TEER und reduzierte Permeabilitätsmesswerte induzierten, konnten diese pro-BHS Eigenschaften bei Evi KO AZ nicht beobachtet werden. Diese Ergebnisse zeigten deutlich, dass Wnts sekretiert von AZ den BHS Phenotyp positive beeinflussen, indem sie die Zell-Zell-Verbindung verstärken, was wiederum zu erhöhtem Zellwiderstand und reduzierter transzellulärer Permeabilität führt. Die Analyse des in vivo Phänotyps von Evi KO Mäusen ergab, dass mit fortschreitendem, postnatalem Alter makroskopisch erkennbare zerebrale Blutungen auftraten. Ausserdem konnte ich zeigen, dass eine Subpopulation von Blutgefässen Malformationen aufwies, die mit reduzierter Astrozytenendfuss-Assoziierung einhergingen.
Das Wissen um die Beteiligung des Wnt Signalweges an der Regulation der BHS auch im adulten Organismus kann in Zukunft von wichtiger Bedeutung sein, da es potentielle therapeutische Anwendungen ermöglicht.
Heterodyne array receivers are employed in radio astronomy to reduce the observing time needed for mapping extended sources. One of the main factors limiting the amount of pixels in terahertz receivers is the difficulty of generating a sufficient amount of local oscillator power. Another challenge is efficient diplexing and coupling of local oscillator and signal power to the detectors. These problems are attacked in this dissertation by proposing the application of two vacuum electronic terahertz amplifier types for the amplification of the LO-signal and by introducing a new method for finding the defects in a quasioptical diplexer.
A traveling wave tube (TWT) design based on a square helix slow wave structure (SWS) at 825 GHz is introduced. It exhibits a simulated small-signal gain of 18.3 dB and a 3-dB bandwidth of 69 GHz. In order to generate LO-power at even higher frequencies, the operation of an 850-GHz square helix TWT as a frequency doubler has been studied. A simulated conversion efficiency of 7% to 1700 GHz, comparable with the state-of-art solid-state doublers, has been achieved for an input power of 25 mW.
The other amplifier type discussed in this work is a 1-THz cascade backward wave amplifier based on a double corrugated waveguide SWS. Specifically, three input/output coupler types between a rectangular waveguide and the SWS are presented. The structures have been realized with microfabrication, and the results of loss measurements at 1 THz will be shown.
Diplexing of the LO- and signal beams is often performed with a Martin-Puplett interferometer. Misalignment and deformation of the quasioptical components causes the polarization state of the output signal to be incorrect, which leads to coupling losses. A ray-tracing program has been developed for studying the influence of such defects. The measurement results of the diplexer of a multi-pixel terahertz receiver operated at the APEX telescope have been analyzed with the program, and the results are presented. The program allows the quasioptical configuration of the diplexer to be corrected in order to obtain higher receiver sensitivity.
This work presents a biochemical, functional and structural characterization of Aquifex aeolicus F1FO ATP synthase obtained using both a native form (AAF1FO) and a heterologous form (EAF1FO) of this enzyme.
F1FO ATP synthases catalyze the synthesis of ATP from ADP and inorganic phosphate driven by ion motive forces across the membrane and therefore play a key cellular function. Because of their central role in supporting life, F1FO ATP synthases are ubiquitous and have been remarkably conserved throughout evolution. For their biological importance, F1FO ATP synthases have been extensively studied for many decades and many of them were characterized from both a functional and a structural standpoint. However, important properties of ATP synthases – specifically properties pertaining to their membrane embedded subunits – have yet to be determined and no structures are available to date for the intact enzyme complex. Therefore, F1FO ATP synthases are still a major focus of research worldwide. Our research group had previously reported an initial characterization of AAF1FO and had indicated that this enzyme presents unique features, i.e. a bent central stalk and a putatively heterodimeric peripheral stalk. Based on such a characterization, this enzyme revealed promising for structural and functional studies on ATP synthases and became the focus of this doctoral thesis. Two different lines of research were followed in this work.
First, the characterization of AAF1FO was extended by bioinformatic, biochemical and enzymatic analyses. The work on AAF1FO led to the identification of a new detergent that maintains a higher homogeneity and integrity of the complex, namely the detergent trans-4-(trans-4’-propylcyclohexyl)cyclohexyl-α-D-maltoside (α-PCC). The characterization of AAF1FO in this new detergent showed that AAF1FO is a proton-dependent, not a sodium ion-dependent ATP synthase and that its ATP hydrolysis mechanism needs to be triggered and activated by high temperatures, possibly inducing a conformational switch in subunit γ. Moreover, this approach suggested that AAF1FO may present unusual features in its membrane subunits, i.e. short N-terminal segments in subunits a and c with implications for the membrane insertion mechanism of these subunits.
Investigating on these unique features of A. aeolicus F1FO ATP synthase could not be done using A. aeolicus cells, because these require a harsh and dangerous environment for growth and they are inaccessible to genetic manipulations. Therefore, a second approach was pursued, in which an expression system was created to produce the enzyme in the heterologous host E. coli. This second approach was experimentally challenging, because A. aeolicus F1FO ATP synthase is a 500-kDa multimeric membrane enzyme with a complicated and still not entirely determined stoichiometry and because its encoding genes are scattered throughout A. aeolicus genome, rather than being organized in one single operon. However, an artificial operon suitable for expression was created in this work and led to the successful production of an active and fully assembled form of Aquifex aeolicus F1FO ATP synthase. Such artificial operon was created using a stepwise approach, in which we expressed and studied first individual subunits, then subcomplexes, and finally the entire F1FO ATP synthase complex. We confirmed experimentally that subunits b1 and b2 form a heterodimeric subcomplex in the E. coli membranes, which is a unique case among ATP synthases of non-photosynthetic organisms. Moreover, we determined that the b1b2 subcomplex is sufficient to recruit the soluble F1 subcomplex to the membranes, without requiring the presence of the other membrane subunits a and c. The latter subunits can be produced in our expression system only when the whole ATP synthase is expressed, but not in isolation nor in the context of smaller FO subcomplexes. These observations led us to propose a novel mechanism for the assembly of ATP synthases, in which first the F1 subcomplex attaches to the membrane via subunit b1b2, and then cring and subunits a assemble to complete the FO subcomplex. Furthermore, we could purify the heterologous ATP synthase (EAF1FO) to homogeneity by chromatography and electro-elution. Enzymatic assays showed that the purified form of EAF1FO is as active as AAF1FO. Peptide mass fingerprinting showed that EAF1FO is composed of the same subunits as AAF1FO and all soluble and membrane subunits could be identified. Finally, single-particle electron microscopy analysis revealed that the structure of EAF1FO is identical to that of AAF1FO. Therefore, the EAF1FO expression system serves as a reliable platform for investigating on properties of AAF1FO.
Specifically, in this work, EAF1FO was used to study the membrane insertion mechanism of rotary subunit c. Subunits c possess different lengths and levels of hydrophobicity across species and by analyzing their N-terminal variability, four phylogenetic groups of subunits c were distinguished (groups 1 to 4). As a member of group 2, the subunit c from A. aeolicus F1FO ATP synthase is characterized by an N-terminal segment that functions as a signal peptide with SRP recognition features, a unique case for bacterial F1FO ATP synthases. By accurately designing mutants of EAF1FO, we determined that such a signal peptide is strictly necessary for membrane insertion of subunit c and we concluded that A. aeolicus subunit c inserts into E. coli membranes using a different pathway than E. coli subunit c. Such a property may be common to other ATP synthases from extremophilic organisms, which all cluster in the same phylogenetic group.
In conclusion, the successful production of the fully assembled and active F1FO ATP synthase from A. aeolicus in E. coli reported in this work provides a novel genetic system to study A. aeolicus F1FO ATP synthase. To a broader extent, it will also serve in the future as a solid reference for designing strategies aimed at producing large multi-subunit complexes with complicated stoichiometry.
In this thesis, the asymptotic behaviour of Pólya urn models is analyzed, using an approach based on the contraction method. For this, a combinatorial discrete time embedding of the evolution of the composition of the urn into random rooted trees is used. The recursive structure of the trees is used to study the asymptotic behavior using ideas from the contraction method.
The approach is applied to a couple of concrete Pólya urns that lead to limit laws with normal distributions, with non-normal limit distributions, or with asymptotic periodic distributional behavior.
Finally, an approach more in the spirit of earlier applications of the contraction method is discussed for one of the examples. A general transfer theorem of the contraction method is extended to cover this example, leading to conditions on the coefficients of the recursion that are not only weaker but also in general easier to check.
The economic success of the World Wide Web makes it a highly competitive environment for web businesses. For this reason, it is crucial for web business owners to learn what their customers want. This thesis provides a conceptual framework and an implementation of a system that helps to better understand the behavior and potential interests of web site visitors by accounting for both explicit and implicit feedback. This thesis is divided into two parts.
The first part is rooted in computer science and information systems and uses graph theory and an extended click-stream analysis to define a framework and a system tool that is useful for analyzing web user behavior by calculating the interests of the users.
The second part is rooted in behavioral economics, mathematics, and psychology and is investigating influencing factors on different types of web user choices. In detail, a model for the cognitive process of rating products on the Web is defined and an importance hierarchy of the influencing factors is discovered.
Both parts make use of techniques from a variety of research fields and, therefore, contribute to the area of Web Science.
Driven by rapid technological advancements, the amount of data that is created, captured, communicated, and stored worldwide has grown exponentially over the past decades. Along with this development it has become critical for many disciplines of science and business to being able to gather and analyze large amounts of data. The sheer volume of the data often exceeds the capabilities of classical storage systems, with the result that current large-scale storage systems are highly distributed and are comprised of a high number of individual storage components. As with any other electronic device, the reliability of storage hardware is governed by certain probability distributions, which in turn are influenced by the physical processes utilized to store the information. The traditional way to deal with the inherent unreliability of combined storage systems is to replicate the data several times. Another popular approach to achieve failure tolerance is to calculate the block-wise parity in one or more dimensions. With better understanding of the different failure modes of storage components, it has become evident that sophisticated high-level error detection and correction techniques are indispensable for the ever-growing distributed systems. The utilization of powerful cyclic error-correcting codes, however, comes with a high computational penalty, since the required operations over finite fields do not map very well onto current commodity processors. This thesis introduces a versatile coding scheme with fully adjustable fault-tolerance that is tailored specifically to modern processor architectures. To reduce stress on the memory subsystem the conventional table-based algorithm for multiplication over finite fields has been replaced with a polynomial version. This arithmetically intense algorithm is better suited to the wide SIMD units of the currently available general purpose processors, but also displays significant benefits when used with modern many-core accelerator devices (for instance the popular general purpose graphics processing units). A CPU implementation using SSE and a GPU version using CUDA are presented. The performance of the multiplication depends on the distribution of the polynomial coefficients in the finite field elements. This property has been used to create suitable matrices that generate a linear systematic erasure-correcting code which shows a significantly increased multiplication performance for the relevant matrix elements. Several approaches to obtain the optimized generator matrices are elaborated and their implications are discussed. A Monte-Carlo-based construction method allows it to influence the specific shape of the generator matrices and thus to adapt them to special storage and archiving workloads. Extensive benchmarks on CPU and GPU demonstrate the superior performance and the future application scenarios of this novel erasure-resilient coding scheme.
Paging is one of the most prominent problems in the field of online algorithms. We have to serve a sequence of page requests using a cache that can hold up to k pages. If the currently requested page is in cache we have a cache hit, otherwise we say that a cache miss occurs, and the requested page needs to be loaded into the cache. The goal is to minimize the number of cache misses by providing a good page-replacement strategy. This problem is part of memory-management when data is stored in a two-level memory hierarchy, more precisely a small and fast memory (cache) and a slow but large memory (disk). The most important application area is the virtual memory management of operating systems. Accessed pages are either already in the RAM or need to be loaded from the hard disk into the RAM using expensive I/O. The time needed to access the RAM is insignificant compared to an I/O operation which takes several milliseconds.
The traditional evaluation framework for online algorithms is competitive analysis where the online algorithm is compared to the optimal offline solution. A shortcoming of competitive analysis consists of its too pessimistic worst-case guarantees. For example LRU has a theoretical competitive ratio of k but in practice this ratio rarely exceeds the value 4.
Reducing the gap between theory and practice has been a hot research issue during the last years. More recent evaluation models have been used to prove that LRU is an optimal online algorithm or part of a class of optimal algorithms respectively, which was motivated by the assumption that LRU is one of the best algorithms in practice. Most of the newer models make LRU-friendly assumptions regarding the input, thus not leaving much room for new algorithms.
Only few works in the field of online paging have introduced new algorithms which can compete with LRU as regards the small number of cache misses.
In the first part of this thesis we study strongly competitive randomized paging algorithms, i.e. algorithms with optimal competitive guarantees. Although the tight bound for the competitive ratio has been known for decades, current algorithms matching this bound are complex and have high running times and memory requirements. We propose the algorithm OnlineMin which processes a page request in O(log k/log log k) time in the worst case. The best previously known solution requires O(k^2) time.
Usually the memory requirement of a paging algorithm is measured by the maximum number of pages that the algorithm keeps track of. Any algorithm stores information about the k pages in the cache. In addition it can also store information about pages not in cache, denoted bookmarks. We answer the open question of Bein et al. '07 whether strongly competitive randomized paging algorithms using only o(k) bookmarks exist or not. To do so we modify the Partition algorithm of McGeoch and Sleator '85 which has an unbounded bookmark complexity, and obtain Partition2 which uses O(k/log k) bookmarks.
In the second part we extract ideas from theoretical analysis of randomized paging algorithms in order to design deterministic algorithms that perform well in practice. We refine competitive analysis by introducing the attack rate
parameter r, which ranges between 1 and k. We show that r is a tight bound on the competitive ratio of deterministic algorithms.
We give empirical evidence that r is usually much smaller than k and thus r-competitive algorithms have a reasonable performance on real-world traces. By introducing the r-competitive priority-based algorithm class OnOPT we obtain a collection of promising algorithms to beat the LRU-standard. We single out the new algorithm RDM and show that it outperforms LRU and some of its variants on a wide range of real-world traces.
Since RDM is more complex than LRU one may think at first sight that the gain in terms of lowering the number of cache misses is ruined by high runtime for processing pages. We engineer a fast implementation of RDM, and compare it
to LRU and the very fast FIFO algorithm in an overall evaluation scheme, where we measure the runtime of the algorithms and add penalties for each cache miss.
Experimental results show that for realistic penalties RDM still outperforms these two algorithms even if we grant the competitors an idealistic runtime of 0.
Silicon wafers such as Silicon on Insulator (SOI) and strained silicon on Insulator (sSOI) are the essential and basic materials of advanced microelectronic devices. However, they often show various kinds of crystal defects which impair the function of these devices. The most efficient method to date, for detecting such defects and for determining their density, is to delineate them by etching the wafers with a suitable etching solution and characterise them via light optical microscopy. Etch pits are formed at defect sites which are etched at a faster rate than at the perfect lattice. The standard etching solution used for SOI and sSOI is a dilute version of Secco. As Secco contains carcinogenic and environmentally hazardous chromium (VI), the use of which is or will be restricted by law in many countries, suitable chromium (VI)-free etching solutions like Organic Peracid Etches (OPE), modified Chemical Polishing Etches (CP) like CP4 mod and mixtures with organic oxidizing agents like chloranil (CA) have been developed for the successful delineation of various types of crystal defects.
However there are still nanometer-sized defects which are hard to detect or escape detection by this method. Copper decoration is a well known method to magnify these defects. It consists in applying a copper nitrate solution to the back of the SOI or sSOI wafer. On annealing, copper diffuses through the substrate and the BOX (buried oxide) to the SOI/sSOI film and on quenching to room temperature, copper precipitates as copper silicide, SiCu3, foremost at crystal defects where the lattice strain is greater than at perfect lattice sites. These silicides increase the volume in these parts of the crystal lattice and defect magnification occurs. A considerable disadvantage of this method is its tendency for artefact formation, when the copper concentration used is too high, with the copper precipitating at the film surface. The consequence is a higher density of etch pits whereby true defect etch pits cannot be differentiated from those caused by artefacts.
The aim of this thesis is to show that the processes of decorating and etching can be combined successfully to delineate all crystal defects in SOI and sSOI. An ideal result would have been to find a copper decoration procedure that decorates all existing crystal defects at a copper concentration that avoids artefact formation.
Endocannabinoids (eCB) are signaling lipids and became known for their importance in the central nervous system as well as in immune defense. Beneficial effects of eCB are shown in processes of excitotoxic lesion, secondary damage and neuronal plasticity throughout the last years. Two canabinoid receptors, type 1 (CB1) and type 2 (CB2) as the respective endogenous ligands belong to the endocannabinoid system (eCBS). In 1990, the CB1 could be cloned and was localised mainly on neurons. Shortly thereafter in 1993, the CB2 was characterised and found primarily on cells belonging to the immune system. N-arachidonoylethanolamide (AEA), often called anandamide, and 2-arachidonoylglycerol (2-AG) are the best characterised eCB. N-palmitylethanolamide (PEA) and N-oleoylethanolamide (OEA) have no or only low affinity to CB1 but enhance the affinity of AEA significantly. This group is therefore often summarized as N-ethanolamides (NEA). ECB are derivates of arachidonic acid and are stored in membranes where they become hydrolysed on demand by specific enzymes. Traumatic brain injury altered the levels of eCB in the blood in vivo and when applied in vitro after neuronal damage, eCB could reduce the damaging burden. Further studies demonstrated that eCB are potent to down-regulate pro-inflammatory cytokines and most important to decrease neuronal excitation.
In the present study, the intrinsic regulation of the endocannabinoid system after neuronal damage over time was investigated in rat Organotypic Hippocampal Slice Cultures (OHSC). Temporal and spatial dynamics of eCB levels were analysed after transection of the perforant pathway (PPT) in originating neurons (enthorhinal cortex, EC), areas of deafferentiation/anterograde axonal degeneration (dentate gyrus, DG) and of the synaptically linked cornu ammonis region 1 (CA1) as well as after excitotoxic lesion in the respective regions.
A strong increase of all eCB was observed only in the denervation zone of the DG 24 hours post PPT. In excitotoxic lesioned OHSC all eCB were elevated, in the investigated regions up to 72 hours post lesion (hpl). The responsible enzyme for biosynthesis of the NEA, NAPE-PLD protein, was increased during the early timepoints of measurement (1-6 hpl). The responsible catabolizing enzyme, FAAH, and the CB1 receptor were up-regulated at a later timepoint, 48 hpl, explaining the eCB levels. In the present model, the inhibition of the enzyme responsible for 2-AG hydrolysis (MAGL) was neuroprotective as previously shown and a re-distribution within neurons and astrocytes during neuronal damage could be observed. In primary cell cultures microglia expressed the regulating enzymes of 2-AG and the enzyme responsible for NEA down-regulation, FAAH. Astrocytes expressed mainly the catalyzing enzymes, indicating the role for eCB break-down. All these findings together demonstrate the great capacity of the eCBS to control inflammatory processes and consequently neuronal cell death.
All effects of the known eCB could not be clarified by CB1/CB2 deficient mice. Several G-protein coupled receptors (GPR) are recently in discussion whether they might and should belong to the endocannabinoid system. The GPR55, the not yet cloned abnormal cannabidiol receptor and further GPRs are candidates as potential endocannabinoid receptors. Recently GPR55 has been discussed as a putative cannabinoid receptor type 3 (CB3). Quantitative PCR revealed that Gpr55 is present in primary microglia and the brain, but the exact regional and cellular distribution and the physiological/pathological effects downstream of GPR55 activation in the CNS still remain open. Therefore, the excitotoxic rat OHSC model, previously used to investigate the neuroprotective potency of eCB, was now used to investigate the neuroprotective potency of GPR55. Activation of GPR55 protected dentate gyrus granule cells in vitro after excitotoxic lesion, induced by NMDA. In parallel, GPR55 activation was able to reduce the number of microglia in the dentate gyrus. These neuroprotective effects vanished however in microglia depleted OHSCs as well as in OHSC transfected with Gpr55 siRNA, indicating a strong involvement of microglia in GPR55 mediated neuroprotection.
In summary, the present study found a strong time-dependent and anterograde mechanism of action of eCB after long-range projection damage and provided further evidence for the neuroprotective properties of eCB. The potential cannabinoid receptor 3 (GPR55) mediates neuronal protection on behalf of microglia.
Essays in behavioral economics - evidence on self-selection into jobs, social networks and leniency
(2013)
Die Dissertation mit dem Titel „Essays in Behavioral Economics – Evidence on Self-Selection into Jobs, Social Networks and Leniency“ besteht aus einer Sammlung von vier wissenschaftlichen Abhandlungen. Alle Arbeiten verbindet die Analyse von theoretischen Konzepten und Erkenntnissen der Verhaltensökonomie unter Verwendung der experimentellen Methode. Die erste wissenschaftliche Abhandlung trägt den Titel „Sorting of Motivated Agents - Empirical Evidence on Self-Selection into the German Police“ und untersucht Selbstselektion bestimmter Individuen in den Polizeiberuf. Die experimentelle Studie untersucht die Frage, ob Polizeibewerber sich hinsichtlich ihrer Präferenzen in Bezug auf ihr Normdurchsetzungsverhalten in den Polizeiberuf selektieren. Die zweite Abhandlung greift diese Erkenntnisse auf und untersucht Polizeianwärter in ihrer Berufsausbildung ebenfalls hinsichtlich ihrer Normdurchsetzungsbereitschaft. Die Arbeit trägt den Titel „Selection and formation of motivated agents -- empirical evidence from the German Police”. In der dritten wissenschaftlichen Abhandlung werden geschlechterspezifische Unterschiede bei der Wahl von Partnern und dem Aufbau des sozialen Netzwerkes untersucht. Diese trägt den Titel „Selectivity and opportunism: two dimensions of gender differences in trust games and network formation“ und wurde zusammen mit Guido Friebel, Marie Lalanne, Paul Seabright und Peter Schwardmann verfasst. Die vierte Abhandlung geht einer aktuellen Fragestellung der Industrieökonomie nach und trägt den Titel „Antitrust, auditing and leniency programs: evidence from the laboratory“, verfasst mit Mehdi Feizi and Ali Mazyaki. In ihrer Gesamtheit liefert meine Dissertation Antworten auf personalpolitische, soziale und industrieökonomische Fragestellungen.
Der programmierte Zelltod (Apoptose) ist ein wichtiger Mechanismus zur Eliminierung von beschädigtem Gewebe und entarteten Zellen. Die Deregulierung der Apoptose führt zu zahlreichen Erkrankungen wie neuro-degenerativen Störungen und Krebs. Insbesondere in Tumoren wird der programmierte Zelltod mit Hilfe von hochregulierten, anti-apoptotischen Proteinen umgangen und es entstehen Resistenzen gegen Chemotherapien. Um innovative therapeutische Ansätze zu finden, wurden in diesem Projekt mit Hilfe eines Hefe-Survival-Screens neue, potentiell anti-apoptotische Proteine im Pankreaskarzinom identifiziert. Von den insgesamt 38 identifizierten Genprodukten wurden zwei für eine weiterführende Analyse ausgewählt.
Eins der näher untersuchten Proteine ist die Pyruvoyl-tetrahydrobiopterin-Synthase (PTS), ein wichtiges Enzym für die Biosynthese von Tetrahydrobiopterin (BH4). BH4 ist ein Kofaktor, der von mehreren Enzymen der Zelle für ihre Funktionen benötigt wird. In Zellkultur-Experimenten konnte gezeigt werden, dass eine Überexpression von PTS die Zellen vor Apoptose schützen kann, während eine Herunterregulation durch genetischen knockdown die Zellen gegenüber Apoptose-Stimuli sensibilisiert und ihr Wachstum beeinträchtigt. In Xenograft-Experimenten mit NOD/SCID-Mäusen konnte zudem gezeigt werden, dass Tumore mit einem PTS-Knockdown signifikant langsamer wachsen als die der Kontrollgruppe. Zusammengenommen deuten diese Ergebnisse auf eine Rolle von PTS bei der Apoptose-Regulation und beim Tumorwachstum hin, was das Protein zu einem attraktiven Target für die Krebstherapie macht.
Als zweites wurde ein Protein analysiert, das eine Untereinheit des respiratorischen Komplex I bildet: NDUFB5 (NADH-Dehydrogenase 1 beta Subcomplex, 5). Das besondere an diesem Protein sind die verschiedenen Isoformen, die durch alternatives Splicing zustandekommen. Eine Isoform, der die Exone 2 und 3 fehlen, wurde im Hefe-Survival-Screen identifiziert. Bei Überexpression in Zelllinien konnte sie im Gegensatz zum Volllänge-Protein die Apoptoserate reduzieren. Und auch Ergebnisse aus Versuchen mit Isoformen-spezifischem knockdown deuten an, dass hauptsächlich die verkürzte Isoform sNDUFB5 für die Regulation von Apoptose und Proliferation verantwortlich ist. Diese Beobachtungen konnten mit denselben Zellen im Xenograft-Tiermodell jedoch nicht bestätigt werden. Die Ursachen dafür blieben unklar. Zusätzlich wurden immunhistochemische Analysen von Pankreaskarzinomen und normalem Pankreasgewebe durchgeführt. Sie ergaben, dass die kurze Isoform sNDUFB5 im Tumor stark überexpremiert ist, während die Expression des Volllänge-Proteins in normalem und Tumorgewebe ähnlich hoch ausfällt. Dieser Befund macht NDUFB5 zu einem interessanten therapeutischen Target.
Die näher untersuchten Kandidaten-Gene zeigen beide Potential als neue Angriffspunkte für eine molekulare Krebstherapie. Andere in dem Hefe-Survival-Screen identifizierte Proteine wurden bereits als anti-apoptotisch und/oder in Krebszellen überexprimiert beschrieben. Diese Ergebnisse demonstrieren, dass ein funktionelles, Hefe-basiertes Screeningsystem geeignet ist, neue bisher unbekannte Proteine mit anti-apoptotischer Funktion zu identifizieren. Auch zeigen die Befunde, dass bereits bekannte Proteine weitere bisher unbekannte Funktionen wie z.B. die Inhibition von Apoptose aufweisen können. Basierend auf solchen mehrfachen Proteinfunktionen lassen sich weitere therapeutische Möglichkeiten ableiten.
In this thesis, Hanbury-Brown-Twiss (HBT) interferometry is used together with the Ultrarelativistic Quantum Molecular Dynamics (UrQMD) to analyse the time and space structure of heavy-ion collisions.
The first chapter after the introduction gives an overview of the different types of models used in the field of heavy-ion collisions and a introduction of the UrQMD model in more detail. The next chapter explains the basics of Hanbury-Brown-Twiss correlations, including azimuthally sensitive HBT (asHBT).
Results section:
4. Charged Multiplicities from UrQMD
5. Formation time via HBT from pp collisions at LHC
6. HBT analysis of Pb+Pb collisions at LHC energies
7. HBT scaling with particle multiplicity
8. Compressibility from event-by-event HBT
9. Tilt in non-central collisions
10. Shape analysis of strongly-interacting systems
11. Measuring a twisted emission geometry
This thesis covers the standard integrated HBT analyses, extracting the Pratt-Bertsch radii, at LHC energies. The analyses at these energies showed a too soft expansion in UrQMD probably related to the absence of a partonic phase in UrQMD. The most promising results in this thesis at these energies are the restriction of the formation time to a value smaller than 0.8 fm/c and furthermore, the results from the asHBT analyses. In simulations of non-central heavy-ion collisions at energies of Elab= 6, 8 and 30 AGeV the validity of the formulae to calculate the tilt angle via asHBT has been checked numerically, even for the case of non-Gaussian, flowing sources. On this basis has been developed and test in the course of this thesis that allows to measure a scale dependent tilt angle experimentally. The signal should be strongest at FAIR energies.
Spin waves in yttrium-iron garnet has been the subject of research for decades. Recently the report of Bose-Einstein condensation at room temperature has brought these experiments back into focus. Due to the small mass of quasiparticles compared to atoms for example, the condensation temperature can be much higher. With spin-wave quasiparticles, so-called magnons, even room temperature can be reached by externally injecting magnons. But also possible applications in information technologies are of interest. Using excitations as carriers for information instead of charges delivers a much more efficient way of processing data. Basic logical operations have already been realized. Finally the wavelength of spin waves which can be decreased to nanoscale, gives the opportunity to further miniaturize devices for receiving signals for example in smartphones.
For all of these purposes the magnon system is driven far out of equilibrium. In order to get a better fundamental understanding, we concentrate in the main part of this thesis on the nonequilibrium aspect of magnon experiments and investigate their thermalization process. In this context we develop formalisms which are of general interest and which can be adopted to many different kinds of systems.
A milestone in describing gases out of equilibrium was the Boltzmann equation discovered by Ludwig Boltzmann in 1872. In this thesis extensions to the Boltzmann equation with improved approximations are derived. For the application to yttrium-iron garnet we describe the thermalization process after magnons were excited by an external microwave field.
First we consider the Bose-Einstein condensation phenomena. A special property of thin films of yttrium-iron garnet is that the dispersion of magnons has its minimum at finite wave vectors which leads to an interesting behavior of the condensate. We investigate the spatial structure of the condensate using the Gross-Pitaevskii equation and find that the magnons can not condensate only at the energy minimum but that also higher Fourier modes have to be occupied macroscopically. In principle this can lead to a localization on a lattice in real space.
Next we use functional renormalization group methods to go beyond the perturbation theory expressions in the Boltzmann equation. It is a difficult task to find a suitable cutoff scheme which fits to the constraints of nonequilibrium, namely causality and the fluctuation-dissipation theorem when approaching equilibrium. Therefore the cutoff scheme we developed for bosons in the context of our considerations is of general interest for the functional renormalization group. In certain approximations we obtain a system of differential equations which have a similar transition rate structure to the Boltzmann equation. We consider a model of two kinds of free bosons of which one type of boson acts as a thermal bath to the other one. Taking a suitable initial state we can use our formalism to describe the dynamics of magnons such that an enhanced occupation of the ground state is achieved. Numerical results are in good agreement with experimental data.
Finally we extend our model to consider also the pumping process and the decrease of the magnon particle number till thermal equilibrium is reached again. Additional terms which explicitly break the U(1)-symmetry make it necessary to also extend the theory from which a kinetic equation can be deduced. These extensions are complicated and we therefore restrict ourselves to perturbation theory only. Because of the weak interactions in yttrium-iron garnet this provides already good results.
Ultrarelativistic Quantum Molecular Dynamics is a physics model to describe the transport, collision, scattering, and decay of nuclear particles. The UrQMD framework has been in use for nearly 20 years since its first development. In this period computing aspects, the design of code, and the efficiency of computation have been minor points of interest. Nowadays an additional issue arises due to the fact that the run time of the framework does not diminish any more with new hardware generations.
The current development in computing hardware is mainly focused on parallelism. Especially in scientific applications a high order of parallelisation can be achieved due to the superposition principle. In this thesis it is shown how modern design criteria and algorithm redesign are applied to physics frameworks. The redesign with a special emphasise on many-core architectures allows for significant improvements of the execution speed.
The most time consuming part of UrQMD is a newly introduced relativistic hydrodynamic phase. The algorithm used to simulate the hydrodynamic evolution is the SHASTA. As the sequential form of SHASTA is successfully applied in various simulation frameworks for heavy ion collisions its possible parallelisation is analysed. Two different implementations of SHASTA are presented.
The first one is an improved sequential implementation. By applying a more concise design and evading unnecessary memory copies, the execution time could be reduced to the half of the FORTRAN version’s execution time. The usage of memory could be reduced by 80% compared to the memory needed in the original version.
The second implementation concentrates fully on the usage of many-core architectures and deviates significantly from the classical implementation. Contrary to the sequential implementation, it follows the recalculate instead of memory look-up paradigm. By this means the execution speed could be accelerated up to a factor of 460 on GPUs.
Additionally a stability analysis of the UrQMD model is presented. Applying metapro- gramming UrQMD is compiled and executed in a massively parallel setup. The resulting simulation data of all parallel UrQMD instances were hereafter gathered and analysed. Hence UrQMD could be proven of high stability to the uncertainty of experimental data.
As a further application of modern programming paradigms a prototypical implementa- tion of the worldline formalism is presented. This formalism allows for a direct calculation of Feynman integrals and constitutes therefore an interesting enhancement for the UrQMD model. Its massively parallel implementation on GPUs is examined.
Development of prototype components for the Silicon Tracking System of the CBM experiment at FAIR
(2013)
The CBM experiment at future accelerator facility FAIR will investigate the properties of nuclear matter under extreme conditions. The experimental programm is different from the heavy-ion experiments at RHIC (BNL) and LHC (CERN) that create nuclear matter at high temperatures. In contrast, the study of the QCD phase diagram in the region of the highest net baryon densities and moderate temperatures that is weakly explored will be performed with high precision. For this, collisions of different heavy-ion beams at the energies of 10–45GeV/nucleon with nuclear target will be measured.
The physics programme of the CBM experiment includes measurement of both rare probes and bulk observables that originate from various phases of a nucleus-nucleus collision. In particular, decay of particles with charm quarks can be registered by reconstructing the decay vertex detached from the primary interaction point by several hundreds of micrometers (e.g., decay length c Tau = 123 µm for D0 meson). For this, precise tracking and full event reconstruction with up to 600 charged particle tracks per event within acceptance are required. Other rare probes require operation at interaction rate of up to 10MHz. The detector system that performs tracking has to provide high position resolution on the order of 10 µm, operate at high rates and have radiation tolerant design with low material budget.
The Silicon Tracking System (STS) is being designed for charged-particle tracking in a magnetic field. The system consists of eight tracking station located in the aperture of a dipole magnet with 1T field. For tracks with momentum above 1GeV, momentum resolution of such a system is expected to be about 1%. In order to fulfill this task, thorough optimization of the detector design is required. In particular, minimal material budget has to be achieved.
Production of a detector module requires research and development activities with respect to the module components and their integration. A detector module is a basic functional unit that includes a sensor, an analogue microcable and frontend electronics mounted on a support structure. The objective of the thesis is to perform quality assurance tests of the prototype module components in order to validate the concept of the detector module and to demonstrate its operation using radioactive sources and particle beams.
Double-sided silicon microstrip detectors have been chosen as sensor technology for the STS because of the combination of a good spatial resolution, two-dimensional coordinate measurement achieved within low material budget (0.3%X0), high readout speed and sufficient radiation tolerance. Several generations of double-sided silicon microstrip sensors have been manufactured in order to explore the radiation hard design features and the concept of a large-area sensor compatible with ladder-type structure of the detector module. In particular, sensors with double metal layer on both sides and active area of 62×62mm2 have been produced. Electrical characterization of the sensors has been performed in order to establish the overall operability as well as to extract the device parameters. Current-voltage, capacitance-voltage characteristics and interstrip parameters have been measured.
Readout of the sensors has been done using self-triggering front-end electronics. A front-end board has been developed based on the n-XYTER readout chip with data driven architecture and capable of operating at 32MHz readout rate. The front-end board included an external analog-to-digital converter (ADC). Calibration of the ADC has been performed using both 241Am X-ray source and external pulse generator. Threshold calibration and investigation of temperature dependence of chip parameters has been carried out.
Low-mass support structures have been developed using carbon fibre that has the rigidity to hold the detector modules and introduce minimal Coulomb scattering of the particle tracks. Analogue microcables have been produced with aluminium traces on a polyimide substrate, thus combining good electrical connection with low material budget. Microcable structure includes several layers optimized for low trace capacitance and thus low-noise performance.
A demonstrator tracking telescope has been constructed and operated in several beam tests including 2.5GeV proton beam at COSY synchrotron (Jülich). Three tracking stations have been complemented with several beam hodoscopes. Analysis of the beam data has yielded information on analogue and timing response, beam profile. Tracking and alignment information has been obtained. Beam stability has been evaluated using specially developed monitoring tools.
As a result of conducted studies, performance of the module components have been evaluated and requirements to the detector module have been formulated. Practical suggestions have been made with respect to the structure of the detector module, whereas precise definition of the final detector module design was outside of the scope of this thesis.
Die Adoleszenz, d.h. die Reifungsphase des Jugendlichen zum Erwachsenen, stellt einen zentralen Abschnitt in der menschlichen Entwicklung dar, der mit tief greifenden emotionalen und kognitiven Veränderungen verbunden ist. Neure Studien (Bunge et al., 2002; Durston et al., 2002; Casey et al., 2005; Crone et al., 2006; Bunge and Wright, 2007) machen deutlich, dass sich die funktionelle Architektur des Gehirns während der Adoleszenz grundlegend verändert und dass diese Veränderungen mit der Reifung höherer kognitiven Funktionen in der Adoleszenz assoziiert sein könnten. Messungen des Gehirn-Volumens mit Hilfe der Magnet-Resonanz-Tomographie (MRT) zum Beispiel zeigen eine nicht-lineare Reduktion der grauen und eine Zunahme der weißen Substanz während der Adoleszenz (Giedd et al., 1999; Sowell et al., 1999, 2003). Des weiteren treten in dieser Zeit Veränderungen in exzitatorischen und inhibitorischen Neurotransmitter-Systemen auf (Tseng and O’Donnell, 2005; Hashimoto et al., 2009). Zusammen deuten diese Ergebnisse darauf hin, dass während der Adoleszenz ein Umbau der kortikalen Netzwerke stattfindet, der wichtige Konsequenzen für die Reifung neuronaler Oszillationen haben könnte. Im Anschluss an eine Einführung im Kapitel 2, fasst Kapitel 3 der vorliegenden Dissertation die Vorbefunde bezüglich entwicklungsbedingter Veränderungen in der Amplitude, Frequenz und Synchronisation neuronaler Oszillationen zusammen und diskutiert den Zusammenhang zwischen der Entwicklung neuronaler Oszillationen und der Reifung höhere kognitiver Funktionen während der Adoleszenz. Ebenso werden die anatomischen und physiologischen Mechanismen, die diesen Veränderungen möglicherweise zu Grunde liegen könnten, theoretisch vorgestellt. Die in Kapitel 4-6 vorgestellten eigenen empirischen Arbeiten untersuchen neuronale Oszillationen mit Hilfe der Magnetoencephalographie (MEG), um die Frequenzbänder und die funktionellen Netzwerke zu charakterisieren, die mit höheren kognitiven Prozessen und deren Entwicklung in der Adoleszenz assoziiert sind. Hierzu wurden drei Experimente durchgeführt, bei denen MEG-Aktivität während der Bearbeitung einer Arbeitsgedächtnisaufgabe und im Ruhezustand aufgezeichnet wurde. Die Ergebnisse dieser Experimente zeigen, dass Alpha Oszillationen und Gamma-Band Aktivität sowohl task-abhängig als auch im Ruhezustand gemeinsam auftreten. Darüber hinaus ergänzen die vorliegenden Untersuchungen Vorarbeiten, indem sie eine Wechselwirkung zwischen beiden Frequenzbändern aufgezeigt wird, die als ein Mechanismus für das gezielte Weiterleiten von Informationen dienen könnte. Die in Kapitel 6 vorgestellten Entwicklungsdaten weisen weiterhin darauf, dass in der Adoleszenz späte Veränderungen im Alpha und Gamma-Band stattfinden und dass diese Veränderungen involviert sind in die Entwicklung der Arbeitsgedächtnis-Kapazität und die Entwicklung der Fähigkeit, Distraktoren zu inhibieren. Abschliessend werden in Kapitel 7, die in dieser Dissertation vorgestellten Arbeiten, aus einer übergeordneten Perspektive im Gesamtzusammenhang diskutiert.
In der vorliegenden Arbeit wurden mikroskopische Studien zur Äquilibrierung von partonischer und hadronischer Materie im Rahmen einer Nichtgleichgewichts-Transporttheorie durchgeführt, die sowohl hadronische als partonische Freiheitsgrade enthält und den Übergang zwischen beiden Phasen dynamisch beschreibt. Des Weiteren wurden die thermischen Eigenschaften des Gleichgewichtszustandes der stark wechselwirkenden Materie untersucht, insbesondere Fluktuationen in der Teilchenzahl wie auch höhere Momente von Observablen und deren Verhältnisse. Besonderes Interesse galt dabei den Transportkoeffizienten wie Scher- und Volumenviskosität sowie der elektrischen Leitfähigkeit.
Die Methode der Nichtgleichgewichts-Green'schen Funktionen - initiiert von Schwinger sowie Kadanoff und Baym - wurde vorgestellt um hochenergetische Kern-Kern Kollisionen zu beschreiben. Weiterhin wurde der Schwinger-Keldysh Formalismus benutzt um im Sinne einer Zweiteilchen-irrediziblen Näherung (2PI) die Dynamik von 'resummierten' Propagatoren und Kopplungen in konsistenter Weise zu beschreiben. Des Weiterhin wurden generalisierte Transportgleichungen auf der Basis der Kadanoff-Baym Gleichungen (in Phasenraumdarstellung) abgeleitet und ein Testteilchenverfahren zur Lösung dieser Gleichungen vorgestellt. Damit wurde der formale Rahmen der Parton-Hadron-String Dynamik (PHSD) abgesteckt.
Das PHSD Transportmodell wurde sodann für die Lösung der expliziten Fragestellungen in dieser Arbeit verwendet. Die 'Eingangsgrößen' des Modells wurden in Kapitel 3 aufgeführt. Weiterhin wurde aufgezeigt, dass das Transportmodell alle Phasen einer relativistischen Schwerionenkollision konsistent beschreibt, d.h. angefangen von den primären harten Stoßprozessen und der Bildung von 'Strings' zur Formierung einer partonischen Phase, den Wechselwirkungen in dieser Phase sowie die
dynamische Beschreibung der Hadronisierung. Weiterhin enthält das Modell zudem die hadronischen Endzustandswechselwirkungen bis zum Ausfrieren der hadronischen Freiheitsgrade bei geringer Dichte. ...
Ziel dieser Arbeit war, die Reaktion von biologischen Gewebeproben auf dünn- und dicht-ionisierende Strahlung zu evaluieren. Dafür wurden die Gewebeproben konventioneller Röntgenstrahlung sowie einem ausgedehnten 12C-Ionen Bragg-Peak ausgesetzt. Zur Bestrahlung der biologischen Proben mit 12C wurde mit dem GSI-eigenen Simulationsprogramm TRiP98 ein Tiefendosisprofil eines ausgedehnten Bragg-peaks erstellt. Ein weiteres Ziel dieser Arbeit war, dieses Tiefendosisprofil mit drei anderen Simulationsprogrammen (ATIMA, MCHIT, TRIM) zu reproduzieren und zu vergleichen.
ATIMA und TRIM sind allgemeine Programme für den Energieverlust von Ionen in Materie. Sie können das von TRiP98 berechnetet Tiefendosisprofil nur ungenügend reproduzieren, da sie aufgrund fehlender Fragmentierung ein linear ansteigendes Tiefendosisprofil berechnen. Das Monte Carlo-Programm MCHIT, welches speziell für die Wechselwirkung von Ionen mit Materie in medizinischer Anwendung entwickelt wurde, zeigt die beste Übereinstimmung mit der TRiP98-Referenzkurve. Bis auf eine leicht höhere Durchschnittsdosis um 0.1 Gy konnte das Tiefendosisprofil nahezu exakt reproduziert werden.
Die biologischen Proben bestanden aus Schnittkulturen gesunder Maus-Lebern und Explantatkulturen gesunder Maus-Pankreata, um Nebenwirkungen ionisierender Strahlen abzuschätzen. Zusätzlich wurde die Reaktion auf 12C-Bestrahlung in neoplastischem Lebergewebe transgener c-myc/TGF-α Mäuse mit induzierbarem Lebertumor bestimmt. Um eine mögliche Tageszeitabhängigkeit der Gewebereaktion auf die Bestrahlung zu untersuchen, wurden die Schnitt- und Explantatkulturen zu zwei unterschiedlichen Tageszeiten präpariert: zur Mitte des subjektiven Tages und zur Mitte der subjektiven Nacht.
Die Präparate wurden für mehrere Tage auf einer Membran an einer Grenzschicht von Flüssigkeit und Luft kultiviert. Leber- und Pankreaskulturen gesunder C3H wildtyp Mäuse wurden mit einer Dosis von 2 Gy, 5 Gy oder 10 Gy Röntgenstrahlen bestrahlt. Leber- und Pankreaskulturen transgener Mäuse wurden mit ausgedehnten C-Ionen Bragg Peaks gleicher Dosen bestrahlt. Als Kontrolle dienten unbestrahlte Proben. Alle Proben wurden 1 h bzw. 24 h nach der Bestrahlung fixiert und immunhistochemisch auf Marker für Proliferation (Ki67), Apoptose (Caspase3) und DNA- Doppelstrangbrüche (γH2AX) untersucht.
Während die Pankreas-Präparate im Hinblick auf die untersuchten Parameter leider keine auswertbaren Ergebnisse ergaben, zeigten die untersuchten Parameter im gesunden Lebergewebe deutliche Tag-Nacht Unterschiede: die Proliferationsrate war zur Mitte des subjektiven Tages signifikant höher als zur Mitte der subjektiven Nacht. Umgekehrt waren die Raten für DNA-Doppelstrangbrüche zur Mitte der subjektiven Nacht signifikant erhöht. Diese Tag-Nacht Unterschiede ließen sich in neoplastischem Lebergewebe nicht nachweisen. Unabhängig von der Art und Dosis, hatte die Bestrahlung im gesunden Lebergewebe keinen Einfluss auf die untersuchten Parameter. In neoplastischem Lebergewebe hingegen wird die Rate an DNA-Doppelstrangbrüchen durch eine Bestrahlung dosisabhängig erhöht.
Die Auswirkungen ionisierender Strahlen auf das circadiane Uhrwerk wurden in Gewebeproben transgener Per2luc-Mäuse überprüft. Per2luc-Mäuse exprimieren das Enzym Luziferase unter der Kontrolle des Promoters von Per2, einem wichtigen Bestandteil des circadianen Uhrwerks. Daher erlaubt die Analyse dieser Tiere, den circadianen Rhythmus des molekularen Uhrwerks in Leber und anderen Geweben durch Messung der Luziferase-Aktivität in Echtzeit aufzuzeichnen. Wie in Leber- und Nebennierenkulturen dieser Tiere gezeigt werden konnte, führten ioniserende Strahlen dosisabhängig zu einem Phasenvorsprung des circadianen Uhrwerks.
Die Ergebnisse erlauben die Schlussfolgerung, dass ionisierende Strahlen das circadiane Uhrwerk verstellen, Proliferation und Apoptose in gesundem Lebergewebe jedoch kaum beeinflussen.
Modern computational molecular quantum chemical studies, such as the present one, typically employ a wide range of theoretical techniques. The latter are often rather complicated and one should not generally expect that an experimental scientist in the area of physical chemistry, a potential reader of this work, should be familiar with all these techniques. To simplify the reading of the Thesis and to make it self-sufficient, it is supplied with an overview of the employed theoretical methodologies (Chapter 1). The overview explains basic quantum-chemical terminology referred to throughout the Thesis, introduces theoretical foundations of the methods and outlines their properties and limitations. In Part 1.1 of Chapter 1, methods for the solution of the molecular Schrödinger equation are introduced, while in the subsequent Parts 1.2 and 1.3 methods for the solution of the electronic Schrödinger equation are presented to find the ground and excited states, respectively. Part 1.4 is dedicated to basis-set effects which are omnipresent in electronic-structure calculations. It contains a number of unusual insights and concepts proposed by the author and, thus, may be insightful also to experts in quantum chemistry.
In Chapter 2, the phenomenon of acetone-water proton exchange catalyzed by tubular as well as amorphous aggregates of calix[4]hydroquinone (CHQ) macromolecules, which has been observed previously in NMR experiments (Ref. D1D), is investigated by means of correlated quantum-chemical methods. The first part of the study (Section 2.3-2.7) considers concerted proton transfer, assisted by several initially neutral OH-groups in the hydrogen-bonded networks of CHQ aggregates. The second part of the study (Section 2.8-2.13) is dedicated to a second mechanism of proton exchange: step-wise proton transfer via formation of ionic intermediates resulting from CHQ pre-dissociation. CHQ application-specific as well as general conclusions, relevant to the main topic of the Thesis (i.e. influence of specific microsolvation on the considered proton transfer processes), are presented in Section 2.14.
The phenomenon of dual fluorescence observed in clusters of methyl 4-N,N-dimethylaminobenzoate ester (DMABME) and two water molecules in the gas phase, is studied in Chapter 3. Experimentally, the dual fluorescence was detected in experiments combining optical and ground-state ion-depletion infrared spectroscopies in ultracold molecular beams (Ref. D2D). In Section 3.3, calculated ground-state infrared spectra are presented that allow to identify the structures of those isomers, which are present in the gas-phase, as well as the structure of the isomer responsible for dual fluorescence. To further understand the reaction mechanism of dual fluorescence, excited-state potential energy surfaces of the identified isomers were computed along the relevant twisted intermolecular charge-transfer formation coordinate and the mechanism of energy dissipation in these complexes was investigated (Section 3.4-3.5) (Ref. D3D). A brief summary of the main results of this chapter and conclusions are given in Section 3.6. Finally, in Section 3.7 a complementary benchmark study of the quality of ground-state potential energy surfaces of prototypical hydrogen-bonded systems (ammonia-water and formic acid-water dimers) obtained at the level of BSSE-corrected MP2 combined with moderate basis sets, has been conducted. The quality of potential energy surfaces was tested with respect to basis-set size, level of electron correlation and anharmonicity effects and the applied methodology to identify the IR spectrum of hydrated DMABME complexes (Section 3.3) has been found to be sufficient to uniquely assign the IR spectra.
Das Gehirn ist die wohl komplexeste Struktur auf Erden, die der Mensch erforscht. Es besteht aus einem riesigen Netzwerk von Nervenzellen, welches in der Lage ist eingehende sensorische Informationen zu verarbeiten um daraus eine sinnvolle Repräsentation der Umgebung zu erstellen. Außerdem koordiniert es die Aktionen des Organismus um mit der Umgebung zu interagieren. Das Gehirn hat die bemerkenswerte Fähigkeit sowohl Informationen zu speichern als auch sich ständig an ändernde Bedingungen anzupassen, und zwar über die gesamte Lebensdauer. Dies ist essentiell für Mensch oder Tier um sich zu entwickeln und zu lernen. Die Grundlage für diesen lebenslangen Lernprozess ist die Plastizität des Gehirns, welche das riesige Netzwerk von Neuronen ständig anpasst und neu verbindet. Die Veränderungen an den synaptischen Verbindungen und der intrinsischen Erregbarkeit jedes Neurons finden durch selbstorganisierte Mechanismen statt und optimieren das Verhalten des Organismus als Ganzes. Das Phänomen der neuronalen Plastizität beschäftigt die Neurowissenschaften und anderen Disziplinen bereits über mehrere Jahrzehnte. Dabei beschreibt die intrinsische Plastizität die ständige Anpassung der Erregbarkeit eines Neurons um einen ausbalancierten, homöostatischen Arbeitsbereich zu gewährleisten. Aber besonders die synaptische Plastizität, welche die Änderungen in der Stärke bestehender Verbindungen bezeichnet, wurde unter vielen verschiedenen Bedingungen erforscht und erwies sich mit jeder neuen Studie als immer komplexer. Sie wird durch ein komplexes Zusammenspiel von biophysikalischen Mechanismen induziert und hängt von verschiedenen Faktoren wie der Frequenz der Aktionspotentiale, deren Timing und dem Membranpotential ab und zeigt außerdem eine metaplastische Abhängigkeit von vergangenen Ereignissen. Letztlich beeinflusst die synaptische Plastizität die Signalverarbeitung und Berechnung einzelner Neuronen und der neuronalen Netzwerke.
Der Schwerpunkt dieser Arbeit ist es das Verständnis der biologischen Mechanismen und deren Folgen, die zu den beobachteten Plastizitätsphänomene führen, durch eine stärker vereinheitlichte Theorie voranzutreiben.Dazu stelle ich zwei funktionale Ziele für neuronale Plastizität auf, leite Lernregeln aus diesen ab und analysiere deren Konsequenzen und Vorhersagen.
Kapitel 3 untersucht die Unterscheidbarkeit der Populationsaktivität in Netzwerken als funktionales Ziel für neuronale Plastizität. Die Hypothese ist dabei, dass gerade in rekurrenten aber auch in vorwärtsgekoppelten Netzwerken die Populationsaktivität als Repräsentation der Eingangssignale optimiert werden kann, wenn ähnliche Eingangssignale eine möglichst unterschiedliche Repräsentation haben und dadurch für die nachfolgende Verarbeitung besser unterscheidbar sind. Das funktionale Ziel ist daher diese Unterscheidbarkeit durch Veränderungen an den Verbindungsstärke und der Erregbarkeit der Neuronen mithilfe von lokalen selbst-organisierten Lernregeln zu maximieren. Aus diesem funktionale Ziel lassen sich eine Reihe von Standard-Lernenregeln für künstliche neuronale Netze gemeinsam abzuleiten.
Kapitel 4 wendet einen ähnlichen funktionalen Ansatz auf ein komplexeres, biophysikalisches Neuronenmodell an. Das Ziel ist eine spärliche, stark asymmetrische Verteilung der synaptischen Stärke, wie sie auch bereits mehrfach experimentell gefunden wurde, durch lokale, synaptische Lernregeln zu maximieren. Aus diesem funktionalen Ansatz können alle wichtigen Phänomene der synaptischen Plastizität erklärt werden. Simulationen der Lernregel in einem realistischen Neuronmodell mit voller Morphologie erklären die Daten von timing-, raten- und spannungsabhängigen Plastizitätsprotokollen. Die Lernregel hat auch eine intrinsische Abhängigkeit von der Position der Synapse, welche mit den experimentellen Ergebnissen übereinstimmt. Darüber hinaus kann die Lernregel ohne zusätzliche Annahmen metaplastische Phänomene erklären. Dabei sagt der Ansatz eine neue Form der Metaplastizität voraus, welche die timing-abhängige Plastizität beeinflusst. Die formulierte Lernregel führt zu zwei neuartigen Vereinheitlichungen für synaptische Plastizität: Erstens zeigt sie, dass die verschiedenen Phänomene der synaptischen Plastizität als Folge eines einzigen funktionalen Ziels verstanden werden können. Und zweitens überbrückt der Ansatz die Lücke zwischen der funktionalen und mechanistische Beschreibungsweise. Das vorgeschlagene funktionale Ziel führt zu einer Lernregel mit biophysikalischer Formulierung, welche mit etablierten Theorien der biologischen Mechanismen in Verbindung gebracht werden kann. Außerdem kann das Ziel einer spärlichen Verteilung der synaptischen Stärke als Beitrag zu einer energieeffizienten synaptischen Signalübertragung und optimierten Codierung interpretiert werden.
Diese Dissertation stellt die systematische Einbeziehung von Eichkorrekturen in die Theorie der thermischen Leptogenese vor, welche eine Erklärung für die Frage nach dem Ursprung der Materie in unserem Universum bereitstellt.
Geht man vom weithin anerkannten Urknallmodell aus, so müsste hierbei zu gleichen Teilen Materie sowie Antimaterie entstanden sein. Aufgrund von Annihilationsprozessen sollte demnach die gesamte Materie zerstrahlt sein und ein leeres Universum zurückbleiben. Da dies aber nicht der Fall ist, stellt sich die Frage, wie das Ungleichgewicht zwischen Materie und Antimaterie entstehen konnte. Der Wert der Asymmetrie lässt sich mit Hilfe von Experimenten sehr genau bestimmen. Für eine systematische theoretische Beschreibung dieser Problematik stellte A. Sacharow drei Bedingungen auf: 1. die Verletzung der Baryonenzahl, 2. die Verletzung der Invarianz von Ladungskonjugation C sowie der Zusammensetzung von Ladungskonjugation und Parität CP sowie 3. eine Abweichung vom thermischen Gleichgewicht.
Da das Urknallmodell und das Standardmodell der Teilchenphysik nicht in der Lage sind, diese Asymmetrie zu beschreiben, beschäftigt sich die vorliegende Dissertation mit der Theorie der thermischen Leptogenese, welche statt von einer ursprünglichen Baryonenasymmetrie von einer Leptonenasymmetrie ausgeht. Zu einem späteren Zeitpunkt wird diese dann mittels Sphaleron-Prozesse, welche die Baryonenzahl verletzen, in eine Baryonenasymmetrie übertragen. Hierzu werden neue Teilchen zum Standardmodell hinzugefügt: schwere Majorana-Neutrinos. Diese zerfallen im thermischen Nichtgleichgewicht CP-verletzend in die bekannten Standardmodell-Leptonen und Higgs-Teilchen.
In dieser Arbeit wird eine hierarchische Anordnung der drei schweren Neutrinomassen betrachtet. Dies hat zur Folge, dass zwei der drei Majorana-Neutrinos ausintegriert werden können und eine effektive Theorie aufgestellt werden kann. Dieses Modell wird auch vanilla leptogenesis genannt und im Folgenden verwendet.
Die Dissertation ist wie folgt gegliedert. Die einleitenden Betrachtungen sind Gegenstand der Kapitel 1 und 2. Dort werden weiterhin andere Modelle zur Lösung des Problems der Baryonenasymmetrie kurz vorgestellt. Die thermische Leptogenese wird eingeführt und der See-saw-Mechanismus sowie die CP-Asymmetrie genauer beschrieben. Am Ende des Kapitels wird der klassische Ansatz für Leptogenese über Boltzmann Gleichungen präsentiert.
In Kapitel 3 werden die Grundlagen für Quantenfeldtheorien im Nichtgleichgewicht eingeführt. Die wichtigsten Definitionen im Falle des thermischen Gleichgewichts werden gegeben, anschließend findet sich die Verallgemeinerung auf Nichtgleichgewichtszustände. Die Bewegungsgleichungen, die sogenannten Kadanoff-Baym-Gleichungen, werden im Folgenden sowohl für skalare Teilchen als auch für Fermionen gelöst.
Kapitel 4 stellt die Notwendigkeit der Einbeziehung von Eichkorrekturen im Kontext der thermischen Leptogenese vor. Durch die Definition einer Leptonenzahlmatrix lässt sich die Asymmetrie durch die Kadanoff-Baym Gleichung für Leptonen umschreiben. Da der Vergleich von Boltzmann und Kadanoff-Baym Gleichungen im letzten Teil dieses Kapitels Unterschiede im Zeitverhalten zeigt, werden im Kadanoff-Baym Ansatz thermische Standardmodell-Breiten des Higgsfeldes und der Leptonen per Hand eingeführt. Mit dieser naiven Erweiterung erhält man ein gleiches Verhalten für die Leptonenzahlmatrix, lokal in der Zeit wie die Lösung der Boltzmann Gleichung. Eine systematische Einführung von Standardmodellkorrekturen für thermische Leptogenese ist daher unumgänglich, weshalb im Rahmen der vorliegenden Dissertation von Grund auf Eichkorrekturen der Diagramme, die zur Asymmetrie führen, berücksichtigt werden.
Die vier für diese Arbeit wichtigen Skalenbereich bedingen zwei Resummationsschemata, Hard Thermal Loop (HTL) und Collinear Thermal Loop (CTL), welche in Kapitel 5 vorgestellt werden. Dies führt schließlich auf zwei Differenzialgleichungen für die Berechnung der thermischen Produktionsrate des Majorana-Neutrinos, welche in Kapitel 6 numerisch weiter ausgewertet werden.
In Kapitel 7 erfolgt zunächst eine naive Berechnung aller eichkorrigierter 3-Schleifen-Diagramme, die zu den beiden die Asymmetrie verursachenden Diagrammen gehören. Da eine einfache Berechnung der 3-Schleifen-Diagramme nicht ausreicht, wird an dieser Stelle ein neues, zylindrisches Diagramm eingeführt, welches alle wichtigen Beiträge, insbesondere die HTL- und CTL-resummierten, enthält. Am Ende des Kapitels findet sich der erste geschlossene Ausdruck für die eichkorrigierte Leptonenzahlmatrix in führender Ordnung in allen Kopplungen.
Abschließend gibt es eine kurze Zusammenfassung und einen Ausblick in Kapitel 8. In dieser Dissertation findet sich zum ersten Mal ein systematischer Zugang zur Berücksichtigung aller Eichwechselwirkungen in der Theorie der thermischen Leptogenese. Ein geschlossener Ausdruck für die eichkorrigierte Leptonenasymmetrie konnte vorgestellt werden.
Spin(9)-invariant valuations
(2013)
The first aim of this thesis is to give a Hadwiger-type theorem for the exceptional Lie group Spin(9). The space of Spin(9)-invariant k-homogeneous valuations is studied through the construction of an exact sequence involving some spaces of differential forms. We present then a description of the spin representation using the properties of the 8-dimensional division algebra of the octonions. Using this description as well as representation-theoretic formulas, we can compute the dimensions of the spaces of differential forms appearing in the exact sequence. Hence we obtain the dimensions of the spaces of k-homogeneous Spin(9)-invariant valuations for k=0,1,...,16.
In the second part of this work, we construct one new element for a basis of one of these spaces. It is clear, that the k-th intrinsic volume is also Spin(9)-invariant. The last chapter of this work presents the construction of a new 2-homogeneous Spin(9)-invariant valuation. On a Riemannian manifold (M,g), we construct a valuation by integrating the curvature tensor over the disc bundle. We associate to this valuation on M a family of valuations on the tangent spaces. We show that these valuations are even and homogeneous of degree 2. Moreover, since the valuation on M is invariant under the action of the isometry group of M, the induced valuation on the tangent space in a point p in M is invariant under the action of the stabilisator of p for all p in M. In the special case where M is the octonionic projective plane, this construction yields an even, homogeneous of degree 2, Spin(9)-invariant valuation, whose Klain function is not constant, i.e. which is linearly independent of the second intrinsic volume.
Alzheimer’s disease (AD), which was first reported more than a century ago by Alhzeimer, is one of the commonest forms of dementia which affects >30 million people globally (>8 million in Europe). The origin and pathogenesis of AD is poorly understood and there is no cure available for the disease. AD is characterized by the accumulation of senile plaques composed of amyloid beta peptides (Ab 37-43) which is formed by the gamma secretase (GS) complex by cleaving amyloid precursor protein. Therefore GS can be an attractive drug target. Since GS processes several other substrates like Notch, CD44 and Cadherins, nonspecific inhibition of GS has many side effects. Due to the lack of crystal structure of GS, which is attributed to the extreme difficulties in purifying it, molecular modeling can be useful to understand its architecture. So far only low resolution cryoEM structures of the complex has been solved which only provides a rough structure of the complex at low 12-15 A resolution Furthermore the activity of GS in vitro can be achieved by means of cell-free (CF) expression.
GS comprises catalytic subunits namely presenilins and supporting elements containing Pen-2, Aph-1 and Nicastrin. The origin of AD is hidden in the regulated intramembrnae proteolysis (RIP) which is involved in various physiological processes and also in leukemia. So far growth factors, cytokines, receptors, viral proteins, cell adhesion proteins, signal peptides and GS has been shown to undergo RIP. During RIP, the target proteins undergo extracellular shredding and intramembrane proteolysis.
This thesis is based on molecular modeling, molecular dynamics (MD) simulations, cell-free (CF) expression, mass spectrometry, NMR, crystallization, activity assay etc of the components of GS complex and G-protein coupled receptors (GPCRs).
First I validated the NMR structure of PS1 CTF in detergent micelles and lipid bilayers using coarse-grained MD simulations using MARTINI forcefield implemented in Gromacs. CTF was simulated in DPC micelles, DPPC and DLPC lipid bilayer. Starting from random configuration of detergent and lipids, micelle and lipid bilyer were formed respectively in presence of CTF and it was oriented properly to the micelle and bilyer during the simulation. Around DPC molecules formed micelle around CTF in agreement of the experimental results in which 80-85 DPC molecules are required to form micelles. The structure obtained in DPC was similar to that of NMR structure but differed in bilayer simulations showed the possibility of substrate docking in the conserved PAL motif. Simulations of CTF in implicit membrane (IMM1) in CHAMM yielded similar structure to that from coarse grained MD.
I performed cell-free expression optimization, crystallization and NMR spectroscopy of Pen-2 in various detergent micelles. Additionally Pen-2 was modeled by a combination of rosetta membrane ab-initio method, HHPred distant homology modeling and incorporating NMR constraints. The models were validated by all atom and coarse grained MD simulations both in detergent micelles and POPC/DPPC lipid bilayers using MARTINI forcefield.
GS operon consisting of all four subunits was co-expressed in CF and purified. The presence of of GS subunits after pull-down with Aph-1 was determined by western blotting (Pen-2) and mass spectrometry (Presenilin-1 and Aph-1). I also studied interactions of especially PS1 CTF, APP and NTF by docking and MD.
I also made models and interfaces of Pen-2 with PS1 NTF and checked their stability by MD simulations and compared with experimental results. The goal is to model the interfaces between GS subunits using molecular modeling approaches based on available experimental data like cross-linking, mutations and NMR structure of C-terminal fragment of PS1 and transmembrane part of APP. The obtained interfaces of GS subunits may explain its catalysis mechanism which can be exploited for novel lead design. Due to lack of crystal/NMR structure of the GS subunits except the PS1 CTF, it is not possible to predict the effect of mutations in terms of APP cleavage. So I also developed a sequence based approach based on machine learning using support vector machine to predict the effect of PS1 CTF L383 mutations in terms of Aβ40/Aβ42 ratio with 88% accuracy. Mutational data derived from the Molgen database of Presenilin 1 mutations was using for training.
GPCRs (also called 7TM receptors) form a large superfamily of membrane proteins, which can be activated by small molecules, lipids, hormones, peptides, light, pain, taste and smell etc. Although 50% of the drugs in market target GPCRs , only few are targeted therapeutically. Such wide range of targets is due to involvement of GPCRs in signaling pathways related to many diseases i.e. dementia (like Alzheimer's disease), metabolic (like diabetes) including endocrinological disorders, immunological including viral infections, cardiovascular, inflammatory, senses disorders, pain and cancer.
Cannabinoid and adrenergic receptors belong to the class A (similar to rhodopsin) GPCRs. Docking of agonists and antagonists to CB1 and CB2 cannabinoid receptors revealed the importance of a centrally located rotamer toggle switch, and its possible role in the mechanism of agonist/antagonist recognition. The switch is composed of two residues, F3.36 and W6.48, located on opposite transmembrane helices TM3 and TM6 in the central part of the membranous domain of cannabinoid receptors. The CB1 and CB2 receptor models were constructed based on the adenosine A2A receptor template. The two best scored conformations of each receptor were used for the docking procedure. In all poses (ligand-receptor conformations) characterized by the lowest ligand-receptor intermolecular energy and free energy of binding the ligand type matched the state of the rotamer toggle switch: antagonists maintained an inactive state of the switch, whereas agonists changed it. In case of agonists of β2AR, the (R,R) and (S,S) stereoisomers of fenoterol, the molecular dynamics simulations provided evidence of different binding modes while preserving the same average position of ligands in the binding site. The (S,S) isomer was much more labile in the binding site and only one stable hydrogen bond was created. Such dynamical binding modes may also be valid for ligands of cannabinoid receptors because of the hydrophobic nature of their ligand-receptor interactions. However, only very long molecular dynamics simulations could verify the validity of such binding modes and how they affect the process of activation.
Human N-formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) involved in many physiological processes, including host defense against bacterial infection and resolving inflammation. The three human FPRs (FPR1, FPR2 and FPR3) share significant sequence homology and perform their action via coupling to Gi protein. Activation of FPRs induces a variety of responses, which are dependent on the agonist, cell type, receptor subtype, and also species involved. FPRs are expressed mainly by phagocytic leukocytes. Together, these receptors bind a large number of structurally diverse groups of agonistic ligands, including N-formyl and nonformyl peptides of different composition, that chemoattract and activate phagocytes. For example, N-formyl-Met-Leu-Phe (fMLF), an FPR1 agonist, activates human phagocyte inflammatory responses, such as intracellular calcium mobilization, production of cytokines, generation of reactive oxygen species, and chemotaxis. This ligand can efficiently activate the major bactericidal neutrophil functions and it was one of the first characterized bacterial chemotactic peptides. Whereas fMLF is by far the most frequently used chemotactic peptide in studies of neutrophil functions, atomistic descriptions for fMLF-FPR1 binding mode are still scarce mainly because of the absence of a crystal structure of this receptor. Elucidating the binding modes may contribute to designing novel and more efficient non-peptide FPR1 drug candidates. Molecular modeling of FPR1, on the other hand, can provide an efficient way to reveal details of ligand binding and activation of the receptor. However, recent modelings of FPRs were confined only to bovine rhodopsin as a template.
To locate specific ligand-receptor interactions based on a more appropriate template than rhodopsin we generated the homology models of FPR1 using the crystal structure of the chemokine receptor CXCR4, which shares over 30% sequence identity with FPR1 and is located in the same γ branch of phylogenetic tree of GPCRs (rhodopsin is located in α branch). Docking and model refinement procedures were pursued afterward. Finally, 40 ns full-atom MD simulations were conducted for the Apo form as well as for complexes of fMLF (agonist) and tBocMLF (antagonist) with FPR1 in the membrane. Based on locations of the N- and C-termini of the ligand the FPR1 extracellular pocket can be divided into two zones, namely, the anchor and activation regions. The formylated M1 residue of fMLF bound to the activation region led to a series of conformational changes of conserved residues. Internal water molecules participating in extended hydrogen bond networks were found to play a crucial role in transmitting the agonist-receptor interactions. A mechanism of initial steps of the activation concurrent with ligand binding is proposed.
I accurately predicted the structure and ligand binding pose of dopamine receptor 3 (RMSD to the crystal structure: 2.13 Å) and chemokine receptor 4 (CXCR4, RMSD to the crystal structure 3.21 Å) in GPCR-Dock 2010 competition. The homology model of the dopamine receptor 3 was 8 th best overall in the competition.
A stochastic model for the joint evaluation of burstiness and regularity in oscillatory spike trains
(2013)
The thesis provides a stochastic model to quantify and classify neuronal firing patterns of oscillatory spike trains. A spike train is a finite sequence of time points at which a neuron has an electric discharge (spike) which is recorded over a finite time interval. In this work, these spike times are analyzed regarding special firing patterns like the presence or absence of oscillatory activity and clusters (so called bursts). These bursts do not have a clear and unique definition in the literature. They are often fired in response to behaviorally relevant stimuli, e.g., an unexpected reward or a novel stimulus, but may also appear spontaneously. Oscillatory activity has been found to be related to complex information processing such as feature binding or figure ground segregation in the visual cortex. Thus, in the context of neurophysiology, it is important to quantify and classify these firing patterns and their change under certain experimental conditions like pharmacological treatment or genetical manipulation. In neuroscientific practice, the classification is often done by visual inspection criteria without giving reproducible results. Furthermore, descriptive methods are used for the quantification of spike trains without relating the extracted measures to properties of the underlying processes.
For that reason, a doubly stochastic point process model is proposed and termed 'Gaussian Locking to a free Oscillator' - GLO. The model has been developed on the basis of empirical observations in dopaminergic neurons and in cooperation with neurophysiologists. The GLO model uses as a first stage an unobservable oscillatory background rhythm which is represented by a stationary random walk whose increments are normally distributed. Two different model types are used to describe single spike firing or clusters of spikes. For both model types, the distribution of the random number of spikes per beat has different probability distributions (Bernoulli in the single spike case or Poisson in the cluster case). In the second stage, the random spike times are placed around their birth beat according to a normal distribution. These spike times represent the observed point process which has five easily interpretable parameters to describe the regularity and the burstiness of the firing patterns.
It turns out that the point process is stationary, simple and ergodic. It can be characterized as a cluster process and for the bursty firing mode as a Cox process. Furthermore, the distribution of the waiting times between spikes can be derived for some parameter combination. The conditional intensity function of the point process is derived which is also called autocorrelation function (ACF) in the neuroscience literature. This function arises by conditioning on a spike at time zero and measures the intensity of spikes x time units later. The autocorrelation histogram (ACH) is an estimate for the ACF. The parameters of the GLO are estimated by fitting the ACF to the ACH with a nonlinear least squares algorithm. This is a common procedure in neuroscientific practice and has the advantage that the GLO ACF can be computed for all parameter combinations and that its properties are closely related to the burstiness and regularity of the process. The precision of estimation is investigated for different scenarios using Monte-Carlo simulations and bootstrap methods.
The GLO provides the neuroscientist with objective and reproducible classification rules for the firing patterns on the basis of the model ACF. These rules are inspired by visual inspection criteria often used in neuroscientific practice and thus support and complement usual analysis of empirical spike trains. When applied to a sample data set, the model is able to detect significant changes in the regularity and burst behavior of the cells and provides confidence intervals for the parameter estimates.
In der nuklearen Astrophysik sind Experimente mit hochgeladenen Radionukliden von großer Bedeutung. Diese exotischen Nuklide können in Schwerionenbeschleunigeranlagen hergestellt und in Speicherringen gespeichert werden. Momentan existieren weltweit zwei Anlagen, die solche Experimente ermöglichen: das GSI Helmholtzzentrum für Schwerionenforschung GmbH in Darmstadt und das Institut für moderne Physik (IMP) in Lanzhou, China. Da die Ausbeute dieser Nuklide gering ist, werden zerstörungsfreie Nachweismethoden in den Speicherringen verwendet. Diese machen von den Methoden der Spektralanalyse Gebrauch. Nicht nur die geringe Ausbeute, sondern auch die kurze Lebensdauer dieser Nuklide stellen hohe Anforderungen an die Sensitivität und Geschwindigkeit dieser Detektoren.
Eine übliche Methode ist die Verwendung kapazitiver Schottky-Sonden. Eine solche Sonde ist seit 1991 an der GSI im Speicherring ESR im Einsatz. Um die Empfindlichkeit zu erhöhen, kann man Mikrowellenkavitäten als resonante Pickups verwenden. Die von den Teilchen induzierten elektromagnetischen Felder können resonante Moden im Resonator anregen. Die Geometrie des Pickups und das verwendete Material spielen eine wesentliche Rolle in der Gestaltung der Feldbilder. Die resultierenden Signale, auch Schottky Signale genannt, werden mittels einer Antenne ausgekoppelt und anschliessend an einen Spektrumanalysator angeschlossen. Für die Analyse der gespeicherten Daten können verschiedene Methoden der Spektralschätzung wie z.B. das Multi-Taper angewendet werden. Nachdem eine externe Kalibrierung durchgeführt worden ist, kann das Pickup auch als ein Stromsensor verwendet werden.
Diese Arbeit befasst sich mit der Theorie, dem Aufbau und ersten Anwendungen eines neuen resonanten Pickups, das im Jahr 2010 in den Speicherring ESR eingebaut und in mehreren Experimenten erfolgreich eingesetzt wurde. Ein ähnliches Pickup wurde im Jahr 2011 in den CSRe im IMP Lanzhou eingebaut. Einzelne Schwerionen mit 400 MeV pro Nukleon wurden erfolgreich mit dem GSI-Pickup nachgewiesen. Das Pickup wird regelmässig in Speicherringexperimenten eingesetzt. Ähnliche Experimente sind für CSRe in Lanzhou geplant.
We investigate multivariate Laurent polynomials f \in \C[\mathbf{z}^{\pm 1}] = \C[z_1^{\pm 1},\ldots,z_n^{\pm 1}] with varieties \mathcal{V}(f) restricted to the algebraic torus (\C^*)^n = (\C \setminus \{0\})^n. For such Laurent polynomials f one defines the amoeba \mathcal{A}(f) of f as the image of the variety \mathcal{V}(f) under the \Log-map \Log : (\C^*)^n \to \R^n, (z_1,\ldots,z_n) \mapsto (\log|z_1|, \ldots, \log|z_n|). I.e., the amoeba \mathcal{A}(f) is the projection of the variety \mathcal{V}(f) on its (componentwise logarithmized) absolute values. Amoebas were first defined in 1994 by Gelfand, Kapranov and Zelevinksy. Amoeba theory has been strongly developed since the beginning of the new century. It is related to various mathematical subjects, e.g., complex analysis or real algebraic curves. In particular, amoeba theory can be understood as a natural connection between algebraic and tropical geometry.
In this thesis we investigate the geometry, topology and methods for the approximation of amoebas.
Let \C^A denote the space of all Laurent polynomials with a given, finite support set A \subset \Z^n and coefficients in \C^*. It is well known that, in general, the existence of specific complement components of the amoebas \mathcal{A}(f) for f \in \C^A depends on the choice of coefficients of f. One prominent key problem is to provide bounds on the coefficients in order to guarantee the existence of certain complement components. A second key problem is the question whether the set U_\alpha^A \subseteq \C^A of all polynomials whose amoeba has a complement component of order \alpha \in \conv(A) \cap \Z^n is always connected.
We prove such (upper and lower) bounds for multivariate Laurent polynomials supported on a circuit. If the support set A \subset \Z^n satisfies some additional barycentric condition, we can even give an exact description of the particular sets U_\alpha^A and, especially, prove that they are path-connected.
For the univariate case of polynomials supported on a circuit, i.e., trinomials f = z^{s+t} + p z^t + q (with p,q \in \C^*), we show that a couple of classical questions from the late 19th / early 20th century regarding the connection between the coefficients and the roots of trinomials can be traced back to questions in amoeba theory. This yields nice geometrical and topological counterparts for classical algebraic results. We show for example that a trinomial has a root of a certain, given modulus if and only if the coefficient p is located on a particular hypotrochoid curve. Furthermore, there exist two roots with the same modulus if and only if the coefficient p is located on a particular 1-fan. This local description of the configuration space \C^A yields in particular that all sets U_\alpha^A for \alpha \in \{0,1,\ldots,s+t\} \setminus \{t\} are connected but not simply connected.
We show that for a given lattice polytope P the set of all configuration spaces \C^A of amoebas with \conv(A) = P is a boolean lattice with respect to some order relation \sqsubseteq induced by the set theoretic order relation \subseteq. This boolean lattice turns out to have some nice structural properties and gives in particular an independent motivation for Passare's and Rullgard's conjecture about solidness of amoebas of maximally sparse polynomials. We prove this conjecture for special instances of support sets.
A further key problem in the theory of amoebas is the description of their boundaries. Obviously, every boundary point \mathbf{w} \in \partial \mathcal{A}(f) is the image of a critical point under the \Log-map (where \mathcal{V}(f) is supposed to be non-singular here). Mikhalkin showed that this is equivalent to the fact that there exists a point in the intersection of the variety \mathcal{V}(f) and the fiber \F_{\mathbf{w}} of \mathbf{w} (w.r.t. the \Log-map), which has a (projective) real image under the logarithmic Gauss map. We strengthen this result by showing that a point \mathbf{w} may only be contained in the boundary of \mathcal{A}(f), if every point in the intersection of \mathcal{V}(f) and \F_{\mathbf{w}} has a (projective) real image under the logarithmic Gauss map.
With respect to the approximation of amoebas one is in particular interested in deciding membership, i.e., whether a given point \mathbf{w} \in \R^n is contained in a given amoeba \mathcal{A}(f). We show that this problem can be traced back to a semidefinite optimization problem (SDP), basically via usage of the Real Nullstellensatz. This SDP can be implemented and solved with standard software (we use SOSTools and SeDuMi here). As main theoretic result we show that, from the complexity point of view, our approach is at least as good as Purbhoo's approximation process (which is state of the art).
Ribosome biogenesis is best understood in the yeast Saccharomyces cerevisiae. In human or mammalian ribosome biogenesis, it has been shown that basic principles are conserved to yeast, but additional features have been reported. Our understanding about the interplay between proteins and RNA in human ribosome biogenesis is far from complete.
The present study focused on the analysis of the human ribosome biogenesis co-factors PWP2, EMG1 and Exportin 5 (XPO5) to understand the degree of conservation of ribosome biogenesis. The proteins were characterized in respect to their localization and interaction partners. For the early 90S co-factor, PWP2, it was possible to pull down and identify the human UTP-B complex with MALDI mass spectrometry. Besides the orthologues of the members of this complex known in yeast (TBL3, WDR3, WDR36, UTP6, UTP18), the human UTP-B complex is not only conserved from yeast to humans, but contains also additional components, like the DEAD-box RNA helicase DDX21, which lacks a yeast orthologue. DDX21 was localized to the nucleus, assembled to the native UTP-B complex and co-precipitated also with other UTP-B complex members, presumably extending the functions of this complex in ribosome biogenesis.
This phenomenon was also observed for the 90S co-factor EMG1, an RNA methyltransferase, whose mutant form causes the Bowen-Conradi syndrome, if aspartic acid is mutated to glycine at position 86. This study revealed that the mutant, EMG1-D86G, clearly lost its nucleolar localization and co-precipitated to histones for unknown reasons.
A participation of the nuclear export receptor XPO5 in human ribosome biogenesis was shown in this study. Pulldown analysis, sucrose density gradients and UV crosslinking and analysis of cDNAs of XPO5 revealed the involvement of XPO5 in pre-60S subunit maturation. Moreover, besides the known pre-miRNAs and tRNAs as substrates for nuclear export, XPO5 crosslinked to snoRNAs. XPO5 was further demonstrated to interact with the miRNA Let-7a, which has an important regulatory function for MYC, a transcription factor required for ribosome biogenesis.
All results support a role of these proteins in human ribosome biogenesis and therefore it seems that the biogenesis of ribosomes in human cells requires additional components, like DDX21 and XPO5.