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Membrane proteins (MPs) constitute about 30% of the genome and are essential in many cellular processes. In particular structural characterisation of MPs is challenged by their hydrophobic nature resulting in expression difficulties and structural instability upon extraction from the membrane. Despite these challenges, progress in sample preparation and the techniques to solve MP structures has led to 281 unique MP structures as of January 2011. Through the combination of a cell-free expression system and selective labelling strategies, this thesis aimed to advance the structure determination of α-helical MPs by NMR spectroscopy and resulted in the structure determination of a seven-ransmembrane-helix protein. Results were obtained for the 5-lipoxygenase-activating protein (FLAP) and proteorhodopsin (PR). The detergent-based cell-free expression mode proved most efficient for production of both targets, but optimisation of FLAP and PR followed different routes. The presence of a retinal cofactor in PR greatly facilitated the search for an appropriate hydrophobic environment. For structural studies, NMR spectra of FLAP indicated favourable properties of the lysolipid LPPG. In contrast, PR was stable and homogenous in the short-chain lipid diC7PC. As NMR spectra of α-helical MPs are generally characterised by broad lines and signal overlap, selective labelling strategies were essential in the assignment process of both targets. For the backbone assignment of FLAP the transmembrane segment-enhanced (TMS) labelling was developed, employing the six amino acids AFGILV. These residues cluster predominantly in transmembrane helices and form long stretches allowing a large extent of backbone assignment. Besides that, the combinatorial labelling enables identification of unique pairs in the sequence based on a mixture of 15N and 1-13C-labelled amino acids. To find the optimal labelling pattern for a given primary structure, the UPLABEL algorithm has been made available and successfully applied in the backbone assignment of PR. Both selective labelling approaches greatly benefitted from the use of a cell-free expression system to reduce isotope scrambling. Additionally, the de novo structure of PR was determined with an average backbone rmsd of 1.2 Å based on TALOS-derived backbone torsion angles, intrahelical hydrogen bond restraints and distance restraints from the NOE and paramagnetic relaxation enhancement (PRE). A major bottleneck in the NMR structure determination of MPs concerns the number of long-range distances which are often limited. In PR, side chain assignment was enabled by stereo-array isotope labelling as well as selective labelling which provided 33 long-range NOEs. These NOEs stabilised the symmetry of the seven helix bundle. With a total number of 1031, the majority of long-range distances were derived from PREs. The structure of PR reveals differences to its homologues such as the absence of an anti-parallel β-sheet between helices B and C and allows conclusions towards the mechanism of colour tuning.
Numorous psychotherapeutic approaches have been put forward to better treat patients with chronic psychological problems or severe personality disorders, among them Schema Therapy (Young, Klosko & Weishaar, 2003). Within Schema Therapy, there are two important theoretical constructs: early maladaptive schemas, which are conceptualized as stable and lasting emotional, behavioral and cognitive patterns regarding ourselves, the world and other people that have their origins in childhood or adolescence and schema modes, which are current states that describe the rapid shifts in emotion, cognition and behaviour that can be seen in patients with severe personality disorders. Schema Therapy has growing popularity with clinicians in the last decade, but only a few studies have tested the empirical validity of the schema mode model. Lobbestael, van Vreeswijk, Spinhoven, Schouten & Arntz (2010) developed and evaluated the Schema Mode Inventory to assess schema modes within the framework of a questionnaire. This instrument has been translated to the German language and the psychometric properties of the instrument indicate an adequate fit to the model established in the original Dutch study (paper 1).
In Schema Therapy, on the one hand mode concepts have been proposed without a focus on one specific disorder (Young et al., 2003), but on the other hand disorder-specific concepts have been established that form the basis for manualization and research. The most popular disorder specific mode concept is that of Borderline Personality Disorder (Arntz & van Genderen, 2009). In randomized controlled trials the effectiveness of Schema Therapy for Borderline Personality Disorder in the individual setting (Giesen-Bloo, van Dyck, Spinhoven, van Tilburg, Dirksen et al., 2006) and the group setting (Farrell, Shaw & Webber, 2009) was evaluated. Whereas Schema Therapy for Borderline Personality Disorder in the individual setting has been manualized (Arntz & van Genderen, 2009), a manual for group Schema Therapy is not yet available, Therefore, particularly since this last evaluation study, there has been a need to better describe, how the Schema Therapy mode model for Borderline Personality Disorder has been applied to the group setting (papers 2 & 3) as well as how both modalities of Schema Therapy treatment, individual and group, are combined in intensive psychotherapy settings such as inpatient or day care (papers 4 & 5). Lastly the question has been put forward as to how effective Schema Therapy for Borderline Personality Disorder is in intensive treatment environments, where it is assumed that the severity of the disorders treated is even higher. It is found that a combined program of individual and group Schema Therapy for Borderline Personality Disorder is effective in the inpatient setting. Subtle variations in treatment programs in three pilot studies leave room for the discussion of possible factors influencing treatment effects (paper 6). The results of all studies are discussed on the background of Schema Therapy’s development as a new psychotherapy approach for the treatment of complex disorders.
In recent years using symmetry has proven to be a very useful tool to simplify computations in semidefinite programming. This dissertation examines the possibilities of exploiting discrete symmetries in three contexts: In SDP-based relaxations for polynomial optimization, in testing positivity of symmetric polynomials, and combinatorial optimization. In these contexts the thesis provides new ways for exploiting symmetries and thus deeper insight in the paradigms behind the techniques and studies a concrete combinatorial optimization question.
The miniaturization of electronics is reaching its limits. Structures necessary to build integrated circuits from semiconductors are shrinking and could reach the size of only a few atoms within the next few years. It will be at the latest at this point in time that the physics of nanostructures gains importance in our every day life. This thesis deals with the physics of quantum impurity models. All models of this class exhibit an identical structure: the simple and small impurity only has few degrees of freedom. It can be built out of a small number of atoms or a single molecule, for example. In the simplest case it can be described by a single spin degree of freedom, in many quantum impurity models, it can be treated exactly. The complexity of the description arises from its coupling to a large number of fermionic or bosonic degrees of freedom (large meaning that we have to deal with particle numbers of the order of 10^{23}). An exact treatment thus remains impossible. At the same time, physical effects which arise in quantum impurity systems often cannot be described within a perturbative theory, since multiple energy scales may play an important role. One example for such an effect is the Kondo effect, where the free magnetic moment of the impurity is screened by a "cloud" of fermionic particles of the quantum bath.
The Kondo effect is only one example for the rich physics stemming from correlation effects in many body systems. Quantum impurity models, and the oftentimes related Kondo effect, have regained the attention of experimental and theoretical physicists since the advent of quantum dots, which are sometimes also referred to as as artificial atoms. Quantum dots offer a unprecedented control and tunability of many system parameters. Hence, they constitute a nice "playground" for fundamental research, while being promising candidates for building blocks of future technological devices as well.
Recently Loss' and DiVincenzo's p roposal of a quantum computing scheme based on spins in quantum dots, increased the efforts of experimentalists to coherently manipulate and read out the spins of quantum dots one by one. In this context two topics are of paramount importance for future quantum information processing: since decoherence times have to be large enough to allow for good error correction schemes, understanding the loss of phase coherence in quantum impurity systems is a prerequisite for quantum computation in these systems. Nonequilibrium phenomena in quantum impurity systems also have to be understood, before one may gain control of manipulating quantum bits.
As a first step towards more complicated nonequilibrium situations, the reaction of a system to a quantum quench, i.e. a sudden change of external fields or other parameters of the system can be investigated. We give an introduction to a powerful numerical method used in this field of research, the numerical renormalization group method, and apply this method and its recent enhancements to various quantum impurity systems.
The main part of this thesis may be structured in the following way:
- Ferromagnetic Kondo Model,
- Spin-Dynamics in the Anisotropic Kondo and the Spin-Boson Model,
- Two Ising-coupled Spins in a Bosonic Bath,
- Decoherence in an Aharanov-Bohm Interferometer.
This work deals with the use of dielectrics with high permeability, so-called high-k dielectrics in organic thin-film field-effect transistors (FETs). The central part was the preparation of the high-k dielectric and its implementation in transistors, in which organic semiconductors were used as active layer. A field-effect transistor can be used to measure the charge carrier mobility. Employing high-k dielectrics the carrier concentration in the active layer can be greatly increased. In this way, high charge carrier concentrations in organic layers can be achieved without chemical doping. As high-k dielectric strontium titanate (STO) was selected. It is also available as a niobium-doped and therefore conducting substrate material. Thus, one has an ideal substrate for the growth of the dielectric layer in conjunction with a substrate which acts as gate electrode. As the organic semiconductor the small molecules pentacene and copper phthalocyanine (CuPc) were sublimated, as electrical contacts gold was used. As a key part of this work an ultra high vacuum chamber system was constructed for in situ preparation of field effect transistors. For the deposition of the organic thin films a molecular beam deposition chamber was built, including a manipulator and effusion cells as evaporation sources. For the preparation of the dielectric a sputtering chamber was set-up. Another chamber was used in conjunction with an effusion cell for the deposition of the gold contacts. For the structured deposition of the different layers in the devices a shadow mask system was implemented. Movable masks could be positioned by means of a wobble stick onto the sample carriers. The system thus allowed for the use of masks in all chambers. The different thin films required in the transistor structure were first individually prepared and characterized. For the characterization primarily X-ray diffraction and optical microscopy were used. The growth of pentacene was analyzed on aplha-AlO substrates. With X-ray diffraction the (00l) reflections of the thin film phase were observed. In growth studies of CuPc aplha-AlO and STO substrates were used. With X-ray diffraction the aplha-phase was detected. With increasing substrate temperature an increase in crystallinity, but also an increase in surface roughness was observed. The sputtering of STO as a high-k dielectric was studied and optimized. Simultaneously, a high deposition rate, a smooth film surface and good crystallinity of the layer were required. As the most important parameters the substrate temperature, pressure and sputtering power were identified. Argon and oxygen were employed as sputtering gases, as substrate MgO was used. The films showed in comparison to crystalline STO a distortion to larger lattice constants. The degree of distortion decreased with increasing chamber pressure, on the other hand, deposition rate decreased with increasing chamber pressure as well. By combining the individual deposition processes FETs in bottom-gate geometry were prepared. The first step was always sputtering of the STO dielectric on niobium-doped STO substrates. Subsequently, the electrodes and the organic layer were deposited. For comparison transistors on silicon substrates with silicon dioxide (SiO2) as the dielectric were prepared. To study the transistor properties a measurement setup was build. A dielectric constant of about 190 for the STO in the transistors was achieved. The transistors with CuPc as active layer showed p-type conduction behavior. The transistors with STO as dielectric had a much stronger response than those with SiO2. They reached mobilities of 2E-4 cm2/Vs at very low applied voltages of 3V. It could thus be demonstrated that STO is suitable as a dielectric for organic FETs, and that through the use of high-k dielectrics high charge carrier densities can be achieved.
Ubiquitin is a highly conserved protein involved in several cellular processes like protein degradation, endocytosis, signal transduction and DNA repair. The discovery of ubiquitin-like proteins (UBL) and ubiquitin-like domains (ULD) increases the number of regulation pathways where the property of the ubiquitin-fold is profitable.
Autophagy is the catabolic pathway used in cells to deliver cytosolic components and dysfunctional organelles to the lysosome for degradation. MAP1LC3 proteins are ubiquitin-like proteins involved in one hand for the expansion of the autophagosome, which sequesters cytosolic substrates. In the other hand, these proteins (LC3- and GABARAP- subfamilies) bind to autophagic receptors linked to polyubiquitinated proteins aggregates. For this project, the 3D structure of the GABARAPL-1/NBR1-LIR complex was determined and confirmed that GABARAPL-1 belongs to the MAP1LC3 proteins family, structurally characterized by an ubiquitin-fold, consisting of a central beta-sheet formed by four beta-strands and two alpha-helices on one side of the beta-sheet, preceded N terminally by two alpha-helices, resulting in the formation of two hydrophobic pockets, hp1 and hp2. The autophagic receptor NBR1 interacts with GABARAPL-1 through the hp1 and hp2 with its LIR motif taking an extended beta conformation upon binding, forming an intermolecular beta-sheet with the second beta-strand of GABARAPL 1. This LC3- interacting region (LIR) consists of an Theta XX Gamma sequence preceded by acidic amino acids, with Theta and Gamma represented by any aromatic and hydrophobic residues, respectively. Interaction studies of the LIR domains of p62, Nix and NBR1 with different members of the MAP1LC3 proteins family indicate that the presence of a tryptophan in the LIR motif increases the binding affinity. Substitution to other aromatic amino acids or increasing the number of negatively charged residues at the N-terminus of the LIR motif, however, has little effect on the binding affinity due to enthalpy-entropy compensation, suggesting that effector proteins can interact with a wide variety of different sequences with similar and moderate binding affinities.
Additionally to be present in proteins dealing with protein folding and degradation, ubiquitin-like domain were found protein involved in the regulation of signal transduction like TBK1, a serine/threonine kinase responsible for induction of immune response. In this second project, based on the NMR chemical shifts of the TBK1 domain contained between amino acids 302 and 383, secondary structure prediction programs (TALOS and CSI) confirmed the presence of an Ubiquitin-like domain in TBK1 by identifying one alpha-helix and four beta-strands sequentially aligned like following beta-beta-alpha-beta-beta. This alignment corresponds perfectly with the secondary structure elements of Ubiquitin and proved that TBK1_ULD belongs to the UBL protein superfamily. The similarity to ubiquitin was even bigger by the presence in addition of a small beta-strand and a short helix, which are observed as the beta 5-strand and a 310-helix in Ubiquitin, respectively. The first attempts on the 3D structure determination confirmed the Ub-fold but due to the lack of assignment in TBK1_ULD, only a structure based on ubiquitin as a model was determined. Interaction studies of TBK1_ULD with the IAD-SRR domain of IRF3 showed that both side of the molecule seems involved and that the TBK1/IRF3 interaction is more complex than a one to one binding process. Unfortunately, the instability of TBK1_ULD associated to the difficulty in the purification of IAD-SRR did not allow to further study this interaction more precisely.
Finally, to overcome the difficulty encountered in NMR experiments because of low expression and/or poor solubility, an expression vector using the intrinsic property of ubiquitin was designed. Fused to proteins or peptides targets, this construct produced proteins and peptides in a larger amount than with traditional expression vectors and also with a less cost than chemical synthesis for pure labeled peptides for NMR structural studies. The presence of a hexa histidine tag was useful for the isolation and the purification of the constructs. The existence of a TEV cleavage site was created to keep the possibility of releasing the ubiquitin moiety from the expressed protein or peptide. Moreover, the ubiquitin-tag could also still be attached to the protein/peptide of interest when biophysical methods like NMR, ITC or CD spectroscopy are applied, providing the same results than for the protein/peptide moiety alone.
Design and optimization of the lattice of the superconducting synchrotron SIS300 for slow extraction
(2011)
The superconducting synchrotron SIS300 is planned to be built at the new Facility for Antiproton and Ion Research (FAIR), at GSI-Darmstadt [1]. SIS300 will be a versatile machine, which by means of a low-energy stretcher-mode or a high-energy ramped-mode will provide slowly extracted heavy ion beams towards the experimental areas. To reach the required maximum field of 4.5 T, cos(θ) magnets are necessary. Thus, SIS300 will become the first superconducting synchrotron worldwide with cos(θ) magnets providing resonant slow extraction.
Since SIS300 will be installed in the same tunnel as the SIS100 synchrotron, the dipole layout of SIS300 cannot be freely chosen. Thus, a standard lattice cannot be applied. A redesign of the SIS300 lattice accepting compromises concerning the positions and phase advances between the optical elements has been proposed. Using the analytical model of the slow extraction, firstly proposed by Kobayashi, and the analytical description of the resonance driving modes, a multiobjective optimization algorithm has been developed for the optimization of the lattice under the given boundary conditions. The final goal of the lattice optimization is a higher efficiency of the slow extraction. The results are evaluated by means of tracking simulations performed with the code Elegant.
The field quality in superconducting cos(θ) magnets is determined by the positions of the superconducting cable and the static and time-dependent effects of the current in the cable. Furthermore, the fast ramp rates of 1 T/s in the dipoles, which are fifty times faster than in any other superconducting cos(θ) magnet, together with the fact that the aperture is smaller than in conventional accelerator magnets, makes it extremely difficult to obtain a high-quality magnetic field. The unavoidable field errors affect the beam dynamics and worsen the slow extraction efficiency. Therefore, the field errors in the SIS300 dipoles have been estimated, and their effects have been taken into account in the optimization algorithm. As a result a compensation scheme has been proposed, in which time-dependent gradients in the sextupoles counteract the decay of the sextupole field errors in the dipole magnets during the slow extraction. For the limits where the compensation was no longer possible, tolerances to the magnet field errors have been determined.
We found that the HMTase G9a, that catalyzes H3K9me2 in euchromatin, plays a key modulatory role in type I IFN expression. This finding raises the possibility of targeted intervention with type I IFN expression by using small synthetic inhibitors of G9a. Given the overall minimal negative effect of G9a-deficiency on differentiated cells, the short-term suppression of G9a could be used to potentiate type I IFN expression during chronic viral diseases such as hepatitis C. Accordingly, pharmacological enhancement of methylation, for example by inhibition of the H3K9me2 specific demethylases, could be potentially used to attenuate type I IFN expression and help to control chronic inflammatory and autoimmune conditions. The mechanism responsible for canvassing the epigenetic profile of type I IFN expressing cells are not known. It is plausible, that similar to neurons, where G9a is targeted to specific loci with the help of noncoding RNAs, IFN expressing cells possess similar mechanisms to target H3K9me2 demethylating enzymes to type I IFN loci, thus keeping these loci accessible for IFN-inducing transcription factors. Identification of non-coding RNAs that may contribute to the establishment of the epigenetic state of IFN producing cells will provide a further opportunity for targeted manipulation of IFN expression.
In my thesis, I describe the collaborative experiments that show the ability of synthetic compounds that interfere with the histone readers to suppress inflammation. Our results present a novel concept for the regulation of inflammatory gene expression. The diversity of histone readers and the combinatorial nature of regulation of gene transcription may provide an opportunity for highly selective interference with disease associated transcriptional programs by interfering with specific readers. In the future we plan to address the therapeutic potential of BET antagonists in autoimmune and chronic inflammatory conditions.In summary, the experiments described in my thesis provide an example of how the understanding of the basic mechanisms of chromatin control of gene expression can facilitate novel therapeutic approaches that target chromatin.
Towards a THz Bloch laser
(2011)
The realisation of tunable THz laser sources working at room temperature would give
rise to further applications in this range of the electromagnetic spectrum. The THz
Bloch laser could therefore become the basis for a technological breakthrough. Beside
this practical relevance, the physics of the gain mechanism has been investigated
theoretically for a long time and the experimental implementation of a self-starting
laser still has not been achieved.
At the beginning of this thesis the basic principles of Bloch oscillations and the
related Bloch gain are described. The need of a superlattice structure to make Bloch
oscillations possible in a semiconductor material is discussed. In this context, the effect
of negative differential resistance and its influence on the field distribution due to Gunn
domains is explained. The latter lead to an inhomogeneous field which may suppress
the Bloch gain mechanism. The Krömer criterion is introduced and the concept of
field-pinning layers to improve the field homogeneity is deduced. Finally, the design of
the laser material is shown and different types of laser waveguides are compared.
In chapter 3 detailed recipes for the processing of samples are given. Different types of
contacts (ohmic and Schottky), the wafer bonding process required for double-metal
lasers and the application of different photoresists for different purposes are described.
An explanation of the formation of waveguides due to dry etching, wet etching
and ion implantation follows. Dry etching is an established technique in the field
of microstructure processing but the challenge of etching about 20 μm has led to
problems. The high etching depth also makes wet etching difficult but this method
could be improved due to a hard bake of the photoresist. The protection of critical
areas on the surface of the samples with photoresist during ion implantation was
increased by optimising the spin coating process. However, a full implantation of the
active layer between the waveguides was not achieved which was the reason for the
development of the hybrid technology. Here a prior wet etching of about 10 μm is
performed and the rest of the material is implanted.
The experimental setup is shown in chapter 4. An alternative method for the electrical
contacting with the help of a copper bar is introduced. This improves the current
distribution and the risk of an electrical breakdown during the measurements could
therefore be lowered. Devices for THz beam guidance and spectroscopic measurements
are shown and the method of biasing the samples with pulses below 100 ns and
determining the effective voltage applied to the sample is depicted. These short pulses
are required to prevent the samples heating up drastically due to high power.
Chapter 5 contains the current-voltage characterisation of several structures including
I-V-samples, Bloch laser samples and a quantum cascade laser. Different contacts
(ohmic and Schottky) and different techniques for the formation of the ridges have
been used in the processing of these samples (performed at the University of Frankfurt
in all cases) and their influence on the I-V-dependence is discussed. The properties of
the THz emission of the quantum cascade laser are in good agreement with published
results from lasers processed with the same material. Another important result of
this chapter is that the Bloch laser samples show unstable behaviour compared to the
quantum cascade structure even with short pulses (of about 10 ns) where the risk of an
electrical breakdown or the building of filaments is low. THz radiation emitted from
one of the Bloch laser samples could not be observed.
Two aspects that may have prevented the Bloch laser to emit are discussed in
chapter 6. The saturation of the gain for higher amplitudes of the THz wave is
investigated in single mode and multiple mode operation (the latter could occur due
to the Bloch gain being expected to be broadband). In both cases it is shown that
the saturation effect would limit the output power only to values clearly above the
detection limit. In the subsequent section the distribution of the electric field is
simulated with SILVACO software. Structures with transit layer lengths above the
Krömer criterion are compared with structures which include field-pinning layers. It is
shown that the latter are useful to avoid propagating Gunn domains as they build up
in similar structures without field-pinning layers. Nevertheless, the electric field inside
the superlattice regions is not stable. Beside spatial inhomogeneities also temporal
variations of the field magnitude are observed. The lack of a suitable field distribution
is expected to be the main reason for the samples not to work.
The role of peroxisome proliferator-activated receptor gamma during sepsis-induced lymphopenia
(2011)
Sepsis is one of the most common diseases on intensive care units all over the world and accounts there for the highest mortality rate. One of the hallmarks of sepsis is an accelerated T-cell apoptosis, resulting in a compromised immune state with the inability to eradicate pathogens. This promotes organ damage or even organ failure. A multiple organ dysfunction evolves, which often ends up in septic shock and death. Recently, it was shown that severe T-cell depletion correlates with sepsis mortality. When inhibiting T-cell apoptosis, an increased mouse survival was observed in experimental sepsis. ...
In human neuroscientific research, there has been an increasing interest in how the brain computes the value of an anticipated outcome. However, evidence is still missing about which valuation related brain regions are modulated by the proximity to an expected goal and the previously invested effort to reach a goal. The aim of this dissertation is to investigate the effects of goal proximity and invested effort on valuation related regions in the human brain. We addressed this question in two fMRI studies by integrating a commonly used reward anticipation task in differential versions of a Multitrial Reward Schedule Paradigm. In both experiments, subjects had to perform consecutive reward anticipation tasks under two different reward contingencies: in the delayed condition, participants received a monetary reward only after successful completion of multiple consecutive trials. In the immediate condition, money was earned after every successful trial. In the first study, we could demonstrate that the rostral cingulate zone of the posterior medial frontal cortex signals action value contingent to goal proximity, thereby replicating neurophysiological findings about goal proximity signals in a homologous region in non-human primates. The findings of the second study imply that brain regions associated with general cognitive control processes are modulated by previous effort investment. Furthermore, we found the posterior lateral prefrontal cortex and the orbitofrontal cortex to be involved in coding for the effort-based context of a situation. In sum, these results extend the role of the human rostral cingulate zone in outcome evaluation to the continuous updating of action values over a course of action steps based on the proximity to the expected reward. Furthermore, we tentatively suggest that previous effort investment invokes processes under the control of the executive system, and that posterior lateral prefrontal cortex and the orbitofrontal cortex are involved in an effort-based context representation that can be used for outcome evaluation that is dependent on the characteristics of the current situation.
The canonical Wnt/β-catenin and the Shh pathway as well as the Notch signaling cascade
are key regulators in stem cell biology and are independently associated with the development
of cancer. Despite the knowledge of a balanced signaling for cellular maintenance, the
fundamental biochemical mechanisms of crosstalk are still poorly understood. This study
demonstrates that the outcome of interaction between Wnt and Shh is cell type specific. A
combined inhibitory mechanism of the Shh and Notch2/Jagged2 pathways on dominant
active β-catenin signaling in the adult tongue epithelium keeps Wnt/β-catenin signaling
restricted to physiological tolerable levels. In the opposite crosstalk the activation of
Wnt/β-catenin signaling in medulloblastoma (MB) of the Shh subtype, in turn inhibits the Hh
pathway.
The inhibitory mechanism of Shh and Notch2/Jagged2 on Wnt/β-catenin signaling is
independent of the degradation complex of β-catenin and takes place inside the nucleus.
Furthermore, the negative feedback on Wnt/β-catenin signaling by the Shh pathway relies
on transcriptional activity of Gli1/2A. Inhibition of Gli1/2A with the specific inhibitor GANT61
abrogated the negative impact of Shh on β-catenin signaling in vitro. Although the negative
feedback loop of Shh is still functional in human SCC25 cells, the inhibitory effect of
Notch2/Jagged2 is lost and contributes to the cancerogenic phenotype of these cells. In the
inverse situation, the activation of β−catenin signaling has a negative feedback on
constantly active Shh signaling and significantly inhibits the Hh pathway. This was shown in
Ptch+/- and Math1-Cre:SmoM2Fl/+ MB tumor spheres in vitro, in which inhibition of sphere
formation and growth was observed and Hh target gene transcription was down-regulated.
This demonstrates for the first time that the activation of canonical Wnt/β-catenin signaling
in primary MB cells with a Hh pathway over-activation has a negative effect on the growth of
these cells in vitro.
In summary the results show that crosstalk of Wnt/β-catenin and Shh signaling has context
specific outcome on pathway activity. Elucidation of the molecular interactions will improve
our understanding of Wnt and Hh associated tumors and contribute to the development of
new therapeutic strategies.
This dissertation investigated the development of the complementiser that from the demonstrative pronoun in the Germanic languages; each chapter dealt with a different aspect. In the introduction, the terms ‘reanalysis’ and ‘analogy’ and their relevance for grammaticalisation were explained, and the issues of the chapters were presented. The second chapter introduced some information about the Germanic language family and the languages which were relevant for this investigation, namely Gothic, Old English, Old Icelandic, Old Saxon and Old High German. Previous assumptions about the diachrony of that were presented and discussed. One of these proposals which mainly draws on evidence from West Germanic involves the idea that the source construction contained two independent main clauses with a demonstrative pronoun (that) at the end of the first clause (cf. e.g. Paul 1962, § 248). In contrast to this, the Gothic evidence showed that the source construction of the reanalysis of ϸatei was not a proper paratactic construction (at least in Gothic) but already a complex construction which contained a complementiser (ei) in the appositional subordinate clause (cf. also e.g. Longobardi 1994 for the diachrony of ϸatei). This contradiction raised the question whether the analysis of the Gothic that-complementiser also applies to the diachrony of that in West Germanic. This issue was taken up in the third chapter which presented an overview of subordination and complementisers in Northwest Germanic. The aim was to show that the Northwest Germanic languages also show a subordinating particle, which functions like the Gothic ei, namely ϸe (OE), er/es (OI), the (OHG, OS). As a result, the subordinating particle could be observed in relative and adverbial clauses in all Northwest Germanic languages. In complement clauses, which are most crucial for the argumentation, the subordinating particle is found in Old English and Old Icelandic but not in Old Saxon. In Old High German, there are only combinations of the with a following pronoun, theih and theiz, in ‘Otfrids Evangelienbuch’ (see Wunder 1965). Consequently, the presence of a subordinating particle is confirmed in North and West Germanic. The fact that the patterns of subordination are quite similar in all Germanic languages suggested a unitary analysis of the development of that in Germanic was appropriate. In chapter four, the similarities and differences between the Germanic languages with respect to the development of that were explained. It was argued that the preconditions of the reanalysis were the same, whereas the consequences of the reanalysis are realised differently in each language. The most important precondition was that the appositional source construction (explained in more detail below) was generally available in Germanic. Since the demonstrative pronoun at the end of the matrix clause and the subordinating particle of the subordinate clause were adjacent, phonological combination might have been crucial for the subsequent reanalysis to take place. After reanalysis, however, different changes can be observed in the different languages. For instance, it appears that during the Old English period the final syllable of the form ϸætte was deleted (see chapter 4 for references), whereas the final –ei is still present in the Gothic ϸatei, and completely absent in Old High German and Old Saxon. The source structure of the reanalysis was discussed in detail in a separate subsection. The appositional source construction, which was already assumed for the reanalysis of Gothic ϸatei, was compared with analyses of clitic left dislocation which propose that two constituents with the same theta-role derive from a Big DP (see e.g. Grewendorf 2009, Belletti 2005). Based on the Big DP analysis of Grewendorf (2009), it was claimed that the appositional clause, introduced by the subordinating particle, is generated in the Spec of a DP, and adjoined to this DP on the surface. It was argued that this whole complement DP-node occurred in an extraposed position in OV-languages so that the verb, when it stays in-situ, does not appear between the demonstrative pronoun and the subordinating particle. The structure in (1) illustrates the syntactic source structure which is assumed to apply to the development of the complementiser that in Germanic. ...
In Nervensystemen werden zahlreiche Informationen wahrgenommen und verarbeitet um ein adäquates Verhalten hervorzurufen. Für die Untersuchung der funktionellen Zusammenhänge hierbei wurden verschiedene Methoden entwickelt, die eine gezielte Manipulation neuronaler Prozesse ermöglichen. Durch Analyse der resultierenden Effekte können dabei synaptische Proteine, einzelne Neuronen oder neuronale Netzwerke funktionell charakterisiert werden. Bisherige Ansätze verfügen jedoch nur über eine geringe zeitliche und räumliche Auflösung oder erlauben lediglich eine eingeschränkte Anwendung im frei beweglichen Tier.
Diese Nachteile können durch die heterologe Expression von lichtgesteuerten, mikrobiellen Rhodopsinen zur gezielten Manipulation des Membranpotentials umgangen werden. So induziert die Photoaktivierung des Kationenkanals Channelrhodopsin 2 (ChR2; (Nagel et al., Curr Biol 2005)) eine Depolarisation, während die Chloridpumpe Halorhodopsin (NpHR; (Zhang et al., Nature 2007)) für die Hyperpolarisation verwendet werden kann. Dabei ermöglichen die schnellen Kinetiken der Rhodopsine eine zeitlich präzise Steuerung des Membranpotentials. Durch Auswahl geeigneter Promotoren ist zudem oftmals eine zell spezifische Expression möglich. Dieser Ansatz wird daher allgemein als Optogenetik bezeichnet.
In der vorliegenden Arbeit wurden zunächst konventionelle Techniken genutzt, um die Funktion von zwei assoziierten Proteinen eines Acetylcholin Rezeptors in C. elegans zu untersuchen. Des Weiteren wurden verschiedene Methoden für den Fadenwurm entwickelt und angewendet, die die Vorteile optogenetischer Techniken für die funktionelle Charakterisierung synaptischer Proteine und neuronaler Netzwerke nutzbar machen. Hierbei erlaubt die Transparenz von C. elegans die optogenetische Stimulation im lebenden Organismus unter nicht invasiven Bedingungen. Weitere Vorteile von C. elegans als neurobiologischem Modellorganismus liegen in seiner einfachen Handhabung (Hope, 1999) und der stereotypen Entwicklung seines Nervensystems mit bekannten anatomischen Ausprägungen (Sulston and Horvitz, Dev Biol 1977; Varshney et al., PLoS Comput Biol 2011; White et al., Philos Trans R Soc Lond B Biol Sci 1986). Durch ihre Häufigkeit und die experimentelle Zugänglichkeit wird hierbei die neuromuskuläre Synapse oftmals zur Erforschung der synaptischen Reizweiterleitung genutzt (Von Stetina et al., Int Rev Neurobiol 2006). Durch pharmakologische (Lewis et al., Neuroscience 1980; McIntire et al., Nature 1993; Miller et al., Proc Natl Acad Sci U S A 1996; Richmond and Jorgensen, Nat Neurosci 1999) und elektrische Stimulation (Richmond and Jorgensen, Nat Neurosci 1999) können dabei Defekte der Transmission hervorgehoben werden, während Verhaltensexperimente oder elektrophysiologische Messungen der post synaptischen Ströme in Muskelzellen eine quantitative Analyse ermöglichen (Richmond and Jorgensen, Nat Neurosci 1999).
Diese Methoden wurden für die funktionelle Charakterisierung von NRA 2 und NRA 4 verwendet, die beide als akzessorische Proteine zusammen mit dem Levamisol sensitiven Acetylcholin Rezeptor der Körperwandmuskelzellen aufgereinigt wurden (Gottschalk et al., EMBO J 2005). Dabei konnte gezeigt werden, dass NRA 2 und NRA 4 im Endoplasmatischen Retikulum (ER) der Muskelzellen einen Komplex bilden, der die Sensitivität von beiden nikotinischen Acetylcholin Rezeptoren gegenüber verschiedenen cholinergen Agonisten verändert. In diesem Zusammenhang wurde auch nachgewiesen, dass die Oberflächenexpression einzelner Untereinheiten der beiden Rezeptoren durch NRA 2/4 beeinflusst wird. Diese Resultate legen die Vermutung nahe, dass beide Proteine die Zusammensetzung der Rezeptoren und somit ihre pharmakologischen Eigenschaften modulieren. Denkbar ist dabei eine regulatorische Funktion bei der Assemblierung verschiedener Untereinheiten zu einem funktionellen Rezeptor oder bei der Kontrolle des ER Austritts von Rezeptoren mit bestimmter Zusammensetzung. In dieser Hinsicht konnte jedoch keine Interaktion von NRA 2/4 mit der Notch Signalkaskade nachgewiesen werden, wie sie für die homologen Proteine nicalin und NOMO in Vertebraten gezeigt wurde (Haffner et al., J Biol Chem 2007; Haffner et al., EMBO J 2004).
Für die Untersuchung synaptischer Proteine durch optogenetische Techniken wurde ChR2(H134R) selektiv in cholinergen oder GABAergen Motorneuronen exprimiert, um die akute und lichtgesteuerte Freisetzung des jeweiligen Neurotransmitters zu ermöglichen. Die resultierende Stimulation bzw. Inhibition von Muskelzellen wurde hierbei durch elektrophysiologische Messungen der post synaptischen Ströme und durch Analyse von Kontraktionen respektive Relaxationen untersucht. Dabei wurde gezeigt, dass Störungen der synaptischen Reizweiterleitung die Ausprägung und Dynamik dieser lichtinduzierten Effekte beeinflussen und dadurch charakterisiert werden können. So zeigten beispielsweise Mutanten von Synaptojanin und Endophilin nachlassende Effekte bei anhaltender oder wiederholter Stimulation, was durch die gestörte Regeneration synaptischer Vesikel erklärt werden kann (Harris et al., J Cell Biol 2000; Schuske et al., Neuron 2003; Verstreken et al., Neuron 2003).
Die hohe Sensitivität dieser Methode wurde im Nachfolgenden dazu verwendet, die Inhibition cholinerger Motorneuronen durch den metabotropen GABAB Rezeptor zu untersuchen, der in C. elegans aus den beiden Untereinheiten GBB 1 und GBB 2 gebildet wird (Dittman and Kaplan, J Neurosci 2008; Vashlishan et al., Neuron 2008). Dabei konnte zunächst gezeigt werden, dass diese heterosynaptische Inhibition verschiedene lokomotorische Verhaltensweisen der Tiere beeinflusst. Für die mechanistische Untersuchung wurden anschließend cholinerge Motorneuronen durch ChR2(H134R) photoaktiviert, während resultierende Kontraktionseffekte in Abhängigkeit von GBB 1/2 analysiert wurden. Um hierbei die Funktion von GBB 1/2 durch erhöhte GABA Konzentrationen hervorzuheben, wurden zusätzlich GABAerge Motorneuronen optogenetisch stimuliert oder die Wiederaufnahme von GABA aus dem synaptischen Spalt durch Mutation des Membran ständigen GABA Transporters blockiert. So konnte gezeigt werden, dass GBB 1/2 eine akute Inhibition der cholinergen Motorneuronen bewirken, was vermutlich für die Regulation von Bewegungsabläufen eine wichtige Rolle spielt. Die geringe Dynamik der GBB 1/2 induzierten Effekte deutet allerdings darauf hin, dass die synaptische Aktivität durch den metabotropen Rezeptor kaum nachhaltig moduliert wird.
In nachfolgenden Versuchen wurde die optogenetische Stimulation von Motorneuronen außerdem mit der elektronenmikroskopischen Analyse der präsynaptischen Feinstruktur kombiniert. Dadurch konnte die Dynamik der Exozytose und Endozytose synaptischer Vesikel (SV) in Abhängigkeit von neuronaler Aktivität untersucht werden. So wurde gezeigt, dass synaptische Vesikel nahe der aktiven Zone während einer 30 sekündigen Hyperstimulation nahezu komplett aufgebraucht waren. Die vollständige Regeneration der SV Pools benötigte anschließend etwa 12 Sekunden und erfolgte zunächst in der Peripherie der aktiven Zone, was auf eine laterale Heranführung der Vesikel schließen lässt. Nach etwa 20 Sekunden erholte sich ebenfalls die Wirksamkeit der Stimulation von Muskelzellen durch die Motorneuronen, was durch elektrophysiologische Messungen der photo induzierten post synaptischen Ströme gezeigt wurde. Während der Hyperstimulation bildeten sich außerdem große vesikuläre Strukturen, die sich anschließend nach etwa acht Sekunden wieder aufgelöst hatten. In Analogie zu vergleichbaren Experimenten in anderen Organismen liegt die Vermutung nahe, dass es sich dabei um Zwischenprodukte der so genannten Bulk Phase Endozytose handelt, die das Clathrin abhängige Recycling von synaptischen Vesikeln bei starker neuronaler Aktivität ergänzt (Heuser and Reese, J Cell Biol 1973; Miller and Heuser, J Cell Biol 1984; Richards et al., Neuron 2000). Bemerkenswerterweise war der Abbau der vesikulären Strukturen in Synaptojanin und Endophilin defizienten Tieren stark verzögert. Denkbar ist, dass beide Proteine für die Synthese von synaptischen Vesikeln aus den vesikulären Zwischenprodukten der Bulk Phase Endozytose wichtig sind, analog zur ihrer Funktion bei der Clathrin abhängigen Endozytose an der Plasmamembran.
Durch die zielgerichtete Manipulation der Zellaktivität ermöglichen optogenetische Techniken außerdem die funktionelle Charakterisierung von Neuronen und neuronalen Netzwerken. Um die zelluläre Spezifität dieses Ansatzes zu erhöhen, wurde ein Tracking System entwickelt das die Position frei beweglicher Tiere in Echtzeit bestimmt und nachverfolgt. Dadurch konnte die Photoaktivierung optogenetischer Proteine auf definierte Bereiche der Fadenwürmer und somit auf ausgewählte Neuronen innerhalb der Expressionsmuster von verwendeten Promotoren eingeschränkt werden. Des Weiteren ermöglichte hierbei die Auswertung translatorischer Parameter die Analyse verschiedener lokomotorischer Merkmale wie Geschwindigkeit, Bewegungsbahn oder Ausprägung der Körperbiegungen. Dieses System wurde beispielhaft für die konzertierte Photoaktivierung durch ChR2(H134R) bzw. Photoinhibition durch MAC von zwei verschiedenen Gruppen von Neuronen angewendet, um die Integration mechanosensorischer Informationen durch Command Interneuronen zu untersuchen. In diesem Zusammenhang wurde zudem eine Rekombinase basierte Methode für optogenetische Proteine adaptiert, die die Transkription auf die zelluläre Schnittmenge von zwei verschiedenen Promotoren einschränkt und somit die Spezifität der Expression erhöht. Idealerweise kann dieser Ansatz außerdem mit der gezielten Photoaktivierung kombiniert werden, um die zelluläre Selektivität optogenetischer Anwendungen weiter zu verbessern.
Weiterhin ist die Anwendung optogenetischer Techniken bisher durch intrinsische Eigenschaften der verwendeten Rhodopsine auf die relativ kurzzeitige Manipulation des Membranpotentials von Zellen beschränkt. So benötigt ChR2 durch die schnelle Schließung seines offenen Kanals eine kontinuierliche Photoaktivierung, um eine andauernde Depolarisation hervorzurufen. Dies ist jedoch potentiell mit phototoxischen und – besonders bei C. elegans – phototaktischen Nebeneffekten verbunden. Deswegen wurden diverse Mutanten von ChR2 mit stark verlangsamter Inaktivierung (Berndt et al., Nat Neurosci 2009) für ihren Nutzen zur Langzeit Stimulation von erregbaren Zellen im Nematode getestet. Dabei wurde gezeigt, dass ChR2(C128S) durch einen kurzen Photostimulus mit vergleichsweise niedriger Intensität eine anhaltende Depolarisation über mehrere Minuten auslösen kann. Die wiederholte Stimulation in ASJ Neuronen ermöglichte zudem eine langzeitige Depolarisation über mehrere Tage, wodurch die genetisch veranlagte Entwicklung von Tieren manipuliert werden konnte. Durch gezielte Punktmutation konnten außerdem relevante Eigenschaften von ChR2(C128S) für die Langzeit Stimulation weiter verbessert werden.
Als weiteres optogenetisches Werkzeug wurde zudem die Photoaktivierbare Adenylatzyklase alpha (PACa) aus Euglena gracilis (Iseki et al., Nature 2002; Ntefidou et al., Plant Physiol 2003; Schroder-Lang et al., Nat Methods 2007) für die akute und lichtgetriebene Synthese des sekundären Botenstoffs cAMP in C. elegans etabliert. Die Photoaktivierung von PACa in cholinergen Motorneuronen verstärkte dabei die Neurotransmitterfreisetzung und induzierte hyperlokomotorische Phänotypen, vergleichbar zu Mutanten mit erhöhten cAMP Konzentrationen.
Zusammengefasst wurden diverse optogenetische Techniken für C. elegans entwickelt und optimiert, die die zellspezifische und nicht invasive Manipulation des Membranpotentials beziehungsweise die Synthese des sekundären Botenstoffs cAMP durch Licht im frei beweglichen Tier ermöglichen. Diese Methoden können zur gezielten Störung neuronaler Aktivität angewendet werden, um dadurch neurobiologische Fragestellungen im Fadenwurm zu untersuchen. Dies wurde beispielhaft für die Erforschung der synaptischen Reizweiterleitung und die funktionelle Analyse neuronaler Netzwerke demonstriert. Denkbar ist außerdem, diese für C. elegans etablierten Methoden vergleichbar in anderen Modellorganismen anzuwenden. So sind die Fruchtfliege ebenso wie der Zebrafisch Embryo bereits für optogenetische Techniken erprobt (Arrenberg et al., Proc Natl Acad Sci U S A 2009; Schroll et al., Curr Biol 2006). Für Säugetiere wie die Maus, die Ratte und den Makaken wurden zudem bereits Ansätze entwickelt, die die gezielte Photostimulation in lebenden und frei beweglichen Tieren ermöglichen (Han et al., Neuron 2009; Wentz et al., J Neural Eng 2011; Yizhar et al., Nature 2011; Zhang et al., Nat Rev Neurosci 2007).
Savannas are the most important timber and non-timber forest products (NTFPs) providing ecosystems in West Africa. They have been shaped by traditional human land-use (i.e. agriculture, grazing, and harvesting) for thousands of years. In the last decades, land-use has drastically changed due to the rapid population growth and the growing production of cash-crop in West Africa and this process is still continuing. The percentage of land intensively used for agriculture has increased, while the length of fallow periods has decreased. Such changes have enormous ecological, economic, and social consequences. In the context of land-use changes, there is an urgent need to better understand and evaluate the impact of land-use on savannas. Such an understanding provides insights on appropriate management activities that ensure the maintenance of savannas and guarantee the availability of savanna products for subsistence and commercial use of rural West African people.
The major objective of the present thesis was to study the impact of land-use on savanna vegetation and diversity as well as on populations of two important NTFP-providing tree species in a semi-arid area in West Africa. The study area was located in the south-eastern part of Burkina Faso and comprised the protected W National Park and its adjacent communal area.
In the first study (chapter 2), I investigated in cooperation with a colleague from Burkina Faso (Blandine Nacoulma) the impact of land-use on the savanna vegetation. We analyzed which environmental factors determine the occurrence of the vegetation types and investigated the effect of land-use on vegetation structure and the occurrence of life forms and highly valued tree species. Furthermore, we tested whether land-use has an impact on plant diversity pattern and if this impact differed between the vegetation types and layers (woody and herb layer). Vegetation relevés were performed and the vegetation and plant diversity of the protected W National Park were compared with those of its surrounding communal area. Our results reveal five vegetation types occurring in both areas. Elevation and physical soil characteristics and thus soil water availability for plants played the most important role for the occurrence of the vegetation types. The influence of land-use on plant diversity differed between the five vegetation types and the two layers. The impact was highest on the vegetation types with the most favorable soil conditions for cultivation and lowest on rocky habitats with poor soils. While the diversity of the woody layer was increased under human land-use, the diversity of the herb layer was diminished. Overall, as land-use effects were not only negative, our findings suggest that land-use does not automatically lead to a loss of plant species and to a degradation of savanna habitats. We conclude that both protected and communal areas are of great importance for the conservation of savanna vegetation and diversity. Our study highlights furthermore the importance of different management strategies for each vegetation type.
In the following two studies (chapter 3 and 4), the impact of land-use - and in particular of harvesting - on populations of Adansonia digitata L., the baobab tree, and Anogeissus leiocarpa (DC.) Guill. & Perr. was examined. These two tree species were chosen as they provide several NTFPs for the local population and as they show different levels of human protection and opposed life histories. Thus, they may react differently to land-use. Stands of the protected W National Park were compared with those of its surrounding communal area (in fallows, croplands, and villages). I applied dendrometric methods to study the population structures and combined it with rates and patterns of NTFP-harvesting (debarking and chopping/pruning). Furthermore, the impact of land-use and harvesting on the fruit production of A. digitata and on the sprouting ability of A. leiocarpa were studied. The inverse J-shaped size class distribution curve indicates that the stands of A. digitata were in a healthy state in the park, while the low number of smaller size classes in fallows, croplands, and villages may give evidence of an ageing population. However, a high number of seedlings were recorded in villages. The stands of A. leiocarpa were also in healthy states in the park and likewise in fallows. In contrast, the absence of saplings gives evidence of a declining population in croplands. Both species were strongly harvested by local people and harvesting was tree size-specific. Pruning in interaction with tree-size had a significant impact on fruit production of A. digitata. While smaller trees were more vulnerable to pruning, bigger trees benefited from slight-pruning. A. leiocarpa had a great ability to respond to chopping by sprouting. The sprouting ability increased even with higher chopping intensity. Results suggest that despite the intense harvesting and the land-use impact, populations of both species are still well preserved. While A. digitata can withstand the harvesting and land-use pressure by its longevity, extremely low adult mortality rates, and particularly due to positive human influences, A. leiocarpa is able to withstand the use pressure by its fast growing, high recruitment, and high sprouting ability. I conclude that a none protected tree species (A. leiocarpa) might not necessarily be at higher risk to the harvesting and land-use impact than a protected tree species (A. digitata) as the adverse impact of harvesting and land-use can be compensated by its specific life history.
Important additional information to such ecological findings can be provided by local people. Learning from traditional knowledge and management systems of local people will help to produce culturally and ecologically reasonable conservation and management strategies. Thus, I investigated local uses and management strategies of A. digitata and A. leiocarpa in the last two studies (chapter 5 and 6). Quantitative ethnobotanical surveys among the Gulimanceba people were conducted in the communal area in order to document uses of the different plant parts, harvesting modes, perceptions about the population status, and conservation status of both species. Hereby, differences in knowledge between gender, generations, and people from different villages were tested. Interviews reveal that both species are harvested for multipurpose and emphasize the high importance of both species for local people. Especially the leaves and fruits of A. digitata add valuable minerals and vitamins to the otherwise micronutrient-“poor” staple crops of the Gulimanceba people. In comparison with other studies in West Africa, it has turned out that people in this area could benefit even more from A. leiocarpa, e.g. for dyeing of clothes, for treatment of malaria and skin problems. Local knowledge did not differ between genders and generations, while it slightly differed between people from different villages. The lack of age differences suggests that the traditional knowledge about these two species is passed on from one generation to another. Differences between people from different villages might be explained by influences from the neighboring countries Niger and Benin. Current local harvesting modes and management strategies of both species resulted in sustainable use. However, ongoing land-use intensifications require adapted harvesting and management techniques to guarantee the persistence of these economically important species. These results provide, in combination with the ecological findings (chapter 3 and 4), appropriate management recommendations for A. digitata and A. leiocarpa that are reliable under currently practiced management strategies.
Which factors determine whether a stimulus is consciously perceived or unconsciously processed? Here, I investigate how previous experience on two different time scales – long term experience over the course of several days, and short term experience based on the previous trial – impact conscious perception. Regarding long term experience, I investigate how perceptual learning does not only change the capacity to process stimuli, but also the capacity to consciously perceive them. To this end, subjects are trained extensively to discriminate between masked stimuli, and concurrently rate their subjective experience. Both the ability to discriminate the stimuli as well as subjective awareness of the stimuli increase as a function of training. However, these two effects are not simple byproducts of each other. On the contrary, they display different time courses, with above chance discrimination performance emerging before subjective experience; importantly, the two learning effects also rely on different circuits in the brain: Moving the stimuli outside the trained receptive field size abolishes the learning effects on discrimination ability, but preserves the learning effects on subjective awareness.
This indicates that the receptive fields serving subjective experience are larger than the ones serving objective performance, and that the channels through which they receive their information are arranged in parallel. Regarding short term experience, I investigate how memory based predictions arising from information acquired on the trial before affect visibility and the neural correlates of consciousness. To this end, I vary stimulus evidence as well as predictability and acquire electroencephalographic data.
A comparison of the neural processes distinguishing consciously perceived from unperceived trials with and without predictions reveals that predictions speed up processing, thus shifting the neural correlates forward in time. Thus, the neural correlates of consciousness display a previously unappreciated flexibility in time and do not arise invariably late as had been predicted by some theorists.
Admittedly, however, previous experience does not always stabilize perception. Instead, previous experience can have the reverse effect: Seeing the opposite of what was there, as in so-called repulsive aftereffects. Here, I investigate what determines the direction of previous experience using multistable stimuli. In a functional magnetic resonance imaging experiment, I find that a widespread network of frontal, parietal, and ventral occipital brain areas is involved in perceptual stabilization, whereas the reverse effect is only evident in extrastriate cortex. This areal separation possibly endows the brain with the flexibility to switch between exploiting already available information and emphasizing the new.
Taken together, my data show that conscious perception and its neuronal correlates display a remarkable degree of flexibility and plasticity, which should be taken into account in future theories of consciousness.
Dynamics of chaotic strings
(2011)
The main topic of this thesis is the investigation of dynamical properties of coupled Tchebycheff map networks. At every node of the network the dynamics is given by the iteration of a Tchebycheff map, which shows strongest possible chaotic behaviour. By applying a coupling between the various individual dynamics along the links of the network, a rich structure of complex dynamical patterns emerges. Accordingly, coupled chaotic map networks provide prototypical models for studying the interplay between local dynamics, network structure, and the emergent global dynamics. An exciting application of coupled Tchebycheff map lattices in quantum field theory has been proposed Beck in Spatio-temporal chaos and vacuum fluctuations of quantized fields' (2002). In this so-called chaotic string model, the coupled map lattice dynamics generates the noise needed for the Parisi-Wu approach of stochastic quantization. The remarkable obversation is that the respective dynamics seems to reproduce distinguished numerical values of coupling constants that coincide with those observed in the standard model of particle physic. The results of this thesis give insights into the chaotic string model and its network generalization from a dynamical point of view. This leads to a deeper understanding of the dynamics, which is essential for a critical discussion of possible physical embeddings. Apart from this specific application to particle physics, the investigated concepts like synchronization or a most random behaviour of the dynamics are of general interest for dynamical system theory and the science of complex networks. As a first approach, discrete symmetry transformations of the model are studied. These transformations are formulated in a general way in order to be also applicable to similar dynamics on bipartite network structures. An observable of main interest in the chaotic string model is the interaction energy. In Spatio-temporal chaos and vacuum fluctuations of quantized fields' (2002) it has been observed that certain chaotic string couplings, corresponding to a vanishing interaction energy, coincide with coupling constants of the standard model of elementary particle physics. Since the interaction energy is basically a spatial correlation measure, an interpretation of the respective dynamical states in terms of a most random behaviour is tempting. In order to distinguish certain states as most random', or evoke another dynamical principle, a deeper understanding of the dynamics essential. In the present thesis the dynamics is studied numerically via Lyapunov measures, spatial correlations, and ergodic properties. It is shown that the zeros of the interaction energy are distinguished only with respect to this specific observable, but not by a more general dynamical principle. The original chaotic string model is defined on a one-dimensional lattice (ring-network) as the underlying network topology. This thesis studies a modification of the model based on the introduction of tunable disorder. The effects of inhomogeneous coupling weights as well as small-world perturbations of the ring-network structure on the interaction energy are discussed. Synchronization properties of the chaotic string model and its network generalization are studied in later chapters of this thesis. The analysis is based on the master stability formalism, which relates the stability of the synchronized state to the spectral properties of the network. Apart from complete synchronization, where the dynamics at all nodes of the network coincide, also two-cluster synchronization on bipartite networks is studied. For both types of synchronization it is shown that depending on the type of coupling the synchronized dynamics can display chaotic as well as periodic or quasi-periodic behaviour. The semi-analytical calculations reveal that the respective synchronized states are often stable for a wide range of coupling values even for the ring-network, although the respective basins of attraction may inhabit only a small fraction of the phase space. To provide analytical results in closed form, for complete synchronization the stability of all fixed points and period-2 orbits of all chaotic string networks are determined analytically. The master stability formalism allows to treat the ring-network of the chaotic string model as a special case, but the results are valid for coupled Tchebycheff maps on arbitrary networks. For two-cluster synchronization on bipartite networks, selected fixed points and period-2 orbits are analyzed.
The adaptive immune system of jawed vertebrates is based on recognition and elimination of cells that are either invaded by intracellular pathogens or malignantly transformed. One essential component of these processes is the cell surface presentation of antigenic peptides via major histocompatibility complex (MHC) class I molecules to cytotoxic T-cells (CTLs). Cells degrade defective ribosomal products and misfolded or unwanted proteins by the ubiquitin-proteasome pathway. The resulting degradation products are recognized and translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum (ER) lumen, where they are loaded onto MHC I molecules. Assembled peptide-MHC complexes are then shuttled by the secretory pathway to the cell surface for antigen presentation to CTLs, leading in the case of viral infection or malignant transformation to lysis and apoptosis of the target cell. Due to the fact that the TAP complex represents a key control point within the antigen presentation pathway, several viruses have evolved sophisticated strategies to evade immune surveillance by interfering with TAP function.
Detailed studies of the TAP mechanism or its viral inhibition have been severely impeded by difficulties in expressing sufficient amounts of functional heterodimeric TAP complex. Thus, the overexpression of TAP in the methylotrophic yeast Pichia pastoris was established for functional analysis of this important ABC complex. Biomass production was scaled up by fermentation using classical batch and feed methods. Extensive screening of optimal solubilization and purification conditions allowed the isolation of the heterodimeric transport complex. Notably, only the very mild detergent digitonin preserved TAP function. Hereby, the optimal solubilization and purification strategy yielded in 30 mg TAP transporter per liter culture. Remarkably, the protein amount was 50-fold increased compared to previously described expression/purification in cultured insect cells.
The high yield and quality of TAP produced in P. pastoris allowed an extensive analysis of substrate binding and transport kinetics of the transport complex in the membrane, its solubilized and purified state, as well as the reconstituted state. Thereby, a strong and direct effect of the lipid bilayer on ATP hydrolysis and peptide transport was discovered. These important results were extended further by successful functional reconstitution of the antigen translocation machinery in different lipid environments. For the first time, a stimulation of the transport activity by phosphatidylinositol (PI) and phosphatidylethanolamine (PE) was observed, whereas cholesterol was identified as an inhibitor of TAP activity.
Purification of TAP and subsequent thin-layer chromatography (TLC)/liquid chromatography Fourier transform-mass spectrometry (LC FT-MS) fingerprinting of residual lipids exhibited specifically associated glycerophospholipids; mainly PC, PE, and PI species. Strikingly, these lipids not only represent the primary class of phospholipids of the ER but were also shown to be essential for functional reactivation of delipidated, and thus inactive, TAP. The results demonstrate that transport of antigenic peptides by the ABC transporter TAP strictly requires specific glycerophospholipids.
In addition to the biochemical characterization of heterologous produced TAP, the soluble domain of the viral inhibitor US6 from human cytomegalovirus was expressed in E. coli. Optimization of the purification and refolding strategy yielded in functional protein, with a 35-fold increased protein amount compared to previous purification procedures. Protein activity was analyzed by specific inhibition of ATP binding to TAP. Furthermore, high protein yields allowed detailed investigation of TAP-dependent spatial and mechanistic separation of MHC I restricted cross-presentation in professional antigen presenting cells (pAPC).
Organische Materialien haben bis zur Mitte des 20. Jahrhunderts hinsichtlich ihrer elektronischen Eigenschaften keine besondere Aufmerksamkeit auf sich gezogen. Größeres Interesse an diesen Materialien entstand erst durch die Entdeckung einer ungewöhnlich hohen elektrischen Leitfähigkeit des organischen Perylen-Bromin Ladungstransfer-Komplexes durch Inokuchi et al. im Jahr 1954. Diese neue Klasse von Materialien besteht typischerweise aus Donor- und Akzeptor-Molekülen, die in einer bestimmten Stöchiometrie aneinander gebunden sind. Elektrische Ladung wird zwischen den Donor- und Akzeptor-Molekülen transferiert. Um diesen Prozess zu beschreiben, entwickelte Robert Mulliken in den 60er Jahren ein theoretisches Gerüst. Abhängig von der Anordnung der Moleküle und transferierten elektrischen Ladung kann der Ladungstransfer-Komplex (oder Salz) ein Isolator, ein Halbleiter, ein Metall oder sogar ein Supraleiter sein. Noch mehr Aufmerksamkeit erhielten Ladungstransfer-Materialien mit der Entdeckung des ersten quasi-eindimensionalen organischen Metalls TTF-TCNQ (tetrathiafulvalene-tetracyanoquinodimethane) im Jahr 1973. ...
Working memory (WM) contributes to countless activities during everyday live: reading, holding a conversation, making tea and so on. The core processes of WM comprise the phases of encoding, maintenance and retrieval. Successful recognition of stored objects requires several subprocesses such as stimulus encoding and evaluation, memory search and the organisation of a decision and a response. Much research has focused on encoding and maintenance of information but little interest has been directed to the retrieval of information. This is why the present dissertation investigated the neuronal correlates of retrieval of previously stored information and its modulation by load and probe-item similarity.
Here memory load and probe-item similarity were manipulated in order to investigate the neuronal correlates of the recognition process using electroencephalography (EEG). We tested the hypothesis recognition is influenced differently by probe-item similarity and by memory load and that these factors are re Effected by distinct neuronal correlates. Furthermore we tested whether distinct neuronal responses could be related to a summed similarity model.
The analysis of high-density ERP recordings showed both a load effect (load 1>load 3) and a similarity effect In addition, there was an interaction between load and similarity. The load effect was present during the whole epoch and did not change over time, whereas the similarity effect showed two distinct components between 300-600ms. In contrast to the load effect the similarity effect changed its sign over time. For the rest component, match probes elicited the strongest ERP responses, whereas for the second component dissimilar probes yielded the strongest ERP responses. The timing of the similarity effect corresponded well with the early and late P3b complex. The P3b complex is associated with stimulus categorisation and evaluation (early subcomponent) and memory search and criterion testing (late subcomponent).
The results suggest that the difficulty of a task is not only determined by load but also enhanced by probe-item similarity. Since increasing the number of samples (i.e. memory load) can also increase the probe-item similarity (i.e. the probability that one of the samples is perceptually similar to the probe), an independent manipulation of both factors is indispensable to disentangle their particular impact on short-term recognition. Furthermore, I propose that the two distinct neural correlates of the P3b complex reeffects different stages of task processing connected with probe-item similarity. As suggested by summed similarity VI models, these components might reflect the subprocesses of similarity summation (early P3b) and criterion testing (late P3b).