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In this thesis a new and powerful approach for modeling laser cavity eigenmodes is presented. This approach is based on an eigenvalue problem for singularly perturbed partial differential operators with complex coefficients; such operators have not been investigated in detail until now. The eigenvalue problem is discretized by finite elements, and convergence of the approximate solution is proved by using an abstract convergence theory also developed in this dissertation. This theory for the convergence of an approximate solution of a (quadratic) eigenvalue problem, which particularly can be applied to a finite element discretization, is interesting on its own, since the ideas can conceivably be used to handle equations with a more complex nonlinearity. The discretized eigenvalue problem essentially is solved by preconditioned GMRES, where the preconditioner is constructed according to the underlying physics of the problem. The power and correctness of the new approach for computing laser cavity eigenmodes is clearly demonstrated by successfully simulating a variety of different cavity configurations. The thesis is organized as follows: Chapter 1 contains a short overview on solving the so-called Helmholtz equation with the help of finite elements. The main part of Chapter 2 is dedicated to the analysis of a one-dimensional model problem containing the main idea of a new model for laser cavity eigenmodes which is derived in detail in Chapter 3. Chapter 4 comprises a convergence theory for the approximate solution of quadratic eigenvalue problems. In Chapter 5, a stabilized finite element discretization of the new model is described and its convergence is proved by applying the theory of Chapter 4. Chapter 6 contains computational aspects of solving the resulting system of equations and, finally, Chapter 7 presents numerical results for various configurations, demonstrating the practical relevance of our new approach.
Subject of this work was to investigate the influence of nonadiabatic coupling on the dynamical changes of electron and nuclear density. The properties of electron density have neither been discussed in the stationary case, nor for excited electronic states or for a coupled electronic and nuclear motion. In order to remove these restrictions one must describe the quantum mechanical motion of all particles in a system at the same level. This is only possible for very small systems. A model system developed by Shin and Metiu [1, 2] contains all necessary physical ingredients to describe a combined electronic and nuclear motion. It consists of a single nuclear and electronic degree of freedom and the particle interaction is parameterized in such a way as to allow for a facile switching between and adiabatic (Born-Oppenheimer type) and a strongly coupled dynamics. The first part of the work determined the “static” properties of the model system: The calculation of electronic eigenfunctions, adiabatic potential curves, kinetic coupling elements and transition dipole moments allowed for a prediction of the coupled dynamics. The potentials obtained from different parameterization showed two distinct cases: In the first case the ground and first excited state are separated by a large energy gap which is the typical Born-Oppenheimer case; the second one exhibits an avoided crossing which results in a breakdown of the adiabatic approximation. Due to the electronic properties of the system, the quantum dynamics in the two distinct situations is very different. This was illustrated by calculating nuclear and electron densities as a function of time. In the Born-Oppenheimer case, the electron density followed the vibrational motion of the nucleus. This was demonstrated in two examples. In the strongly coupled case the wave packet did not exhibit features caused by nonadiabatic coupling. However, projections of the wave function onto the electronic states revealed the usual picture obtained from solutions of the nuclear Schrödinger equation involving coupled electronic states. In that case the nuclear motion triggered charge transfer via nonadiabatic coupling. The second part of the work demonstrated that the model system can easily be modified to yield binding situations often found in diatomic molecules. The different situations can be characterized in terms of bound and dissociative adiabatic potential curves. The investigation focussed on the case of an electronic predissociation, where the ground state is dissociative in the asymptotic limit of large internuclear distances. Within our model system we were able to demonstrate how the character of the electron density changes during the fragmentation process. In the third part we investigated the influence of external fields on the correlated dynamics of electron and nucleus. Employing adiabatic potential curves, the structure of absorption spectra can be understood within the weak-field limit. In the above described Born-Oppenheimer case the adiabatically calculated spectrum was in very good agreement with the exact one, whereas in the strongly coupled case the obtained spectrum was not able to resemble the exact one. Regarding the dynamics during a laser excitation process the time-dependent electron and nuclear densities nicely illustrated the famous Franck-Condon principle. The interaction with strong laser pulses lead to an excitation of many bound electronic and vibrational states. The electron density reflected the classical-like quiver motion of the electron induced by the fast variations of the electric field. The nucleus did not follow these fast oscillations because of its much larger mass. The last part of the work extended the original model system by including an additional electron. As a consequence of the Pauli principle, the spatial electronic wave function has to be either symmetric or anti-symmetric with respect to exchange of the two electrons. This corresponds to anti-parallel or parallel electron spins, respectively. The extended model already contains the physical properties of a many-electron system. Solving the time-dependent Schrödinger equation for a typical vibrational wave packet motion clearly indicated that the electron density is no longer suited to “localize” single electrons. We extended the definition of the electron localization function (ELF) to an exact, time-dependent wave function and demonstrated, how the ELF can be used to further characterize a coupled electron and nuclear motion. Finally, we gave an outlook of how to define electron localization in the case of anti-parallel electron spins. We derived a quantity similar to the ELF denoted “anti-parallel spin electron localization function” (ALF) and demonstrated that the ALF allows to follow time-dependent changes of the electron localization in a numerical example. [1] S. Shin, H. Metiu, J. Chem. Phys. 1995, 102, 9285. [2] S. Shin, H. Metiu, J. Phys. Chem. 1996, 100, 7867.
This work is subdivided into two main areas: resilient admission control and resilient routing. The work gives an overview of the state of the art of quality of service mechanisms in communication networks and proposes a categorization of admission control (AC) methods. These approaches are investigated regarding performance, more precisely, regarding the potential resource utilization by dimensioning the capacity for a network with a given topology, traffic matrix, and a required flow blocking probability. In case of a failure, the affected traffic is rerouted over backup paths which increases the traffic rate on the respective links. To guarantee the effectiveness of admission control also in failure scenarios, the increased traffic rate must be taken into account for capacity dimensioning and leads to resilient AC. Capacity dimensioning is not feasible for existing networks with already given link capacities. For the application of resilient NAC in this case, the size of distributed AC budgets must be adapted according to the traffic matrix in such a way that the maximum blocking probability for all flows is minimized and that the capacity of all links is not exceeded by the admissible traffic rate in any failure scenario. Several algorithms for the solution of that problem are presented and compared regarding their efficiency and fairness. A prototype for resilient AC was implemented in the laboratories of Siemens AG in Munich within the scope of the project KING. Resilience requires additional capacity on the backup paths for failure scenarios. The amount of this backup capacity depends on the routing and can be minimized by routing optimization. New protection switching mechanisms are presented that deviate the traffic quickly around outage locations. They are simple and can be implemented, e.g, by MPLS technology. The Self-Protecting Multi-Path (SPM) is a multi-path consisting of disjoint partial paths. The traffic is distributed over all faultless partial paths according to an optimized load balancing function both in the working case and in failure scenarios. Performance studies show that the network topology and the traffic matrix also influence the amount of required backup capacity significantly. The example of the COST-239 network illustrates that conventional shortest path routing may need 50% more capacity than the optimized SPM if all single link and node failures are protected.
Identification of rat NKT cells and molecular analysis of their surface receptor mediated activation
(2004)
Summary: Originally, NKT cells have been defined by their expression of T-cell receptor (TCR) and NK cell markers NKRP1A in human and NK1.1 (NKRP1C) in mouse. Most of these cells express CD1d-restricted TCR with a characteristic rearrangement- Va24JaQ/Vb11 in human and Va14Ja18/Vb8.2 in mouse, and have been implicated in playing an important role in first line defence and immunoregulation. The subject of this thesis was the characterisation of the hypothetical rat NKT cell population. In the mouse system, CD1d-restricted NK1.1+ T cells represented around 30% of intrahepatic and around 3% of splenic lymphocytes and could be visualised by staining with a-GalCer-loaded mouse CD1d tetramer. Rat NKRP1A+TCR+ cells, similar to mouse NKT cells, were predominantly expressed in the liver. However, their frequency was around 5 fold lower than the frequency of mouse intrahepatic lymphocytes. F344 rat NKT cells, in contrast to mouse CD4+ or DN NK1.1+ T lymphocytes, were of CD8 rather than CD4 phenotype, and did not bind to mCD1d-a-GalCer-tetramer. Since human hepatic CD1d-restricted Va24JQ+ T cells are not as frequent as their mouse counterparts and may express CD8- a marker not expressed by mouse CD1d-restricted cells, it is possible that the phenotype of F344 rat NKT cells corresponds more to the phenotype of human than mouse NKT cells. Similar to mouse NKT cells, F344 rat liver- and spleen-derived lymphocytes were able to produce IL-4 and IFN-g; when stimulated with the synthetic ligand a-GalCer in vitro. Therefore, the lack of binding of rat lymphocytes to mouse CD1d tetramer could not be due to their inability to respond to a-GalCer. To better characterise the reactivity of rat NKRP1A+TCR+ cells to a-GalCer, the rat invariant TCR was analysed. RT-PCR of liver lymphocytes with Va14-specific primers and subsequent cloning revealed a much weaker PCR signal for rat lymphocyte cDNA than for mouse cDNA. Furthermore the analysis of rat AV14JA18 sequences showed that the rat Va14+TCR invariant could be rearranged not only with AJ18 but also with other AJ segments. The low number of clones with in frame Va14Ja18 rearrangement could suggest that only a small proportion of liver lymphocytes were CD1d restricted NKT cells. Mouse and human NKT cells are able to recognise a-GalCer presented by the CD1d-b2 microglobulin complex, leading to their activation, proliferation and cytokine secretion. In order to compare the capacity of mouse and rat CD1d to present a-GalCer, rat CD1d was cloned. Sequence analysis and functional tests in vitro confirmed the structural and functional homology of rat CD1d with mouse CD1d. In parallel, to characterise the reactivity of rat NKRP1A+TCR+ cells to a-GalCer, rat Va14+TCR invariant was cloned and expressed in the TCR- T cell hybridoma BWr/mCD28. Rat Va14TCR+CD28+ transgenic cells secreted IL-2 upon aTCR/CD3 antibody stimulation, but were not specific for a-GalCer. Such cells were also negative in staining with mCD1d-a-GalCer tetramer. The lack of reactivity to a-GalCer and the lack of binding to mouse tetramer were probably caused by amino acid alterations, particularly at position 72 (51 according to IMTG nomenclature) of cloned rat TCRinv. Reversal of these “alterations” using molecular biology techniques was performed but the expression of this TCR on the surface of BWr/mCD28 cells could not be achieved. In contrast to rat TCRinv, mouse Va14+TCR was fully functional and was specific for mouse CD1d tetramer. KT12 hybridoma and BWr/mCD28 cells expressing mouse TCRinv, when stimulated with a-GalCer presented by primary CD1d+ cells or rCD1d transgenic cell lines, produced IL-2 in an Ag- and CD1d-dependent manner. Transgenic lines expressing TCR comprising mouse Va14 and rat Vb8.4 responded to a-GalCer presented by rat and mouse CD1d, and bound mCD1 tetramer. By contrast, cell lines expressing TCR comprising mouse Va14 and rat Vb8.2 responded only to a-GalCer presented by rCD1d and bound weakly to mCD1d tetramer. This suggests that germ line encoded regions of the b-chain (CDR2 or CDR4) bind to species-specific determinants of CD1d. The cytokine secretion of the cell lines was inhibited by anti-CD80 mAb, indicating the importance of CD80-CD28 costimulation in their activation. To check whether rat NKT cells may exist in other rat strains, the frequency and functions of NKRP1A+TCR+ in F344 and LEW rat were compared. F344 and LEW, two rat strains expressing different allelic CD1d forms, varied slightly in the level of CD1d expression, as assessed by staining with a newly generated CD1d specific monoclonal antibody. By contrast, these rat strains differed in terms of a-GalCer recognition. NKRP1A+TCR+ cells were less frequent in LEW than in F344 rats, and did not respond to a-GalCer or the analogue OCH in vitro, a result which is of special interest considering the susceptibility of LEW but not F344 rats to experimentally induced organ specific autoimmune diseases. In summary, the rat and mouse CD1d-invariant TCR systems show a high degree of structural and functional homology, but it seems that invariant NKT cells in rat, similar to such cells in human, occur at lower frequency than in mice. TCR transgenic cell line species-specific patterns of CD1d a-GalCer reactivity will provide a valuable tool for the mapping of CD1d/TCR contacts. Also monoclonal antibodies specific for rat and mouse CD1d, generated in this study, provide valuable tools to determine CD1d protein expression in various rat tissues and will help to better characterise functions of CD1d-restricted rat T cells.
The role of DNA supercoiling in the coordinated regulation of gene expression in Helicobacter pylori
(2004)
Summary Mechanisms of global gene regulation in bacteria are not well characterized yet. Changes in global or local supercoiling of chromosomal DNA are thought to play a role in global gene silencing and gene activation. In Helicobacter pylori, a bacterium with few dedicated transcriptional regulators, the structure of some promoters indicates a dependency on DNA topology. For example, the promoter of the major flagellar subunit gene flaA (ó28-dependent) has a shorter spacing of 13 nucleotides (nt) in comparison to the consensus promoter (15 nt). Supercoiling changes might be a mechanism of gene-specific and global transcriptional regulation in this bacterium. The aim of this study was to elucidate, if changes in global supercoiling have an influence on global gene regulation in H. pylori, and on the temporal regulation of the flagellar biosynthesis pathway in this organism. In the present work, global DNA supercoiling in H. pylori was visualized for the first time, by determining the supercoiling state of plasmids under different growth conditions. Using this method, we showed that cellular supercoiling was clearly growth phase-dependent in H. pylori. Coinciding with increased supercoiling during the growth phases, transcription of the flaA gene was increased, while the transcription of a second ó28-dependent gene with regular promoter spacing (HP0472) was reduced, supporting the hypothesis that growth phase-dependency of promoters might be mediated by changes of DNA topology. Supercoiling in H. pylori could be influenced in a reproducible fashion by inhibition of gyrase using novobiocin, which led to DNA relaxation and to a concomitant decrease of flaA transcript levels. Promoter spacer mutagenesis of the flaA promoter was performed. With flaA promoters of increased or reduced length, transcription of flaA was reduced, less susceptible to supercoiling changes, and, under specific conditions, inverted as compared to the wild type promoter. Transcriptional interdependence between the coupled topA-flaB genes and flaA was found by analysis of the flaA promoter mutants. Chromosomally linked gyrA-flgR, and topA-flaB genes were all dependent on supercoiling and coregulated with each other. Comprehensive transcript profiling (DNA microarrays) of wildtype H. pylori with and without novobiocin treatment identified a number of genes (10% of total genes), including flagellin, virulence and housekeeping genes, which were strongly dependent on and appeared to be synchronized by supercoiling changes (transcriptional up- or downregulation). These findings indicate a tightly coupled temporal regulation of flagellar biogenesis and metabolism in H. pylori, dependent on global supercoiling. A specific group of genes was also regulated in H. pylori by overexpression of Topoisomerase I, as detected by genome-wide analysis (DNA microarray). The DNA-bending protein HU is thought to be responsible for influencing the negative supercoiling of DNA, through its ability to wrap DNA. HU is encoded by the hup single gene in H. pylori, and constitutively expressed during the whole growth curve. An H. pylori hup mutant was constructed. H. pylori cells lacking HU protein were viable, but exhibited a severe growth defect. Our data indicate that the lack of HU dramatically changes global DNA supercoiling, indicating an important function of HU in chromosome structuring in H. pylori. Transcriptome analyses were performed and demonstrated that a total of 66 genes were differentially transcribed upon hup deletion, which include virulence genes and many other cell functions. The data indicate that HU might act as further important global regulator in H. pylori. Increased gene expression of heat shock proteins and a decreased transcription of the urease gene cluster may indicate a co-ordinated response of H. pylori to changes of environmental conditions in its specific ecological niche, mediated by HU. After the whole genomic sequences of H. pylori strains 26695 and J99 were published, two ORFs (HP0116 and HP0440) were presumptively annotated as topoisomerase I orthologs. HP0116 is the functional H. pylori topoisomerase I (TopA). HP0440 (topA2) was found in only few (5 of 43) strains. Western blot analysis indicated that TopA2 is antigenically different from TopA. TopA2 is transcribed in H. pylori, but the protein must be functionally different from TopA, since it is lacking one functionally essential zinc finger motif, and was not able to functionally complement a TopA-deficient E. coli. Like topA, topA2 was also transcribed in a growth phase-dependent manner. We did not find a function of TopA2 in DNA structuring or topology, but, in the present study, we were able for the first time to establish a unique function for TopA2 in global gene regulation, by comprehensive transcriptome analysis (DNA microarray). Transcriptome analysis showed that a total of 46 genes were differentially regulated upon topA2 deletion, which included flagellar genes and urease genes. These results suggest that TopA2 might act as a novel important regulator of both flagellar biosynthesis and urease in H. pylori.
Summary Using the facultative root hemiparasite Rhinanthus minor and Hordeum vulgare as a host, several aspects of water relations, the flows and partitioning of mineral nutrients, the flows, depositions and metabolism of abscisic acid (ABA) and zeatin type cytokinins (zeatin Z, zeatin riboside ZR, zeatin nucleotide ZN) within the host, the parasite and between host and parasite and the flows and partitioning of the transport metabolites mannitol in the parasite, and of sucrose in the host, have been studied during the study period 41 to 54 days after planting, i.e about 30 to 43 days after successful attachment of the parasite to the host. Water relations Extraction of xylem sap by the parasite from the host’s roots is facilitated by considerably higher transpiration per leaf area in the parasite than in the host and by the fact that stomata of attached Rhinanthus were wide open all day and night despite extremely high ABA concentrations in the leaves. By comparison, another related root hemiparasite, Melampyrum arvense, parasitising on various grasses in the field (botanic garden), showed normal diurnal stomatal behaviour. The abnormal behaviour of Rhinanthus stomata was not due to anatomical reasons as closure could be induced by applying high external ABA concentrations. Remarkable differences have been detected between the hydraulic conductance of barley seminal roots showing relatively low values, and that of Rhinanthus the seminal root showing very high values. The latter could be related to the observed high ABA concentrations in these roots. Whole plant water uptake, transpirational losses, growth-dependent deposition and the flows of water within the plants have been measured in singly growing Rhinanthus and Hordeum plants and in the parasitic association between the two. Water uptake, deposition and transpiration in Rhinanthus were dramatically increased after attachment to the barley host; most of the water used by the parasite was extracted as xylem sap from the host, thereby scavenging 20% of the total water taken up by the host’s roots. This water uptake by the parasitised host, however, due to a parasite induced reduction in the hosts growth, was decreased by 22% as compared to non- parasitised barley. The overall changes in growth-related water deposition in host and parasite pointed to decreased shoot and relatively favoured root growth in the host and to strongly favoured shoot growth and less strongly increased root growth only in the parasite. These changes in the host became more severe, when more than one Rhinanthus was parasitising one barley plant. Mineral nutrients relations 5 mM NO3- supply In parasitising Rhinanthus shoot growth was 12-fold, but root growth only twofold increased compared to the non-parasitising (very small) plants. On the other hand, in the Hordeum host, shoot dry matter growth was clearly reduced, by 33% in leaf laminae and by 52% in leaf sheaths, whereas root growth was only slightly reduced as a consequence of parasitism. Growth-dependent increments of total N and P and of K, Ca and Mg in parasitising Rhinanthus shoot were strongly increased, particularly increments of total N and P, which were 18 and 42 times, respectively, higher than in the small solitary Rhinanthus. On the other hand, increments of the above mineral nutrients in leaf sheaths of parasitised Hordeum vulgare were more strongly decreased than in leaf laminae in response to parasitic attack. Estimation of the flows of nutrients revealed that Rhinanthus withdrew from the host xylem sap about the same percentage of each nutrients: 18% of total N, 22% of P and 20% of K. Within the host almost all net flows of nutrient ions were decreased due to parasitism, but retranslocation from shoot to root-as related to xylem flow-was somewhat increased for all nutrients. Quantitative information is provided to show that the substantially increased growth in the shoot of attached Rhinanthus and the observed decrease in Hordeum shoot growth after infection were related to strongly elevated supply of nitrogen and phosphorus in the parasite and to incipient deficiency of these nutrients in the parasitised host. The flows of nutrients between host and parasite are discussed in terms of low selectivity of nutrient abstraction from the host xylem by the hemiparasite Rhinanthus minor. 1 mM NO3- or 1 mM NH4+ supply Rhinanthus shoot growth as measured by dry matter increase, was 19-fold (1 mM NO3-) and 15-fold (1 mM NH4+), but root growth only twofold (1 mM NO3-) and 2.9-fold (1 mM NH4+) increased-relative to singly growing Rhinanthus-when parasitising on host barley. In the Hordeum host, shoot dry matter growth was clearly reduced, whereas root growth was only slightly affected. Growth-dependent increments of total N and P and of K, Ca and Mg in parasitising Rhinanthus shoot were strongly increased, particularly increments of total N or of P, which were 20 or 53 times (1 mM NO3-) and 18 or 51 times (1 mM NH4+) , respectively, higher than those in solitary Rhinanthus. Within the host almost all net flows of nutrient ions were decreased due to parasitism. Flows of mannitol in parasite and sucrose flows in host barley When the plants were supplied with 5 mM NO3-, the biosynthesis of mannitol in Rhinanthus shoots increased 16-fold by parasitism, resulting in a 15-fold higher mannitol flow in the phloem and a 10-fold higher deposition in the shoot. Also the backward transport of mannitol in the xylem were increased 10-fold after attachment. Lower level nitrogen supply increased the deposition of mannitol in both single and attached Rhinanthus shoot and root. No mannitol was found in barley roots even in the direct vicinity of the haustoria. This indicates there are no backward transport of xylem sap from parasite to host. Compared to unparasitised barley, the net biosynthesis and deposition of sucrose in the shoot and the phloem flow was decreased substantially when plants were supplied with 5 mM NO3- or 1 mM NO3-. No sucrose has been detected in barley xylem sap and consequently there was no indication of a sucrose transfer from the host to the parasite. A possible involvement of mannitol in the abscisic acid relations of the parasite is discussed. ABA relations When the plants were supplied with 5 mM NO3-, there were weak or no effects of parasitism on ABA flows, biosynthesis and ABA degradation in barley. However, ABA growth-dependent deposition was significantly increased in the leaf laminae (3 fold) and in leaf sheath (2.4 fold), but not in roots. Dramatic changes in ABA flows, metabolism and deposition on a per plant basis, however, have been observed in Rhinanthus. Biosynthesis in the roots was 12-fold higher after attachment resulting in 14-fold higher ABA flows in the xylem. A large portion of this ABA was metabolised, a small portion was deposited. Phloem flows of ABA were increased 13-fold after attachment. The concentrations of ABA in tissues and xylem sap were higher in attached Rhinanthus by an order of magnitude than in host tissues and xylem sap. Similar dramatic difference existed when comparing the high concentrations in the xylem sap of single Rhinanthus with unparasitised barley. As compared to 5 mM NO3-, lower NO3- or 1 mM NH4+ supply doubled the ABA concentrations in barley leaf laminae, while having only small or no significant effects in the other organs. The possible special functions of ABA for the parasite are discussed. Zeatin type cytokinins relations Parasitism decreased, in the case of zeatin (Z), the synthesis (by 57%) in the root, xylem flows (by 56%) and metabolism (by 71%) in leaf laminae, however, increased the phloem flows of zeatin massively (3-fold) in host barley. The deposition of zeatin in the root of Rhinanthus and the flowing in xylem and phloem were 24, 12, 29-fold, respectively, increased after successfully attaching to the host barley. However, net biosynthesis of zeatin in Rhinanthus roots decreased by 39% after attachment. This indicates that a large portion (70%) of xylem flow of zeatin in attached Rhinanthus was extracted from the host. In singly growing Rhinanthus plants, the balance of zeatin deposition in the shoot was negative, i.e. zeatin was metabolised and exported back to root in the phloem. The xylem flows of zeatin riboside (ZR) in barley decreased by 39% after infected by Rhinanthus; phloem flow, which was 117% relative to xylem flow was less decreased (by 13%) after infection. Deposition of ZR has not been significantly affected in the leaf laminae, in leaf sheaths and roots. After parasitising on the host barley depositions in root, xylem flow and phloem flow increased 12, 18, 88–fold respectively in Rhinanthus. A large portion (57%) of xylem flow of ZR in attached Rhinanthus was extracted from the host. In single Rhinanthus increament of shoot zeatin riboside was negative and a substantial portion was degraded in shoot and the rest was retranslocated back to the root in the phloem. A significant depositions of Z and ZR were detected in the haustoria of the Rhinanthus/barley association. Flows and deposition of zeatin nucleotides also have been investigated. The possible physiological functions of the large quantities of Z and ZR derived from the host barley, for the improved growth and the stomatal opening in the parasitising Rhinanthus are discussed.
The availability of coherent soft x-rays through the nonlinear optical process of high-harmonic generation allows for the monitoring of the fastest events ever observed in the laboratory. The attosecond pulses produced are the fundamental tool for the time-resolved study of electron motion in atoms, molecules, clusters, liquids and solids in the future. However, in order to exploit the full potential of this new tool it is necessary to control the coherent soft x-ray spectra and to enhance the efficiency of conversion from laser light to the soft x-ray region in the harmonic-generation process. This work developed a comprehensive approach towards the optimization of the harmonic generation process. As this process represents a fundamental example of \emph{light}--\emph{matter} interaction there are two ways of controlling it: Shaping the generating laser \emph{light} and designing ideal states of \emph{matter} for the conversion medium. Either of these approaches was closely examined. In addition, going far beyond simply enhancing the conversion process it could be shown that the qualitative spectral response of the process can be modified by shaping the driving laser pulse. This opens the door to a completely new field of research: Optimal quantum control in the attosecond soft x-ray region---the realm of electron dynamics. In the same way as it is possible to control molecular or lattice vibrational dynamics with adaptively shaped femtosecond laser pulses these days, it will now be feasible to perform real-time manipulation of tightly bound electron motion with adaptively shaped attosecond light fields. The last part of this work demonstrated the capability of the herein developed technique of coherent soft-x-ray spectral shaping, where a measured experimental feedback was used to perform a closed-loop optimization of the interaction of shaped soft x-ray light with a sulfur hexafluoride molecule to arrive at different control objectives. For the optimization of the high-harmonic-generation process by engineering the conversion medium, both the gas phase and the liquid phase were explored both in experiment and theory. Molecular media were demonstrated to behave more efficiently than commonly used atomic targets when elliptically polarized driving laser pulses are applied. Theory predicted enhancement of harmonic generation for linearly polarized driving fields when the internuclear distance is increased. Reasons for this are identified as the increased overlap of the returning electron wavefunction due to molecular geometry and the control over the delocalization of the initial electronic state leading to less quantum-mechanical spreading of the electron wavepacket during continuum propagation. A new experimental scheme has been worked out, using the method of molecular wavepacket generation as a tool to enhance the harmonic conversion efficiency in `pump--drive' schemes. The latter was then experimentally implemented in the study of high-harmonic generation from water microdroplets. A transition between the dominant laser--soft-x-ray conversion mechanisms could be observed, identifying plasma-breakdown as the fundamental limit of high-density high-harmonic generation. Harmonics up to the 27th order were observed for optimally laser-prepared water droplets. To control the high-harmonic generation process by the application of shaped laser light fields a laser-pulse shaper based on a deformable membrane mirror was built. Pulse-shape optimization resulted in increased high-harmonic generation efficiency --- but more importantly the qualitative shape of the spectral response could be significantly modified for high-harmonic generation in waveguides. By adaptive optimization employing closed-loop strategies it was possible to selectively generate narrow (single harmonics) and broad bands of harmonic emission. Tunability could be demonstrated both for single harmonic orders and larger regions of several harmonics. Whereas any previous experiment reported to date always produced a plateau of equally intense harmonics, it has been possible to demonstrate ``untypical'' harmonic soft x-ray spectra exhibiting ``switched-off'' harmonic orders. The high degree of controllability paves the way for quantum control experiments in the soft x-ray spectral region. It was also demonstrated that the degree of control over the soft x-ray shape depends on the high-harmonic generation geometry. Experiments performed in the gas jet could not change the relative emission strengths of neighboring harmonic orders. In the waveguide geometry, the relative harmonic yield of neighboring orders could be modified at high contrast ratios. A simulation based solely on the single atom response could not reproduce the experimentally observed contrast ratios, pointing to the importance of propagation (phase matching) effects as a reason for the high degree of controllability observed in capillaries, answering long-standing debates in the field. A prototype experiment was presented demonstrating the versatility of the developed soft x-ray shaping technique for quantum control in this hitherto unexplored wavelength region. Shaped high-harmonic spectra were again used in an adaptive feedback loop experiment to control the gas-phase photodissociation reaction of SF$_6$ molecules. A time-of-flight mass spectrometer was used for the detection of the ionic fragments. The branching ratios of particular fragmentation channels could be varied by optimally shaped soft x-ray light fields. Although in one case only slight changes of the branching ratio were possible, an optimal solution was found, proving the sufficient technical stability of this unique coherent soft-x-ray shaping method for future applications in optimal control. Active shaping of the spectral amplitude in coherent spectral regions of $\sim$10~eV bandwidth was shown to directly correspond to shaping the temporal features of the emerging soft x-ray pulses on sub-femtosecond time scales. This can be understood by the dualism of frequency and time with the Fourier transformation acting as translator. A quantum-mechanical simulation was used to clarify the magnitude of temporal control over the shape of the attosecond pulses produced in the high-harmonic-generation process. In conjunction with the experimental results, the first attosecond time-scale pulse shaper could thus be demonstrated in this work. The availability of femtosecond pulse shapers opened the field of adaptive femtosecond quantum control. The milestone idea of closed-loop feedback control to be implemented experimentally was expressed by Judson and Rabitz in their seminal work titled ``Teaching lasers to control molecules''. This present work extends and turns around this statement. Two fundamentally new achievements can now be added, which are ``Teaching molecules to control laser light conversion'' and ``Teaching lasers to control coherent soft x-ray light''. The original idea thus enabled the leap from femtosecond control of molecular dynamics into the new field of attosecond control of electron motion to be explored in the future. The \emph{closed}-loop approach could really \emph{open} the door towards fascinating new perspectives in science. Coming back to the introduction in order to close the loop, let us reconsider the analogy to the general chemical reaction. Photonic reaction control was presented by designing and engineering effective media (catalysts) and controlling the preparation of educt photons within the shaped laser pulses to selectively produce desired photonic target states in the soft x-ray spectral region. These newly synthesized target states in turn could be shown to be effective in the control of chemical reactions. The next step to be accomplished will be the control of sub-femtosecond time-scale electronic reactions with adaptively controlled coherent soft x-ray photon bunches. To that end a time-of-flight high-energy photoelectron spectrometer has recently been built, which will now allow to directly monitor electronic dynamics in atomic, molecular or solid state systems. Fundamentally new insights and applications of the nonlinear interaction of shaped attosecond soft x-ray pulses with matter can be expected from these experiments.
This thesis is concerned with the development of an on-line in-situ device for a chemical characterisation of flowing aerosols. The thesis describes the principles and most important features of such a system, allowing also on-line measurements using Raman spectroscopy as a diagnostic technique An analysis of the effect of forced oscillations on the motion of the particle dispersed in a gas flow is given in Chapter 2. Also the most important particle parameters are introduced. A review of the particle/fluid interaction in laminar air flows and the response of the particle is presented. In Chapter 3 the behaviour of the particle under different external conditions (ion bombardment and electric fields) is extended. A brief review of the most important particle charging theories (diffusion, field, and alternating potential charging) shows, that the effect of the electrical properties (represented by the dielectric constant) of the particles affects the charging process. A non-contact method for particle charge measurement was also presented. In the second part of the chapter, the interaction between the electric field and the charged particle for the purpose of particle trapping is illustrated. The most common systems like the two or four ring electrodynamic balance and the quadrupole trap are pointed out. In Chapter 4 a short review of the possibility of using scattered light to study aerosol particles is presented. First, the conditions and the facilities of using the Mie theory for particle size and refractive index determination are mentioned, then some features concerning the classical treatment of the Raman effect are presented Supported by the theoretical considerations exposed in Chapter 2, 3, and 4 the construction and the tests of different devices are presented in Chapter 5. Following the goal of the thesis, first an overview of the used materials and methods for particle generation is presented. Then, the constructed charging devices are described (from the mechanical and electrical point of view) and compared by measuring the acquired charge on the particle. Charged particles can be trapped in different containers. Two types of axially symmetric electrodynamic balances (two ring or an extended four ring configuration) were presented. For a deeper understanding these systems were studied using analytic and numerical methods. Considering the presented purpose of the work another type of trapping system has been developed, namely the quadrupole trap. A similar theoretical characterisation (in term’s of Mathieu equation) as for the electrodynamic balance was presented pointing out some specific features of this system. The incoming particle stream will be focused to the centre of the system simultaneously also the applied DC and AC potential onto the tube electrodes, yields a stable trapping of one or more particles. Chapter 6 consists of two parts: the system for single particle and for many particles investigation. The individual devices presented in Chapter 5 are now put together. The first part presents the method and the experimental realisation of a set-up for solid particle injection. In order to suppress the phase injection disadvantage found for the electrodynamic balance a developed program processes the information obtained from a particle cloud through an adequate electronic detection system, and reduces the number of particles until just one single particle is trapped. The method for one particle investigation can be extended for many particles. Using the presented set-up the particles are moved from one quadrupole to another and transformed from a particle cloud to a particle stream. A linearity between an external vertical mounted detector and the formed image of the particle stream on the CCD camera has been observed and used for simultaneous detection of many particles by Raman spectroscopy. For both methods Raman results are presented. One limitation of Raman Spectroscopy is the relatively long integration time needed for adequate signal-to-noise ratio. There are two factors which influence the integration time: first the incident radiation and the detector sensitivity, and second the intensity of the Raman bands. Using a CCD detector, the desired detector sensitivity should be achieved. So, the improvement of the signal-to-noise ratio should be the next goal in the system development. In order to reduce the integration time an optical system including optic fibres and the integration of an FT-Raman module operating in the visible region is planed. The goal of this work was to develop and construct an instrument for on-line in-situ single particle investigation by Raman spectroscopy. With the presented experimental set-up and the developed program the purpose of the work, the on-line in-situ near atmospheric pressure aerosol investigation was achieved. The Raman spectroscopy has been used successfully for a chemical characterisation of the aerosol particles.
The success of diagnostic knowledge systems has been proved over the last decades. Nowadays, intelligent systems are embedded in machines within various domains or are used in interaction with a user for solving problems. However, although such systems have been applied very successfully the development of a knowledge system is still a critical issue. Similarly to projects dealing with customized software at a highly innovative level a precise specification often cannot be given in advance. Moreover, necessary requirements of the knowledge system can be defined not until the project has been started or are changing during the development phase. Many success factors depend on the feedback given by users, which can be provided if preliminary demonstrations of the system can be delivered as soon as possible, e.g., for interactive systems validation the duration of the system dialog. This thesis motivates that classical, document-centered approaches cannot be applied in such a setting. We cope with this problem by introducing an agile process model for developing diagnostic knowledge systems, mainly inspired by the ideas of the eXtreme Programming methodology known in software engineering. The main aim of the presented work is to simplify the engineering process for domain specialists formalizing the knowledge themselves. The engineering process is supported at a primary level by the introduction of knowledge containers, that define an organized view of knowledge contained in the system. Consequently, we provide structured procedures as a recommendation for filling these containers. The actual knowledge is acquired and formalized right from start, and the integration to runnable knowledge systems is done continuously in order to allow for an early and concrete feedback. In contrast to related prototyping approaches the validity and maintainability of the collected knowledge is ensured by appropriate test methods and restructuring techniques, respectively. Additionally, we propose learning methods to support the knowledge acquisition process sufficiently. The practical significance of the process model strongly depends on the available tools supporting the application of the process model. We present the system family d3web and especially the system d3web.KnowME as a highly integrated development environment for diagnostic knowledge systems. The process model and its activities, respectively, are evaluated in two real life applications: in a medical and in an environmental project the benefits of the agile development are clearly demonstrated.
One primary source for self-knowledge is social comparison. Often objective criteria for self-evaluations are not available or useful and therefore comparisons with other people play a crucial role in self-evaluations. But the question is whether social comparisons could indeed provide information about the self without consuming too much cognitive resources or time. Therefore, in this research I wanted to look at practice effects in social comparison and the particular significance of routine standards. Whereas traditional research on standard selection mostly focused on goal-oriented and strategic standard selection processes, this research sets out to integrate social cognitive knowledge, ideas, and methods. Researchers from many different fields agree that people’s behavior and thinking is not fully determined by rational choices or normative considerations. Quite the contrary, factors like knowledge accessibility, habits, procedural practice, stereotyping, categorization, and many more cognitive processes play an important role. The same may be true in social comparison and standard selection. In my research I demonstrate that efficiency concerns play an important role in social comparison. Since people may not be able to engage in a strategic standard selection whenever they engage in social comparison processes, there has to be a more efficient alternative. Using routine standards would be such an alternative. The efficiency advantage of routine standards may thereby be founded not only in the abandonment of a strategic but arduous standard selection process, but also in a higher efficiency of the comparison process itself. I therefore set out to show how the use of routine standards facilitates the social comparison processes. This was done in three steps. First, I replicated and improved our former research (Mussweiler & Rüter, 2003, JPSP) indicating that people really do use their best friends as routine standards to evaluate themselves. Second, I demonstrated that it is more efficient to compare with a routine standard than with another standard. In Studies 2 and 3 I therefore show that comparisons between the self and a routine standard (either a natural routine standard like the best friend or a experimentally induced routine standard based on practice) are faster and more efficient than comparisons with other standards. Finally, I looked at the underlying mechanism of the efficiency advantage of routine standards. The results of Studies 4 and 5 point out, that both general as well as specific practice effects occur with repeated comparisons. Whereas a specific practice effect implies the repeated processing of the same content (i.e., knowledge about the routine standard), general practice effects indicate that the pure process (i.e., comparing the self with a routine standard) becomes more efficient regardless whether new content (i.e., comparison relevant knowledge) has to be processed. Taken together, the efficiency advantage of routine standards during self-evaluation is based not only on the lack of necessity for an arduous standard selection, but is additionally supported by the facilitation of the comparison process itself. The efficiency of routine standards may provide an explanation as to why people base self-evaluations on comparisons with these standards and dispense with strategic considerations to select the most suitable standard.
Cellular proliferation, differentiation and survival in response to extracellular signals are controlled by the signal transduction pathway of Ras, Raf and MAP kinase. The Raf proteins are serine/threonine kinases with essential function in growth/differentiation/survival - related signal transduction events. In mammals, three functional (A-, B-, and C-Raf) genes were described. Biochemical studies suggest overlapping and differential utilization of Raf isozymes. However, the frequent co-expression of Raf isozymes and their multiple activators and effectors impedes the full understanding of their specific roles. The elucidation of these roles is important due to the involvement of the Ras/Raf/MEK/MAP kinase cascade in human disorders especially in tumor development and progression. B-Raf was shown to posses the strongest kinase activity among Raf kinases and display antiapoptotic properties. Mice deficient in B-Raf show overall growth retardation and die between E10.5 and E12.5 of vascular defects caused by excessive death of differentiated endothelial cells. To elucidate the redundancy of Raf isozymes during embryonic development and to rescue B-Raf-/- (KO) phenotype, B-Raf alleles were disrupted by introducing A-Raf cDNA under the control of endogenous B-Raf promoter. The resulting BRaf A-Raf/A-Raf (KIN) phenotype depends on genetic background. The living embryos displaying normal development but size reduction were found with low incidence at E12.5d-16.5d. All of them displayed the rescue of vascular system. One adult p20 mouse without any visible defects in development and behavior was obtained. On the other hand, the processes of neurogenesis and neural precursors migration in survived embryos were disturbed which led in some cases to underdevelopment of different brain compartments. TUNEL and cell proliferation (PCNA staining) assays revealed more apoptotic (E13.5d) and less proliferating(E12.5d cells within ventricular and sub-ventricular zones of brain ventricles and in striatum of KIN embryos. In addition, more apoptotic cells were detected in many other tissues of E13.5d and in lung of E16.5d KIN embryos but not in adult KIN mouse. p20 KIN mouse demonstrated reduced fraction of neural precursor cells in sub-granular zone of hippocampus and mature neurons in olfactory bulb. The other processes of neurogenesis were not disturbed in adult KIN animal. Fibroblasts obtained from KIN embryos demonstrated less proliferative ability and were more susceptible to apoptotic stimuli compared to WT. This was accompanied by the reduction of active ERK and Akt required for survival, and with decrease of inactive phosphorylated BAD. The kinetic of both ERK and Akt phosphorylation upon serum stimulation was delayed. All these data indicate that moderate A-Raf kinase activity can prevent the endothelial apoptosis but is not enough to completely rescue the other developmental consequences.
The development and in-depth characterization of new fluoroaryl functionalized ORMOCER® materials (inorganic-organic hybrid polymers) for optical waveguide applications in telecommunication is presented. The preparation of the materials included precursor silane synthesis, hydrolysis/polycondensation of organoalkoxysilane mixtures, and photolithographic processing of the resulting oligosiloxane resins in order to establish the inorganic-organic hybrid network. During all stages of ORMOCER® preparation, structure-property relations were deduced from characterization data, particularly with respect to low optical loss in the important near-infrared spectral region as well as refractive index. With the aid of molecular modeling, structural characteristics of oligomeric intermediates were visualized, which was found valuable in the fundamental understanding of the material class. The material development started with the syntheses of a variety of commercially unavailable fluorinated and unfluorinated arylalkoxysilanes by means of Grignard and hydrosilylation pathways, respectively. A survey of silane optical properties, particularly their absorptions at the telecom wavelengths 1310 nm and 1550 nm, gave an impulse to the choice of suitable precursors for the preparation of low-loss ORMOCER® resins. Accordingly, precursor silane mixtures and hydrolysis/polycondensation reaction conditions were chosen and optimized with regard to low contents of C-H and Si-OH functions. Thus, absorptions as low as 0.04 dB/cm at 1310 nm and 0.18 dB/cm at 1550 nm, respectively, could be obtained from an oligosiloxane resin based on pentafluorophenyltrimethoxysilane (1) mixed with pentafluorophenyl(vinyl)-dimethoxysilane (5). In order to improve the organic crosslinkability under photolithographic processing conditions, further resins on the basis of the aforementioned were prepared, which additionally incorporated the styrene-analogous precursor 4-vinyltetrafluorophenyl-trimethoxysilane (4). Thus, ORMOCER® resins with low optical losses of 0.28 dB/cm at 1310 nm and 0.42 dB/cm at 1550 nm, respectively, were prepared, which exhibited excellent photopatternability. The manufacture of micropatterns such as optical waveguide structures by UV-photolithography under clean room conditions was the final stage of material synthesis. The optimization of processing parameters allowed the preparation of test patterns for the determination of optical, dielectrical and mechanical properties. A low optical loss of 0.51 dB/cm at 1550 nm could be measured on a waveguide manufactured from a photopatternable fluoroaryl functionalized ORMOCER®. The structural characterization of liquid resins as well as cured ORMOCER® samples was accomplished chiefly with solution and solid state 29Si-NMR spectroscopy, respectively. Particularly for polycondensates incorporating species based on more than one precursor silane, the spectra showed a high degree of complexity. An additional challenge arouse from the partial loss of fluoroaryl groups during ORMOCER® condensation and curing, which resulted in even more condensation products. Thus, in order to provide a basis for resin analysis, first the hydrolysis/condensation reactions of the isolated precursors were investigated under reaction time-resolution with NMR spectroscopy at low temperature. Backed by signal assignments in these single-precursor systems, the respective species could also be identified in the complex resin spectra, allowing for their quantitative interpretation. The structural characterization was rounded out by IR spectroscopy and SAXS analyses. With the help of molecular modeling, the experimental data were finally transferred into a three-dimensional image of an organosiloxane oligomer, which is representative for a photopatternable fluoroaryl functionalized ORMOCER® resin. The combination of low-temperature NMR, which made the characterization of polycondensates possible, with oligomer modeling paved the way to a further understanding of ORMOCER® resin systems. On the basis of this visualization of structural characteristics, e.g. properties such as organic crosslinkability of oligomers were discussed in the light of steric features within the molecular structure. Thus, new possibilities were established for the systematic optimization of ORMOCER® formulations. Structure-property relations with respect to optical loss and refraction, as determined within this work, follow trends, which are in accordance with the literature. Particularly the direct comparison of data derived from analogous fluorinated and unfluorinated ORMOCER® resins showed that fluorination results in significant decrease in NIR optical loss. Additionally, different unfluorinated aryl functionalized systems with varying aliphatic C-H content were compared. In case of a lower aliphatic content, a widening effect on the 1310 nm window was found. This is due to a shift of arylic C-H vibrations (1145 nm) towards lower wavelengths compared to aliphatic C-H (1188 nm). Finally, on the basis of NIR spectra of analogous fluorinated resins with low and high silanol content, respectively, a significant impact of (Si)O-H groups on the 1550 nm window was demonstrated, while the 1310 nm window was unaffected. This is due to O-H vibrations with a maximum at 1387 nm and further bands at higher wavelength. The index of refraction was drastically lowered due to fluorination. Thus, the analogous fluorinated and unfluorinated ORMOCER® resins had indices of 1.497 and 1.570, respectively, in the VIS region. For the fluorinated systems, refraction did not change significantly during organic cross-connection and hardbake. In conclusion, the new fluoroaryl functionalized ORMOCER® systems represent low-loss materials for telecom applications. In addition, in-depth characterization during material development allowed the proposal of structure-property relations, particularly with respect to optical properties, which are of considerable importance for future developments.
The present work consist of two major parts. The first part, extending over chapters 1, 2, 3 and 4, addresses the design and construction of a device capable of determining the shell thickness and the core size for monolayer spherical particles in a flow. The second part containing chapters 5, 6, 7, 8, 9 and 10, concentrate on the use of Raman spectroscopy as a space application, namely for use as a tool for in situ planetary investigations. This part directly addresses the MIRAS project, a study run under the auspices of Federal Ministry of Education and Research, BMBF and German Aerospace Center, DLR under national registration number 50OW0103. MIRAS stands for "Mineral Investigation by in situ Raman Spectroscopy". Microcapsule Sizing by Elastic Light Scattering The industrial development of processes based on microcapsules depends on the possibility to provide clear and complete information about the properties of these microcapsules. However, the tools for an easy and efficient determination of the microcapsule properties are lacking, several methods being often required to describe adequately the microcapsule behavior. Methods for evaluating the individual size and size distribution of both the core and the shell are required together with methods for measuring the mechanical strength, stability in appli-cation media, permeability of the shell, etc. Elastic light scattering measurements provide a possible way of determining properties such as core size, shell size and refractive index. The design and con-struction of a device capable of measuring the above mentioned parameters for a core-shell particle is the subject of the first part of this thesis. The basic principle of measurement for the device proposed here consists of an-alyzing one particle at a time by recording the elastic light scattering pattern at angles between approx. 60 and 120 grad. By comparing the experimentally recorded phase functions with the previously calculated phase functions stored in a database, the geometry of the scattering object can be identified. In our case the geometry is characterized by two parameters: the shell thickness and the core radius. In chapter 2 a short overview on the methods used for sizing microparticles is given. Different sizing methods are compared, and the advantages and disadvan-tages for the general problem of sizing are shortly discussed. It is observed that all sizing methods that are based on elastic light scattering theories are ensemble methods. Chapter 3 focusses on the theories used for calculating the theoretical scattering patterns with emphasize on the Mie theory. The generalization of Mie theory for layered particles is shortly presented and the far field intensity approximations are discussed. The last chapter (4) of this first part describes the experimental approach for building an automatic microcapsule sizer. The approach started by O. Sbanski [76] with the development of a software packet for calculating and storing theoret-ical phase functions for core-shell particles was continued with the designing and construction of a measuring device. The hardware construction and the software with all implemented corrections imposed by the individual setup components are described in detail. For the laser, the monochromaticity, the intensity profile of the beam as well as the planarity of the equi-phase fronts are taken into consid-eration. The flow cell with three different designs is described, and the influences of the employed design on the light scattering patterns are discussed together with the optical system used for recording the experimental phase functions. The detection system formed by two identical linear CCD arrays is presented together with the software approach used for data acquisition. Ways of improving the quality and the speed of the analyzing process are discussed. The final section presents measurements run on samples made of homogeneous spheres and also on samples containing industrial microcapsules. Mineral Investigation by in situ Raman Spectroscopy The envisaged future planetary missions require space-born instruments, which are highly miniaturized with respect to volume and mass and which have low needs of power. A micro Raman spectrometer as a stand alone device on a planetary surface (e.g. Mars) offers a wide spectrum of possibilities. It can assess the chemical analysis via determination of the mineral composition, detect organic molecules in the soil, identify the principal mineral phases, etc. The technical developments in the last years have introduced a new generation of small Raman systems suitable for robotic mineral characterization on planetary surfaces [20, 95]. Two different types of spectrometer were considered for the MIRAS study. As supporting laboratory experiments for the MIRAS study, the measure-ments on standard minerals and on SNC Mars meteorites are discussed in chapter 6. The following SNC meteorites have been investigated: Sayh al Uhaymir 060, Dar al Gani 735, Dar al Gani 476, Northwest Africa 856, Los Angeles, Northwest Africa 1068 and Zagami. Pyrite as a hitherto undescribed phase in the picritic (olivin-phyric) shergottite NWA 1068 as well as reduced carbon (e.g. graphite) and anatase in the shergottite Say al Uhaymir 060 are new findings for this class of meteorites. A detailed description of the proposed designs for MIRAS, with the compo-nents used for building the test version on a breadboard is covered in chapter 7. The scientific as well as the mission requirements imposed on the instrument are discussed. The basic design is presented and the main components that are brought together to build the device being the laser unit, the Raman head, the Rayleigh filtering box, and the spectral sensor (spectrometer with a matching de-tector) are described. The two proposed designs, one based on an acousto-optic tunable filter (AOTF) and the other based on a dispersive hadamard transform spectrometer are compared to each other. The actual breadboard setup with the detailed description of the components follows in Section 7.3. Further de-velopment of a Raman spectrometer for planetary investigations is proposed in combination with a microscope as part of the Extended-MIRAS project. The software developed for controlling the breadboard version of MIRAS is described in chapter 8 together with a short description of the structure of a relational database used for in house spectra management. The measuring pro-cedures and the data processing steps are presented. Spectra acquired with the MIRAS breadboard version based on the AOTF are shown in chapter 9. The final chapter addresses a rather different possibility of using Raman spectroscopy for planetary investigations. The chapter summarizes the content of four tech-nical notes that were established within the study contracted by the European Space Agency with firma Kayser-Threde in Munich concerning the possibility of applying Raman spectroscopy in the field of remote imaging.
The present thesis encompasses two parts. The first supramolecular part focuses on the development of new flexible self-assembling zwitterions as building blocks for supramolecular polymers. In the second part, the aim was to develop bioorganic receptors for amino acids and dipeptides in aqueous media. Both research projects are based on the guanidiniocarbonyl pyrrole 1 as a new efficient binding motif for the complexation of carboxylates in polar solution.A necessary requirement for the realization of these research projects was to develop an efficient and mild synthetic approach for the cationic guanidiniocarbonyl pyrroles in general. The harsh reaction conditions of the previously used method and the problematic purification of the cationic guanidinocarbonyl pyrroles so far prevented a more extensive exploration in bioorganic and supramolecular research. In the course of this work I successfully developed a new synthesis starting with mono tBoc-protected guanidine that was coupled with a benzyl protected pyrrole carboxylic acid. After deprotection of the benzyl group, a key intermediate in the newly developed synthesis, the tBoc-protected guanidinocarbonyl pyrrole acid, was obtained. This new, mild and extremely efficient synthetic approach for the introduction of acyl guanidines is now the standard procedure in our group for the preparation of both solution and solid-phase guanidiniocarbonyl pyrroles. With this facile method at hand, a new class of flexible zwitterions, in which a carboxylate is linked via an alkyl chain to a guanidiniocarbonyl pyrrole cation was synthesized. The self-aggregation and the influence of the length and therefore flexibility of the alkyl spacer on the structure and stability of the formed aggregates were studied in solution and gas phase. In solution the aggregation was studied by NMR-dilution experiments in DMSO which suggest that flexible zwitterions with n = 1, 3 and 5 form oligomers. For n = 1, highly stable helical aggregates with nanometer size are formed. In the gas phase studies the stability and the fragmentation kinetics of a series of sodiated dimeric zwitterions with n = 2, 3 and 5 were investigated. This was done by infrared multiphoton dissociation Fourier transform ion cyclotron resonance mass spectrometry (IRMPD-FT-ICR-MS). These kinds of studies can be used in the future for a more directed design of supramolecular building blocks The bioorganic research part comprises three different projects. In a first project I synthesized four new arginine analogues which can be implemented in peptides as a substitute for arginine. Therefore, I developed the new multi-step synthesis shown below for these arginine analogues. As a test for their application in normal solid phase synthesis, I successfully prepared a tripeptide sequence Ala-AA1-Val (AA: arginine analogue. In a second project I studied the influence of additional ionic interactions within our binding motif. I synthesized a di-cationic and a tris-cationic receptor and evaluated the binding properties via NMR titration experiments against a variety of amino acids. Especially, the tris-cationic receptor was capable to strongly complex amino acids. The association constants were about a factor of 100 higher than those for the guanidiniocarbonyl pyrroles known so far. Even in 90 %water/10 % DMSO the association constants determined by NMR titration were extremely high with values around Kass = 2000 M-1. In the third project I developed a de-novo designed receptor for C-terminal dipeptides in a beta-sheet conformation based on molecular calculations. This receptor was studied in NMR and also UV titration experiments. In 40 % water/ 60 % DMSO the association constants were too strong to be measured by NMR titration experiments. Therefore, the complexation properties of 12 were studied by UV titration in water (with 10 % DMSO added for solubility reasons) with various dipeptides and amino acids as substrates. The data show that 12 binds dipeptides very efficiently even in water with association constants Kass > 10000 M-1, making 12 one of the most effective dipeptide receptors known so far. In contrast to that, simple amino acids are bound up to ten times less efficiently (Kass > 1000 M-1) than dipeptides. In the series of dipeptides studied the complex stability increases depending on the side chains present in the order Gly < Ala < Val which is a result of the decreasing flexibility of the peptide and the increasing hydrophobicity of the side chains. The binding properties of this receptor are superior to any other dipeptide receptor reported so far. Within my thesis I have not only developed an essential, mild and efficient synthetic approach for guanidiniocarbonyl pyrroles in general, but also a new binding motif for the complexation of amino acids 15, 11 and in addition a dipeptide receptor 12 that is superior to all dipeptides receptors known so far.
Nitric oxide production by tobacco plants and cell cultures under normal conditions and under stress
(2004)
Nitric oxide (NO) is a gaseous free radical involved in the regulation of diverse biochemical and physiological processes in animals. During the last decade, evidence has accumulated that NO might also play an important role as a second messenger in plants. Of special interest were observations that NO was involved in a signal chain leading to the hypersensitive response (HR) in incompatible plant-pathogen interactions. In contrast to animals, plants have probably several enzymes that may produce NO. Potential candidates are: Cytosolic nitrate reductase (NR; EC 1.6.6.1), plasma-membrane (PM)-nitrite: NO reductase (Ni:NOR), nitric oxide synthase (NOS; EC 1.14.13.39) and Xanthine dehydrogenase (XDH; EC 1.1.1.204). The major goal of this work was to quantify NO production by plants, and to identify the enzymes responsible for NO production. As a major method, NO production by tobacco leaves or cell suspensions was followed under normal, non-stress conditions, and under biotic stress, through on-line measurement of NO emission into the gas phase (chemiluminescence). Plants used were tobacco wild-type (N. tabacum cv Xanthi or cv Gatersleben), NR-free mutants grown on ammonium in order to prevent NR induction, plants grown on tungstate to inhibit synthesis of functional MoCoenzymes, and a NO-overproducing nitrite reductase (NiR)-deficient transformant. Induction of HR in tobacco leaves and in cell suspensions was achieved using the fungal peptide elicitor cryptogein. Non-elicited leaves from nitrate-grown plants showed a typical NO-emission pattern where NO-emission was low in dark, higher in the light and very high under dark-anaerobic conditions. Even at maximum rates, NO production in vivo was only a few percent of total NR activity (NRA). Consistent with that, with a solution of purified NR as a simple, “low quenching” system, NO-emission was also about 1 % of NRA. Thus, NO scavenging by leaves and stirred cell suspensions appeared small and NO-emission into purified air should give a reliable estimate of NO production. NO-emission was always high in a NiR-deficient transformant which accumulated nitrite, and NO-emission was completely absent in plants or cell suspensions which did not contain NR. Thus, in healthy plants or cell suspensions, NO-emission was exclusively due to the reduction of nitrite to NO, mainly by cytosolic NR. In addition to nitrite, cytosolic NADH appears as an important factor limiting NO production. Unexpectedly, plants (in absence of NR) were able to reduce nitrite to NO under anaerobic conditions through an unknown enzyme system that was not a MoCo-enzyme and was cyanide-sensitive. When infiltrated into leaves at nanomolar concentrations, the fungal elicitor cryptogein provoked cell death in tobacco leaves and cell suspensions. The HR could be prevented by the NO-scavengers PTIO or c-PTIO, suggesting that NO production was indeed required for the HR. However, the product of the reaction of c-PTIO with NO, c-PTI, also prevented cell death without quenching NO emission. Thus, prevention of cell death by c- PTIO is no proof for an involvement of NO. No differences were found in the HR induction between NR-free plants and/or cell suspensions and WT plants. Thus, NR appears not necessary for the HR. Further, and in contrast to literature suggestions, a continuously high NO-overproduction by a NiR-free mutant did not interfere with the development of the HR. Most surprisingly, no additional NO-emission from tobacco leaves was induced by cryptogein at any phase of the HR. In contrast, some NO-emission, paralleled by nitrite accumulation, was detected 3-6 h after cryptogein addition with nitrate grown cell suspensions, but not with NR free, ammonium- grown cells. Thus, induction of NO-emission by cryptogein appeared somehow correlated with NR and nitrite, at least in cell suspensions. But since cryptogein induced the HR even in NR-free cell suspensions, this nitrite-related NO- emission was not required for cell death. NOS inhibitors neither prevented cell death nor did they affect nitrite-dependent NO-emission. Thus, in total these data question the often proposed role of NO as a signal in the HR, and of NOS as source for NO.
The point of departure for the present work has been the following free boundary value problem for analytic functions $f$ which are defined on a domain $G \subset \mathbb{C}$ and map into the unit disk $\mathbb{D}= \{z \in \mathbb{C} : |z|<1 \}$. Problem 1: Let $z_1, \ldots, z_n$ be finitely many points in a bounded simply connected domain $G \subset \mathbb{C}$. Show that there exists a holomorphic function $f:G \to \mathbb{D}$ with critical points $z_j$ (counted with multiplicities) and no others such that $\lim_{z \to \xi} \frac{|f'(z)|}{1-|f(z)|^2}=1$ for all $\xi \in \partial G$. If $G=\mathbb{D}$, Problem 1 was solved by K?nau [5] in the case of one critical point, and for more than one critical point by Fournier and Ruscheweyh [3]. The method employed by K?nau, Fournier and Ruscheweyh easily extends to more general domains $G$, say bounded by a Dini-smooth Jordan curve, but does not work for arbitrary bounded simply connected domains. In this paper we present a new approach to Problem 1, which shows that this boundary value problem is not an isolated question in complex analysis, but is intimately connected to a number of basic open problems in conformal geometry and non-linear PDE. One of our results is a solution to Problem 1 for arbitrary simply connected domains. However, we shall see that our approach has also some other ramifications, for instance to a well-known problem due to Rellich and Wittich in PDE. Roughly speaking, this paper is broken down into two parts. In a first step we construct a conformal metric in a bounded regular domain $G\subset \mathbb{C}$ with prescribed non-positive Gaussian curvature $k(z)$ and prescribed singularities by solving the first boundary value problem for the Gaussian curvature equation $\Delta u =-k(z) e^{2u}$ in $G$ with prescribed singularities and continuous boundary data. This is related to the Berger-Nirenberg problem in Riemannian geometry, the question which functions on a surface R can arise as the Gaussian curvature of a Riemannian metric on R. The special case, where $k(z)=-4$ and the domain $G$ is bounded by finitely many analytic Jordan curves was treated by Heins [4]. In a second step we show every conformal pseudo-metric on a simply connected domain $G\subseteq \mathbb{C}$ with constant negative Gaussian curvature and isolated zeros of integer order is the pullback of the hyperbolic metric on $\mathbb{D}$ under an analytic map $f:G \to \mathbb{D}$. This extends a theorem of Liouville which deals with the case that the pseudo-metric has no zeros at all. These two steps together allow a complete solution of Problem 1. Contents: Chapter I contains the statement of the main results and connects them with some old and new problems in complex analysis, conformal geometry and PDE: the Uniformization Theorem for Riemann surfaces, the problem of Schwarz-Picard, the Berger-Nirenberg problem, Wittich's problem, etc.. Chapter II and III have preparatory character. In Chapter II we recall some basic results about ordinary differential equations in the complex plane. In our presentation we follow Laine [6], but we have reorganized the material and present a self-contained account of the basic features of Riccati, Schwarzian and second order differential equations. In Chapter III we discuss the first boundary value problem for the Poisson equation. We shall need to consider this problem in the most general situation, which does not seem to be covered in a satisfactory way in the existing literature, see [1,2]. In Chapter IV we turn to a discussion of conformal pseudo-metrics in planar domains. We focus on conformal metrics with prescribed singularities and prescribed non-positive Gaussian curvature. We shall establish the existence of such metrics, that is, we solve the corresponding Gaussian curvature equation by making use of the results of Chapter III. In Chapter V we show that every constantly curved pseudo-metric can be represented as the pullback of either the hyperbolic, the euclidean or the spherical metric under an analytic map. This is proved by using the results of Chapter II. Finally we give in Chapter VI some applications of our results. [1,2] Courant, H., Hilbert, D., Methoden der Mathematischen Physik, Erster/ Zweiter Band, Springer-Verlag, Berlin, 1931/1937. [3] Fournier, R., Ruscheweyh, St., Free boundary value problems for analytic functions in the closed unit disk, Proc. Amer. Math. Soc. (1999), 127 no. 11, 3287-3294. [4] Heins, M., On a class of conformal metrics, Nagoya Math. J. (1962), 21, 1-60. [5] K?nau, R., L?gentreue Randverzerrung bei analytischer Abbildung in hyperbolischer und sph?ischer Geometrie, Mitt. Math. Sem. Giessen (1997), 229, 45-53. [6] Laine, I., Nevanlinna Theory and Complex Differential Equations, de Gruyter, Berlin - New York, 1993.
The experimental work of this thesis addresses the questions of whether established cell lines injected into murine blastocysts find their way back home and seed preferentially at the site of their origin. Furthermore, can they change their fate and differentiate to unrelated cell types when exposed to the embryonic environment. This survey was based on the fact that different cell lines have different potentials in developing embryos, dependent on their cellular identity. The cell lines used in this survey were AGM region-deriving DAS 104-4, DAS 104-8 cells, yolk sac-deriving YSE cells and bone marrow-deriving FDCP mix cells. These cells were injected into mouse blastocysts. Donor cells were traced in developing embryos via specific markers. Analysis of the embryos revealed that DAS cells are promiscuous in their seeding pattern, since they were found in all analysed tissues with similar frequencies. YSE cells showed preferences in seeding yolk sac and liver. YSE donor cells in chimaeric tissues were not able to change their immuno-phenotype, indicating that they did not change their destiny. Analysis of adult mice did not reveal any of YSE-derived cells donor contribution. In contrast, FDCP mix cells mostly engrafted haematopoietic tissues, although the embryos analysed by in situ hybridization had donor signals frequently in cartilage primordia, heads, and livers. Analysis of whether FDCPmix-derived cells found in foetal livers were of haematopoietic or hepatocytes nature showed that progeny of injected FDCP mix cells do not differentiate into cells that express a hepatocyte-specific marker. Further analysis showed that FDCPmix-derived donor cells found in brain express neural or haematopoietic markers. In order to reveal if they transdifferentiate to neurons or fuse with neurons/glial cells, nuclear diameters of donor and recipient cells were determined. Comparison of the nuclear diameters of recipient and donor cells revealed no differences. Therefore this suggests that progeny of FDCP mix in brain are not fusion products. Analysis of adult mice tissues revealed that presence of FDCP mix-derived cells was the highest in brains. These results confirmed the assumption that the developmental potential of the analysed cells cannot be easily modified, even when exposed to early embryonic environment. Therefore one can conclude that the analysed cell types had different homing patterns depending on their origins.
Although the role of B-cells in autoimmunity is not completely understood, their importance in the pathogenesis of autoimmune diseases has been more appreciated in the past few years. It is now well known that they have roles in addition to (auto) antibody production and are involved by different mechanisms in the regulation of T-cell mediated autoimmune disorders. The evolution of an autoimmune disease is a dynamic process, which takes a course of years during which complex immunoregulatory mechanisms shape the immune repertoire until the development of clinical disease. During this course, the B-cell repertoire itself is influenced and a change in the distribution of immunoglobulin heavy and light chain genes can be observed. B-cell depletive therapies have beneficial effects in patients suffering from rheumatoid arthritis (RA), highlighting also the central role of B-cells in the pathogenesis of this disease. Nevertheless, the mechanism of action is unclear. It has been hypothesised that B-cell depletion is able to reset deviated humoral immunity. Therefore we wanted to investigate if transient B-cell depletion results in changes of the peripheral B-cell receptor repertoire. To address this issue, expressed immunoglobulin genes of two patients suffering from RA were analysed; one patient for the heavy chain repertoire (patient H), one patient for the light chain repertoire (patient L). Both patients were treated with rituximab, an anti-CD20 monoclonal antibody that selectively depletes peripheral CD20+ B-cells for several months. The B-cell repertoire was studied before therapy and at the earliest time point after B-cell regeneration in both patients. A longer follow-up (up to 27 months) was performed in patient H who was treated a second time with rituximab after 17 months. Heavy chain gene analysis was carried out by nested-PCR on bulk DNA from peripheral B-cells using family-specific primers, followed by subcloning and sequencing. During the study, patient H received two courses of antibody treatment. B-cell depletion lasted 7 and 10 months, respectively and each time was accompanied by a clinical improvement. Anti-CD20 therapy induced two types of changes in this patient. During the early phase of B-cell regeneration, we noticed the presence of an expanded and recirculating population of highly mutated B-cells. These cells expressed very different immunoglobulin VH genes compared before therapy. They were class-switched and could be detected for a short period only. The long-term changes were more subtle. Nevertheless, characteristic changes in the VH2 family, as well as in specific mini-genes like VH3-23, 4-34 or 1-69 were noticed. Some of these genes have already been reported to be biased in autoimmune diseases. Also in autoimmune diseases, in particular in RA, clonal B-cells have been frequently found in the repertoire. B-cell depletion with anti-CD20 antibody resulted in a long term loss of clonal B-cells in patient H. Thus, temporary B-cell depletion induced significant changes in the heavy chain repertoire. For the light chain gene analysis, the repertoire changes were analysed separately for naive (CD27-) and memory (CD27+) B-cells. Individual CD19+ B-cells were sorted into CD27- and CD27+ cells and single cell RT-PCR was performed, followed by direct sequencing. During the study, patient L received one course of antibody treatment. B-cell depletion lasted 10 months and the light chain repertoire was studied before and after therapy. Before therapy, some differences in the distribution of VL and JL genes were observed between naive and memory B-cells. In particular, the predominant usage of Jk-proximal Vk genes by the CD27- naive B-cells indicated that the receptor editing was less frequent in this population compared to memory cells. In VlJl rearrangements also, some evidence for decreased receptor editing was noticed, with the overrepresentation of the Jl2/3 gene segments. The CDR3 regions of naive and memory cells showed different characteristics: the activity of the terminal deoxynucleotidyl transferase and exonuclease in Vl(5’) side was greater in memory cells. Also in the light chain repertoire, we observed some changes induced by the B-cell depletive therapy. There was a tendency of a less frequent usage of Jk-proximal Vk genes in the naive population. Some Vl genes, previously described in autoimmune diseases and connected to rheumatoid factor activity, such as 3p, 3r, 1g, were not found after therapy. The different characteristics of the CDR3 regions of VlJl rearrangements were not observed anymore. Very significantly, the ratio Vk to Vl was shifted toward a greater usage of Vk genes in the naive population after therapy. Taken together, these results indicate that therapeutic transient B-cell depletion by anti-CD20 antibody therapy modulates the immunoglobulin gene repertoire in the two RA patients studied. Measurable changes were observed in the heavy chain as well as in the light chain repertoire, which may be relevant to the course of the disease. This also supports the notion that the composition of the B-cell repertoire is influenced by the disease and that B-cell depletion can reset biases that are typically found in autoimmune diseases.
In the last years more than one hundred microbial genomes have been sequenced, many of them from pathogenic bacteria. The availability of this huge amount of sequence data enormously increases our knowledge on the genome structure and plasticity, as well as on the microbial diversity and evolution. In parallel, these data are the basis for the scientific “revolution” in the field of industrial and environmental biotechnology and medical microbiology – diagnostics and therapy, development of new drugs and vaccines against infectious agents. Together with the genomic approach, other molecular biological methods such as PCR, DNA-chip technology, subtractive hybridization, transcriptomics and proteomics are of increasing importance for research on infectious diseases and public health. The aim of this work was to characterize the genome structure and -content of the probiotic Escherichia coli strain Nissle 1917 (O6:K5:H31) and to compare these data with publicly available data on the genomes of different pathogenic and non-pathogenic E. coli strains and other closely related species. A cosmid genomic library of strain Nissle 1917 was screened for clones containing the genetic determinants contributing to the successful survival in and colonization of the human body, as well as to mediate this strain’s probiotic effect as part of the intestinal microflora. Four genomic islands (GEI I-IVNissle 1917) were identifed and characterized. They contain many known fitness determinants (mch/mcm, foc, iuc, kps, ybt), as well as novel genes of unknown function, mobile genetic elements or newly identified putative fitness-contributing factors (Sat, Iha, ShiA-homologue, Ag43-homologues). All islands were found to be integrated next to tRNA genes (serX, pheV, argW and asnT, respectively). Their structure and chromosomal localization closely resembles those of analogous islands in the genome of uropathogenic E. coli strain CFT073 (O6:K2(?):H1), but they lack important virulence genes of uropathogenic E. coli (hly, cnf, prf/pap). Evidence for instability of GEI IINissle 1917 was given, since a deletion event in which IS2 elements play a role was detected. This event results in loss of a 30 kb DNA region, containing important fitness determinants (iuc, sat, iha), and therefore probably might influence the colonization capacity of Nissle 1917 strain. In addition, a screening of the sequence context of tRNA-encoding genes in the genome of Nissle 1917 was performed to identify genome wide potential integration sites of “foreign” DNA. As a result, similar “tRNA screening patterns” have been observed for strain Nissle 1917 and for the uropathogenic E. coli O6 strains (UPEC) 536 and CFT073. I. Summary 4 The molecular reason for the semi-rough phenotype and serum sensitivity of strain Nissle 1917 was analyzed. The O6-antigen polymerase-encoding gene wzy was identified, and it was shown that the reason for the semi-rough phenotype is a frame shift mutation in wzy, due to the presence of a premature stop codon. It was shown that the restoration of the O side-chain LPS polymerization by complementation with a functional wzy gene increased serumresistance of strain Nissle 1917. The results of this study show that despite the genome similarity of the E. coli strain Nissle 1917 with the UPEC strain CFT073, the strain Nissle 1917 exhibits a specific set of geno- and phenotypic features which contribute to its probiotic action. By comparison with the available data on the genomics of different species of Enterobacteriaceae, this study contributes to our understanding of the important processes such as horizontal gene transfer, deletions and rearrangements which contribute to genome diversity and -plasticity, and which are driving forces for the evolution of bacterial variants. At last, the fim, bcs and rfaH determinats whose expression contributes to the mutlicellular behaviour and biofilm formation of E. coli strain Nissle 1917 have been characterized.
Summary The nature of the chemical bond is a topic under constant debate. What is known about individual molecular properties and functional groups is often taught and rationalized by explaining Lewis structures, which, in turn, make extensive use of the valence concept. The valence concept distinguishes between electrons, which do not participate in chemical interactions (core electrons) and those, which do (single, double, triple bonds, lone-pair electrons, etc.). Additionally, individual electrons are assigned to atomic centers. The valence concept is of paramount success: It allows the successful planning of chemical syntheses and analyses, it explains the behavior of individual functional groups, and, moreover, it provides the “language” to think of and talk about molecular structure and chemical interactions. The resounding success of the valence concept may be misleading to forget its approximative character. On the other hand, quantum mechanics provide in principle a quantitative description of all chemical phenomena, but there is no discrimination between electrons in quantum mechanics. From the quantum mechanical point of view there are only indistinguishable electrons in the field of the nuclei, i.e., it is impossible to assign a given electron to a particular center or to ascribe a particular purpose to individual electrons. The concept of indistinguishability of micro particles is founded on the Heisenberg uncertainty relation, which states, that wavepackets diverge in the 6N dimensional phase space, such that individual trajectories can not be identified. Hence it is a deep-rooted and approved physical concept. As an introduction to the present work density partitioning schemes were discussed, which divide the total molecular density into chemically meaningful areas. These partitioning schemes are intimately related to either the concepts of bound atoms in a molecule (as in the Atoms In Molecules theory (AIM) according to Bader or as in the Hirshfeld partitioning scheme) or to the concept of chemical structure in the sense of Lewis structures, which divide the total molecular density into core and valence density, where the valence density is split up again into bonding and non-bonding electron densities. Examples are early and recent loge theories, the topological analysis by means of the Electron Localization Function (ELF), and the Natural Bond Orbital (NBO) approach. Of these partitioning schemes, the theories according to Bader (AIM), to Becke and Edgecomb (ELF) and according to Weinhold (NBO and Natural Resonance Theory, NRT), respectively, were reviewed in detail critically. Points of criticism were explicated for each of the mentioned theories. Since theoretically derived electron densities are to be compared to experimentally derived densities, a brief introduction into the theory of X-ray di®raction experiments was given and the multipole formalism was introduced. The procedure of density refinement was briefly discussed. Various suggestions for improvements were developed: One strategy would be the employment of model parameters, which are to a maximum degree mutually orthogonal, with the object of minimizing correlations among the model parameters, e.g., to introduce nodal planes into the radial functions of the multipole model. A further suggestion involves the guidance of the iterative refinement procedure by an extremum principle, which states, that when di®erent solutions to the least squares minimization problem are available with about the same statistical measures of quality and with about the same residual density, then the solution is to prefer, which yields a minimum density at the bond critical point (BCP) and a maximum polarity in terms of the ratio of distances between the BCP and the nuclei. This suggestion is based on the well known fact, that the bond polarity (in terms of the ratio of distances between the BCP and the respective nuclei) is underestimated in the experiment. Another suggestion for including physical constraints is the explicit consideration of the virial theorem, e.g., by evaluating the integration of the Laplacian over the entire atomic basins and comparing this value to zero and to the value obtained from the integration of the electron gradient field over the atomic surface. The next suggestion was to explicitly use the electrostatic theorem of Feynman (often also denoted as Hellmann-Feynman theorem), which states, that the forces onto the nuclei can be calculated from the purely classical electrostatic forces of the electron distribution and the nuclei distribution. For a stationary system, these forces must add to zero. This also provides an internal quality criterion of the density model. This can be performed in an iterative way during the refinement procedure or as a test of the final result. The use of the electrostatic theorem is expected to reduce significantly correlations among static density parameters and parameters describing vibrations, since it is a valuable tool to discriminate between physically reasonable and artificial static electron densities. All of these mentioned suggestions can be applied as internal quality criteria. The last suggestion is based on the idea to initiate the experimental refinement with a set of model parameters, which is, as much as possible close to the final solution. This can be achieved by performing periodic boundary conditions calculations, from which theoretically created files are obtained, which contain the Miller indices (h, k, l) and the respective intensity I. This file is used for a model parameter estimation (refinement), which excludes vibrations. The resulting parameters can be used for the experimental refinement, where, in a first step, the density parameters are fixed to determine the parameters describing vibrations. For a fine tuning, again the electrostatic theorem and the other above mentioned suggestions could be applied. Theoretical predictions should not be biased by the method of computation. Therefore the dependence of the density analyzing tools on the level of calculation (method of calculation/basis set) and on the substituents in complex chemical bonding situations were evaluated in the second part of the present work. A number of compounds containing formal single and double sulfur nitrogen bonds was investigated. For these compounds, experimental data were also available. The calculated data were compared internally and with the experimental results. The internal comparison was drawn with regard to questions of convergency as well as with regard to questions of consistency: The resulting molecular properties from NBO/NRT analyses were found to be very stable, when the geometries were optimized at the respective level of theory. This stability is valid for variations in the methods of calculation as well as for variations in the basis set. Only the individual resonance weights of the contributing Natural Lewis Structures differed considerably depending on the level of calculation and depending on the substituents. However, the deviations were in both cases to a large extent within a limit which preserves the descending order of the leading resonance structure weights. The resulting bond orders, i.e., the total, covalent and ionic bond order from NRT calculations, were not affected by the shift in the resonance weights. The analysis of the bond topological parameters resulted in a discrimination between insensitive parameters and sensitive parameters. The stable parameters do neither depend strongly on the method of calculation nor on the basis set. Only minor variation occurs in the numerical values of these parameters, when the level of calculation is changed or even when other functional groups (H, Me, or tBu) are employed, as long as the methods of calculation do not drop considerably below a standard level. The bond descriptors of the sulfur nitrogen bonds were found to be also stable with respect to the functional groups R = H, R = Me, and R = tBu. Stable parameters are the bond distance, the density at the bond critical point (BCP) and the ratio of distances between the BCP and the nuclei A and B, which varies clearly when considering the formal bond type. For very small basis sets like the 3-21G basis set, this characteristic stability collapses. The sensitive parameters are based on the second derivatives of the density with respect to the coordinates. This is in accordance with the well known fact, that the total second derivative of the density with respect to the coordinates is a strongly oscillating function with positive as well as negative values. A profound deviation has to be anticipated as a consequence of strong oscillations. lambda3, which describes the local charge depletion in the direction of the interaction line, is the most varying parameter. A detailed analysis revealed that the position of the BCP in the rampant edge of the Laplacian distribution is responsible for the sensitivity of the numerical value of lambda3 in formal double bonds. Since the slope of the Laplacian assumes very high values in its rampant edge, a tiny displacement of the BCP leads already to a considerable change in lambda3. This instability is not a failure of the underlying theory, but it yields de facto to a considerable dependence of sensitive bond topological properties on the method of calculation and on the applied basis sets. Since the total second derivative is important to judge on the nature of the bond in the AIM theory (closed shell interactions versus shared interactions), the changes in lambda3 can lead to differing chemical interpretations. The comparison of theoretically derived bond topological properties of various sulfur nitrogen bonds provides the possibility to measure the self consistency of this data set. All data sets clearly exhibit a linear correlation between the bond distances and the density at the BCP on one hand and between the bond distances and the Laplacian values at the BCP on the other hand. These correlations were almost independent of the basis set size. In this context, the linear regression has to be regarded exclusively as a descriptive statistics tool. There is no correlation anticipated a priori. The formal bond type was found to be readily deducible from the theoretically obtained bond topological descriptors of the model systems. In this sense, the bond topological properties are self consistent despite of the numerical sensitivity of the derivatives, as exemplified above. Often, calculations are performed with the experimentally derived equilibrium geometries and not with optimized ones. Applying this approach, the computationally costly geometry optimizations are saved. Following this approach the bond topological properties were calculated using very flexible basis sets and employing the fixed experimental geometry (which, of course, includes the application of tBu groups). Regression coe±cients similar to those from optimized geometries were obtained for correlations between bond distances and the densities at the BCP as well as for the correlation between bond distances and the Laplacian at the BCP, i.e. the approach is valid. However, the data points scattered less and the coe±cient of correlation was clearly increased when geometry optimizations were performed beforehand. The comparison between data obtained from theory and experiment revealed fundamental discrepancies: In the data set of bond topological parameters from the experiment, the behavior of only 2 out of 3 insensitive parameters was comparable to the behavior of the theoretically obtained values, i.e. theoretical and experimental bond distances as well as theoretical and experimental densities at the BCP correlate. From the theoretically obtained data it was easy to deduce the formal bond type from the position of the BCP, since it changed in a systematic manner. The respective experimentally obtained values were almost constant and did not change systematically. For the SN bonds containing compounds, the total second derivative assumes exclusively negative values in the experiment. Due to the different internal behavior, experimentally and theoretically sensitive bond topological values could not be compared directly. The qualitative agreement in the Laplacian distribution, however, was excellent. In the third and last part of this work, the application to chemical systems follows. Formal hypervalent molecules, i.e. molecules where some atoms are considered to hold more than 8 electrons in their valence shell, were investigated. These were compounds containing sulfur nitrogen bonds (H(NtBu)2SMe, H2C{S(NtBu)2(NHtBu)}2, S(NtBu)2 and S(NtBu)3) and a highly coordinated silicon compound. The set of sulfur nitrogen compounds also contained a textbook example for valence expansion, the sulfur triimide. For these molecules, experimental reference values were available from high resolution X-ray experiments. The experimental results were in the case of the sulfur triimide not unique. Furthermore, from the experimental bond topological data no definite conclusion about the formal bonding type could be drawn. The situation of sulfur nitrogen bonds in the above mentioned set of molecules was analyzed in terms of a geometry discussion and by means of a topological analysis. The methyl-substituted isolated molecules served as model compounds. For the interpretation of the bonding situation additional NBO/NRT calculations were preformed for the sulfur nitrogen compounds and an ELF calculation and analysis was performed for the silicon compound. The ELF analysis included not only the presentation and discussion of the ELF-isosurfaces (eta = 0.85), but also the investigation of populations of disynaptic valence basins and the percentage contributions to these populations of the individual atoms when the disynaptic valence basins are split into atomic contributions according to Bader’s partitioning scheme. The question of chemical interest was whether hypervalency is present in the set of molecules or not. In the first case the octet rule would be violated, in the second case Pauling’s verdict would be violated. While the concept of hypervalency is well established in chemistry, the violation of Pauling’s verdict is not. The quantitative numbers of the sensitive bond topological values from theory and experiment were not comparable, since no systematic relationship between the experimentally and theoretically determined sensitive bond descriptors was found. However, the insensitive parameters are in good agreement and the qualitative Laplacian distribution is, with few exceptions, in excellent agreement. The formal bonding type was deduced from experimental and theoretical topological data by considering the number and shape of valence shell charge concentrations in proximity to the sulfur and nitrogen centers. The results from NBO/NRT calculations confirmed the findings. All employed density analyzing tools AIM, ELF and NBO/NRT coincided in describing the bonding situation in the formally hypervalent molecules as highly polar. A comparison and analysis of experimentally and theoretically derived electron densities led consistently to the result, that regarding this set of molecules, hypervalency has to be excluded unequivocally.