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Background
Suicide and non-fatal suicidal behaviour are significant public health issues in Europe requiring effective preventive interventions. However, the evidence for effective preventive strategies is scarce. The protocol of a European research project to develop an optimized evidence based program for suicide prevention is presented.
Method
The groundwork for this research has been established by a regional community based intervention for suicide prevention that focuses on improving awareness and care for depression performed within the European Alliance Against Depression (EAAD). The EAAD intervention consists of (1) training sessions and practice support for primary care physicians,(2) public relations activities and mass media campaigns, (3) training sessions for community facilitators who serve as gatekeepers for depressed and suicidal persons in the community and treatment and (4) outreach and support for high risk and self-help groups (e.g. helplines). The intervention has been shown to be effective in reducing suicidal behaviour in an earlier study, the Nuremberg Alliance Against Depression. In the context of the current research project described in this paper (OSPI-Europe) the EAAD model is enhanced by other evidence based interventions and implemented simultaneously and in standardised way in four regions in Ireland, Portugal, Hungary and Germany.
The enhanced intervention will be evaluated using a prospective controlled design with the primary outcomes being composite suicidal acts (fatal and non-fatal), and with intermediate outcomes being the effect of training programs, changes in public attitudes, guideline-consistent media reporting. In addition an analysis of the economic costs and consequences will be undertaken, while a process evaluation will monitor implementation of the interventions within the different regions with varying organisational and healthcare contexts.
Discussion
This multi-centre research seeks to overcome major challenges of field research in suicide prevention. It pools data from four European regions, considerably increasing the study sample, which will be close to one million. In addition, the study will gather important information concerning the potential to transfer this multilevel program to other health care systems. The results of this research will provide a basis for developing an evidence-based, efficient concept for suicide prevention for EU-member states.
Platelet activation induces cytoskeletal rearrangements involving a change from discoid to spheric shape, secretion, and eventually adhesion and spreading on immobilized ligands. Small GTPases of the Rho family, such as Rac1 and Cdc42, are known to be involved in these processes by facilitating the formation of lamellipodia and filopodia, respectively. This thesis focuses on the role Rac1 and Cdc42 for platelet function and formation from their precursor cells, the megakaryocytes (MKs), using conditional knock-out mice. In the first part of the work, the involvement of Rac1 in the activation of the enzyme phospholipase (PL) C2 in the signaling pathway of the major platelet collagen receptor glycoprotein (GP) VI was investigated. It was found that Rac1 is essential for PLC2 activation independently of tyrosine phosphorylation of the enzyme, resulting in a specific platelet activation defect downstream of GPVI, whereas signaling of other activating receptors remains unaffected. Since Rac1-deficient mice were protected from arterial thrombosis in two different in vivo models, the GTPase might serve as a potential target for the development of new drugs for the treatment and prophylaxis of cardio- and cerebrovascular diseases. The second part of the thesis deals with the first characterization of MK- and platelet-specific Cdc42 knock-out mice. Cdc42-deficient mice displayed mild thrombo-cytopenia and platelet production from mutant MKs was markedly reduced. Unexpectedly, Cdc42-deficient platelets showed increased granule content and release upon activation, leading to accelerated thrombus formation in vitro and in vivo. Furthermore, Cdc42 was not generally required for filopodia formation upon platelet activation. Thus, these results indicate that Cdc42, unlike Rac1, is involved in multiple signaling pathways essential for proper platelet formation and function. Finally, the outcome of combined deletion of Rac1 and Cdc42 was studied. In contrast to single deficiency of either GTPase, platelet production from double-deficient MKs was virtually abrogated, resulting in dramatic macrothrombocytopenia in the animals. Formed platelets were largely non-functional leading to a severe hemostatic defect and defective thrombus formation in double-deficient mice in vivo. These results demonstrate for the first time a functional redundancy of Rac1 and Cdc42 in the hematopoietic system.
The scope of this work is to develop a novel single-molecule imaging technique by combining atomic force microscopy (AFM) and optical fluorescence microscopy. The technique is used for characterizing the structural properties of multi-protein complexes. The high-resolution fluorescence microscopy and AFM are combined (FIONA-AFM) to allow for the identification of individual proteins in such complexes. This is achieved by labeling single proteins with fluorescent dyes and determining the positions of these fluorophores with high precision in an optical image. The same area of the sample is subsequently scanned by AFM. Finally, the two images are aligned and the positions of the fluorophores are displayed on top of the topographical data. Using quantum dots as fiducial markers in addition to fluorescently labeled proteins, fluorescence and AFM information can be aligned with an accuracy better than 10 nm, which is sufficient to identify single fluorescently labeled proteins in most multi-protein complexes. The limitations of localization precision and accuracy in fluorescence and AFM images are investigated, including their effects on the overall registration accuracy of FIONA-AFM hybrid images. This combination of the two complementary techniques opens a wide spectrum of possible applications to the study of protein interactions, because AFM can yield high resolution (5–10 nm) information about the conformational properties of multi-protein complexes while the fluorescence can indicate spatial relationships of the proteins within the complexes. Additionally, computer simulations are performed in order to validate the accuracy of the registration algorithm.
Phytohormones are known for their pivotal roles in promoting normal growth and development of the plants and contributing to the mechanism of defense. Although an over simplification, however, they may be categorized as stress specific and growth promoting. SA and JA/Ethylene are implicated in stress responses while auxins, cytokinins and gibberellins are involved in developmental processes. Phytohormones from the above perspective got much attention in the last few decades; however their reciprocal role is currently in focus. It is because of the reason that plant pathogens cause overall hormonal imbalance at host pathogen interface and alter host physiology for the sake of pathogenecity. Despite their importance in growth and development, cytokinins are among the most neglected phytohormones that are usually noticed as consequence rather than a cause of pathogen infection. Results presented in this thesis are based on the hypothesis that elevated levels of CKs embody plants with resistance against hemibiotrophic pathogens. To explore a connection between the spread of P. syringae and its tobacco host, CKs over producing transgenic plants were investigated whereby bacterial IPT gene was expressed under the control of pathogen inducible, tetracycline inducible and developmentally inducible promoters. To further validate the out-come of transgenic plants, various types of cytokinins were exogenously fed to detached tobacco leaves. Mentioned transgenics and exogenous CKs feeding approaches unanimously resulted in, “more cytokinins less disease symptoms” and vice versa. This state of cytokinins mediated resistance was further substantiated with various cellular, signaling, biochemical and microbial approaches wherein levels of SA and JA remained unaffected. Conversely, PR1 gene expression was strongly up-regulated in enhanced cytokinins accumulating samples. Moreover, less accumulation of ROS was observed in IPT expressing sites of the plants as compared to their corresponding controls. Additionally, we neither noticed any direct effect of cytokinins on the growth of P. syringae pv. tabaci nor found presence of anti-microbial peptides in cytokinins enriched extracts. Interestingly, enhanced accumulation of phtyoalexins in elevated CKs status of the plant proved to be a possible gesture in jeopardizing the spread of pathogen. Contrarily, no reduction was observed in the spread of fungal necrotrophic pathogen Sclerotinia sclerotiorum when leaves of elevated CKs were inoculated. Besides host-pathogen interaction in perspective of elevated cytokinins, impact of modulated sugar status of the plant on the spread of pathogen was also investigated. For this purpose, previously generated modulated invertase enzyme tobacco transgenic plants were analyzed. We showed that repression and de-repression of CIN1 gene under the control of tetracycline inducible-promoter did not affect the growth of P. syrinage pv. tabaci in Tet::CIN1 transgenic plants. Moreover, invertase inhibitor tobacco lines expressing NtCIF gene under the control of the same promoter failed to exhibit differential pathogenic responses in induced and non induced status of the plant. Similar was the case of tomato transgenic plants expressing NtCIF gene under the control of invertase gene Lin6 promoter in Lin6:: NtCIF plants for P.syringae pv. tomato DC 3000. Interestingly, when challenged Lin6:: NtCIF tomato plants with Botrytis cinerea, severe disease symptoms were observed on transgenic leaves as compared to control plants. To dissect a potential link between cytokinins and sugar metabolism with its effect on the growth of pathogen, invertase transgenic plants with elevated CKs were probed. When expressed exogenous IPT gene under the control of pathogen inducible promoter (4xJERE::IPT) in transgenic background of Tet::CIN1, we observed localized differences in symptom development for P.syringae pv. tabaci. Similarly, when exogenously fed with kinetin, detached leaves of Tet::CIN1 exhibited retarded growth of P.syringae pv. tabaci as compared to the tetracycline induced leaves. These results led to the conclusion that extracellular invertase may not play an essential role in cytokinins mediated disease resistance against hemibiotrophic pathogens.
In the context of the indirect search for non-standard physics in the flavour sector of the Standard Model (SM), one of the most interesting processes is the rare inclusive B -> X_s gamma decay. On the one hand, being a flavour-changing neutral current, this B decay is sensitive to new physics, as it is loop-suppressed in the SM. On the other hand, it is only mildly affected by non-perturbative effects, and thus allows for precise theoretical predictions in the framework of renormalization-group improved perturbation theory. Accurate measurements as well as precise theoretical predictions with a good control over both perturbative and non-perturbative contributions have to be provided in order to derive stringent constraints on the parameter space of physics beyond the SM. On the experimental side, an outstanding accuracy in the measurement of the B -> Xs gamma decay rate has been achieved, which is mainly due the specialized experiments BaBar and Belle at the so-called B factories. To match the small experimental uncertainty, higher order computations within an effective low-energy theory of the SM are mandatory. In fact, next-to-next-to-leading order (NNLO) QCD corrections are required to provide a prediction for the decay rate with the same precision as the measurement. The NNLO evaluation of the B -> Xs gamma decay rate has been pursued by various groups over the last decade. The project was completed to a large extent and a first estimate at this level of perturbation theory was obtained in 2006. This prediction, however, lacks important contributions from yet unknown matrix elements, that were estimated from results which are only partially known to date. In this work, we provide a framework for the systematic study of the missing matrix elements at the NNLO. As main results of this thesis, we determine fermionic corrections to the charm quark mass dependent matrix elements of four-quark operators in the effective theory at NNLO. For the first time, the full mass dependence was kept. Moreover, we evaluate both bosonic and fermionic corrections to the decay rate in the limit of vanishing charm quark mass. These findings, combined with yet unknown remaining real contributions, will help to reduce the uncertainty of the NNLO branching ratio estimate considerably. Another central topic of the present work is the development of an automatic high-precision computation of multi-loop multi-scale integrals, a crucial ingredient for the here presented results.
In this thesis, we present novel approaches for formation driving of nonholonomic robots and optimal trajectory planning to reach a target region. The methods consider a static known map of the environment as well as unknown and dynamic obstacles detected by sensors of the formation. The algorithms are based on leader following techniques, where the formation of car-like robots is maintained in a shape determined by curvilinear coordinates. Beyond this, the general methods of formation driving are specialized and extended for an application of airport snow shoveling. Detailed descriptions of the algorithms complemented by relevant stability and convergence studies will be provided in the following chapters. Furthermore, discussions of the applicability will be verified by various simulations in existing robotic environments and also by a hardware experiment.
In conclusion, the present thesis demonstrates that the highly desired J-type aggregation of functional perylene bisimide chromophores can be achieved by proper design of monomeric building blocks that direct self-assemble by mutual effects of hydrogen bonding and pi-pi interaction, and on the other hand, are prevented to assemble in columnar stacks owing to their twisted pi-conjugated core and sterically demanding substituents. Furthermore, the self-assembly studies gave new insights into the dynamic aggregation process of low-dimensional extended assemblies with strongly excitonically coupled chromophores. The relationship between commonly known cyanine dye aggregates like that of THIATS and that of the present PBI 1a was investigated by absorption and fluorescence spectroscopy at low temperatures down to 5 K. The formerly unprecedented functional properties of PBI aggregates that are expressed in J-type excitonic coupling hold promise for application in optoelectronic and photovoltaic devices.
The thesis investigates the electrical transport properties of different π-conjugated organic semiconductors applied as active semiconducting material in organic field-effect transistor (OFET) devices. Theses organic materials are αω-dihexylquaterthiophene (DH4T), the tetrathiafulvalene (TTF) derivatives dibenzene-tetrathiafulvalene (DB-TTF) and dithiophene-tetrathiafulvalene (DT-TTF), and polytriarylamine (PTAA). The latter material is an amorphous polymer, the three others are small molecule oligomer materials. Different deposition methods were applied and compared. The investigations in the thesis treat the pure characterisation of the above materials with their different properties in OFET devices. Furthermore, the aim was to observe and analyse general rules and effects in OFETs depending on the structure, previous history, and the device scaling. Therefore, different tools and special analysing methods were developed and applied. These are a standard characterisation method for the classification of the used organic semiconductor, temperature dependent electrical characterisation investigating the electrical transport properties, the newly developed in situ measurement method of OFET devices, the downscaling of the OFET devices of channel length below 100 nm, and the lithographical structuring of a PTAA film.
We consider competitive location problems where two competing providers place their facilities sequentially and users can decide between the competitors. We assume that both competitors act non-cooperatively and aim at maximizing their own benefits. We investigate the complexity and approximability of such problems on graphs, in particular on simple graph classes such as trees and paths. We also develop fast algorithms for single competitive location problems where each provider places a single facilty. Voting location, in contrast, aims at identifying locations that meet social criteria. The provider wants to satisfy the users (customers) of the facility to be opened. In general, there is no location that is favored by all users. Therefore, a satisfactory compromise has to be found. To this end, criteria arising from voting theory are considered. The solution of the location problem is understood as the winner of a virtual election among the users of the facilities, in which the potential locations play the role of the candidates and the users represent the voters. Competitive and voting location problems turn out to be closely related.
Plants must respond to multiple stimuli in a natural environment. Therefore they need the ability to rapidly reorganise and specifically build up appropriate metabolites to adapt to their environment. Abiotic cues, such as ambient solar radiation, influence the next trophic level directly, but also an altered plant composition triggered by these environmental cues can have an effect on the behaviour of herbivores. The aim of this study was to test effects of the important ultraviolet (UV) radiation on plants and on plant-insect interactions using multi-level investigations. The focus was on the conduction of controlled experiments with broccoli plants in highly engineered greenhouses covered with innovative materials, which only differed in their UV-B transmission. For the first time in this controlled environment the plant-mediated UV-B effects on phloem-feeding aphids were studied. Broccoli plants (Brassica oleracea L. convar. botrytis, Brassicaceae) were under filter tents either exposed to (inclusion, +UV) or not exposed to (exclusion, -UV) UV-A / UV-B radiation. In greenhouses covered with new, innovative materials transmitting high (80%), medium (23%) or low (4%) levels of ambient solar UV-B radiation, in particular the influence of UV-B radiation on broccoli was examined. Plants respond highly specific to environmental stimuli such as UV-B radiation and herbivory. UV-B radiation has a strong impact on the plants’ architecture and flavonoid contents, which can in turn influence plant-insect interactions. Phloem-feeding aphids can be negatively affected by UV-B mediated plant changes. However, a direct effect of UV radiation on the behaviour of herbivores is also evident. Mainly the number, composition and quality of herbivorous species as well as an exceeding of a certain infestation threshold determine the mode of plant changes. In conclusion, UV-B radiation has the potential to harden plants against herbivores and simultaneously increases the concentrations of valuable secondary metabolites for human nutrition in important crop species such as broccoli.
Vegetation structure can profoundly influence patterns of abundance, distribution, and reproduction of herbivorous insects and their susceptibility to natural enemies. The three main structural traits of herbaceous vegetation are density, height, and connectivity. This study determined the herbivore response to each of these three parameters by analysing oviposition patterns in the field and studying the underlying mechanisms in laboratory bioassays. The generalist leaf beetle, Galeruca tanaceti L. (Coleoptera: Chrysomelidae), preferentially deposits its egg clutches on non-host plants such as grasses. Earlier studies revealed that oviposition within structurally complex vegetation reduces the risk of egg parasitism. Consequently, leaf beetle females should prefer patches with dense, tall, or connected vegetation for oviposition in order to increase their reproductive success. In the present study, we tested the following three hypotheses on the effect of stem density, height, and connectivity on oviposition: (1) Within habitats, the number of egg clutches in areas with high stem densities is disproportionately higher than in low-density areas. The number of egg clutches on (2) tall stems or (3) in vegetation with high connectivity is higher than expected for a random distribution. In the field, stem density and height were positively correlated with egg clutch presence. Moreover, a disproportionately high presence of egg clutches was determined in patches with high stem densities. Stem height had a positive influence on oviposition, also in a laboratory two-choice bioassay, whereas stem density and connectivity did not affect oviposition preferences in the laboratory. Therefore, stem height and, potentially, density, but not connectivity, seem to trigger oviposition site selection of the herbivore. This study made evident that certain, but not all traits of the vegetation structure can impose a strong influence on oviposition patterns of herbivorous insects. The results were finally compared with data on the movement patterns of the specialised egg parasitoid of the herbivore in comparable types of vegetation structure.
Controllability Aspects of the Lindblad-Kossakowski Master Equation : A Lie-Theoretical Approach
(2009)
One main task, which is considerably important in many applications in quantum control, is to explore the possibilities of steering a quantum system from an initial state to a target state. This thesis focuses on fundamental control-theoretical issues of quantum dynamics described by the Lindblad-Kossakowski master equation which arises as a bilinear control system on some underlying real vector spaces, e.g controllability aspects and the structure of reachable sets. Based on Lie-algebraic methods from nonlinear control theory, the thesis presents a unified approach to control problems of finite dimensional closed and open quantum systems. In particular, a simplified treatment for controllability of closed quantum systems as well as new accessibility results for open quantum systems are obtained. The main tools to derive the results are the well-known classifications of all matrix Lie groups which act transitively on Grassmann manifolds, and respectively, on real vector spaces without the origin. It is also shown in this thesis that accessibiity of the Lindblad-Kossakowski master equation is a generic property. Moreover, based on the theoretical accessibility results, an algorithm is developed to decide when the Lindblad-Kossakowski master equation is accessible.
Abstract: Inbreeding depression, asymmetries in costs or benefits of dispersal, and the mating system have been identified as potential factors underlying the evolution of sex-biased dispersal. We use individual-based simulations to explore how the mating system and demographic stochasticity influence the evolution of sex-specific dispersal in a metapopulation with females competing over breeding sites, and males over mating opportunities. Comparison of simulation results for random mating with those for a harem system (locally, a single male sires all offspring) reveal that even extreme variance in local male reproductive success (extreme male competition) does not induce male-biased dispersal. The latter evolves if the between-parch variance in reproductive success is larger for males than females. This can emerge due to demographic stochasticity if the habitat patches are small. More generally, members of a group of individuals experiencing higher spatio-temporal variance in fitness expectations may evolve to disperse with greater probability than others.
This study presents new petrological results obtained from high-grade metamorphic rocks of the Beit Bridge, Mahalapye and Phikwe Complexes, which constitute the Central Zone of the Limpopo Belt in southern Africa. These results provide detailed information about the prograde and retrograde pressure-temperature (P-T) evolution of the three investigated complexes and, in concert with geochronological data, form the basis for the development of a coherent geodynamic model for the evolution of the Limpopo’s Central Zone. The P-T paths were inferred by the thorough investigation of silica-saturated and silica- undersaturated metapelitic and metabasic rocks, comprising six sillimanite-garnet-cordierite gneisses, four (garnet)-biotite-plagioclase gneisses, two garnet-orthopyroxene-biotite-Kfeldspar-plagioclase gneisses, one garnet- cordierite-orthoamphibole fels, one garnet-biotite amphibolite, and one garnet-clinopyroxene amphibolite. P-T points and P-T evolutions were derived by the application of conventional geothermobarometers, and quantitative phase diagrams in the systems Na2O - CaO - K2O - FeO - MgO - Al2O3 - SiO2 - H2O - TiO2 - O (NCKFMASHTiO), and MnO - TiO2 - Na2O - CaO - K2O - FeO - MgO - Al2O3 - SiO2 - H2O (MnTiNCKFMASH) - using the computer software THERMOCALC and THERIAK-DOMINO. The petrological information, in particular those obtained by comparison between observed and thermodynamically calculated mineral assemblages, zonations and modes, in combination with new and existing geochronological data provide evidence that rocks from the three investigated complexes underwent slightly different P-T evolutions at different times. The samples from the Bulai Pluton area (Beit Bridge Complex) provide evidence for a Neoarchean high-grade metamorphic event at ~2.64 Ga (M2), with peak P-T conditions of ~850°C at 8-9 kbar, and a decompression-cooling path to ~750°C at 5-6 kbar. This metamorphic evolution perhaps took place in a magmatic arc setting. In contrast, samples from the Mahalapye and Phikwe Complex document a Palaeoproterozoic event at ~2.03-2.05 Ga (M3), and were subject to different styles of prograde metamorphism. Metamorphic rocks from the Mahalapye Complex experienced a high-temperature low-pressure (HT-LP) metamorphic overprint, accompanied by the emplacement of voluminous granite bodies between 2.06 and 2.02 Ga, and provide evidence for a slightly prograde decompression from ~650°C/7 kbar to ~800°C/5.5 kbar. In contrast, the metamorphic rocks from the Phikwe Complex provide evidence for a simultaneous pressure and temperature increase from ~600°C/6 kbar to ~750°C/8 kbar, in the absence of significant Palaeoproterozoic magmatism. The HT-LP metamorphic evolution of the Mahalapye Complex is interpreted to be initiated by the underplating of hot mafic melts, either formed in response to SE-subduction during the Kheis-Magondi orogeny, and/or by contemporaneous mantle plume activities related to the formation of the Bushveld Complex. In contrast, the prograde pressure and temperature increase reflected by the rocks from the Phikwe Complex rather reflects successive crustal stacking at ~2.03 Ga. This stacking, which is also reported from many other units throughout the Limpopo Belt, is interpreted to result from the final convergence between the Kaapvaal and Zimbabwe Cratons, perhaps caused by SE-directed compression in response to the Kheis-Magondi orogeny between ~2.06 and 1.90 Ga.
Abstract: 1. The response of dispersal towards evolution largely depends on its heritability for which upper limits are determined by the trait's repeatability. 2. In the Linyphiid spider E. atra, we were able to separate long- and short-distance dispersal behaviours (respectively ballooning and rappelling) under laboratory conditions. By performing repeated behavioural trials for females, we show that average dispersal trait values decrease with increasing testing days. By comparing mated and unmated individuals during two periods (before and after mating for the mated group, and the same two periods for the unmated group), we show that mating has no effect on the mean displayed dispersal behaviour or its within-individual variation. Repeatabilities were high and consistent for ballooning motivation, but not for rappelling. 3. Ballooning motivation can be regarded as highly individual-specific behaviour, while general pre-dispersal and rappelling behaviours showed more individual variation. Such difference in repeatability between long-and short-distance dispersal suggests that short-and long-distance dispersal events are triggered by different ecological and evolutionary mechanisms.
Background: Male killing endosymbionts manipulate their arthropod host reproduction by only allowing female embryos to develop into infected females and killing all male offspring. Because the resulting change in sex ratio is expected to affect the evolution of sex-specific dispersal, we investigated under which environmental conditions strong sex-biased dispersal would emerge, and how this would affect host and endosymbiont metapopulation persistence. Results: We simulated host-endosymbiont metapopulation dynamics in an individual-based model, in which dispersal rates are allowed to evolve independently for the two sexes. Prominent male-biased dispersal emerges under conditions of low environmental stochasticity and high dispersal mortality. By applying a reshuffling algorithm, we show that kin-competition is a major driver of this evolutionary pattern because of the high within-population relatedness of males compared to those of females. Moreover, the evolution of sex-specific dispersal rescues metapopulations from extinction by (i) reducing endosymbiont fixation rates and (ii) by enhancing the extinction of endosymbionts within metapopulations that are characterized by low environmental stochasticity. Conclusion: Male killing endosymbionts induce the evolution of sex-specific dispersal, with prominent male-biased dispersal under conditions of low environmental stochasticity and high dispersal mortality. This male-biased dispersal emerges from stronger kin-competition in males compared to females and induces an evolutionary rescue mechanism.
Bispecific T cell engager (BiTE) display a novel design among the class of bispecific antibodies and hold great promise to fight diverse cancers. BiTE molecules consist of two different binding entities derived from two human IgG antibodies connected by a short peptide linker. Their binding arms are directed against the CD3e chain of the T cell receptor on T cells and against an antigen that is specific for (e.g., CD19 for lymphoma in MT103) or over-expressed on (e.g., EpCAM for epithelial cancer in MT110) tumor cells. Without requirement for pre- or co-stimulation, BiTE molecules efficiently redirect CD3+ T cells towards tumor cells expressing the relevant target antigen. Only a BiTE molecule simultaneously bound to both tumor cell and T cell activates the T cell to exert its cytolytic function resulting in tumor cell death. In T cells stimulated with both BiTE and target cells, elevated levels of caspase activation and increased expression of cytotoxic and signaling proteins are observed. These include cytolytic proteins granzyme B and perforin, activation markers CD69 and CD25 and adhesion molecules CD2 and LFA-1. Activated T cells secrete the usual mix of cytokines, among them pro-inflammatory cytokines IFN-g and TNF-a. The membrane of tumor cells expressing the relevant target antigen is perforated during the attack of BiTE-stimulated effector cells as can be concluded from adenylate kinase release from the cytosol of tumor cells. Ca2+-chelator EGTA completely blocked BiTE-mediated activation of caspases and tumor cell lysis. As perforin is strictly Ca2+-dependent, a major role for this pore-forming protein is assumed for the elimination of tumor cells via BiTE-stimulated T cells. Granzyme B and caspases are main players in BiTE-mediated elimination of tumor cells. Inhibitors of granzyme B or caspases reduce or block, respectively the activation of caspases. However, other signals of apoptosis (cleavage of PARP and fragmentation of DNA) were only reduced by granzyme B inhibitor or caspase inhibitor. Most interestingly, the lytic capacity of BiTE molecules was not impaired by granzyme B inhibitor or caspase inhibitor. It seems that there is no requirement for granzyme B and caspases to be present simultaneously. Instead the data presented provide evidence that they can be replaced one at a time by related proteins. Pre-incubation of effector cells with the glucocorticoids dexamethasone or methylprednisolone resulted in markedly decreased secretion of cytokines by T cells yet only a small reduction in the expression of activation markers and adhesion molecules on T cells and specific lysis of tumor cells upon BiTE stimulation. Soluble factors secreted in an undirected manner by BiTE-stimulated T cells do not mediate tumor cell death by themselves. Bystander cells negative for the antigen that is recognized by the BiTE molecule will not be compromised by BiTE activity. The cytokine TGF-b reduced proliferation as well as granzyme B and perforin expression of BiTE-stimulated T cells. Redirected lysis by BiTE-activated T cells was also decreased under the influence of TGF-b, however lysis was still performed at a reasonable rate (72 % of target cells). TGF-b does not exert a deleterious effect on lytic potential of BiTE-stimulated T cells. The minimal anticipated biological effect level for the BiTE MT110 was determined for the entry of MT110 into phase I clinical studies. Experiments analyzing redirected lysis of tumor cells, expression of activation marker CD25 and cytokine release by T cells revealed a MABEL value of 50 pg/ml for MT110.
Studies on platelet cytoskeletal dynamics and receptor regulation in genetically modified mice
(2009)
Platelets are produced by bone marrow megakaryocytes in a process involving actin dynamics. Actin-depolymerizing factor (ADF) and cofilin are actin-binding proteins that act as key regulators in actin turnover by promoting filament severing and depolymerization. The overall significance of ADF/cofilin function and actin turnover in platelet formation is presently unclear. In the first part of this thesis, platelet formation and function were studied in mice constitutively lacking ADF and/or mice with a conditional deficiency (Cre/loxP) in n-cofilin. To delete cofilin exclusively in megakaryocytes and platelets, cofilinfl/fl mice were crossed with PF4 (platelet factor 4)-Cre mice. While a single-deficiency in ADF or n-cofilin resulted in no or only a minor platelet formation defect, respectively, a double-deficiency in ADF and n-cofilin led to an almost complete loss of platelets. Bone marrow megakaryocytes of ADF/n-cofilin-deficient mice showed defective platelet zone formation. Interestingly, in vitro and ex vivo megakaryocyte differentiation revealed reduced proplatelet formation and absence of platelet-forming swellings. These data establish that ADF and n-cofilin have redundant but essential roles in the terminal step of platelet formation in vitro and in vivo. In the second part of the thesis, mechanisms underlying cellular regulation of the major platelet collagen receptor, glycoprotein VI (GPVI), were studied. GPVI mediates platelet activation on exposed subendothelial collagens at sites of vascular injury, and thereby contributes to normal hemostasis but also to occlusion of diseased vessels in the setting of myocardial infarction or stroke. Thus, GPVI is an attractive target for anti-thrombotic therapy, particularly because previous studies have shown that anti-GPVI antibodies induce irreversible down-regulation of the receptor in circulating platelets by internalization and ectodomain shedding. Metalloproteinases of the ADAM (a disintegrin and metalloproteinase domain) family are suspected to mediate this ectodomain shedding, but in vivo evidence for this is lacking. To study the mechanism of GPVI regulation in vivo, two mouse lines, Gp6 knock-out and Adam10fl/fl, PF4-Cre mice, were generated and in addition low TACE (TNFalpha converting enzyme) mice were analyzed. It was shown that GPVI can be cleaved in vitro by ADAM10 or TACE depending on the shedding-inducing signaling pathway. Moreover, GPVI was down-regulated in vivo upon antibody injection in ADAM10-deficient and low TACE mice suggesting that either both or an additional metalloproteinase is involved in GPVI regulation in vivo.
Members of the enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family are important regulators of the actin cytoskeleton dynamics. VASP functions as well as its interactions with other proteins are regulated by phosphorylation at three sites - serine157 (S157), serine239 (S239), and threonine278 (T278) in humans. cAMP- and cGMP- dependent protein kinases phosphorylate S157 and S239, respectively. In contrast, the kinase responsible for T278 was as yet unknown and identified in the first part of this thesis. In a screen for T278 phosphorylating kinases using a phospho-specific antibody against phosphorylated T278 AMP-activated protein kinase (AMPK) was identified in endothelial cells. Mutants of AMPK with altered kinase-activity modulate T278-phosphorylation levels in cells. AMPK-driven T278-phosphorylation impaired stress fiber formation and changed cell morphology in living cells. AMPK is a fundamental sensor of cellular and whole body energy homeostasis. Zucker Diabetic Fatty (ZDF) rats, which are an animal model for type II diabetes mellitus, were used to analyze the impact of phosphorylated T278 in vivo. AMPK-activity and T278-phosphorylation were substantially reduced in arterial vessel walls of ZDF rats in comparison to control animals. These findings demonstrate that VASP is a new AMPK substrate, that VASP phosphorylation mediates the effects of metabolic regulation on actin cytoskeleton rearrangements, and that this signaling system becomes down-regulated in diabetic vessel disorders in rats. In the second part of this thesis, a functional analysis of differential VASP phosphorylations was performed. To systematically address VASP phosphorylation patterns, a set of VASP phosphomimetic mutants was cloned. These mutants enable the mimicking of defined phosphorylation patterns and the specific analysis of single kinase-mediated phosphorylations. VASP localization to the cell periphery was increased by S157- phosphorylation and modulated by phosphorylation at S239 and T278. Latter phosphorylations synergistically reduced actin polymerization. In contrast, S157- phosphorylation had no effect on actin-dynamics. Taken together, the results of the second part show that phosphorylation of VASP serves as a fine regulator of localization and actin polymerization activity. In summary, this study revealed the functions of VASP phosphorylations and established novel links between signaling pathways and actin cytoskeleton rearrangement.
Many organisms show polymorphism in dispersal distance strategies. This variation is particularly ecological relevant if it encompasses a functional separation of short- (SDD) and long-distance dispersal (LDD). It remains, however, an open question whether both parts of the dispersal kernel are similarly affected by landscape related selection pressures. We implemented an individual-based model to analyze the evolution of dispersal traits in fractal landscapes that vary in the proportion of habitat and its spatial configuration. Individuals are parthenogenetic with dispersal distance determined by two alleles on each individual‘s genome: one allele coding for the probability of global dispersal and one allele coding for the variance of a Gaussian local dispersal with mean value zero. Simulations show that mean distances of local dispersal and the probability of global dispersal, increase with increasing habitat availability, but that changes in the habitat's spatial autocorrelation impose opposing selective pressure: local dispersal distances decrease and global dispersal probabilities increase with decreasing spatial autocorrelation of the available habitat. Local adaptation of local dispersal distance emerges in landscapes with less than 70% of clumped habitat. These results demonstrate that long and short distance dispersal evolve separately according to different properties of the landscape. The landscape structure may consequently largely affect the evolution of dispersal distance strategies and the level of dispersal polymorphism.
Genome sequence analysis A combination of genome analysis application has been established here during this project. This offers an efficient platform to interactively compare similar genome regions and reveal loci differences. The genes and operons can be rapidly analyzed and local collinear blocks (LCBs) categorized according to their function. The features of interests are parsed, recognized, and clustered into reports. Phylogenetic relationships can be readily examined such as the evolution of critical factors or a certain highly-conserved region. The resulting platform-independent software packages (GENOVA and inGeno), have been proven to be efficient and easy to handle in a number of projects. The capabilities of the software allowed the investigation of virulence factors, e.g., rsbU, strains’ biological design, and in particular pathogenicity feature storage and management. We have successfully investigated the genomes of Staphylococcus aureus strains (COL, N315, 8325, RN1HG, Newman), Listeria spp. (welshimeri, innocua and monocytogenes), E.coli strains (O157:H7 and MG1655) and Vaccinia strains (WR, Copenhagen, Lister, LIVP, GLV-1h68 and parental strains). Metabolic network analysis Our YANAsquare package offers a workbench to rapidly establish the metabolic network of such as Staphylococcous aureus bacteria in genome-scale size as well as metabolic networks of interest such as the murine phagosome lipid signalling network. YANAsquare recruits reactions from online databases using an integrated KEGG browser. This reduces the efforts in building large metabolic networks. The involved calculation routines (METATOOL-derived wrapper or native Java implementation) readily obtain all possible flux modes (EM/EP) for metabolite fluxes within the network. Advanced layout algorithms visualize the topological structure of the network. In addition, the generated structure can be dynamically modified in the graphic interface. The generated network as well as the manipulated layout can be validated and stored (XML file: scheme of SBML level-2). This format can be further parsed and analyzed by other systems biology software, such as CellDesigner. Moreover, the integrated robustness-evaluation routine is able to examine the synthesis rates affected by each single mutation throughout the whole network. We have successfully applied the method to simulate single and multiple gene knockouts, and the affected fluxes are comprehensively revealed. Recently we applied the method to proteomic data and extra-cellular metabolite data of Staphylococci, the physiological changes regarding the flux distribution are studied. Calculations at different time points, including different conditions such as hypoxia or stress, show a good fit to experimental data. Moreover, using the proteomic data (enzyme amounts) calculated from 2D-Gel-EP experiments our study provides a way to compare the fluxome and the enzyme expression. Oncolytic vaccinia virus (VACV) We investigated the genetic differences between the de novo sequence of the recombinant oncolytic GLV-1h68 and other related VACVs, including function predictions for all found genome differences. Our phylogenetic analysis indicates that GLV-1h68 is closest to Lister strains but has lost several ORFs present in its parental LIVP strain, including genes encoding CrmE and a viral Golgi anti-apoptotic protein, v-GAAP. Functions of viral genes were either strain-specific, tissue-specific or host-specific comparing viral genes in the Lister, WR and COP strains. This helps to rationally design more optimized oncolytic virus strains to benefit cancer therapy in human patients. Identified differences from the comparison in open reading frames (ORFs) include genes for host-range selection, virulence and immune modulation proteins, e.g. ankyrin-like proteins, serine proteinase inhibitor SPI-2/CrmA, tumor necrosis factor (TNF) receptor homolog CrmC, semaphorin-like and interleukin-1 receptor homolog proteins. The contribution of foreign gene expression cassettes in the therapeutic and oncolytic virus GLV-1h68 was studied, including the F14.5L, J2R and A56R loci. The contribution of F14.5L inactivation to the reduced virulence is demonstrated by comparing the virulence data of GLV-1h68 with its F14.5L-null and revertant viruses. The comparison suggests that insertion of a foreign gene expression cassette in a nonessential locus in the viral genome is a practical way to attenuate VACVs, especially if the nonessential locus itself contains a virulence gene. This reduces the virulence of the virus without compromising too much the replication competency of the virus, the key to its oncolytic activity. The reduced pathogenicity of GLV-1h68 was confirmed by our experimental collaboration partners in male mice bearing C6 rat glioma and in immunocompetent mice bearing B16-F10 murine melanoma. In conclusion, bioinformatics and experimental data show that GLV-1h68 is a promising engineered VACV variant for anticancer therapy with tumor-specific replication, reduced pathogenicity and benign tissue tropism.
This thesis deals with three selected dimensions of strategic behavior, namely investment in R&D, mergers and acquisitions, and inventory decisions in dynamic oligopolies. The question the first essay addresses is how the market structure evolves due to innovative activities when firms' level of technological competence is valuable for more than one project. The focus of the work is the analysis of the effect of learning-by-doing and organizational forgetting in R&D on firms' incentives to innovate. A dynamic step-by-step innovation model with history dependency is developed. Firms can accumulate knowledge by investing in R&D. As a benchmark without knowledge accumulation it is shown that relaxing the usual assumption of imposed imitation yields additional strategic effects. Therefore, the leader's R&D effort increases with the gap as she is trying to avoid competition in the future. When firms gain experience by performing R&D, the resulting effect of knowledge induces technological leaders to rest on their laurels which allows followers to catch up. Contrary to the benchmark case the leader's innovation effort declines with the lead. This causes an equilibrium where the incentives to innovate are highest when competition is most intense. Using a model of oligopoly in general equilibrium the second essay analyzes the integration of economies that might be accompanied by cross-border merger waves. Studying economies which prior to trade were in stable equilibrium where mergers were not profitable, we show that globalization can trigger cross-border merger waves for a sufficiently large heterogeneity in marginal cost. In partial equilibrium, consumers benefit from integration even when a merger wave is triggered which considerably lowers intensity of competition. Welfare increases. In contrast, in general equilibrium where interactions between markets and therefore effects on factor prices are considered, gains from trade can only be realized by reallocation of resources. The higher the technological dissimilarity between countries the better can efficiency gains be realized in integrated general equilibrium. The overall welfare effect of integration is positive when all firms remain active but indeterminate when firms exit or are absorbed due to a merger wave. It is possible for decreasing competition to dominate the welfare gain from more efficient resource allocation across sectors. Allowing for firms' entry alters results as in an integrated world coexistence of firms of different countries is never possible. Comparative advantages with respect to entry and production are important for realizing efficiency gains from trade. The third essay analyzes the interaction between price and inventory decisions in an oligopoly industry and its implications for the dynamics of prices. The work extends existing literature and especially the work of Hall and Rust (2007) to endogenous prices and strategic oligopoly competition. We show that the optimal decision rule is an (S,s) order policy and prices and inventories are strategic substitutes. Fixed ordering costs generate infrequent orders. Additionally, with strategic competition in prices, (S,s) inventory behavior together with demand uncertainty generates cyclical pattern in prices The last chapter presents some concluding remarks on the results of the essays.
Since its popularization due to Randall and Sundrum (RS) one decade ago, and in connection with the $AdS/CFT$ correspondence in particular, 5D warped background spacetime has been one of the most fruitful new ideas in physics beyond the standard model (SM), leading to new insights into symmetry breaking and the properties of strongly interacting theories inaccessible to direct perturbative calculations, while at the same time relating gravity to phenomenological model building. This has, among others, led to a renewed interest in models of electroweak symmetry breaking without physical scalar fields in the guise of so-called 'warped higgsless' models, which could provide an alternative to the famed Higgs mechanism of electroweak symmetry breaking which is part of the Standard Model of particle physics. However, little emphasis was put on reconciling these models with the strong evidence from astrophysical observations that one or several new, as yet unknown, stable particle species exist which form the cold dark matter content of the universe. The nature of dark matter and electroweak symmetry breaking are among the most prominent puzzles subject to experimental scrutiny at the Tevatron, direct search experiments, and in the near future at the LHC, which compels us the believe that both issues should be addressed together in any alternative scenario beyond the Standard Model. In this thesis we have investigated phenomenological implications which arise for cosmology and collider physics when the electroweak symmetry breaking sector of warped higgsless models is extended to include warped supersymmetry with conserved $R$ parity. The goal was to find the simplest supersymmetric extension of these models which still has a realistic light spectrum including a viable dark matter candidate. To accomplish this, we have used the same mechanism which is already at work for symmetry breaking in the electroweak sector to break supersymmetry as well, namely symmetry breaking by boundary conditions. While supersymmetry in five dimensions contains four supercharges and is therefore directly related to 4D $\mathcal{N}=2$ supersymmetry, half of them are broken by the background leaving us with ordinary $\mathcal{N}=1$ theory in the massless sector after Kaluza-Klein expansion. We thus use boundary conditions to model the effects of a breaking mechanism for the remaining two supercharges. The simplest viable scenario to investigate is a supersymmetric bulk and IR brane without supersymmetry on the UV brane. Even though parts of the light spectrum are effectively projected out by this mechanism, we retain the rich phenomenology of complete $\mathcal{N}=2$ supermultiplets in the Kaluza-Klein sector. While the light supersymmetric spectrum consists of electroweak gauginos which get their $\mathcal{O}(100\mbox{ GeV})$ masses from IR brane electroweak symmetry breaking, the light gluinos and squarks are projected out on the UV brane. The neutralinos, as mass eigenstates of the neutral bino-wino sector, are automatically the lightest gauginos, making them LSP dark matter candidates with a relic density that can be brought to agreement with WMAP measurements without extensive tuning of parameters. For chargino masses close to the experimental lower bounds at around $m_{\chi^+}\approx 100\dots 110$ GeV, the dark matter relic density points to LSP masses of around $m_\chi\approx 90$ GeV. At the LHC, the standard particle content of our model shares most of the key features of known warped higgsless models. We have performed Monte Carlo simulations of warped higgsless LSP and NLSP production at a benchmark point using \nameomega/\namewhizard, concentrating on $\ptmiss$ in association with third generation quarks. After background reduction cuts on the quark momenta and angles, we get hadronic cross sections of $\sigma>100\mbox{ fb}$ at $14\mbox{ TeV}$ with characteristic $\ptmiss$ distributions for $\chi\chi t\overline{t}$ final states, while the final states with $b\overline{b}$ pairs have much lower event rates and shapes which are hard to discern in experiments. Our results suggest that the discovery of warped higgsless LSP dark matter at the LHC via missing energy is within reach for the first few $\mbox{ fb}^{-1}$ at $14$ TeV if $b$ and in particular $t$ identification is reliable.
Fragmentation, deterioration, and loss of habitat patches threaten the survival of many insect species. Depending on their trophic level, species may be differently affected by these factors. However, studies investigating more than one trophic level on a landscape scale are still rare. In the present study we analyzed the effects of habitat size, isolation, and quality for the occurrence and population density of the endangered leaf beetle Cassida canaliculata Laich. (Coleoptera: Chrysomelidae) and its egg parasitoid, the hymenopteran wasp Foersterella reptans Nees (Hymenoptera: Tetracampidae). C. canaliculata is strictly monophagous on meadow sage (Salvia pratensis), while F. reptans can also parasitize other hosts. Both size and isolation of habitat patches strongly determined the occurrence of the beetle. However, population density increased to a much greater extent with increasing host plant density ( = habitat quality) than with habitat size. The occurrence probability of the egg parasitoid increased with increasing population density of C. canaliculata. In conclusion, although maintaining large, well-connected patches with high host plant density is surely the major conservation goal for the specialized herbivore C. canaliculata, also small patches with high host plant densities can support viable populations and should thus be conserved. The less specialized parasitoid F. reptans is more likely to be found on patches with high beetle density, while patch size and isolation seem to be less important.
The olfactory system of leafcutting ants: neuroanatomy and the correlation to social organization
(2009)
In leaf-cutting ants (genera Atta and Acromyrmex), the worker caste exhibits a pronounced size-polymorphism, and division of labor is largely dependent on worker size (alloethism). Behavioral studies have shown a rich diversity of olfactory-guided behaviors, and the olfactory system seems to be highly developed and very sensitive. To allow fine-tuned behavioral responses to different tasks, adaptations within the olfactory system of different sized workers are expected. In a recent study, two different phenotypes of the antennal lobe of Atta vollenweideri workers were found: MG- and RG-phenotype (with and without a macroglomerulus, MG). The existence of the macroglomerulus is correlated to the body size of workers, with small workers showing the RG-phenotype and large workers showing the MG-phenotype. In the MG, the information about the releaser component of the trail-pheromone is processed. In the first part of my PhD-project, I focus on quantifying behavioral differences between different sized workers in Atta vollenweideri. The study analyzes the trail following behavior; which can be generally performed by all workers. An artificial trail consisting of the releaser component of the trail-pheromone in decreasing concentration was used to test the trail-following performance of individual workers. The trail-following performance of the polymorphic workers is depended of the existence of the MG in the antennal lobe. Workers possessing the MG-phenotype were significantly better in following a decreasing trail then workers showing the RG-phenotype. In the second part I address the question if there are more structural differences, besides the MG, in the olfactory system of different sized workers. Therefore I analyze whether the glomerular numbers are related to worker size. The antennal lobes of small workers contain ~390 glomeruli (low-number; LN-phenotype), and in large workers I found a substantially higher number of ~440 glomeruli (high-number; HN-phenotype). All LN-phenotype workers and some of the small HN-phenotype workers do not possess an MG (LN-RG-phenotype and HN-RG-phenotype) at all, whereas the remaining majority of HN-phenotype workers do possess an MG (HN-MG-phenotype). Mass-stainings of antennal olfactory receptor neurons revealed that the sensory tracts divide the antennal lobe into six clusters of glomeruli (T1-T6). In the T4-cluster ~50 glomeruli are missing in the LN-phenotype workers. Selective staining of single sensilla and their associated receptor neurons showed that T4-glomeruli are innervated by receptor neurons from the main type of olfactory sensilla, the Sensilla trichodea curvata which are also projecting to glomeruli in all other clusters. The other type of olfactory sensilla, the Sensilla basiconica, exclusively innervates T6-glomeruli. Quantitative analyses revealed a correlation between the number of Sensilla basiconica and the volume of T6 glomeruli in different sized workers. The results of both behavioral and neuroanatomical studies in Atta vollenweideri suggest that developmental plasticity of antennal-lobe phenotypes promotes differences in olfactory-guided behavior which may underlie task specialization within ant colonies. The last part of my project focuses on the evolutionary origin of the macroglomerulus and the number of glomeruli in the antennal lobe. I compared the number, volumes and position of the glomeruli of the antennal lobe of 25 different species from all three major Attini groups (lower, higher and leaf-cutting Attini). The antennal lobes of all investigated Attini comprise a high number of glomeruli (257-630). The highest number was found in Apterostigma cf. mayri. This species is at a basal position within the Attini phylogeny, and a high number of glomeruli might have been advantageous in the evolution of the advanced olfactory systems of this Taxa. The macroglomerulus can be found in all investigated leaf-cutting Attini, but in none of the lower and higher Attini species. It is found only in large workers, and is located close to the entrance of the antennal nerve in all investigated species. The results indicate that the presence of a macroglomerulus in large workers of leaf-cutting Attini is a derived overexpression of a trait in the polymorphic leaf-cutting species. It presumably represents an olfactory adaptation to elaborate foraging and mass recruitment systems, and adds to the complexity of division of labor and social organization known for this group.
In the generalized Nash equilibrium problem not only the cost function of a player depends on the rival players' decisions, but also his constraints. This thesis presents different iterative methods for the numerical computation of a generalized Nash equilibrium, some of them globally, others locally superlinearly convergent. These methods are based on either reformulations of the generalized Nash equilibrium problem as an optimization problem, or on a fixed point formulation. The key tool for these reformulations is the Nikaido-Isoda function. Numerical results for various problem from the literature are given.
An increase in cytosolic Ca2+ levels ([Ca2+]i) is a key event that occurs downstream of many signaling cascades in response to an external stimulus and regulates a wide range of cellular processes, including platelet activation. Eukaryotic cells increase their basal [Ca2+]i allowing extracellular Ca2+ influx into the cell, which involves different mechanisms. Store-operated Ca2+ entry (SOCE) is considered the main mechanism of extracellular Ca2+ influx in electrically non-excitable cells and platelets, and comprises an initial Ca2+ depletion from intracellular Ca2+ stores prior to activation of extracellular Ca2+ influx. Although the close relation between Ca2+ release from intracellular stores and extracellular Ca2+ influx was clear, the nature of the signal that linked both events remained elusive until 2005, when Stromal Interaction Molecule 1 (STIM1) was identified as an endoplasmic reticulum (ER) Ca2+ sensor essential for inositol (1,4,5)-trisphosphate (IP3)-mediated SOCE in vitro. However, the function of its homologue STIM2 in Ca2+ homeostasis was in general unknown. Therefore, mice lacking STIM2 (Stim2-/-) were generated in this work to study initially STIM2 function in platelets and in cells of the immune system. Stim2-/- mice developed normally in size and weight to adulthood and were fertile. However, for unknown reasons, they started to die spontaneously at the age of 8 weeks. Unexpectedly, Stim2-/- mice did not show relevant differences in platelets, revealing that STIM2 function is not essential in these cells. However, STIM2 seems to be involved in mammary gland development during pregnancy and is essential for mammary gland function during lactation. CD4+ T cells lacking STIM2 showed decreased SOCE. Our data suggest that STIM2 has a very specific function in the immune system and is involved in Experimental Autoimmune Encephalomyelitis (EAE) at early stages of the disease progression. Stim2-/- neurons were also defective in SOCE. Surprisingly, our results evidenced that STIM2 participates in mechanisms of neuronal damage after ischemic events in brain. This is the first time that the involvement of SOCE in ischemic neuronal damage has been reported. This finding may serve as a basis for the development of novel neuroprotective agents for the treatment of ischemic stroke, and possibly other neurodegenerative disorders in which disturbances in cellular Ca2+ homeostasis are considered a major pathophysiological component.
Glacier outlines during the ‘Little Ice Age’ maximum in Jotunheimen were mapped by using remote sensing techniques (vertical aerial photos and satellite imagery), glacier outlines from the 1980s and 2003, a digital terrain model (DTM), geomorphological maps of individual glaciers, and field-GPS measurements. The related inventory data (surface area, minimum and maximum altitude) and several other variables (e.g. slope, range) were calculated automatically by using a geographical information system. The length of the glacier flowline was mapped manually based on the glacier outlines at the maximum of the ‘Little Ice Age’ and the DTM. The glacier data during the maximum of the ‘Little Ice Age’ were compared with the Norwegian glacier inventory of 2003. Based on the glacier inventories during the maximum of the ‘Little Ice Age’, the 1980s and 2003, a simple parameterization after HAEBERLI & HOELZLE (1995) was performed to estimate unmeasured glacier variables, as e.g. surface velocity or mean net mass balance. Input data were composed of surface glacier area, minimum and maximum elevation, and glacier length. The results of the parameterization were compared with the results of previous parameterizations in the European Alps and the Southern Alps of New Zealand (HAEBERLI & HOELZLE 1995; HOELZLE et al. 2007). A relationship between these results of the inventories and of the parameterization and climate and climate changes was made.
Chlamydia are Gram-negative obligate intracellular bacteria responsible for a wide spectrum of relevant diseases. Due to their biphasic developmental cycle Chlamydia depend on an intact host cell for replication and establishment of an acute infection. Chlamydia have therefore evolved sophisticated strategies to inhibit programmed cell death (PCD) induced by a variety of stimuli and to subvert the host immune system. This work aimed at elucidating whether an infection with C. trachomatis can influence the cellular response to double-stranded RNA (dsRNA). The synthesis of dsRNA is a prominent feature of viral replication inside infected cells that can induce both PCD and the activation of a cellular innate immune response. In order to mimic chlamydial and viral co-infections, Chlamydia-infected cells were transfected with polyinosinic:polycytidylic acid (polyI:C), a synthetic dsRNA. In the first part of this work it was investigated whether C. trachomatis-infected host cells could resist apoptosis induced by polyI:C. A significant reduction in apoptosis, determined by PARP cleavage and DNA fragmentation, could be observed in infected cells. It could be shown that processing of the initiator caspase-8 was inhibited in infected host cells. This process was dependent on early bacterial protein synthesis and was specific for dsRNA because apoptosis induced by TNFalpha was not blocked at the level of caspase-8. Interestingly, the activation of cellular factors involved in apoptosis induction by dsRNA, most importantly PKR and RNase L, was not abrogated in infected cells. Instead, RNA interference experiments revealed the crucial role of cFlip, a cellular caspase-8 inhibitor, for chlamydial inhibition of dsRNA-induced apoptosis. First data acquired by co-immunoprecipitation experiments pointed to an infection-induced concentration of cFlip in the dsRNA-induced death complex of caspase-8 and FADD. In the second part of this work, the chlamydial influence on the first line of defense against viral infections, involving expression of interferons and interleukins, was examined. Activation of the interferon regulatory factor 3 (IRF-3) and the NF-kappaB transcription factor family member p65, both central regulators of the innate immune response to dsRNA, was altered in Chlamydia-infected epithelial cells. polyI:C-induced degradation of IkappaB-alpha, the inhibitor of NF-kappaB, was accelerated in infected cells which was accompanied by a change in nuclear translocation of the transcription factor. Translocation of IRF-3, in contrast, was significantly blocked upon infection. Together the data presented here demonstrate that infection with C. trachomatis can drastically alter the cellular response to dsRNA and imply an impact of chlamydial infections on the outcome of viral super-infections.
This work focuses on coordination methods and the control of motion in groups of nonholonomic wheeled mobile robots, in particular of the car-like type. These kind of vehicles are particularly restricted in their mobility. In the main part of this work the two problems of formation motion coordination and of rendezvous in distributed multi-vehicle systems are considered. We introduce several enhancements to an existing motion planning approach for formations of nonholonomic mobile robots. Compared to the original method, the extended approach is able to handle time-varying reference speeds as well as adjustments of the formation's shape during reference trajectory segments with continuously differentiable curvature. Additionally, undesired discontinuities in the speed and steering profiles of the vehicles are avoided. Further, the scenario of snow shoveling on an airfield by utilizing multiple formations of autonomous snowplows is discussed. We propose solutions to the subproblems of motion planning for the formations and tracking control for the individual vehicles. While all situations that might occur have been tested in a simulation environment, we also verified the developed tracking controller in real robot hardware experiments. The task of the rendezvous problem in groups of car-like robots is to drive all vehicles to a common position by means of decentralized control laws. Typically there exists no direct interaction link between all of the vehicles. In this work we present decentralized rendezvous control laws for vehicles with free and with bounded steering. The convergence properties of the approaches are analyzed by utilizing Lyapunov based techniques. Furthermore, they are evaluated within various simulation experiments, while the bounded steering case is also verified within laboratory hardware experiments. Finally we introduce a modification to the bounded steering system that increases the convergence speed at the expense of a higher traveled distance of the vehicles.
From the introduction: It is not always easy to define what a word means. We can choose between a variety of possibilities, from simply pointing at the correct object as we say its name to lengthy definitions in encyclopaedias, which can sometimes fill multiple pages. Although the former approach is pretty straightforward and is also very important for first language acquisition, it is obviously not a practical solution for defining the semantics of the whole lexicon. The latter approach is more widely accepted in this context, but it turns out that defining dictionary and encyclopaedia entries is not an easy task. In order to simplify the challenge of defining the meaning of words, it is of great advantage to organize the lexicon in a way that the structure in which the words are integrated gives us information about the meaning of the words by showing their relation to other words. These semantic relations are the focal point of this paper. In the first chapter, different ways to describe meaning will be discussed. It will become obvious why semantic relations are a very good instrument to organizing the lexicon. The second chapter deals with WordNet, an electronic lexical database which follows precisely this approach. We will examine the semantic relations which are used in WordNet and we will study the distinct characteristics of each of them. Furthermore, we will see which contribution is made by which relation to the organization of the lexicon. Finally, we will look at the downside of the fact that WordNet is a manually engineered network by examining the shortcomings of WordNet. In the third chapter, an alternative approach to linguistics is introduced. We will discuss the principles of corpus linguistics and, using the example of the British National Corpus, we will consider possibilities to extract semantic relations from language corpora which could help to overcome the deficiencies of the knowledge based approach. In the fourth chapter, I will describe a project the goal of which is to extend WordNet by findings from cognitive linguistics. Therefore, I will discuss the development process of a piece of software that has been programmed in the course of this thesis. Furthermore, the results from a small‐scale study using this software will be analysed and evaluated in order to check for the success of the project.
Extracellular signals are translated and amplified via cascades of serially switched protein kinases, MAP kinases (MAPKs). One of the MAP pathways, the classical RAS/RAF/MEK/ERK pathway, transduces signals from receptor tyrosine kinases and plays a central role in regulation of cell proliferation. RAF kinases (A-, B- and C-RAF) function atop of this cascade and convert signals emanating from conformational change of RAS GTPases into their kinase activity, which in turn phosphorylates their immediate substrate, MEK. Disregulated kinase activity of RAF can result in tumor formation, as documented for many types of cancer, predominantly melanomas and thyroid carcinomas (B-RAF). A-RAF is the least characterized RAF, possibly due to its low intrinsic kinase activity and comparatively mild phenotype of A-RAF knockout mice. Nevertheless, the unique phenotype of araf -/- mice, showed predominantly neurological abnormalities such as cerebellum disorders, suggesting that A-RAF participates in a specific process not complemented by activities of B- and CRAF. Here we describe the role of A-RAF in membrane trafficking and identify its function in a specific step of endocytosis. This work led to the discovery of a C-terminally truncated version of A-RAF, AR149 that strongly interfered with cell growth and polarization in yeast and with endocytosis and actin polymerization in mammalian cells. As this work was in progress two splicing isoforms of ARAF, termed DA-RAF1 and DA-RAF2 were described that act as natural inhibitors of RAS-ERK signaling during myogenic differentiation (Yokoyama et al., 2007). DA-RAF2 contains the first 153 aa of A-RAF and thus is nearly identical with AR149. AR149 localized specifically to the recycling endosomal compartments as confirmed by colocalization and coimmunoprecipitation with ARF6. Expression of AR149 interferes with recycling of endocytosed transferrin (Tfn) and with actin polymerization. The endocytic compartment, where internalized Tfn is trapped, was identified as ARF6- and RAB11- positive endocytic vesicles. We conclude that the inhibition of Tfn trafficking in the absence of A-RAF or under overexpression of AR149 occurs between tubular- and TGNassociated recycling endosomal compartments. siRNA-mediated depletion of endogenous A-RAF or inhibition of MEK by U0126 mimic the AR149 overexpression phenotype, supporting a role of ARAF regulated ERK signalling at endosomes that is controlled by AR149 and targets ARF6. Our data additionally suggest EFA6 as a partner of A-RAF during activation of ARF6. The novel findings on the A-RAF localization and the interaction with ARF6 have led to a new model of ARAF function were A-RAF via activation of ARF6 controls the recycling of endocytic vesicles.Endocytosis and rapid recycling of synaptic vesicles is critically important for the physiological function of neurons. The finding, that A-RAF regulates endocytic recycling open a new perspective for investigation of the role of A-RAF in the nervous system.
Insects have evolved an astonishing array of defences to ward off enemies. Well-known and widespread is the regurgitation of oral secretions (OS), fluids that repel attacking predators. In herbivores, the effectiveness of OS has been ascribed so far to the presence of deterrent secondary metabolites sequestered from the host plant. This notion implies, however, that generalists experience less protection on plants with low amounts of secondary metabolites or with compounds ineffective against potential enemies. Resolving the dilemma, we describe a novel defence mechanism that is independent of deterrents as it relies on the OS’ intrinsic detergent properties. The OS of Spodoptera exigua (and other species) was found to be highly amphiphilic and well capable of wetting the hydrophobic cuticle of predatory ants. As a result, affected ants stopped attacking and engaged in extensive cleansing. The presence of surfactants was sufficient to explain the defensive character of herbivore OS. We hypothesize that detergency is a common but unrecognised mode of defence which provides a base level of protection that may or may not be further enhanced by plant-derived deterrents. Our study also proves that insects ‘invented’ the use of defensive surfactants long before modern agriculture had started applying them as insecticides.
Land plants must control the transpiration water stream and balance it with carbon dioxide uptake for optimal photosynthesis. A highly specialized type of plant cell called guard cells have evolutionary appeared which are suited for this complicated purpose. Guard cells are located by pairs on aerated plant surface and form stomata – structural units, which represent highly regulated “watergate” (Roelfsema and Hedrich, 2005). Guard cells sense many environmental and internal plant-derived stimuli and by changing degree of their swelling tightly regulate diffusion of water vapor and other gases. Cell processes taking place in stomata during their movements had been a subject of intensive investigation for more than three decades (Schroeder et al., 2001; Assmann and Shimazaki, 1999). With use of electrophysiological technique the basic processes underlying stomatal movements were described (Thiel et al., 1992; Dietrich et. al., 2001; Roelfsema and Hedrich, 2005). Another set of questions arised between plant biologists is how the signals affecting stomatal aperture are transduced in guard cells starting from perception by receptor structures and ending on the osmodynamic motor components. Introduction of fluorescent microspectroscopy technique allowed to characterize some Ca2+ and H+-based signaling events, taking place in the cytoplasm during stomata function. Most of the processes, taking place in stomata were characterized in guard cell preparations, such as strips of isolated leaf epidermis or guard cell protoplasts, - cells with enzymaticaly digested cell walls. Some experimental observations although point that reactions of guard cells located in their natural environment, leaves of intact plants can differ from those could be registered in preparations. These deviations might be explained by the modulation of guard cell function by apoplastic factors originating from surrounding tissues like mesophyll or leaf epidermis (Roelfsema and Hedrich, 2002). On the other hand registration of physiological responses in prepared tissues may also contain possible artifacts, related to the preparation procedures. The aim of the experimental work presented here was to investigate the cell signaling events, taking place in guard cells upon plant stress hormone abscisic acid (ABA) and some other stimuli action. Abscisic acid is a compound that synthesized in plant roots upon drought and closes stomata in the leaf to prevent the plant organism from excessive water loss. Previous studies on guard cell of isolated epidermis and guard cell protoplasts showed, that ABA induces stomatal closure via activation of plasma membrane anion channels (Grabov et al., 1997; Pei et al, 1997). Anion channels are known to be activated by elevated 2 concentrations of cytoplasmic Ca2+ [Ca2+]cyt (Schroeder and Hagiwara, 1989; Hedrich et al., 1990). Application of Ca2+-sensitive fluorescent probes revealed [Ca2+]cyt increases in guard cells upon ABA action (McAinsh et al., 1990). This observation led to suggestion that [Ca2+]cyt directly participate in the transduction of ABA signal in guard cells. Although no direct evidences for co-occurrence of [Ca2+]cyt rises and following activation of anion channels upon ABA action was not presented until yet. Results of experimental work performed on intact Vicia faba, Commelina communis and Nicotiana plumbagnifolia plants showed that guard cells of intact plant leaves respond with transient activation of plasma membrane anion channels upon perception of ABA. Kinetics of the response is highly reproducible and seemed to be conserved between species. Although despite clear generation of anion current transients, no [Ca2+]cyt increases could be recorded with using fluorescent probe Fura-2 microinjected into the cytoplasm. Together with results of later study on intact Nicotiana tabacum guard cells, reported obligatory [Ca2+]cyt increases which were desynchronized with anion current transients (Marten et al., 2007b) this, may indicate that [Ca2+]cyt increases are not necessary component of ABA signal transduction pathway. Together with absence of the effect of cytoplasm-delivered Ca2+- mobilizing agents IP3, IP6 and NAADP on anion currents these data may suppose that role of [Ca2+]cyt in ABA signaling must be reassessed. Further interest represented characterization of [Ca2+]cyt signaling and homeostasis in intact guard cells comparing with those in prepared cells. Experiments revealed strong deviations in [Ca2+]cyt behavior between different measuring systems. While guard cells of intact plants were able to strictly maintain [Ca2+]cyt level upon experimental shifting of [Ca2+]cyt level in either direction of elevation or decrease, cells of isolated epidermis showed complete absence of such ability. Guard cell protoplasts showed even weaker [Ca2+]cyt regulation ability and were capable of low physiological [Ca2+]cyt levels maintaining only at depolarized membrane potentials. Apart to these differences, prepared guard cells showed also for-time less activation of anion currents by experimentally imposed [Ca2+]cyt increases. These data strongly suggest that registered in guard cell preparations [Ca2+]cyt signals may contain significant part of artifacts and must be carefully used for the building of models of guard cells signaling. Further experimental investigations are strongly required for understanding guard cell functioning, especially with relation of vacuoles participation. The experimental work was done by the author in the period from october 2001 until november 2004 under supervision of Professor Dr. Rainer Hedrich in laboratory of molecular plant physiology and biophysics at Julius-Maximillians University of Würzburg, Würz3 burg, Federal Republic of Germany. Scientific coordinator of the Ph. D. project is Dr. Max Robert Gustaaf Roelfsema, University of Würzburg. Most of experimental results, presented here (chapter III) are also published elsewhere (Roelfsema et al., 2004; Langer et al., 2004; Levchenko et al., 2005, 2008). Chapter I intend to shortly introduce the reader into the field of guard cell research and point out the current level of understanding regarding this branch of plant research. Special attention is given to description of guard cell ion channels, their function and regulation, including the mechanisms of Ca2+-, H+- and phosphorylation-based signaling. This section is preceded by a short history of guard cell research and explains the actuality of presented work. In chapter II experimental techniques, methods and data processing approaches, used in the presented work are described. Technique used for electrophysiological registrations on intact plant leaves were used before and described in more details by Roelfsema et al. (2001). Fluorescent microspectroscopy technique was for the first time applied to intact plant leaves in this work and described in more details including calibration of Fura-2 based measurements. Chapter III presents the major results of the experimental work. In chapter IV the experimental results are discussed and put into context with current knowledge of guard cell function knowledge. Finally, remarks on perspectives of guard cell signaling research are drawn.
Prion diseases such as scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle or Creutzfeldt-Jakob disease (CJD) in humans are fatal neurodegenerative disorders characterized by brain lesions and the accumulation of a disease-associated protein, designated PrPSc. How prions proceed to damage neurons and whether all or only subsets of neurons have to be affected for the onset of the clinical disease is still elusive. The manifestation of clinical prion disease is characterized by motor dysfunctions, dementia and death. Furthermore loss of motor neurons (MN) in the spinal cord is a constant finding in different mouse models of prion disease, suggesting that MN are vulnerable cells for triggering the onset of clinical symptoms. To determine whether the protection of MN against prion induced dysfunctions is an approach for holding the disease at the sub-clinical level, we established a novel conditional model for Cre-mediated expression of a dominant-negative PrP mutant (PrPQ167R) in the cells of interest. Dominant-negative PrP mutants provide protection of prion induced dysfunctions by inhibiting prion replication. Transgenic mice were generated carrying a floxed LacZ marker gene followed by the coding sequence of PrPQ167R under control of the human ubiquitin C promoter. Two Cre strains have been used to direct PrPQ167R expression either to a subset of MN of the spinal cord (Hb9-Cre) or to various neuronal cell populations of the spinal cord and brain (NF-L-Cre). Transgenic mice were infected with mouse-adapted prions via different inoculation routes (intranerval, intracerebral and intraperitoneal) and monitored for effects on incubation time and pathology. Tg floxed LacZ-PrPQ167R/NF-L-Cre mice showed about 15% prolonged survival upon intraperitoneal low dose prion infection, whereas survival of Tg floxed LacZ-PrPQ167R/Hb9-Cre mice was comparable to control littermates. The results suggest that the protection of spinal MN prolongs the incubation period but is not sufficient to completely inhibit clinical prion disease. In a second approach, Cre was transferred into the hind limb muscles of transgenic mice via a double-stranded adeno-associated virus vector (dsAAV2-Cre). The goal of this strategy was to target a broader cell population and thus to enhance expression levels of protective PrPQ167R in the spinal cord of Tg floxed-LacZ-PrPQ167R mice. After intramuscular (i.m.) application of dsAAV2-Cre, exhibiting a physical titer of 5x1010 GP/ml, recombinant transgenic DNA was detected only in the muscle tissue, pointing out that functional Cre-recombinase was expressed at the side of virus application. However, dsAAV2-Cre did neither induce recombination of transgenic DNA in the spinal cord or brain nor expression of dominant-negative PrPQ167R. In conclusion the dsAAV2-Cre vectors system needs further improvement to achieve efficient transport from muscle tissue to the central nervous system (CNS). 105 7 SUMMARY The lymphoreticular system (LRS) is an early site of prion replication. In splenic tissue prion infectivity is associated with follicular dendritic cells (FDC) as well as with Band T-lymphocytes. However, it is still unknown if those cell types are able to replicate the infectious agent or if other PrP-expressing cell types are engaged. To investigate if neurons and in particular MN are involved, transgenic mice carrying one allele of floxed Prnp (lox2+=��) and either one allele of Hb9-Cre or NF-L-Cre were generated on a Prnp0=0 background. Therefore a conditional PrP knockout was established in a subset of MN of the spinal cord (Hb9-Cre) or in various neuronal populations of the spinal cord and brain (NF-L-Cre). Transgenic mice were inoculated with prions to study the accumulation of PrPSc and prion infectivity in spleen and spinal cord at an early time point after infection. The findings show that PrPSc accumulation in mice with MN-specific PrP depletion (lox2+=��/ Hb9-Cre) was comparable to control littermates, while pan-neuronal PrP deficient mice (lox2+=��/NF-L-Cre) were not able to accumulate PrPSc in splenic tissue until 50 days post inoculation. Moreover spleens of lox2+=��/NF-L-Cre mice exhibited a clearly reduced prion infectivity titer, suggesting that accumulation of prions in the spleen is dependent on PrP expression in the nervous tissue.
The Three-Site Higgsless Model is alternative implementation of electroweak symmetry breaking which in the Standard Model is mediated by the Higgs mechanism. The main features of this model is the appearance of two new heavy vector resonances W' and Z' with masses > 380 GeV as well as a set of new heavy fermions (> 1.8 TeV). In this model, unitarity of the amplitudes for the scattering of longitudinal gauge bosons is maintained by the exchange of the W' and Z' up to a scale of ~2 TeV. Consistency with the electroweak precision observables from the LEP / LEP-II experiments implies an exceedingly small coupling of the new vector bosons to the light Standard Model fermions (about 3% of the isospin gauge coupling). In this thesis, the LHC phenomenology of this scenario is explored. To this end, we calculated the couplings and widths of all the new particles and implemented the model into the Monte-Carlo eventgenerator WHIZARD / O'Mega. With this implementation, we simulated the parton-level production of the gauge boson and fermion partners in different channels possibly suitable for their discovery at the LHC. The results are presented together with an introduction to the model and a discussion of its properties. We find that, while the fermiophobic nature of the new heavy gauge bosons does make them intrinsically difficult to observe at a collider, the LHC should be able to establish the existence of both resonances and even give some hints about the properties of their couplings which would be a vital test of the consistency of such a scenario. For the heavy fermions, we find that their large mass is accompanied by relative widths of more than $10\%$, making them ill-suited for a direct discovery at the LHC. Nevertheless, our simulations reveal that there is a part of parameter space where, given enough time, patience and a good understanding of detector and backgrounds, a direct discovery might be possible.
Mediators of Social Anxiety - External Social Threat-Cues vs. Self-Related Negative Cognitions
(2009)
Based on a review of models and empirical findings a working model is proposed, suggesting that self-related negative cognitions and biased processing of external social threat-cues are mediators of social anxiety. Hypotheses derived from this model were tested in three experiments. The first experiment examined whether levels of trait social anxiousness predicted fearful responding to external social threat-cues (angry vs. neutral and happy facial expressions) during social evaluation. Higher trait social anxiousness predisposes to an inward focus on one’s fear reaction to social threat. Using this strategy was expected to enhance fearful responding to angry facial expressions. A strategy of identifying with angry faces was expected to counteract fearful responding, but was expected to fail more often with increasing levels of trait social anxiousness. To examine these hypotheses, affective modulation of the startle eye-blink was assessed in forty-four undergraduate students. This measure served as a probe into the activation of brain structures involved in the automatic evaluation of environmental threat-cues. Trait and state anxiety as well as explicit emotional responding to the stimuli were assessed with questionnaires and ratings. Processing angry faces potentiated startle amplitudes as expected. Low arousal induced by the stimuli was a probable reason, why startle potentiation to happy faces emerged instead of attenuation. Trait social anxiousness and the cognitive strategies did not influence these effects. Yet, increased trait social anxiousness predicted decreased startle latency, indicating motor hyper-responsivity, which is part of the clinical representation of social anxiety disorder (SAD). Processing facial expressions and identifying with them disrupted this association. Previous studies support that similar strategies may enhance treatment of SAD. Individuals with SAD were expected to respond with increased arousal to external social threat-cues. Therefore, the second experiment examined whether nine individuals with SAD showed attentional (prepulse inhibition, PPI) or affective startle modulation to angry as compared to neutral and happy facial expressions. Corrugator supercilii activity was assessed as a behavioral indicator for effects of facial expressions. The remaining setup resembled the first experiment. Facial expressions did not modulate the startle reflex, but corrugator supercilii activity was sensitive to facial valence. However, the effects were not related to trait social anxiousness. Apparently, angry facial expressions do not act as phobic stimuli for individuals with SAD. The third experiment examined whether focusing on self-related negative cognitions or biased processing of external social threat-cues mediates relationships between trait social anxiety and anxious responding in a socially challenging situation. Inducing self-related negative cognitions vs. relaxation was expected to reveal a functional dependency on the supposed mediation in a multivariate assessment of criteria of the working model. Within this design, the impact of external social threat-cues (facial expressions and emotional words) was compared to control stimuli and context effects, using the startle paradigm. The findings provide first evidence for full statistical mediation of the associations between trait social anxiety and self-reported anxiety as well as parasympathetic withdrawal by self-related negative cognitions, when thirty-six undergraduate students anticipated public speaking. Apprehensive arousal, as indicated by increased skin conductance levels and heart rate, was present in all participants. Observer ratings of behavior during public speaking matched the self-rated quality of the performance. None of these measures were correlated with trait social anxiousness. Startle amplitude correlated with state and trait social anxiety, but was no mediator. Finally, there was no affective modulation of the startle amplitude by external social threat-cues. These studies advance both our current understanding of the factors that mediate social anxiety responses to situations and our knowledge of the physiological and anatomical mechanisms involved in social anxiety. Based on these findings a revised version of the working model on mediators of social anxiety is proposed in the hope it may aid further research for the ultimate goal of developing an empirically validated functional anatomical model of social anxiety.
TWEAK is a typical member oft he TNF ligand family. Therefore it is initially expressed as a type II transmembrane protein, but a soluble variant can be released by proteolytic processing. In this work it is shown that oligomerized TWEAK is more competent than soluble, trimeric TWEAK regarding the activation of classical NFκB signaling pathway. However, both TWEAK variants are able to induce depletion of TRAF2 and processing of p100, which are hallmarks for the activation of the noncanonical NFκB pathway. Like other solube TNF ligands with no or poor activity on their corresponding receptor, TWEAK gains high activity upon oligomerization resembling the activity of the transmembrane ligand. TRAF2 has a key role in TWEAK-induced NFκB signaling. Depletion or degradation of TRAF2 is crucial for activation of the noncanonial or both, the classical and the noncanonical NFκB pathway. Blocking the TWEAK receptor Fn14 inhibits the activation of NFκB signaling, irrespective of the TWEAK form used for stimulation. This indicates that the different activities of the two TWEAK variants in activation of classical and noncanonical NFκB signaling are not caused by the use of different receptors. Therefore this study on TWEAK is the first reported case where one TNF ligand in different variants induces qualitatively different activities of the corresponding TNF receptor.
Adducing a number of illustrations of Shakespeare's "The Tempest" and "Othello", the purpose of this study is to analyze the transformative processes that took place in the 18th and 18th centuries in Great Britain, regarding the cultural, political and social aspects of its encounters with the other. In this context, especially the depiction of the characters Caliban and Othello are taken into consideration.
This work deals with the sintering of multi-material composites. It aims at the establishment of an alternative to the existing complex models for sintering. The development of the associated experimental procedure is also included in this work. The developed material model must be able to predict (i) the sintering kinetics and (ii) the viscous moduli of a material. An experimental approach with free sintering and hot-forging measurements is favoured in this work. The prediction of the sintering kinetics is addressed with the construction of a map of sintering kinetics data: the Master Sintering Diagram (MSD). The MSD is based on a generalized equation for solid-state diffusion, thus is suitable for any thermal activated diffusion. The MSD allows the prediction of sintering kinetics for a large range of temperatures and external loads. A novel approach to the determination of the viscous moduli is developed in this work: the cyclic unloading method. It is a hot-forging measurement (sintering under uniaxial compression) where the applied load is released for short periods. The measurements are carried out with continuous heating, so that the viscous moduli are determined over large ranges of temperatures and densities. The advantage of this method is the measurement of the viscous moduli in anisotropic microstructures. The material model is validated in two steps. Firstly, the predictions of sintering kinetics with the MSD are compared with experimental results: changes of thermal profile and changes of load are predicted with a maximum deviation of 10%. Secondly, the experimentally determined viscous moduli are used for the prediction of a bi-layer curvature using models for warpage from literature. The prediction is qualitatively good for a maximum deviation of 27%. The study of a sintering glass-ceramic tape on a rigid substrate is presented. It shows that this co-sintering problem can be qualitatively investigated with requirement of the material model. The formation of anisotropy intrinsic to the hot-forging experiments is also reported in this work. It appears to be a important point to address in the future for a better understanding of the cosintering.
Investigation of Intermolecular Interaction in Organic Thin Films by means of NEXAFS Spectroscopy
(2009)
The present work reports on the electron–vibron coupling in large organic molecules and particularly on the intermolecular interaction in molecular condensates. The optical and electrical properties of these organic systems are in the focus of attention due to their crucial importance for the development of (hybrid) organic electronic devices. In particular, the charge transport mechanism and hence the interaction between condensed molecules is a matter of debate [1–4]. In order to shed light on this interaction, the spectroscopic signatures of isolated molecules in the gas phase and their condensed counterparts have been studied. The applied technique, near–edge x–ray absorption fine structure (NEXAFS) spectroscopy, is a local probe with high chemical selectivity, well suited for the investigation of the electronic structure of molecular valence levels [5]. In the experimental part, the experimental set–up developed in this work is described with special attention to the characteristic issues of gas phase measurements, energy calibration and the subsequent data evaluation. The high quality gas phase and solid state NEXAFS spectra are analysed with respect to energy positions, shape and intensity of the sharp pi*–resonances characteristic for these aromatic molecules. Where applicable, a detailed Franck–Condon (FC) analysis of the vibronic fine structure has been performed, yielding additional information on the changes that occur upon solid state formation. Together with former results on vibrational features in large organic molecules, this information has been used to investigate the correlation of vibrational energies in the ground and electronically excited state. We find a relatively good agreement with other empirical studies on vibronic structures in photoelectron spectroscopy (PES) spectra of small molecules [6]. The molecular compounds investigated are in general believed to interact via weak van–der–Waals forces only. The present results however reveal distinct differences between the spectra of the gas and solid phase that can not be explained within the context of a mere interaction by dispersive forces. In detail, differential red–shifts of 0.1 to 0.3eV of transitions assigned to the aromatic system have been observed in the C–K spectra of benzene–tetracarboxylic acid dianhydride (BTCDA), 1,4,5,8–naphthalene–tetracarboxylic acid dianhydride (NTCDA), and 3,4,9,10–perylene–tetracarboxylic acid dianhydride (PTCDA) upon solid state formation. From BTCDA to PTCDA the shift increases, indicating an improving intermolecular interaction with molecular size or a closer molecular packing. In contrast, all transitions assigned to the anhydride carbon atom (C1) do not show any shift. For the O–K spectra, small changes in relative intensity have been observed for BTCDA and NTCDA. In case of PTCDA, a blue–shift of up to 0.2eV is evident for the OB 1sLEMO+1 transition. Theoretical models for the intermolecular interaction have been proposed in this work, based on a change of molecular geometry and interaction of adjacent molecules in the ground and excited state, respectively. While an interaction of adjacent molecular orbitals may explain the experimental findings for one particular molecule, this model falls short for a comprehensive explanation of all three dianhydrides. For an interaction in the excited state, the excitonic coupling with the neighbours attached at an angle, quantum chemical calculations yield no significant change in peak positions for NTCDA. Unfortunately, results for the stacked neighbours as well as the larger compound PTCDA are still lacking. For tris (8–quinolinol) aluminum (Alq3), the observed peak–shifts are restricted to just one unoccupied orbital, the LEMO+2, which is mainly localised at the phenoxide side of the quinolinol ligands. Although the shifts differ for the individual edges, the main interaction can therefore be assigned to this orbital. In summary, NEXAFS spectroscopy, if performed with great care in terms of experimental details and data analysis especially for the gas phase data, provides very detailed and highly interesting data on the changes of the electronic structure of organic molecules upon condensation. The present data can be applied as a reference for further experimental and (highly desired) theoretical investigations, which are needed for a comprehensive understanding of the complex interaction mechanisms between organic molecules.
This thesis analyzes the 2001-2006 labor market reforms in Germany. The aim of this work is twofold. First, an overview of the most important reform measures and the intended effects is given. Second, two specific and very fundamental amendments, namely the merging of unemployment assistance and social benefits, as well as changes in the duration of unemployment insurance benefits, are analyzed in detail to evaluate their effects on individuals and the entire economy. Using a matching model with optimal search intensity and Semi-Markov methods, the effects of these two amendments on the duration of unemployment, optimal search intensity and unemployment are analyzed.
Mycobacterium tuberculosis is the causative agent of tuberculosis and responsible for more than eight million new infections and about two million deaths each year. Novel chemotherapeutics are urgently needed to treat the emerging threat of multi drug resistant and extensively drug resistant strains. Cell wall biosynthesis is a widely used target for chemotherapeutic intervention in bacterial infections. In mycobacteria, the cell wall is comprised of mycolic acids, very long chain fatty acids that provide protection and allow the bacteria to persist in the human macrophage. The type II fatty acid biosynthesis pathway in Mycobacterium tuberculosis synthesizes fatty acids with a length of up to 56 carbon atoms that are the precursors of the critical mycobacterial cell wall components mycolic acids. KasA, the mycobacterial ß-ketoacyl synthase and InhA, the mycobacterial enoyl reductase, are essential enzymes in the fatty acid biosynthesis pathway and validated drug targets. In this work, KasA was expressed in Mycobacterium smegmatis, purified and co-crystallized in complex with the natural thiolactone antibiotic thiolactomycin (TLM). High-resolution crystal structures of KasA and the C171Q KasA variant, which mimics the acyl enzyme intermediate of the enzyme, were solved in absence and presence of bound TLM. The crystal structures reveal how the inhibitor is coordinated by the enzyme and thus specifically pinpoint towards possible modifications to increase the affinity of the compound and develop potent new drugs against tuberculosis. Comparisons between the TLM bound crystal structures explain the preferential binding of TLM to the acylated form of KasA. Furthermore, long polyethylene glycol molecules are bound to KasA that mimic a fatty acid substrate of approximately 40 carbon atoms length. These structures thus provide the first insights into the molecular mechanism of substrate recognition and reveal how a wax-like substance can be accommodated in a cytosolic environment. InhA was purified and co-crystallized in complex with the slow, tight binding inhibitor 2-(o-tolyloxy)-5-hexylphenol (PT70). Two crystal structures of the ternary InhA-NAD+-PT70 were solved and reveal how the inhibitor is bound to the substrate binding pocket. Both structures display an ordered substrate binding loop and corroborate the hypothesis that slow onset inhibition is coupled to loop ordering. Upon loop ordering, the active site entrance is more restricted and the inhibitor is kept inside more tightly. These studies provide additional information on the mechanistic imperatives for slow onset inhibition of enoyl ACP reductases.
This thesis was dedicated to the studies of the electronic and chemical properties of liquids and solutions using soft x-ray spectroscopies. The used photon-in-photon-out methods namely x-ray absorption spectroscopy (XAS), x-ray emission spectroscopy (XES), and resonant inelastic x-ray scattering (RIXS) appeared to be an excellent choice for these studies. In the framework of this thesis, the necessary experimental setup for using the above mentioned experimental techniques on liquids was developed. Hereby, a new flow-through liquid cell was introduced which simplifies the studies of liquids and solutions. The cell design is very flexible and thus can be modified for gases and liquid/solid interfaces. With this cell it is possible to study the samples under well-controlled conditions (temperature and flow rate). The novel flow-through liquid cell is part of the new SALSA synchrotron endstation including an electron analyzer and a novel high-resolution, high-transmission soft x-ray spectrometer. The latter makes it possible to measure two-dimensional RIXS maps in a very short time, which include the full excitation and emission information in one plot. Making use of the new instrumentation, a variety of different liquids and solutions were investigated. As first system, aqueous solutions of sodium hydroxide (NaOH) and sodium deuteroxide (NaOD) were investigated. In the XAS as well as in the XES spectra a pronounced concentration dependence was found. At non-resonant energies, the spectra are dominated by the solvent and thus look similar to water. Making use of the pre-pre-edge in the absorption spectra which can exclusively be attributed to OH- / OD- it was possible to extract the resonant emission spectra of the ions which show an indication for proton dynamics during the core-hole lifetime. For the solid state NaOH XES spectra it was possible to reveal a high energetic shoulder and a low energetic shoulder at the high energy emission feature. These shoulders can be assigned to self-dissociation processes where OH- forms O2- ions and H2O. The study of NaOH was also of interest for the studies of the amino acids, which were in the focus of the next part, since the pH-values of the respective solutions were controlled by NaOH. In the next part of this thesis, amino acid solutions were investigated. Amino acids are the building blocks of peptides and proteins and thus important for life science. The investigated representatives were glycine, the simplest amino acid, and lysine, an amino acid with two amine groups. Both amino acids react on pH-value changes at the amine group where the local environment at the nitrogen atom changes (NH2 ↔ NH3+). A strong change of the spectra induced by this protonation/deprotonation could be found. Furthermore, for low pH-values (protonated amine groups) the amine groups are influenced by strong proton dynamics. First DFT calculations confirm the dissociation model of the amino acids. Qualitatively the high energy peak in the N K XES spectra can be attributed to the deprotonated amine group and the low energy area for the protonated amine group. Besides amino acids, alcohols and acids are important in biological processes. Therefore, the smallest alcohol (methanol) and the smallest carboxylic acid (acetic acid) were under investigation. For the liquid methanol XES spectra a very good agreement with DFT calculations of gas phase methanol could be found. This observation suggests that the influence of the environment (hydrogen bonding) on the spectra is small. The achieved spectra are in good agreement with DFT calculations found in literature. It was possible to selectively excite the two non-equivalent oxygen atoms in acetic acid and to reveal the carboxyl specific C K XES. The carbon XAS spectra showed strong differences compared to gas phase measurements which might be a hint for the influence of the hydrogen bond network. The investigation of the electronic and chemical properties of liquids and solutions is a very young field of research and the results presented in this thesis show that it is a very interesting topic. The presented results can be seen as the fundamental frame work for all following studies. With the understanding of basic, i.e., simple, systems as shown in this work it will be possible to understand complex biological systems in their native environment, e.g., peptides and proteins, which are the building blocks of life.
Perylene bisimide (PBI) dyes are a widely used class of industrial pigments, and currently have gained significant importance for organic-based electronic and optical devices. Structural modification at the PBI core results in changes of the optical and electronic properties, which enable tailored functions. Moreover, the aggregation behavior of PBIs is alterable and controllable to achieve new materials, among which organogels are of particular interest because of their potential for applications as supramolecular soft materials. In this work, new PBI-based organic gelators were designed, synthesized, and characterized, and the aggregation behaviors under different conditions were intensively studied by various spectroscopic and microscopic methods. In chapter 2, a brief overview is given on the structural and functional features of organogel systems. The definition, formation and reversibility of organogels are introduced. Some examples on dye based organogel are selected, among which PBI-based organogelators reported so far are especially emphasized. Some basic knowledges of supramolecular chirality are also overviewed such as characterization, amplification, and symmetry breaking of the chiral aggregates. According to our former experiences, PBIs tend to form aggregates because the planer aromatic cores interact with one another by pi-pi interaction. In chapter 3, a new PBI molecule is introduced which possesses amide groups between the conjugated core and periphery alkyl chains. It is found that well oriented aggregates are formed by hydrogen bonding and the pi-pi interaction of the cores. These interactions enable the aggregates to grow in one-dimension forming very long fibers, and these fibers further intercross to 3D network structures, e.g., organogels. In comparison to the very few PBI-based gelators reported before, one advantage of this gelator is that, it is more versatile and can gelate a wide range of organic solvents. Moreover, the well-organized fibers that are composed of extended π-stacks provide efficient pathways for n-type charge carriers. Interestingly, AFM studies reveal that the PBI molecules form well-defined helical fibers in toluene. Both left-handed (M) and right-handed (P) helicities can be observed without any preference for one handedness because the building block is intrinsically achiral. In chapter 4, we tried to influence the M/P enantiomeric ratio by applying external forces. For example, we utilized chiral solvents to generate chiral aggregates with a preferential handedness. AFM analysis of the helices showed that a enantiomeric ratio of about 60: 40 can be achieved by aggregation in chiral solvents R- or S-limonene. Moreover, the long aggregated fibres can align at macroscopic level in vortex flows upon rotary stirring In chapter 5, bulky tetra-phenoxy groups are introduced in the bay area of the PBI gelator. The conjugated core of the new molecule is now distorted because of the steric hindrance. UV/Vis studies reveal a J-type aggregation in apolar solvents like MCH due to intermolecular pi-pi-stacking and hydrogen-bonding interactions. Microscopic studies reveal formation of columnar aggregates in apolar solvent MCH, thus this molecule lacks the ability to form gels in this solvent, but form highly fluorescent lyotropic mesophases at higher concentration. On the other hand, in polar solvents like acetone and dioxane, participation of the solvent molecules in hydrogen bonding significantly reduced the aggregation propensity but enforced the gel formation. The outstanding fluorescence properties of the dye in both J-aggregated viscous lyotropic mesophases and bulk gel phases suggest very promising applications in photonics, photovoltaics, security printing, or as fluorescent sensors. In chapter 6, we did some studies on combining PBI molecules with inorganic gold nanorods. Gold nanorods were synthesized photochemically. By virtue of the thioacetate functionalized PBIs, the rods were connected end to end to form gold nanochains, which were characterized by absorption spectra and TEM measurement. Such chromophore-nanorod hybrids might be applied to guide electromagnetic radiation based on optical antenna technology.
In this work, a behavioural analysis of different mutants of the fruit fly Drosophila melanogaster has been carried out. Primarily, the gap climbing behaviour (Pick & Strauss, 2005) has been assayed as it lends itself for the investigation of decision making processes and the neuronal basis of adaptive behaviour. Furthermore it shows how basic motor actions can be combined into a complex motor behaviour. Thanks to the neurogenetic methods, Drosophila melanogaster has become an ideal study object for neurobiological questions. Two different modules of climbing control have been examined in detail. For the decision making, the mutant climbing sisyphus was analysed. While wild-type flies adapt the initiation of climbing behaviour to the width of the gap and the probability for a successful transition. climbing sisyphus flies initiate climbing behaviour even at clearly insurmountable gap widths. The climbing success itself is not improved in comparison to the wild-type siblings. The mutant climbing sisyphus is a rare example of a hyperactive mutant besides many mutants that show a reduced activity. Basic capabilities in vision have been tested in an optomotor and a distance-estimation paradigm. Since they are not affected, a defect in decision making is most probably the cause of this behavioural aberration. A second module of climbing control is keeping up orientation towards the opposite side of the gap during the execution of climbing behaviour. Mutants with a structural defect in the protocerebral bridge show abnormal climbing behaviour. During the climbing attempt, the longitudinal body axis does not necessarily point into the direction of the opposite side. Instead, many climbing events are initiated at the side edge of the walking block into the void and have no chance to ever succeed. The analysed mutants are not blind. In one of the mutants, tay bridge1 (tay1) a partial rescue attempt used to map the function in the brain succeeded such that the state of the bridge was restored. That way, a visual targeting mechanism has been activated, allowing the flies to target the opposite side. When the visibility of the opposing side was reduced, the rescued flies went back to a tay1 level of directional scatter. The results are in accord with the idea that the bridge is a central constituent of the visual targeting mechanism. The tay1 mutant was also analysed in other behavioural paradigms. A reduction in walking speed and walking activity in this mutant could be rescued by the expression of UAS-tay under the control of the 007Y-GAL4 driver line, which concomitantly restores the structure of the protocerebral bridge. The separation of bridge functions from functions of other parts of the brain of tay1 was accomplished by rescuing the reduced optomotor compensation in tay1 by the mb247-GAL4>UAS-tay driver. While still having a tay1-like protocerebral bridge, mb247-GAL4 rescue flies are able to compensate at wild-type levels. An intact compensation is not depended on the tay expression in the mushroom bodies, as mushroom body ablated flies with a tay1 background and expression of UAS-tay under the control of mb247-GAL4 show wild-type behaviour as well. The most likely substrate for the function are currently unidentified neurons in the fan-shaped body, that can be stained with 007Y-GAL4 and mb247-GAL4 as well.
trans-1,1,1,3-Tetrafluoropropene (HFO-1234ze) and 2,3,3,3-tetrafluoropropene (HFO-1234yf) are non-ozone-depleting fluorocarbon replacements with low global warming potentials and short atmospheric lifetimes. They are developed as foam blowing agent and refrigerant, respectively. Investigations on biotransformation in different test species and in vitro systems are required to assess possible health risks of human exposure and needed for commercial development. The biotransformation of HFO-1234ze and HFO-1234yf was therefore investigated after inhalation exposure. Male Sprague-Dawley rats were exposed to air containing 2 000; 10,000; or 50,000 ppm (n=5/concentration) HFO-1234ze or HFO-1234yf. Male B6C3F1 mice were only exposed to 50,000 ppm HFO-1234ze or HFO-1234yf. Due to lethality observed in a developmental study with rabbits after exposure to high concentrations of HFO-1234yf, the metabolic fate of the compound was tested by whole body inhalation exposure of female New Zealand White rabbits to air containing 2 000; 10,000; or 50,000 ppm (n=3/concentration) HFO-1234yf. All inhalation exposures were conducted for 6 h in a dynamic exposure chamber. After the end of the exposures, animals were individually housed in metabolic cages and urines were collected at 6 or 12 h intervals for 48 h (rats and mice) or 60 h (rabbits). For metabolite identification, urine samples were analyzed by 1H-coupled and 1H-decoupled 19F-NMR and by LC/MS-MS or GC/MS. Metabolites were identified by 19F-NMR chemical shifts, signal multiplicity, 1H-19F coupling constants and by comparison with synthetic reference compounds. Biotransformation of HFO-1234ze in rats exposed to 50,000 ppm yielded S-(3,3,3-trifluoro-trans-propenyl)mercaptolactic acid as the predominant metabolite which accounted for 66% of all integrated 19F-NMR signals in urines. No 19F-NMR signals were found in spectra of rat urine samples collected after inhalation exposure to 2 000 or 10,000 ppm HFO-1234ze likely due to insufficient sensitivity. S-(3,3,3-Trifluoro-trans-propenyl)-L-cysteine, N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine, 3,3,3-trifluoropropionic acid and 3,3,3-trifluorolactic acid were also present as metabolites in urine samples of rats and mice at the 50,000 ppm level. A presumed amino acid conjugate of 3,3,3-trifluoropropionic acid was the major metabolite of HFO-1234ze in urine samples of mice exposed to 50,000 ppm and related to 18% of total integrated 19F-NMR signals. Quantitation of three metabolites in urines of rats and mice was performed, using LC/MS-MS or GC/MS. The quantified amounts of the metabolites excreted with urine in both mice and rats, suggest only a low extent (<<1% of dose received) of biotransformation of HFO-1234ze and 95% of all metabolites were excreted within 18 h after the end of the exposures (t1/2 approx. 6 h). Due to its low boiling point of −22 °C, most of the inhaled HFO-1234ze is expected to be readily exhaled. Moreover, steric and electronic factors may decrease the reactivity of the parent compound with soft nucleophiles such as glutathione. The obtained results suggest that HFO-1234ze is subjected to an addition-elimination reaction with glutathione and to a cytochrome P450-mediated epoxidation at low rates. The extent of a direct addition reaction of HFO-1234ze with glutathione is negligible, compared to that of the observed addition-elimination reaction. The results of in vivo testing of HFO-1234ze could not be supported by in vitro investigations, since HFO-1234ze was not metabolized in incubations with either liver microsomes or subcellular fractions from rat and human. Regarding the structures delineated in the biotransformation scheme of HFO-1234ze, 1,1,1,3-tetrafluoroepoxypropane and 3,3,3-trifluoropropionic acid are toxic intermediates which, however, are not supposed to display toxicity in the species after exposure to HFO-1234ze, due to the low extent of formation and an efficient detoxification of the epoxide by hydrolysis and glutathione conjugation. The findings of biotransformation of HFO-1234ze in rats and mice correlate with the absence of adverse effects in the toxicity testings and indicate their innocuousness to a human exposure. Biotransformation of HFO-1234yf yielded N-acetyl-S-(3,3,3-trifluoro-2-hydroxypropanyl)-L-cysteine as predominat metabolite which accounted for approx. 44, 90 and 32% (50,000 ppm) of total 19F-NMR signal intensities in urine samples from rabbits, rats and mice, respectively. S-(3,3,3-Trifluoro-2-hydroxypropanyl)mercaptolactic acid and the sulfoxides of mercapturic acid and mercaptolactic acid S-conjugate were identified as minor metabolites of HFO-1234yf in urine samples from rabbits, rats and mice, whereas trifluoroacetic acid, 3,3,3-trifluorolactic acid and 3,3,3-trifluoro-1-hydroxyacetone were present as minor metabolites only in urine samples from rats and mice. The absence of these metabolites in rabbit urine samples...
Evaluation of 1H-NMR and GC/MS-based metabonomics for the assessment of liver and kidney toxicity
(2009)
For the assessment of metabonomics techniques for the early, non-invasive detection of toxicity, the nephrotoxins gentamicin (s.c. administration of 0, 60 and 120 mg/kg bw 2x daily for 8 days), ochratoxin A (p.o. administration of 0, 21, 70 and 210 µg/kg bw 5 days/week for 90 days) and aristolochic acid (p.o. administration of 0, 0.1, 1.0 and 10 mg/kg bw for 12 days) were administered to rats and urine samples were analyzed with 1H-NMR and GC/MS. Urine samples from the InnoMed PredTox project were analyzed as well, thereby focusing on 1H-NMR analysis and bile duct necrosis as histopathological endpoint. 1H-NMR analysis used water supression with the following protocol: 1 M phosphate buffer, D2O as shift lock reagent, D4-trimethylsilylpropionic acid as chemical shift reference, noesygppr1d pulse sequence (Bruker). For multivariate data analysis, spectral intensity was binned into 0.04 ppm wide bins. GC/MS analysis of urine was carried out after protein precipitation with methanol, drying, derivatization with methoxyamine hydrochloride in pyridine and with methyl(trimethylsilyl)trifluoroacetamide on a DB5-MS column using EI ionization. The chromatograms were prepared for multivariate data analysis using the R-program based peak picking and alignment software XCMS version 2.4.0. Principal component analysis (PCA) to detect and visualize time-point and dose-dependent differences between treated animals and controls and orthogonal projection to latent structures discriminant analysis (OPLS-DA) for identification of potential molecular markers of toxicity was carried out using SIMCA P+ 11.5 1H-NMR-based markers were identified and quantified with the Chenomx NMR Suite, GC/MS based markers were identified using the NIST Mass Spectral Database and by co-elution with authentic reference standards. PCA of urinary metabolite profiles was able to differentiate treated animals from controls at the same time as histopathology. An advantage over classical clinical chemistry parameters regarding sensitivity could be observed in some cases. Metabonomic analysis with GC/MS and 1H-NMR revealed alterations in the urinary profile of treated animals 1 day after start of treatment with gentamicin, correlating with changes in clinical chemistry parameters and histopathology. Decreased urinary excretion of citrate, 2-oxoglutarate, hippurate, trigonelline and 3-indoxylsulfate increased excretion of 5-oxoproline, lactate, alanine and glucose were observed. Ochratoxin A treatment caused decreased excretion of citrate, 2-oxoglutarate and hippurate and and increased excretion of glucose, myo-inositol, N,N-dimethylglycine, glycine, alanine and lactate as early as 2 weeks after start of treatment with 210µg OTA/kg bw, correlating with changes in clinical chemistry parameters and histopathology. Integration of histopathology scores increased confidence in the molecular markers discovered. Aristolochic acid treatment resulted in decreased urinary excretion of citrate, 2-oxoglutarate, hippurate and creatinine as well as increased excretion of 5-oxoproline, N,N-dimethylglycine, pseudouridine and uric acid. No alterations in clinical chemistry parameters or histopathology were noted.Decreased excretion of hippurate indicates alterations in the gut microflora, an effect that is expected as pharmacological action of the aminoglycoside antibiotic gentamicin and that can also be explained by the p.o. administration of xenobiotica. Decreased Krebs cycle intermediates (citrate and 2-oxoglutarate) and increased lactate is associated with altered energy metabolism. Increased pseudouridine excretion is associated with cell proliferation and was observed with aristolochic acid and ochratoxin A, for which proliferative processes were observed with histopathology. 5-oxoproline and N,N-dimethylglycine can be associated with oxidative stress. Glucose, a marker of renal damage in clinical chemistry, was observed for all three nephrotoxins studied. Single study analysis with PCA of GC/MS chromatograms and 1H-NMR spectra of urine from 3 studies conducted within the InnoMed PredTox project showing bile duct necrosis revealed alterations in urinary profiles with the onset of changes in clinical chemistry and histopathology. Alterations were mainly decreased Krebs cycle intermediates and changes in the aromatic gut flora metabolites, an effect that may result as a secondary effect from altered bile flow. In conclusion, metabonomics techniques are able to detect toxic lesions at the same time as histopathology and clinical chemistry. The metabolites found to be altered are common to most toxicities and are not organ-specific. A mechanistic link to the observed toxicity has to be established in order to avoid confounders such as body weight loss, pharmacological effects etc. For pattern recognition purposes, large databases are necessary.
Charcot-Marie-Tooth disease (CMT) is a cohort of human hereditary disorders of the peripheral nervous system (PNS) which exhibit symptoms like sensory dysfunction, muscle weakness and gait disturbances. Different mutations are described as causation for this neuropathy, such as a duplication of chromosome 17 comprising the gene for the peripheral myelin protein-22 (PMP22). Based on different animal models former studies identified immune cells, i.e. macrophages and T-lymphocytes, as crucial mediators of pathology in these neuropathies. In this study, PMP22-overexpressing mice (PMP22tg, C61), serving as a model for a specific type of CMT – CMT1A – were crossbred with immune-deficient mutant mice to examine the impact of the immune system on nerve pathology. Crossbreeding of PMP22tg mice with recombination activating gene-1 (RAG-1) deficient mice, lacking mature T- and B-lymphocytes, caused no striking alterations of pathogenesis in peripheral nerves of mutant mice. In contrast, crossbreeding of PMP22tg myelin mutants with mice deficient in the chemokine monocyte chemoattractant protein-1 (MCP-1, CCL2) caused an amelioration of the demyelinating phenotype of peripheral nerves when MCP-1 was either reduced or completely absent. Furthermore, functional investigations, i.e. neurographic recordings and examinations of the grip strength of the extremities, revealed an amelioration in PMP22tg/MCP-1-/- mice in regard to a symptomatic improvement in the compound action muscle potential (CMAP) and stronger grip strength of the hindlimbs. Interestingly, peripheral nerves of PMP22tg mice showed an irregular distribution of potassium channels in presence of MCP-1, whereas the absence of MCP-1 in the myelin mutants rescued the ion channel distribution and resulted in a more wild type-like phenotype. Having shown the impact of MCP-1 as an important mediator of nerve pathology in PMP22/MCP-1 double mutants, the regulation of this chemokine became an important target for potential treatment strategies. We found that the signaling cascade MEK1/2/ERK1/2 was more strongly activated in peripheral nerves of PMP22tg mice compared to nerves of wild type mice. This activation corresponded to an increase in MCP-1 mRNA expression in peripheral nerves at the same age. Furthermore, a MEK1/2-inhibitor was used in vivo to confirm the regulation of MCP-1 by the MEK1/2/ERK1/2 pathway. After a treatment period of three weeks, a clear reduction of ERK1/2-phosphorylation as well as a reduction of MCP-1 mRNA expression was observed, accompanied by a decline in macrophage number in peripheral nerves of PMP22tg mice. These observations suggest that the expression of MCP-1 is crucial for the neuropathological progression in a mouse model for CMT1A. Therefore, this chemokine could provide a basis for a putative treatment strategy of inherited neuropathies.
The aim of the current work was to enhance the understanding of the relationship between goals and the self. More specifically, I wanted to achieve three things. First, I developed an implicit measure of self-activation (SA) based on response latencies to avoid the problems of traditional measures of self-activation (i.e., demand effects, self-presentation concerns). Therefore, two studies were conducted in which increased self-activation, induced by classic self-manipulations, was measured with a newly developed picture task. Thereby it was assumed that individuals would react faster to photographs of themselves when the self was activated than when it was not. Second, I aimed to demonstrate that there exists a close connection between personal goals and the self. Despite being inherent in several theories, this assumption has never been tested directly before. It was hypothesized that thinking about personal goals should activate the self, resulting in faster reactions in the newly developed measure of SA, i.e., quicker responses to the self-pictures. Third, it was investigated whether goals and the self are linked in a bidirectional fashion; according to the reported findings, it seems to be functional for individuals’ self-regulation and goal pursuit to develop such a link. To provide evidence for the bidirectionality of the relationship, it was hypothesized that in conditions of high SA, it should be more likely personal evaluations to be construed as goals; this goal activation should result in higher accessibility of goal-related knowledge, stronger approach motivational tendencies towards goal-related targets, and more goal-directed behavior. The obtained results endorse the applicability of the picture task as implicit method to measure increased SA and also corroborate the core hypothesis, namely that personal goals and the self are inherently connected and that they are linked in a bidirectional fashion.
It is well-known that a multivariate extreme value distribution can be represented via the D-Norm. However not every norm yields a D-Norm. In this thesis a necessary and sufficient condition is given for a norm to define an extreme value distribution. Applications of this theorem includes a new proof for the bivariate case, the Pickands dependence function and the nested logistic model. Furthermore the GPD-Flow is introduced and first insights were given such that if it converges it converges against the copula of complete dependence.
Macromolecular complexes, also termed molecular machines, facilitate a large spectrum of biological reactions and tasks crucial to the survival of cells. These complexes are composed of either protein only, or proteins bound to nucleic acids (DNA or RNA). Prominent examples for each class are the proteosome, the nucleosome and the ribosome. How such units are assembled within the context of a living cell is a central question in molecular biology. Earlier studies had indicated that even very large complexes such as ribosomes could be reconstituted from purified constituents in vitro. The structural information required for the formation of macromolecular complexes, hence, lies within the subunits itself and, thus, allow for self- assembly. However, increasing evidence suggests that in vivo many macromolecular complexes do not form spontaneously but require assisting factors (“assembly chaperones”) for their maturation. In this thesis the assembly of RNA-protein (RNP) complexes has been studied by a combination of biochemical and structural approaches. A resourceful model system to study this process is the biogenesis pathway of the uridine-rich small nuclear ribonucleoproteins (U snRNPs) of the spliceosome. This molecular machine catalyzes pre-mRNA splicing, i.e. the removal of non-coding introns and the joining of coding exons to functional mRNA. The composition and architecture of U snRNPs is well defined, also, the nucleo-cytoplasmic transport events enabling the formation of these particles in vivo have been analyzed in some detail. Furthermore, recent studies suggest that the formation of U snRNPs in vivo is mediated by an elaborate assembly machinery consisting of protein arginine methyltransferase (PRMT5)- and survival motor neuron (SMN)-complexes. The elucidation of the reaction mechanism of cellular U snRNP assembly would serve as a paradigm for our understanding of how RNA-protein complexes are formed in the cellular environment. The following key findings were obtained as part of this study: 1) Efforts were made to establish a full inventory of the subunits of the SMN-complex. This was achieved by the biochemical definition and characterization of an atypical component of this complex, the unrip protein. This protein is associated with the SMN-complex exclusively in the cytoplasm and influences its subcellular localization. 2) With a full inventory of the components in hand, the architecture of the SMN-complex was defined on the basis of an interaction map of all subunits. This study elucidated that the proteins SMN, Gemin7 and Gemin8 form a backbone, onto which the remaining subunits adhere in a modular manner. 3) The two studies mentioned above formed the basis to elucidate the reaction mechanism of cellular U snRNP assembly. Initially, an early phase in the SMN-assisted formation of U snRNPs was analyzed. Two subunits of the U7 snRNP (LSm10 and 11) were found to interact with the PRMT5-complex, without being methylated. This report suggests that the stimulatory role of the PRMT5-complex is independent of its methylation activity. 4) Key reaction intermediates in U snRNP assembly were found and characterized by a combination of biochemistry and structural studies. Initially, a precursor to U snRNPs with a sedimentation coefficient of 6S is formed by the pICln subunit of the PRMT5-complex and Sm proteins. This intermediate was shown to constitute a kinetic trap in the U snRNP assembly reaction. Progression towards the assembled U snRNP depends on the activity of the SMN-complex, which acts as a catalyst. The formation of U snRNPs is shown to be structurally similar to the way clamps are deposited onto DNA to tether poorly processive polymerases. 5) The human SMN-complex is composed of several subunits. However, it is unknown whether all subunits of this entity are essential for U snRNP assembly. A combination of bioinformatics and biochemistry was applied to tackle this question. By mining databases containing whole-genome assemblies, the SMN-Gemin2 heterodimer is recognized as the most ancestral form of the SMN-complex. Biochemical purification of the Drosophila melanogaster SMN-complex reveals that this complex is composed of the same two subunits. Furthermore, evidence is provided that the SMN-Gemin2 heterodimer is necessary and sufficient to promote faithful U snRNP assembly. Future studies will adress further details in the reaction mechanism of cellular U snRNP assembly. The results obtained in this thesis suggest that the SMN and Gemin2 subunits are sufficient to promote U snRNP formation. What then is the function of the remaining subunits of the SMN-complex? The reconstitution schemes established in this thesis will be instrumental to address this question. Furthermore, additional mechanistic insights into the U snRNP assembly reaction will require the elucidation of structures of the assembly machinery trapped at various states. The prerequisite for these structural studies, the capability to generate homogenous complexes in sufficient amounts, has been accomplished in this thesis.
Several lines of evidence implicate a dysregulation of tryptophan hydroxylase (TPH)-dependent serotonin (5-HT) synthesis in emotions and stress and point to their potential relevance to the etiology and pathogenesis of various neuropsychiatric disorders. However, the differential expression pattern of the two isoforms TPH1 and TPH2 which encode two forms of the rate-limiting enzyme of 5-HT synthesis is controversial. Here, a comprehensive spatio-temporal analysis clarifies TPH1 and TPH2 expression during pre- and postnatal development of the mouse brain and in adult human brain as well as in peripheral organs including the pineal gland. Four different methods (real time PCR, in situ hybridization, immunohistochemistry and Western blot analysis) were performed to systematically control for tissue-, species- and isoform-specific expression on both the pre- and posttranslational level. TPH2 expression was consistently detected in the raphe nuclei, as well as in fibres in the deep pineal gland and in the gastrointestinal tract. Although TPH1 expression was found in these peripheral tissues, no significant TPH1 expression was detected in the brain, neither during murine development, nor in mouse and human adult brain. Also under conditions like stress and clearing the tissue from blood cells, no changes in expression levels were detectable. Furthermore, the reuptake of 5-HT into the presynaptic neuron by the serotonin transporter (SERT) is the major mechanism terminating the neurotransmitter signal. Thus, mice with a deletion in the Sert gene (Sert KO mice) provide an adequate model for human affective disorders to study lifelong modified 5-HT homeostasis in interaction with stressful life events. To further explore the role of TPH isoforms, Tph1 and Tph2 expression was studied in the raphe nuclei of Sert deficient mice under normal conditions as well as following exposure to acute immobilization stress. Interestingly, no statistically significant changes in expression were detected. Moreover, in comparison to Tph2, no relevant Tph1 expression was detected in the brain independent from genotype, gender and treatment confirming expression in data from native animals. Raphe neurons of a brain-specific Tph2 conditional knockout (cKO) model were completely devoid of Tph2-positive neurons and consequently 5-HT in the brain, with no compensatory activation of Tph1 expression. In addition, a time-specific Tph2 inducible (i) KO mouse provides a brain-specific knockdown model during adult life, resulting in a highly reduced number of Tph2-positive cells and 5-HT in the brain. Intriguingly, expression studies detected no obvious alteration in expression of 5-HT system-associated genes in these brain-specific Tph2 knockout and knockdown models. The findings on the one hand confirm the specificity of Tph2 in brain 5-HT synthesis across the lifespan and on the other hand indicate that neither developmental nor adult Tph2-dependent 5-HT synthesis is required for normal formation of the serotonergic system, although Tph1 does not compensate for the lack of 5-HT in the brain of Tph2 KO models. A further aim of this thesis was to investigate the expression of the neuropeptide oxytocin, which is primarily produced in the hypothalamus and released for instance in response to stimulation of 5-HT and selective serotonin reuptake inhibitors (SSRIs). Oxytocin acts as a neuromodulator within the central nervous system (CNS) and is critically involved in mediating pain modulation, anxiolytic-like effects and decrease of stress response, thereby reducing the risk for emotional disorders. In this study, the expression levels of oxytocin in different brain regions of interest (cortex, hippocampus, amygdala, hypothalamus and raphe nuclei) from female and male wildtype (WT) and Sert KO mice with or without exposure to acute immobilization stress were investigated. Results showed significantly higher expression levels of oxytocin in brain regions which are involved in the regulation of emotional stimuli (amygdala and hippocampus) of stressed male WT mice, whereas male Sert KO as well as female WT and Sert KO mice lack these stress-induced changes. These findings are in accordance with the hypothesis of oxytocin being necessary for protection against stress, depressive mood and anxiety but suggest gender-dependent differences. The lack of altered oxytocin expression in Sert KO mice also indicates a modulation of the oxytocin response by the serotonergic system and provides novel research perspectives with respect to altered response of Sert KO mice to stress and anxiety inducing stimuli.
A new class of optimization problems name 'mathematical programs with vanishing constraints (MPVCs)' is considered. MPVCs are on the one hand very challenging from a theoretical viewpoint, since standard constraint qualifications such as LICQ, MFCQ, or ACQ are most often violated, and hence, the Karush-Kuhn-Tucker conditions do not provide necessary optimality conditions off-hand. Thus, new CQs and the corresponding optimality conditions are investigated. On the other hand, MPVCs have important applications, e.g., in the field of topology optimization. Therefore, numerical algorithms for the solution of MPVCs are designed, investigated and tested for certain problems from truss-topology-optimization.
Semiconductor Quantum Dots (QDs) have been attracting immense interest over the last decade from both basic and application-orientated research because of their envisioned use as fundamental building blocks in non-classical device architectures. Their presumable ease of integration into existing semiconductor technology has bought them the reputation of being cost-efficiently scalable and renders them a place among the top candidates in a wide range of proposed quantum logic and quantum information processing schemes. These include the highly acclaimed use of QD as triggered sources of single pairs of entangled photons, which is a key ingredient of most of the intensivly investigated optical quantum cryptography operations. A big obstacle towards these goals are the pronounced asymmetries that are intrinsically present in all currently availabe semiconductor QD systems. They are a natural by-product that stems from the employed self-assembled growth methods and manifest in various forms such as shape-asymmetry, inhomogeneous strain distribution within the QD and concomittant piezo-elecric fields. These asymmetries in return give rise to distinct anisotropies in the optical properties of QDs, which in fact render their optical response classic. For device oriented research these anisotropies are therefore typically considered unwanted and actively researched to be controlled. They are, however, interesting from a fundamental point of view, as anisotropic QDs basically provide a testbed system for fundamental atom-like quantum physics with non-centrosymmetric potentials. As shall be shown in the current work, this gives rise to novel and interesting physics in its own right. Employing photoluminescence spectroscopy (PL) we investigate the optical anisotropy of the radiative recombination of excitons confined to CdSe/ZnSe QDs. This is done by angle-dependent polarization-resolved PL. We demonstrate experimentally that the electron-hole exchange interaction in asymmetric QDs gives rise to an effective conversion of the optical polarization from linear to circular and vice versa. The experiment is succesfully modeled in the frame of an exciton pseudospin-formalism that is based on the exchange induced finestructure splitting of the radiative excitonic states and unambiguously proves that the observed polarization conversion is the continuous-wave equivalent to quantum beats between the exchange split states in the time domain. These results indicate that QDs may offer extended functionality beyond non-classical light sources in highly integrated all-optical device schemes, such as polarization converters or modulators. In a further extension we apply the exciton pseudospin-formalism to optical alignment studies and demonstrate how these can be used to directly measure the otherwise hidden symmetry distribution over an ensemble of QDs. This kind of measurement may be used on future optical studies in order to link optical data more directly to structural investigations, as it yields valuable information on capped QDs that cannot be looked at directly by topological methods. In the last part of this work we study the influence of an in-plane magnetic field on the optical anisotropy. We find that the optical axis of the linear polarization component of the photoluminescence signal either rotates in the opposite direction to that of the magnetic field or remains fixed to a given crystalline direction. A qualitative theoretical analysis based on the exciton pseudospin Hamiltonian unambiguously demonstrates that these effects are induced by isotropic and anisotropic contributions to the heavy-hole Zeeman term, respectively. The latter is shown to be compensated by a built-in uniaxial anisotropy in a magnetic field B=0.4 T, resulting in an optical response that would be expected for highly symmetric QDs. For a comprehensive quantitative analysis the full heavy-hole exciton k.p-Hamiltonian is numerically calculated and the resulting optical polarization is modeled. The model is able to quantitatively describe all experimental results using a single set of parameters. From this model it is explicitly seen that a optical response characteristic for high symmetry QDs may be obtained from an ensemble of asymmetric QDs without a crossing of the zero-field bright exciton states, which was required for application of QDs in non-classical light sources. It is clearly demonstrated that any scheme using in-plane magnetic fields to symmetrize the optical response has to take into account at least four optically active states instead of the two observed in the absence of magnetic fields. These findings may explain some of the major disagreement on recent entanglement studies in asymmetric QDs, as models that do not take the above result into account cannot be a priori expected to provide reliable results on excitonic Bell states.
This work focuses on a fundamental problem in modern numerical rela- tivity: Extracting gravitational waves in a coordinate and gauge independent way to nourish a unique and physically meaningful expression. We adopt a new procedure to extract the physically relevant quantities from the numerically evolved space-time. We introduce a general canonical form for the Weyl scalars in terms of fundamental space-time invariants, and demonstrate how this ap- proach supersedes the explicit definition of a particular null tetrad. As a second objective, we further characterize a particular sub-class of tetrads in the Newman-Penrose formalism: the transverse frames. We establish a new connection between the two major frames for wave extraction: namely the Gram-Schmidt frame, and the quasi-Kinnersley frame. Finally, we study how the expressions for the Weyl scalars depend on the tetrad we choose, in a space-time containing distorted black holes. We apply our newly developed method and demonstrate the advantage of our approach, compared with methods commonly used in numerical relativity.
The approach of using the combination of Ultraviolet (UPS) and Inverse Photoemission (IPS) to determine the transport levels in thin films of organic semiconductors is the scope of this work. For this matter all influences on the peak position and width in Photoelectron Spectroscopy are discussed with a special focus on organic semiconductors. Many of these influences are shown with experimental results of the investigation of diindenoperylene on Ag(111). These findings are applied to inorganic semiconductors silicon in order to establish the use of UPS and IPS on a well-understood system. Finally, the method is used to determine the transport level of several organic semiconductors (PTCDA, Alq3, CuPc, DIP, PBI-H4) and the corresponding exciton binding energies are calculated by comparison to optical absorption data.
Simulations (MASim) and non-rational behaviour. This non-rational behaviour is here based on the Prospect Theory [KT79] (PT), which is compared to the rational behaviour in the Expected Utility Theory [vNM07] (EUT). This model was used to design a modified Q-Learning [Wat89, WD92] algorithm. The PT based Q-Learning was then integrated into a proposed agent architecture. Because much attention is given to a limited interpretation of Simon's definition of bounded-rationality, this interpretation is broadened here. Both theories, rationality and the non-rationality, are compared and the discordance in their results discussed. The main contribution of this work is to show that an alternative is available to the EUT that is more suitable for human decision-makers modelling. The evidences show that rationality is not appropriated for modelling persons. Therefore, instead of fine-tuning the existent model the use of another one is proposed and evaluated. To tackle this, the route choice problem was adopted to perform the experiments. To evaluate the proposed model three traffic scenarios are simulated and their results analysed.
This thesis analyzes the relationship between market concentration and efficiency of the market outcome in a differentiated good context from different points of view. The first chapter introduces the objectives of competition policy and antitrust authorities and outlines the importance of market concentration. Chapter 2 analyzes the relationship between social surplus and market heterogeneity in a differentiated Cournot oligopoly. Market heterogeneity is due to differently efficient firms, each of them producing one variety of a differentiated good. All firms exhibit constant but different marginal costs without fixed costs. Consumers preferences are given by standard quadratic utility originated by Dixit (1979). Since preferences are quasi-linear social surplus is the measure for Pareto-optimality. The main finding is that consumer suprlus as well as producer surplus increase with the variance of marginal costs. The third chapter analyzes the relationship between the cost structure and market concentration measured by the Herfindahl-Hirschman Index. Market concentration increases with the variance of marginal costs as well as the mean of marginal costs. Chapter four analyzes welfare implications of present antitrust enforcement policy on basis of the same theoretical model. European as well as the US Merger Guidelines presume a negative impact of market concentration on the competitiveness of the market and, therefore, on the efficiency of the market outcome. The results of the previous chapters indicate that this assumption is false. The main finding is that post-merger joint profit of the insider increase with the size of the merger. Moreover, there is a negative relationship between the size of the merger and efficiency of the market outcome. Present antitrust enforcement policy increases the disparity of output levels and enforces the removal of the least efficient firm of the market. The welfare gains can be traced back on these two effects. Therefore, neither a minimum of market concentration nor a maximum of product diversity is necessarily welfare enhancing even in absence of fixed costs.
Nuclear magnetic resonance has numerous applications for in vivo diagnostics. However, methods requiring homogeneous magnetic fields, particularly magnetic resonance spectroscopy (MRS) techniques, have limited applicability in regions near or on anatomical boundaries that cause strong inhomogeneities. In cases where the shim system can not or just partly correct for these inhomogeneities, methods based on intermolecular multiple quantum coherence (iMQC) detection can provide an alternative solution for in vivo MRS. This dissertation presented the development, validation and application potential of a novel MRS pulse sequence detecting intermolecular zero-quantum coherences (iZQC) with special emphasis on in vivo experiments. In addition, the detection limit and spectral behaviour of iZQC-MRS under modelled realistic conditions were systematically approached for the first time. Based on the original sequence used to detect two dimensional (2D) iZQC-spectra, dubbed HOMOGENIZED, methodological development led to increased sensitivity and water suppression, and decreased T2-relaxation effects through the application of a frequency selective 90° RF-pulse in place of a non selective beta-pulse. Best water suppression was achieved by placing a pair of selective refocusing units immediately prior to the acquisition window. The same placement was found to be optimal also for single voxel localization units based on slice selective spin echo refocusing. By voxel selection before the iZQC-MRS sequence, the chemical shift artefact could be avoided. However, this led to significant residual signal from outside the voxel. Analytical derivations of signal evolution for several sequences presented in this dissertation provide useful additions to the iZQC MRS theory. In vivo applications of the developed sequence provided high quality spectra in the central nervous system of the rat, the mouse brain and in subcutaneous xenograft tumor grown on the thigh of the mouse. In all these 2D spectra, the limiting factor of the resolution in the indirect dimension was the digital sampling rate, rather than inhomogeneous line broadening. Nevertheless, linewidths of the cross-peaks were similar or narrower than along the direct axis, where the sampling rate was about ten times higher. The first MR spectroscopic investigation of the rat spinal cord at 17.6 T was performed. Through its insensitivity to macroscopic field inhomogeneities, the localized iZQC method allowed for the selection of larger voxels than conventional methods and still provided the same spectral resolution. This property was used also in tumor tissue to propel the relative signal to noise (SNR) efficiency of the iZQC spectroscopy for the first time above the SNR efficiency of a conventional sequence. Future applications for fast metabolite count in large inhomogeneous organs, like a tumor, are thinkable. Extensive simulations and phantom experiments assessed the limit of iZQC cross-peak detection in presence of local field distortions. The order of maximum volume ratio between dipole source and voxel was found to be between 0.1 % and 1 %. It is an essential conclusion of this study that the dominant effect of microscopic to mesoscopic inhomogeneities on iZQC spectra under general in vivo conditions, like for voxels greater than (1 mm)³ and metabolite concentrations in the millimolar range, is a cross-peak intensity reduction and not line broadening. The iZQC method provided resolution enhancement in comparison to conventional MRS even in the presence of clustered paramagnetic microparticles. However, the vision of iZQC spectroscopy in green leafs or the lung epithelium has to be, unfortunately, abandoned, because cross-peaks can be observed until the volume of the separating medium is much larger than the volume of local dipole sources. Intermolecular zero-quantum coherence spectroscopy remains an exciting field in NMR research on living organisms. It provides access to the monitoring of relative metabolite concentration changes in the presence of microscopic iron particles, which raises realistic hopes for new applications in studies using stained stem cells.
Regulation of effector T cells is an important mechanism to control organ-specific inflammation. Thereby regulatory T cells (Treg cells) are essential for maintaining peripheral immune tolerance and for establishing parenchyma immune homeostasis. A novel population of natural human Treg characterized by the constitutive expression of the immune-tolerogenic human HLA-G molecule has been identified. In the first part of the study, we elucidated the mechanism(s) by which CD4+ HLA-Gpos Treg modulates their cellular targets namely autologous HLA-G negative responder T cells (HLAGneg Tresp). Using a suppression system free of antigen-presenting cells (APC), we demonstrate a T-T cell interaction resulting in suppression of HLA-Gneg Tresp. We could also show that this suppression was independent of cell-cell contact. Importantly, stimulus of T cell receptor (TCR) on HLA-Gpos Treg facilitated their suppressive capacity. We also observed that removal of HLA-Gpos Treg from the established co-cultures could restore the ability of HLA-Gneg Tresp to proliferate upon TCR re-stimulation, indicating that the suppression was reversible. Further, HLA-Gpos Treg–mediated suppression was critically depending on the secretion of IL-10 but not TGF-β. Taken together, this part of the work provides an in-depth characterization of the mechanisms of how HLA-Gpos Treg suppresses T responder cells in direct T-T interactions. Understanding the suppressive mechanism used by HLA-Gpos Treg may help to develop therapeutic strategies to modulate regulatory arms of T-cell suppression. In the second part of this study, the potential role of HLA-Gpos Treg in the pathophysiological process of Multiple Sclerosis (MS), a prototypic autoimmune inflammatory central nervous system (CNS), has been investigated. We found that HLA-Gpos Treg are enriched in the cerebrospinal fluid (CSF) from MS patients, but not in non-inflammatory controls. CSFderived HLA-Gpos Treg showed predominance of central memory (CD45RA-CD27+) phenotype, exhibited markers of activation (ICOS), and had significantly higher expression of the inflammatory chemokine receptor CCR5. Importantly, these cells demonstrated as potent suppressors to autologous CD4+ T-cell proliferation. Using an in vitro model of human blood brain barrier, we showed that HLA-Gpos Treg have a strong propensity to migrate, which could be facilitated by MIP1α and RANTES (ligands of CCR5) but not MIP3β (a ligand of CCR7). The HLA-Gpos Treg migration triggered by chemokines was also associated with a gain of suppressive capacity upon cellular transmigration. In contrast to CD4+CD25+ naturally occurring FoxP3-expressing Treg, HLA-Gpos Treg from patients with MS did not exhibit impaired function, suggesting that HLA-Gpos Treg are selectively recruited to the sites of CNS inflammation in an effort to combat destructive inflammation during MS. Our results contribute to the understanding of the role and function of HLA-Gpos Treg and provide an important example of “beneficial” T-cell inflammation in CNS autoimmunity- interesting both from a patho/-physiological and a therapeutically point of view.
The Upper Bajocian-Bathonian Kashafrud Formation is a thick package of siliciclastic sediments that crops out in NE Iran from the southeast, near the Afghanistan border, to north- northwestern areas around the city of Mashhad. The thickness ranges from less than 300 m in a deltaic succession (Kuh-e-Radar) to more than 2500 m in the Maiamay area, but the normal thickness in Ghal-e-Sangi, Kol-e-Malekabad, and Fraizi areas is about 1200-1300 m. It is the fill of an elongated basin, which extended for more than 200 km in NW-SE direction and a width of at least 50 km along the southern margin of the Koppeh Dagh. Prior to this study, little information existed about the sedimentary environments and other characters, especially the geometry of the basin. Exact biostratigraphic data from the top of the Kashafrud Formation were rare. Based on the macrofauna from the lower part of the overlying Chamanbid Formation the upper boundary of the Kashafrud Formation had been attributed to the Late Bathonian and/or Early Callovian, but now the upper limit of the Kashafrud Formation is defined as Late Bathonian in age, based on ammonite biostratigraphy. Except for chapter one, which deals with the introduction and related sub-titles, in the following chapters, step by step, field observations and data were surveyed according to the questions to solve. In order to reconstruct the facies architecture and the geometry of the basin, a number of sections have been logged in detail (see chapter 3, “The sections”). The exact biostratigraphic setting is discussed in chapter 4 (“Biostratigraphy”). Sedimentary environments range from non-marine alluvial fans and braided rivers in the basal part of the succession to deltas, storm-dominated shelf, slope and deep-marine basin. The latter comprises the largest part of the basin fill, consisting of monotonous mudstones, siltstones and proximal to distal turbidities. The only continuous carbonate unit (~30 m) locally formed at Tappenader. Other localities in which thin fossil-bearing carbonate strata occur are Torbat-e-Jam (benthic fauna) and, to a lesser extent, Ghal-e-Sangi. These rare shallow-water carbonates, which also contain corals, represent only short intervals (see chapter 5,” Facies association and sedimentary environments”). Relative changes in sea level were reconstructed on the basis of deepening- and shallowing-upward trends. Sequence boundaries and parasequences have been distinguished and analyzed in chapter 6 (“Sequence stratigraphy”). In most areas, the basin rapidly evolved from a shallow marine, transgressive succession to a deep-marine, basinal succession. The only area where shallow conditions persisted from the Late Bajocian to the Late Bathonian, and even into the Early Callovian is the Kuh-e-Radar area which corresponds to a fan-delta setting. A trace fossil analysis has been carried out to obtain additional evidence on the bathymetry of the basin (see chapter 7, “Ichnology”). Altogether 29 ichnospecies belonging to 15 ichnogenera have been identified, as well as 10 ichnogenera, which were determined only at genus level. They can be grouped in the well-known “Seilacherian ichnofacies”. Very high subsidence rates and strong lateral thickness variations suggest that the Kashafrud Formation is a rift related basin that formed as the eastern extension of the South Caspian Basin. The basin evolution is reviewed, the eastern and western continuations of the basin were checked in the field and also in the literature (see chapter 8, “Basin evolution”). In all, the present study provided new insights into the development of the Kashafrud Formation, e.g. more biostratigraphic data from the base and the top of the succession, a relatively complete picture of the trace fossil associations, a better recognition and reconstruction of the sedimentary environments in different parts of the basin. Finally this research project will be a good basis for further investigations, especially towards the west, as parts of the Kashafrud Formation are source rocks of a hydrocarbon reservoir in NE Iran.
Four sections of the Galala and Maghra El Hadida formations on the footwalls of the slopes of the northern and southern Galala plateaus in Wadi Araba (Eastern Desert) have been measured and sampled in great detail. The Galala Formation is ranging in thickness from 55 to 95 meters. It unconformably overlies the Malha Formation which forms the base of the studied sections. The upper boundary of the Galala Formation is characterized by a major unconformity which separates it from the overlying the Maghra El Hadida Formation. The Galala Formation can be subdivided into five shallowing-upward cycles, each cycle starting with deep-lagoonal, marly-silty deposits at the base and grading into highly fossiliferous shallow-lagoonal limestones at the top. Only the basal part of the Galala Formation consists of unfossiliferous, greenish sandy siltstones intercalated with thin cross-bedded, bioturbated, fine- to medium-grained sandstones. Despite the lack of biostratigraphic markers in that lower part, its age can be assigned to the late Middle Cenomanian, since the conformably overlying strata contain the ammonite Neolobites vibrayeanus (D’ORBIGNY), the index marker of the early Upper Cenomanian which extends into the top of the formation. The measured thickness of the overlying Maghra El Hadida Formation is ranging from 59 to 118 meters. This formation starts with the Ghonima Member, introduced in this work to distinguish a brown, fine- to medium-grained calcareous sandstone unit in its lower part. The Ghonima Member is erosionally incised into the Galala Formation, explaining its strong lateral variability in thickness, ranging from 3 to 21 meters. It is mostly unfossiliferous except for irregular bioturbation in its upper part. The Ghonima Member is assigned to the middle Upper Cenomanian, based on its stratigraphic position between the lower Upper Cenomanian Neolobites vibrayeanus Zone and the overlying upper Upper Cenomanian Metoicoceras geslinianum and Vascoceras cauvini zones. This means that the lower part of the Maghra El Hadida Formation, about 20 – 30 m thick, accumulated during the latest Cenomanian and that the base of the formation does not coincide with the base of the Turonian as commonly believed. The overlying succession of the Maghra El Hadida Formation is characterized by an increase of carbonate content, represented by yellow, soft marls intercalated with fine-grained wacke- to packstones containing a highly fossiliferous ammonite assemblage of the upper Upper Cenomanian and Lower Turonian (zones of Vascoceras proprium, Choffaticeras spp., and Wrightoceras munieri). The Middle Turonian part of the Maghra El Hadida Formation consists of poorly fossiliferous, thick-bedded yellowish marls with upward-increasing silt content, showing occasional intercalations of medium- to coarse-grained sandstones with hummocky cross-stratification. The topmost part of the Maghra El Hadida Formation consists of brownish, medium-grained sandstones topped by fossiliferous marly limestones yielding the Upper Turonian zonal ammonite Coilopoceras requienianum (D’ORBIGNY). Based on sequence stratigraphic analyses, four complete 3rd order depositional sequences and the lower part of a fifth one, each bounded by major unconformities, can be recognized: depositional sequence DS WA 1 (upper Middle – lower Upper Cenomanian) includes the entire Galala Formation, while the Maghra El Hadida Formation comprises all the overlying depositional sequences: DS WA 2 (upper Upper Cenomanian – Lower Turonian) reaches from the base of the Metoicoceras geslinianum Zone to the top of Wrightoceras munieri Zone, DS WA 3 and DS WA 4 comprise the Middle Turonian, while Upper Turonian sequence DS WA 5 is not complete. The stratigraphic positions of the recognized sequence 2 boundaries SB WA 1 to SB WA 5 match well with contemporaneous sequence boundaries known from Europe and elsewhere. The stacking pattern of the basic cycles and bundles of the Galala Formation (5:1) and the Maghra El Hadida Formation (4:1) strongly suggest an orbital forcing by MILANKOVITCH periodicities. The Galala Formation is composed of five 5th-order bundles which equal to ~500 kyr, each bundle equals to ~100 kyr (short eccentricity). Every bundle has five basic (6th-order) cycles, each one representing ~20 kyr (precession). Based on this precession-short eccentricity syndrome, the accumulation rate of the Galala Formation therefore accounts for about 19 cm/kyr. The rate of sea-level fall at sequence boundary SB WA 2 (equivalent to the quasi-global mid-Late Cenomanian SB Ce V) estimated is with 35 cm/kyr which can be explained only by glacio-eustasy. The Upper Cenomanian and Lower Turonian part of the Maghra El Hadida Formation is considered to equal to ~1200 kyr, based on the existence of three 4th-order bundles with an inferred duration of ~400 kyr for each bundle (long eccentricity of the MILANKOVITCH Band). Every bundle consists of four basic cycles with a duration of ~100 kyr. This means that the upper Cenomanian part of the Maghra El Hadida Formation is equivalent to ~400 kyr, while the Lower Turonian (consisting of the two upper bundles) lasted 800 kyr. This matches well with the recently proposed 785 kyr duration of the Early Turonian (SAGEMAN et al., 2006; VOIGT et al., 2008) and contradicts the 1300 kyr according to the standard time scale of GRADSTEIN et al. (2004). According to this temporal constrains, the accumulation rate of the Maghra El Hadida Formation is about 4.25 cm/kyr. In addition, based on the cyclostratigraphic analysis, the range of the Early Turonian genus Choffaticeras (HYATT) is equivalent to ~325 kyr and morphological changes within its lineage can be quantified. The macrobenthos (bivalves, gastropods, echinoids) and cephalopods of the Galala and Maghra El Hadida formations were identified and illustrated in 24 figures. The ammonite taxonomy and palaeobiogeographic distribution is discussed in detail. Four genera and eight ammonite species are recorded from Egypt for the first time. The microfloral and -faunal assemblage identified in thin sections revealed two species of dasycladalean algae, two species of udoteacean algae, five species of benthic foraminifera, and two species of crustacean microcoprolites. The six facies types of the upper Middle – Upper Cenomanian Galala Formation document largely open-lagoonal, warm water conditions, while the depositional environment of the Upper Cenomanian – Turonian Maghra El Hadida Formation (16 facies types) is suggested to range from a deep-subtidal to intertidal.
Recent progresses and developments in molecular biology provide a wealth of new but insufficiently characterised data. This fund comprises amongst others biological data of genomic DNA, protein sequences, 3-dimensional protein structures as well as profiles of gene expression. In the present work, this information is used to develop new methods for the characterisation and classification of organisms and whole groups of organisms as well as to enhance the automated gain and transfer of information. The first two presented approaches (chapters 4 und 5) focus on the medically and scientifically important enterobacteria. Its impact in medicine and molecular biology is founded in versatile mechanisms of infection, their fundamental function as a commensal inhabitant of the intestinal tract and their use as model organisms as they are easy to cultivate. Despite many studies on single pathogroups with clinical distinguishable pathologies, the genotypic factors that contribute to their diversity are still partially unknown. The comprehensive genome comparison described in Chapter 4 was conducted with numerous enterobacterial strains, which cover nearly the whole range of clinically relevant diversity. The genome comparison constitutes the basis of a characterisation of the enterobacterial gene pool, of a reconstruction of evolutionary processes and of comprehensive analysis of specific protein families in enterobacterial subgroups. Correspondence analysis, which is applied for the first time in this context, yields qualitative statements to bacterial subgroups and the respective, exclusively present protein families. Specific protein families were identified for the three major subgroups of enterobacteria namely the genera Yersinia and Salmonella as well as to the group of Shigella and E. coli by applying statistical tests. In conclusion, the genome comparison-based methods provide new starting points to infer specific genotypic traits of bacterial groups from the transfer of functional annotation. Due to the high medical importance of enterobacterial isolates their classification according to pathogenicity has been in focus of many studies. The microarray technology offers a fast, reproducible and standardisable means of bacterial typing and has been proved in bacterial diagnostics, risk assessment and surveillance. The design of the diagnostic microarray of enterobacteria described in chapter 5 is based on the availability of numerous enterobacterial genome sequences. A novel probe selection strategy based on the highly efficient algorithm of string search, which considers both coding and non-coding regions of genomic DNA, enhances pathogroup detection. This principle reduces the risk of incorrect typing due to restrictions to virulence-associated capture probes. Additional capture probes extend the spectrum of applications of the microarray to simultaneous diagnostic or surveillance of antimicrobial resistance. Comprehensive test hybridisations largely confirm the reliability of the selected capture probes and its ability to robustly classify enterobacterial strains according to pathogenicity. Moreover, the tests constitute the basis of the training of a regression model for the classification of pathogroups and hybridised amounts of DNA. The regression model features a continuous learning capacity leading to an enhancement of the prediction accuracy in the process of its application. A fraction of the capture probes represents intergenic DNA and hence confirms the relevance of the underlying strategy. Interestingly, a large part of the capture probes represents poorly annotated genes suggesting the existence of yet unconsidered factors with importance to the formation of respective virulence phenotypes. Another major field of microarray applications is gene expression analysis. The size of gene expression databases rapidly increased in recent years. Although they provide a wealth of expression data, it remains challenging to integrate results from different studies. In chapter 6 the methodology of an unsupervised meta-analysis of genome-wide A. thaliana gene expression data sets is presented, which yields novel insights in function and regulation of genes. The application of kernel-based principal component analysis in combination with hierarchical clustering identified three major groups of contrasts each sharing overlapping expression profiles. Genes associated with two groups are known to play important roles in Indol-3 acetic acid (IAA) mediated plant growth and development as well as in pathogen defence. Yet uncharacterised serine-threonine kinases could be assigned to novel functions in pathogen defence by meta-analysis. In general, hidden interrelation between genes regulated under different conditions could be unravelled by the described approach. HMMs are applied to the functional characterisation of proteins or the detection of genes in genome sequences. Although HMMs are technically mature and widely applied in computational biology, I demonstrate the methodical optimisation with respect to the modelling accuracy on biological data with various distributions of sequence lengths. The subunits of these models, the states, are associated with a certain holding time being the link to length distributions of represented sequences. An adaptation of simple HMM topologies to bell-shaped length distributions described in chapter 7 was achieved by serial chain-linking of single states, while residing in the class of conventional HMMs. The impact of an optimisation of HMM topologies was underlined by performance evaluations with differently adjusted HMM topologies. In summary, a general methodology was introduced to improve the modelling behaviour of HMMs by topological optimisation with maximum likelihood and a fast and easily implementable moment estimator. Chapter 8 describes the application of HMMs to the prediction of interaction sites in protein domains. As previously demonstrated, these sites are not trivial to predict because of varying degree in conservation of their location and type within the domain family. The prediction of interaction sites in protein domains is achieved by a newly defined HMM topology, which incorporates both sequence and structure information. Posterior decoding is applied to the prediction of interaction sites providing additional information of the probability of an interaction for all sequence positions. The implementation of interaction profile HMMs (ipHMMs) is based on the well established profile HMMs and inherits its known efficiency and sensitivity. The large-scale prediction of interaction sites by ipHMMs explained protein dysfunctions caused by mutations that are associated to inheritable diseases like different types of cancer or muscular dystrophy. As already demonstrated by profile HMMs, the ipHMMs are suitable for large-scale applications. Overall, the HMM-based method enhances the prediction quality of interaction sites and improves the understanding of the molecular background of inheritable diseases. With respect to current and future requirements I provide large-scale solutions for the characterisation of biological data in this work. All described methods feature a highly portable character, which allows for the transfer to related topics or organisms, respectively. Special emphasis was put on the knowledge transfer facilitated by a steadily increasing wealth of biological information. The applied and developed statistical methods largely provide learning capacities and hence benefit from the gain of knowledge resulting in increased prediction accuracies and reliability.
Like many other social insect societies, honeybees collectively share the resources they gather by feeding each other. These feeding contacts, known as trophallaxis, are regarded as the fundamental basis for social behavior in honeybees and other social insects for assuring the survival of the individual and the welfare of the group. In honeybees, where most of the trophallactic contacts are formed in the total darkness of the hive, the antennae play a decisive role in initiation and maintenance of the feeding contact, because they are sensitive to gustatory stimuli. The sequences of behaviors performed by the receiver bees at the beginning of a feeding contact includes the contact of one antenna with the mouthparts of a donor bee where the regurgitated food is located. The antennal motor action is characterized by behavioral asymmetry, which is novel among communicative motor actions in invertebrates. This preference of right over left antenna is without exception even after removal of the antennal flagellum. This case of laterality in basic social interaction might have its reason in the gustatory asymmetry in the antennae, because the right antenna turns out to be significantly more sensitive to stimulation with sugar water of various concentrations than the left one. Trophallactic contacts which guarantee a constant access to food for every individual in the hive are vitally important to the honeybee society, because honeybees are heterothermic insects which actively regulate their thoracic temperature. Even though the individual can regulate its body temperature, its heating performance is strictly limited by the amount of sugar ingested. The reason for this is that honeybees use mostly the glucose in their hemolymph as the energy substrate for muscular activity, and the heat producing flight muscles are among the metabolically most active tissues known. The fuel for their activity is honey; processed nectar with a sugar content of ~80% stored in the honeycomb. The results show that the sugar content of the ingested food correlates positively with the thoracic temperature of the honeybees even if they are caged and show no actual heating-related behavior as in brood warming or heating in the centre of the winter cluster. Honeybees actively regulate their brood temperature by heating to keep the temperature between 33 °C to 36 °C if ambient temperatures are lower. Heating rapidly depletes the worker’s internal energy; therefore the heating performance is limited by the honey that is ingested before the heating process. This study focused on the behavior and the thoracic temperature of the participants in trophallactic food exchanges on the brood comb. The brood area is the centre of heating activity in the hive, and therefore the region of highest energy demand. The results show that the recipients in a trophallactic food exchange have a higher thoracic temperature during feeding contacts than donors, and after the feeding contact the former engage in brood heating more often. The donor bees have lower thoracic temperature and shuttle constantly between honey stores and the brood comb, where they transfer the stored honey to heating bees. In addition, the results show a heat-triggered mechanism that enables donor and recipient to accomplish trophallactic contacts without delay in the total darkness of the hive in the brood area as the most energy consuming part of the hive. Providing heat-emitting workers with small doses of high performance fuel contributes to an economic distribution of resources consistent with the physiological conditions of the bees and the ecological requirements of the hive, resulting in a highly economical resource management system which might be one of the factors favouring the evolution of perennial bee colonies in temperate regions. The conclusion of these findings suggests a resource management strategy that has evolved from submissive placation behavior as it is seen in honeybees, bumblebees and other hymenopterans. The heat-triggered feedback mechanism behind the resource management of the honeybee´s thermoregulatory behavior reveals a new aspect of the division of labor and a new aspect of communication, and sheds new light on sociality in honeybees.
The work of the previous chapters describes the role of Nipah virus (NiV) V and W proteins regarding their role in interferon antagonism and regulation of viral replication. Previous publications have shown that NiV encodes IFN antagonist activity in its V, W and C protein (Park et al., 2003b; Rodriguez et al., 2002). In order to study the effect of both NiV proteins in the context of a virus infection, recombinant Newcastle disease viruses (rNDVs) expressing NiV V or NiV W were constructed. As a control virus served rNDV expressing NDV V proteins, which behaved like wildtype NDV. Growth kinetic experiments demonstrated that rNDVs expressing NiV V or W grew to higher titers than rNDV expressing NDV V in human A549 cells. This result suggested that both NiV V and W were able to render the avian virus, which normally does not replicate well in human cells, into a better growing virus. This hypothesis was supported by the fact that all rNDVs grew similarly in avian DF1 or Vero cells. When rNDV-infected A549 cells were specifically stained for NiV V or W protein it was observed that V is localized in the cytoplasm whereas W could be predominantly found in the nucleus. This observation was in agreement with previous studies reporting a nucleus export signal (NES) for NiV V and a nuclear localization signal (NLS) for NiV W (Rodriguez et al., 2004; Shaw et al., 2005). The specific localization of each NiV protein has also been shown to contribute to different functions in terms of IFN antagonism (Shaw et al., 2005). Here, NiV V and W proteins caused a severe attenuation of the immune response in rNDV-infected human A549 and dendritic cells. The transcription of type I interferons and ISGs was significantly downregulated in the presence of NiV V and W proteins. As a consequence of the transcriptional block, there was also an inhibition at the level of translation (as seen for A549 cells) and the secretion of IFNs and cytokines/chemokines (as seen for DCs). In contrast, NDV V protein induced a host immune response. Both NiV V and W also displayed a strong inhibitory effect on the function DCs. DCs represent a very important cell class because they link the innate immune response to the adaptive immune response (Banchereau & Steinman, 1998). By downregulating the production and secretion of important cytokines/chemokines that are important for the activation of B and T lymphocytes, NiV V and W were able to disrupt that link. Interestingly, NiV W seemed to be a stronger inhibitor than NiV V in both A549 cells and DCs. Overall, it was demonstrated that NiV V and W were able to prevent the induction of the innate and adaptive host immune response cascade by inhibiting the transcription of immune genes in DCs and A549 cells. The second part of this work addressed the question whether NiV V and W proteins have a regulatory role in viral replication. This has been previously reported for Nipah virus itself (Sleeman et al., 2008) and other viruses (Atreya et al., 1998; Horikami et al., 1996; Witko et al., 2006). In order to study the ability of the V and W proteins of NiV to regulate viral transcription and/or replication, an existing NiV minireplicon assay was used (Halpin et al., 2004). Here, it was shown that NiV V and W (but not C) proteins significantly downregulated NiV minireplicon activity. The common N terminal region was shown to harbor the inhibitory activity. Co-immunoprecipitation experiments showed that both NiV V and W (but not C) were able to interact with NiV N, one component of the NiV polymerase. This result was supported by immunofluorescence experiments that revealed co-localization of NiV N with V and W. The binding of NiV V or W to NiV N occurred via their N terminus and more specifically amino acids 1-50. This suggested that V and W might inhibit viral replication by interacting with the viral polymerase resulting in a loss of function. Exact mechanisms still have to be elucidated.
In the first part of this work we presented the synthesis and photophysical properties of a series of transition metal donor-acceptor Ir(III)complexes of the type [(C^N)2Ir(N^N)][PF6]. The Ir(III) was connected with hole conducting donor-moieties like carbazole (CZ) and triarylamine (TAA) linked via a methylene and ethylene bridge to the cyclometalating C^N ligands phenylpyrazole (ppz) and phenylpyridine (ppy). Bidentate N^N and P^P ligands like 2,2’-bipyridyl (bpy), 3,4,7,8-tetramethyl-1,10-phenanthroline (tmp) and cis-1,2-bis(diphenylphosphino)ethylene (bdppe) were used as acceptor units. In order to analyse the influence of the electron density of the bpy ligand, TAA-complexes with acceptor- and donor-substituted bpy acceptor units were synthesised. Therefore, 4,4’-dinitro-2,2’-bipyridyl, 4,4’-dichloro-2,2’-bipyridyl, 4,4’-dimethoxy-2,2’-bipyridyl and 4,4’-dimethylamino-2,2’bipyridyl were used as neutral N^N ligands. In order to compare the photophysical properties, all reference compounds without hole conducting component were syntesised. All the carbazole compounds, except the bdppe complexes, exhibit emission and transient absorption properties similar to their reference compounds that make them interesting for OLED (organic light emitting device) applications. LEC (light emitting electrochemical cell) studies show a red shifted luminescence. The triarylamine compounds do not luminesce at RT but they exhibit an intense, blue-shifted and long-lived luminescence at 77 K in a rigid matrix. The transient absorption spectra differ strongly from that of their reference compounds. The spectra display characteristic features of the spectra of the isolated radical anions and cations supported by spectroelectrochemical measurements. Thus, it can be assumed that the transient states are charge separated (CS) states in which the positive charge is localised at the TAA donor units and the negative charge at the N^N acceptor units. The decays of the transient states are biexponentially what indicates the presence of two transient states, the 1CS and the 3CS state. To understand this behaviour the differently substituted bipyridyl-complexes were synthesised and analysed. Temperature dependent transient absorption measurements showed that all rate constants are indepentend of the temperature, except for the complex with OMe subsituents at the bpy ligand. The equilibrium constant K = k1 / k2 is nearly one for all complexes. For the OMe-compound it decreases with increasing temperature. Plotting the rate constants vs. the free energy differences (determined by cyclovoltammetry measurements) shows that all constants are decreasing with increasing donor strength of the bpy ligand. DFT calculations on the OMe-compound are already in work. In the second part of this work, neutral Ir(III) and Pt(II) complexes of the type [(O^O)Ir(N^N)2] and [(O^O)Pt(N^N)] were introduced. There, TTA was connected directly or via a CH2 bridge to acectylacetonate (acac = O^O) in order to probe the influence of the different kinds of connection on the photophysics of the complexes. As the bidentate N^N ligand 2,2’-bipyridyl (bpy) was chosen. All the corresponding reference compounds without triarylamine were obtained in order to compare with the TAA substituted analoga. Furthermore, the homoleptic fac Ir(N^N)3 complex with triarylamine connected via a methylene and ethylene bridge to phenylpyrazole as introduced in the first part of this work was synthesised. The synthesis of the Ir(III) compound with the TAA substituted acac ligand connected via the CH2 group was not successful. All the neutral triarylamine-substituted -diketonato Pt(II) and Ir(III) complexes do not luminesce at RT, except the Pt(II)-complex with CH2 bridge. This compound shows transient state characteristics that are in good agreement with the luminescence lifetimes at RT and that are similar to the reference compound, what suggests to a 3Pt(N^N)(O^O) state. The complexes without the CH2 bridging unit show no transient signals what may be caused by charge-transfer quenching due to the direct linkage between donor and acceptor unit. The homoleptic fac Ir(N^N)3 complex exhibits no emission at RT and no transient signals. At 77 K it shows a highly structured emission with 14 s lifetime. Compared to the literature-known reference compound this emission is caused by the population of a 3Ir(ppz)3 state. Our findings are important for designing complexes with stronger acceptor units (i.e. naphthaleneimide) for long CS states lifetimes to be used as photosynthesisers in solar cells and other optoelectronic devices. Besides, LEC and OLED studies on the carbazole complexes are still of interest to analyse the degree of triplet-triplet-annihiliation in these devices.
Tumors of the hematopoietic and lymphoid system are classified into Hodgkin lymphoma and non-Hodgkin lymphoma (NHL). Approximately 80% of non-Hodgkin lymphomas (NHL) are B-cell lymphomas (B-NHL) and the remainder include T-cell and NK-cell lymphomas as well as immunodeficiency-associated lymphoproliferative disorders. The presence of genetic alterations such as translocations involving the immunoglobulin (Ig) receptor loci in B-NHL, e.g. the translocation t(14;18)(q32.33;q21.3) in follicular lymphoma (FL), are of great value for the classification and of importance in the pathogenesis of these neoplasms. In analogy to the Ig receptor genes in B-NHL, the T-cell receptor (TCR) gene loci are targeted by chromosomal breaks in approximately 30% of precursor T-cell lymphoblastic leukemias/lymphomas involving various translocation or inversion partners. Most of these events result in the overexpression of an oncogene by juxtaposing it to the regulatory sequences of the TCR genes. However, the pathogenesis of mature T-cell NHL (T-NHL) and the underlying molecular mechanisms are only poorly understood so far. To determine the exact frequency of breakpoints occurring in the TCR loci of 227 mature T-NHL cases, we designed fluorescence in situ hybridization (FISH) assays for the TCR loci that are applicable for large scale analysis of formalin fixed and paraffin embedded (FFPE) lymphoma specimens in a tissue microarray format. This approach revealed only two mature T-NHL cases with a chromosomal breakpoint in one of the TCR loci making the rearrangement of TCR loci a very rare event in these neoplasms that occurs in less than 1% of cases.FL is the second most frequent type of B-NHL that can show predominantly follicular, combined follicular and diffuse, or predominantly diffuse growth patterns. The characteristic genetic hallmark of FL is the translocation t(14;18)that occurs in approximately 90% of cases and leads to a deregulated expression of the anti-apoptotic BCL2 proto-oncogene. FL has yet been a subject of many studies deciphering morphological, clinical and molecular features of this entity. However, only little information exists about cases lacking this translocation. In this thesis we divided 184 FL cases by polymerase chain reaction (PCR) and by FISH assays into FL cases with and without t(14;18) and investigated their respective gene expression profiles and copy number alterations. For FISH analysis we followed the refined conditions established for the T-NHL study. The only genetic alterations that differed significantly by comparative genomic hybridization (CGH) analysis between FL cases with and without t(14;18) were frequent gains or amplifications in 18q11-q21 in 32% of t(14;18)-positive and 0% of t(14;18)-negative cases. Gene expression profiling and geneset enrichment analysis (GSEA) revealed an enrichment of germinal center B-cell (GCB) signatures in t(14;18)-positive cases whereas an enrichment of activated B-cell (ABC) like, NFkB-, proliferation-, cell cycle-, interferon and bystander cell signatures were observed in t(14;18)-negative cases. A validation approach by immunohistochemistry (IHC) on an independent test set of FL cases (n=84) revealed a more frequent expression of the germinal center (GC) marker CD10/MME in cases with t(14;18) and a higher expression of the post GC marker IRF4/MUM1, the proliferation marker Ki67 and the cytotoxic T-cell marker GZMB in cases without t(14;18). Although these results may suggest a post-GCB phenotype for translocation t(14;18)-negative cases, ongoing somatic hypermutations of the immunoglobulin heavy chain genes in these cases rather point to a late GC stage of B-cell differentiation in FL without t(14;18). In an independent study with 35 predominantly diffuse FL cases, it was furthermore possible to define another subset of t(14;18)-negative FL characterized by a chromosomal deletion (del) in 1p36 and distinct morphological and clinical features by IHC, classical chromosome banding, FISH and gene expression profiling. The gene expression profiles of predominantly diffuse FL cases fell into the spectrum of FL. However, by GSEA they showed a significant enrichment of T-cell, NK-cell- and two dendritic-cell subset signatures, whereas a significant enrichment of GCB cell-, proliferation-, cell cycle- and B-cell signatures was observed in a control group of “classic” FL cases. Remarkably, patients with diffuse FL frequently presented with low clinical stage and large, but localized inguinal tumors. In conclusion, our results suggest that t(14;18)-negative FL are part of the spectrum of FL in general, but nevertheless show distinct molecular and clinical features. In particular, predominantly diffuse FL with (del)1p36, low clinical stage and large but localized inguinal tumors may represent a distinct t(14;18)-negative FL subtype.
Within this thesis, the analysis and hence the better comprehension of the chemical bond within metal–element compounds is the central topic. By use of various DFT methods a selection of M–E interactions have been modeled and analyzed via Bader’s QTAIM, the ELF and NBO techniques. Special focus was set on a series of transition metal borylene and carbene complexes, and the Li–C bonds as representatives for main group organometallics. Therefore, this thesis is split into three parts:(I) An introduction reviewing the quantum chemical machinery as well as the analysis tools applied for the evaluation of chemical bonds. (II) Within the second part the chemical interactions taking place in transition metal complexes are studied focusing on borylenes and cognate carbenes. (III) In Part III, a broad overview of the appropriate modeling and nature of the Li–C bond as well as intermolecular interactions in methyllithium is provided.
Contents List of Publications 1 Introduction 2 Basic concepts and instrumentation 2.1 Mathematical description of femtosecond laser pulses 2.2 Optical quantities and measurements 2.2.1 Intensity 2.2.2 Absorbance and Beer-Lambert law 2.3 Laser system 2.4 General software framework for scientific data acquisition and simulation 2.4.1 Core components 2.4.2 Program for executing a single measurement sequence 2.4.3 Scan program 2.4.4 Evolutionary algorithm optimization program 2.4.5 Applications of the software framework 2.5 Summary 3 Generation of ultrabroadband femtosecond pulses in the visible 3.1 Nonlinear optics 3.1.1 Nonlinear polarization and frequency conversion 3.1.2 Phase matching 3.2 Optical parametric amplification 3.3 Noncollinear optical parametric amplifier 3.4 Considerations and experimental design of NOPA 3.4.1 Options for broadening the NOPA bandwidth 3.4.2 Experimental setup 3.5 NOPA pulse characterization 3.5.1 Second harmonic generation frequency-resolved optical gating 3.5.2 Transient grating frequency-resolved optical gating 3.6 Compression and shaping methods for NOPA pulses 3.6.1 Grating compressor 3.6.2 Prism compressor 3.6.3 Chirped mirrors 3.6.4 Detuned zero dispersion compressor 3.6.5 Deformable mirror pulse shaper 3.6.6 Liquid crystal pulse shaper 3.7 Liquid crystal pulse shaper 3.7.1 Femtosecond pulse shapers 3.7.2 Experimental design and parameters 3.7.3 Optical setup of the LC pulse shaper 3.7.4 Calibrations of the pulse shaper 3.8 Adaptive pulse compression 3.8.1 Closed loop pulse compression 3.8.2 Open loop pulse compression 3.9 Conclusions 4 Coherent optical two-dimensional spectroscopy 4.1 Introduction 4.2 Theory of third order nonlinear optical spectroscopies 4.2.1 Response function, electric fields, and signal field 4.2.2 Signal detection with spectral interferometry 4.2.3 Evaluation of two-dimensional spectra and phasing 4.2.4 Selection and classification of terms in induced nonlinear polarization 4.2.5 Oscillatory character of measured signal 4.3 Previous experimental implementations 4.4 Inherently phase-stable setup using conventional optics only 4.4.1 Manipulation of pulse pairs as a basis for stability 4.4.2 Experimental setup 4.4.3 Measurement procedure 4.4.4 Data evaluation 4.5 First experimental results 4.5.1 Demonstration of phase stability 4.5.2 2D spectrum of Nile Blue at room temperature 4.6 Summary and outlook 5 Product accumulation for ultrasensitive femtochemistry 5.1 The problem of sensitivity in femtochemistry 5.2 Accumulation for increased sensitivity 5.2.1 Comparison of conventional and accumulative sensitivity 5.2.2 Schematics and illustrative example 5.3 Experimental setup 5.4 Calibration and modeling of accumulation 5.5 Experiments on indocyanine green 5.5.1 Calibration of the setup 5.5.2 Chirped pulse excitation 5.5.3 Adaptive pulse shaping 5.6 Conclusions 6 Ultrafast photoconversion of the green fluorescent protein 6.1 Green fluorescent protein 6.2 Experimental setup for photoconversion of GFP 6.3 Calibration of the setup for GFP 6.3.1 Model for concentration dynamics of involved GFP species 6.3.2 Estimate of sensitivity 6.4 Excitation power study 6.5 Time-resolved two-color experiment 6.6 Time-delayed unshaped 400 nm – shaped 800 nm pulse excitation 6.6.1 Inducing photoconversion with chirped pulses 6.6.2 Photoconversion using third order phase pulses 6.7 Conclusions 7 Applications of the accumulative method to chiral systems 7.1 Introduction 7.2 Chiral asymmetric photochemistry 7.2.1 Continuous-wave circularly polarized light 7.2.2 Controlled asymmetric photochemistry using femtosecond laser pulses 7.3 Sensitive and fast polarimeter 7.3.1 Polarimeter setup 7.3.2 Detected signal I(t) 7.3.3 Angular amplification 7.3.4 Performance of the polarimeter 7.4 Molecular systems and mechanisms for enantioselective quantum control 7.4.1 Binaphthalene derivatives 7.4.2 Photochemical helicene formation 7.4.3 Spiropyran/merocyanine chiroptical molecular switches 7.5 Summary 8 Summary Zusammenfassung Bibliography Acknowledgements
Around 10.000 – 150.000 endogenous DNA damage-induced lesions occur in a human body per day and cell. Accumulation of unrepaired lesions can lead to aneuploidy and the loss of genomic integrity which in turn contributes to tumor formation. Therefore, an efficient DNA damage response has to be initiated, in the end leading to cell cycle inhibition and induction of repair. Since it is known that a recently characterized human multiprotein complex named LINC (or human dREAM) together with B-MYB is involved in the regulation of G2/M gene expression (Plk1, cyclin B1, cdc2 etc.), its function in the DNA damage response was analyzed in this study. In growing cells B-MYB is associated to the LIN core complex which consists of 5 different proteins named LIN-9, LIN-54, LIN-52, LIN-37 and RbAp48. After induction of DNA damage B-MYB leaves the complex and binding of E2F4 and p130 to LINC is induced. Importantly, the upstream pathway leading to LINC rearrangement is dependent on the activation of p53 and p21. Interestingly, p53 -/- cells solely have the potential to block in the G2 phase of the cell cycle, thereby making them vulnerable for errors during G2 arrest induction or maintenance. Here I demonstrate that LINC rearrangement is absent in p53 -/- cells and that B-MYB/LINC binding to target gene promoters is increased. This in turn leads to an increased G2/M gene expression after DNA damage induction and triggers premature cell cycle re-entry (checkpoint adaptation). Significantly, B-MYB expression is increased in p53 mutated primary breast cancer tumors and correlates with poor prognosis and reoccurrence probably due to its function in checkpoint adaptation. This study gives evidence that inhibition of B-MYB gene expression or B-MYB function in p53 mutant tumors could be a good choice for adjuvant therapy.
The present approach highlights a procedural account of intuitive judgments. In intuitions of hidden semantic coherence, people can intuitively detect whether a word triad has a common remote associate (coherent) or not (incoherent) before, and independently from actually retrieving the common associate. The present fluency-affect intuition model (FAIM) maintains that semantic coherence increases the processing fluency for coherent compared to incoherent triads, and that this increased fluency triggers brief and subtle positive affect, which is the experiential basis of these intuitions. Published work concerning 25 experiments is reviewed that gathered empirical support for this model. Furthermore, the impact of fluency and affect was also generalized to intuitions of visual coherence, and intuitions of grammaticality in an artificial grammar learning paradigm.
Two-particle excitations, such as spin and charge excitations, play a key role in high-Tc cuprate superconductors (HTSC). Due to the antiferromagnetism of the parent compound the magnetic excitations are supposed to be directly related to the mechanism of superconductivity. In particular, the so-called resonance mode is a promising candidate for the pairing glue, a bosonic excitation mediating the electronic pairing. In addition, its interactions with itinerant electrons may be responsible for some of the observed properties of HTSC. Hence, getting to the bottom of the resonance mode is crucial for a deeper understanding of the cuprate materials . To analyze the corresponding two-particle correlation functions we develop in the present thesis a new, non-perturbative and parameter-free technique for T=0 which is based on the Variational Cluster Approach (VCA, an embedded cluster method for one-particle Green's functions). Guided by the spirit of the VCA we extract an effective electron-hole vertex from an isolated cluster and use a fully renormalized bubble susceptibility chi0 including the VCA one-particle propagators.Within our new approach, the magnetic excitations of HTSC are shown to be reproduced for the Hubbard model within the relevant strong-coupling regime. Exceptionally, the famous resonance mode occurring in the underdoped regime within the superconductivity-induced gap of spin-flip electron-hole excitations is obtained. Its intensity and hourglass dispersion are in good overall agreement with experiments. Furthermore, characteristic features such as the position in energy of the resonance mode and the difference of the imaginary part of the susceptibility in the superconducting and the normal states are in accord with Inelastic Neutron Scattering (INS) experiments. For the first time, a strongly-correlated parameter-free calculation revealed these salient magnetic properties supporting the S=1 magnetic exciton scenario for the resonance mode. Besides the INS data on magnetic properties further important new insights were gained recently via ARPES (Angle-Resolved Photoemission-Spectroscopy) and Raman experiments which disclosed a quite different doping dependence of the antinodal compared to the near-nodal gap. This thesis provides an approach to the Raman response similar to the magnetic case for inspecting this gap dichotomy. In agreement with experiments and one-particle data obtained in the VCA, we recover the antinodal gap decreasing and the near-nodal gap increasing as a function of doping. Hence, our results prove the Hubbard model to account for these salient gap features. In summary, we develop a two-particle cluster approach which is appropriate for the strongly-correlated regime and contains no free parameter. Our results obtained with this new approach combined with the phase diagram and the one-particle excitations obtained in the VCA strongly constitute a Hubbard model description of HTSC cuprate materials.
The RS1 protein (gene RSC1A1) participates in regulation of Na+-D-glucose cotransporter SGLT1 and some other solute carriers. In subconfluent LLC-PK1 cells, RS1 inhibits release of SGLT1 from the trans-Golgi network and transcription of SGLT1. In subconfluent cells, RS1 is localized in the nucleus and the cytoplasm whereas confluent cells contain predominantly cytoplasmic RS1. In the present study, the mechanism and regulation of confluence-dependent nuclear location of RS1 was investigated. Confluence dependent nuclear location of RS1 was shown to be regulated by the cell cycle. A nuclear shuttling signal (NS) in pRS1 was identified that ensures confluence-dependent distribution of pRS1 and comprises nuclear localization signal (NLS) and nuclear export signal (NES). The NLS and NES of RS1 mediate translocation into and out of the nucleus via importin ß1 and CRM1, respectively, and the nuclear/cytoplasmic distribution of the RS1 protein is determined by the nuclear export activity. The adjacent protein kinase C (PKC) phosphorylation site at serine 370 of pRS1 was shown to control nuclear localization driven by NS and is necessary for the differential localization of RS1 in quiescent versus proliferating cells. Basing on the data of site-directed mutagenesis, PKC activation experiments and mass spectrometry analysis of RS1 phosphorylation, the following model of the regulation of RS1 nuclear location in LLC-PK1 cells was proposed. In subconfluent cells, RS1 is actively imported into the nucleus whereas nuclear export of RS1 is not active leading to accumulation of RS1 in the nucleus. After confluence, phosphorylation of serine 370 of pRS1 by PKC takes place leading to enhancement of RS1 nuclear export and predominantly cytoplasmic distribution of the protein in the confluent cells. The confluence-dependent regulation of RS1 localization may control SGLT1 expression during regeneration of enterocytes in small intestine and during regeneration of renal tubular cells after hypoxemic stress. Moreover, the gene expression profiling of mouse embryonic fibroblasts with RS1-/- genotype suggests that transcriptional regulation by RS1 might be important for the cell cycle and cell division. Since RS1 localization depends on the cell cycle, RS1 might play a role in the regulation of the solute carriers during specific phases of the cell cycle.
The observation of neutrino masses and lepton mixing has highlighted the incompleteness of the Standard Model of particle physics. In conjunction with this discovery, new questions arise: why are the neutrino masses so small, which form has their mass hierarchy, why is the mixing in the quark and lepton sectors so different or what is the structure of the Higgs sector. In order to address these issues and to predict future experimental results, different approaches are considered. One particularly interesting possibility, are Grand Unified Theories such as SU(5) or SO(10). GUTs are vertical symmetries since they unify the SM particles into multiplets and usually predict new particles which can naturally explain the smallness of the neutrino masses via the seesaw mechanism. On the other hand, also horizontal symmetries, i.e., flavor symmetries, acting on the generation space of the SM particles, are promising. They can serve as an explanation for the quark and lepton mass hierarchies as well as for the different mixings in the quark and lepton sectors. In addition, flavor symmetries are significantly involved in the Higgs sector and predict certain forms of mass matrices. This high predictivity makes GUTs and flavor symmetries interesting for both, theorists and experimentalists. These extensions of the SM can be also combined with theories such as supersymmetry or extra dimensions. In addition, they usually have implications on the observed matter-antimatter asymmetry of the universe or can provide a dark matter candidate. In general, they also predict the lepton flavor violating rare decays mu -> e gamma, tau -> mu gamma and tau -> e gamma which are strongly bounded by experiments but might be observed in the future. In this thesis, we combine all of these approaches, i.e., GUTs, the seesaw mechanism and flavor symmetries. Moreover, our request is to develop and perform a systematic model building approach with flavor symmetries and to search for phenomenological implications. This provides a new perspective in model building since it allows us to screen models by its predictions on the theoretical and phenomenological side, i.e., we can apply further model constraints to single out a desired model. The results of our approach are, e.g., diverse lepton flavor and GUT models, a systematic scan of lepton flavor violation, new mass matrices, a new understanding of lepton mixing angles, a general extension of the idea of quark-lepton complementarity theta_12=pi/4-epsilon/sqrt{2} and for the first time the QLC relation in an SU(5) GUT.
The main focus of this thesis was the synthesis and analysis of multifunctional oligopeptides. The study of their non-covalent interactions with various counterparts revealed interesting new results, leading to both methodological and application related progress. The first project of this thesis concentrated on the in-depth analysis of the peptide receptor CBS-Lys-Lys-Phe-NH2 to acquire a better understanding of its binding mode upon complexation with a substrate. In this context it was possible to develop—in cooperation with the group of Prof. Sebastian Schlücker—a direct and label free spectroscopic detection of immobilized compounds which are often found in combinatorial libraries. This new screening method utilizes the advantages of the surface enhanced Raman spectroscopy and allowed for the first time a surface mapping of a single polystyrene bead for the identification of peptides in femtomolar concentrations. Hence, this method allows a very fast and sensitive detection of resin bound compounds. The development of this promising new approach set the starting point for future experiments to enable on-bead library screenings and to investigate the complex formation of immobilized compounds. After the comprehensive analysis of the basic structural features of small peptide receptors in the first part of this thesis, the second big block focused on its in vitro evaluation using biological relevant targets. Therefore, several different modifications of the initial peptide structures were synthesized. These modifications provided a molecular toolkit for the tailor made synthesis of structures individually designed for the respective target. The first tests addressed the interaction with Alzheimer’s related amyloid fibrils. During these experiments, the successful SPPS syntheses of tri- and tetravalent systems were achieved. The comparison of the multivalent form with the corresponding monovalent version was then under special investigations. These concentrated mainly on the interaction with various bacteria strains, as well as with different parasites. To localize the compounds within the organisms, the synthesis of fluorescence labelled versions was achieved. In addition, several compounds were tested by the Institute for Molecular Infection Biology of the University of Würzburg for their antibacterial activity. This thorough evaluation of the biological activity generated precious information about the influence of small structural changes in the peptide receptors. Especially the distinct influence of the multivalency effect and the acquired synthetic skills led to the development of an advanced non-covalent recognition event, as described in the final project of this thesis. The last part of this thesis discussed the development of a novel inhibitor for the serine protease beta-tryptase based on a tailor-made surface recognition event. It was possible to study and analyze the complex interaction with the unique structure of tryptase, that features a tetrameric frame and four catalytic cleavage sites buried deep inside of the hollow structure. However, the point of attack were not the four binding pockets, as mostly described in the literature, but rather the acidic areas around the cleavage sites and at the two circular openings. These should attract peptides with basic residues, which then can block the accessibility to the active sites. A combinatorial library of 216 tetravalent peptide compounds was synthesized to find the best structural composition for the non-covalent inhibition of beta-tryptase. For the screening of the library a new on-bead assay was applied. With this method a simultaneous readout of the total inhibition of all library members was possible, thus allowing a fast and direct investigation of the still resin bound inhibitors. Several additional experiments in solution unveiled the kinetics of the inhibition process. In conclusion, both mono- and multivalent inhibitors interact in a non-destructive and reversible way with the tryptase.
In this thesis, soft x-ray absorption spectroscopy (XAS) and resonant inelastic x-ray scattering (RIXS) studies of the electronic structure of selected organic molecules and liquids were carried out. The first part focuses on the used experimental techniques and the development of the instrumentation necessary for these studies, namely a soft x-ray spectrometer, and a temperature-controlled flow-through liquid cell. The former was optimized by a special analytical ray tracing method developed exclusively for this purpose. Due to its high transmission, the spectrometer facilitates a novel experimental approach of recording comprehensive 'RIXS maps', which are 2-dimensional plots of x-ray scattering intensities as a function of both, excitation and emission photon energy. The liquid cell extends these possibilities to the study of liquids, especially the interaction of molecules in liquids and their chemical reactions under well-controlled conditions. Organic molecules have attracted considerable attention in the last decade. The intense research activities related to these materials have two main motivations: on the one hand, organic molecules have a technological application as building blocks of organic semiconductors, while, on the other hand, organic molecules are the functional elements in biological systems. In order to cost-effectively produce optimized organic electronic devices, a fundamental knowledge of the electronic properties of the organic molecules interface is necessary. Therefore, many studies of the electronic structure of potential candidates for organic electronics exist. Two of these candidates, namely C60 and well-ordered multilayers PTCDA on a Ag(111) surface are investigated in this thesis. For the study of C60 molecules, a comprehensive 'RIXS map' was recorded and analyzed. The RIXS map taken in only 25 minutes allows a quantitative analysis of energy losses, yielding for example the HOMO-LUMO distance. It also identifies a core-excitonic state and facilitates a quantitative comparison of its binding energy with that of valence excitons in C60. Furthermore, decay channel-selective partial fluorescence yield XAS spectra can be extracted from the RIXS map, yielding information on the population of the core-excitonic state as a function of excitation energy. As a second model system of organic molecules relevant for organic electronics, PTCDA was chosen. The complex electronic structure of the occupied states of a highly ordered, flat-lying PTCDA multilayer on a Ag(111) surface was investigated by symmetry-resolved resonant x-ray emission spectroscopy. The rapidly occurring beam damage effects were characterized on the basis of irradiation-time dependent series of C and O x-ray emission spectra. Upon varying the excitation energy and emission geometry, atom- and symmetry-specific carbon K emission spectra with negligible beam damage effects were obtained that allow to distinguish between electronic states with sigma and pi symmetry. A density functional theory calculation of the PTCDA molecule reproduces the energy positions of the most prominent emission features remarkably well. In addition, the energy positions of the sigma and pi emissions agree well with the calculated energies of the respective orbitals. In order to shed light on the second aspect of organic molecules, namely their role in biological systems, first a detailed investigation of the electronic structure and proton dynamics of liquid water as the medium of most chemical and biochemical reactions was carried out. Therefore, a comprehensive oxygen K RIXS map of liquid water was recorded and analyzed in great detail. A temperature-dependent comparison with XAS and RIXS data of D2O, NaOH, and NaOD leads to the conclusion, that ultra-fast dissociation takes place in liquid water on the timescale of the oxygen 1s core hole lifetime, resulting in a characteristic spectral contribution in the RIXS spectra. The dissociation is promoted by intact hydrogen bonds with neighboring molecules. In consequence, the rate of dissociation directly depends on the initial hydrogen bond configuration. In the next step towards biologically relevant systems, the nitrogen K edges of the amino acids glycine and histidine were investigated in powderous form as well as in their native environment, namely in aqueous solution. X-ray absorption and emission spectra of the aqueous solutions were analyzed at pH-values of 6 and for glycine also at pH 12 and compared to the spectra of powders. A pH-value of 12 causes deprotonation of the amino group, leading to significant changes in the nitrogen spectra as compared to pH 6. The results from these four examples demonstrate that a wealth of novel information can be obtained by using the new experimental tools developed in this thesis, namely a highly sensitive x-ray spectrometer and a flow-through liquid cell.
In future telecommunication systems, we observe an increasing diversity of access networks. The separation of transport services and applications or services leads to multi-network services, i.e., a future service has to work transparently to the underlying network infrastructure. Multi-network services with edge-based intelligence, like P2P file sharing or the Skype VoIP service, impose new traffic control paradigms on the future Internet. Such services adapt the amount of consumed bandwidth to reach different goals. A selfish behavior tries to keep the QoE of a single user above a certain level. Skype, for instance, repeats voice samples depending on the perceived end-to-end loss. From the viewpoint of a single user, the replication of voice data overcomes the degradation caused by packet loss and enables to maintain a certain QoE. The cost for this achievement is a higher amount of consumed bandwidth. However, if the packet loss is caused by congestion in the network, this additionally required bandwidth even worsens the network situation. Altruistic behavior, on the other side, would reduce the bandwidth consumption in such a way that the pressure on the network is released and thus the overall network performance is improved. In this monograph, we analyzed the impact of the overlay, P2P, and QoE paradigms in future Internet applications and the interactions from the observing user behavior. The shift of intelligence toward the edge is accompanied by a change in the emerging user behavior and traffic profile, as well as a change from multi-service networks to multi-networks services. In addition, edge-based intelligence may lead to a higher dynamics in the network topology, since the applications are often controlled by an overlay network, which can rapidly change in size and structure as new nodes can leave or join the overlay network in an entirely distributed manner. As a result, we found that the performance evaluation of such services provides new challenges, since novel key performance factors have to be first identified, like pollution of P2P systems, and appropriate models of the emerging user behavior are required, e.g. taking into account user impatience. As common denominator of the presented studies in this work, we focus on a user-centric view when evaluating the performance of future Internet applications. For a subscriber of a certain application or service, the perceived quality expressed as QoE will be the major criterion of the user's satisfaction with the network and service providers. We selected three different case studies and characterized the application's performance from the end user's point of view. Those are (1) cooperation in mobile P2P file sharing networks, (2) modeling of online TV recording services, and (3) QoE of edge-based VoIP applications. The user-centric approach facilitates the development of new mechanisms to overcome problems arising from the changing user behavior. An example is the proposed CycPriM cooperation strategy, which copes with selfish user behavior in mobile P2P file sharing system. An adequate mechanism has also been shown to be efficient in a heterogeneous B3G network with mobile users conducting vertical handovers between different wireless access technologies. The consideration of the user behavior and the user perceived quality guides to an appropriate modeling of future Internet applications. In the case of the online TV recording service, this enables the comparison between different technical realizations of the system, e.g. using server clusters or P2P technology, to properly dimension the installed network elements and to assess the costs for service providers. Technologies like P2P help to overcome phenomena like flash crowds and improve scalability compared to server clusters, which may get overloaded in such situations. Nevertheless, P2P technology invokes additional challenges and different user behavior to that seen in traditional client/server systems. Beside the willingness to share files and the churn of users, peers may be malicious and offer fake contents to disturb the data dissemination. Finally, the understanding and the quantification of QoE with respect to QoS degradations permits designing sophisticated edge-based applications. To this end, we identified and formulated the IQX hypothesis as an exponential interdependency between QoE and QoS parameters, which we validated for different examples. The appropriate modeling of the emerging user behavior taking into account the user's perceived quality and its interactions with the overlay and P2P paradigm will finally help to design future Internet applications.
20 years after the discovery of the Crab Nebula as a source of very high energy gamma-rays, the number of sources newly discovered above 100 GeV using ground-based Cherenkov telescopes has considerably grown, at the time of writing of this thesis to a total of 81. The sources are of different types, including galactic sources such as supernova remnants, pulsars, binary systems, or so-far unidentified accelerators and extragalactic sources such as blazars and radio galaxies. The goal of this thesis work was to search for gamma-ray emission from a particular type of blazars previously undetected at very high gamma-ray energies, by using the MAGIC telescope. Those blazars previously detected were all of the same type, the so-called high-peaked BL Lacertae objects. The sources emit purely non-thermal emission, and exhibit a peak in their radio-to-X-ray spectral energy distribution at X-ray energies. The entire blazar population extends from these rare, low-luminosity BL Lacertae objects with peaks at X-ray energies to the much more numerous, high-luminosity infrared-peaked radio quasars. Indeed, the low-peaked sources dominate the source counts obtained from space-borne observations at gamma-ray energies up to 10 GeV. Their spectra observed at lower gamma-ray energies show power-law extensions to higher energies, although theoretical models suggest them to turn over at energies below 100 GeV. This opened the quest for MAGIC as the Cherenkov telescope with the currently lowest energy threshold. In the framework of this thesis, the search was focused on the prominent sources BL Lac, W Comae and S5 0716+714, respectively. Two of the sources were unambiguously discovered at very high energy gamma-rays with the MAGIC telescope, based on the analysis of a total of about 150 hours worth of data collected between 2005 and 2008. The analysis of this very large data set required novel techniques for treating the effects of twilight conditions on the data quality. This was successfully achieved and resulted in a vastly improved performance of the MAGIC telescope in monitoring campaigns. The detections of low-peaked and intermediate-peaked BL Lac objects are in line with theoretical expectations, but push the models based on electron shock acceleration and inverse-Compton cooling to their limits. The short variability time scales of the order of one day observed at very high energies show that the gamma-rays originate rather close to the putative supermassive black holes in the centers of blazars, corresponding to less than 1000 Schwarzschild radii when taking into account relativistic bulk motion.
Leonia cymosa (Violaceae) is a small tree from the under story of the Amazonian rain forest. I investigated the seed dispersal ecology of L. cymosa in plots of old growth terra firme forest located within the Cuyabeno Faunistic Reserve in north-eastern Ecuador. This species offered good conditions to examine the variation of traits of individual trees and the way they are linked with fruit removal from each tree. With this study I aimed to address the question whether frugivores exert selection pressures on fruits and the fruiting regime of fleshy fruited plants. The mean height of a fruiting L. cymosa was 6.6 m (range: 2 - 12.6 m). The median tree density was 11.8 trees per hectare. Trees grew in clusters consisting of different numbers of trees of different heights. L. cymosa flowered two times a year, in late February to March and in October. The respective fruiting seasons occurred in August/September and between March and May. The fruit pulp of L. cymosa contained the sugars fructose, glucose, and sucrose, the total soluble sugar being the first important nutritional compound of the fruit pulp. The second important compound was proteins. No lipids were found in the fruit pulp. The variation of nutritional quality of the fruits was high within trees. Nonetheless, significant differences were found among trees in all nutrient constituents studied. The maximum of ripe fruits produced per season by a single tree was 427. Median productivity of the trees was 45 ripe fruits throughout the fruiting season in 1999 (n=57) and 36 ripe fruits in 2000 (n=92). The maximum standing crop of fruits in a tree was 324 fruits (counted in 2000). Black mantle tamarins, Saguinus nigricollis (Callitrichidae), and squirrel monkeys, Saimiri sciureus (Cebidae), and possibly an unknown nocturnal frugivore consumed the fruits of L. cymosa at my study site. Green-rumped acouchis (Myoprocta pratti, Dasyproctidae) consumed fallen fruits and seeds underneath the trees. Black mantle tamarins and squirrel monkeys differed widely in their effectiveness as seed dispersers. Black mantle tamarins swallowed the seeds together with the fruit pulp and defecated intact seeds far away from the mother tree. Squirrel monkeys opened the fruits to suck and gnaw on the fruit pulp, and then dropped seeds to the forest floor below the tree crowns. Each of my study plots fell into the core home range of one group each of S. nigricollis and S. sciureus. Thus, the frugivore assemblage is small and disperser availability is limited for the individual tree of L. cymosa. In a sample of 6 trees of comparable and high fruit crop size, the total of ripe fruits removed from a tree throughout the whole fruiting season by the reliable seed disperser S. nigricollis was neither significantly correlated with the content of any of the nutrients measured in the fruit pulp (fructose, glucose, sucrose, total protein; pulp does not contain lipids), nor with total metabolisable energy, seed to pulp weight ratio, or water content of the fruit pulp. Feeding preferences for single sugars determined by other laboratory studies were not confirmed by this field study. The reliable seed disperser S. nigricollis does not seem to exert selective pressure on the nutrient content of the fruits of L. cymosa. Seasonal fruit crop size was the main predictor of all aspects of fruit removal by the effective disperser of L. cymosa, Saguinus nigricollis, as well as by the non-disperser, Saimiri sciureus. Trees with larger seasonal fruit crop size had a higher probability to have fruits removed by the disperser than those with small seasonal fruit crop sizes. They also had a higher number of fruits removed by the seed disperser. However, the proportion of fruits removed by the disperser decreased with increasing seasonal fruit crop size. In contrast, probability of fruit removal, the number of fruits removed, and the proportion of fruits removed by the non-disperser increased with increasing seasonal fruit crop sizes. The observed differences between disperser and non-disperser are due to differences in feeding capacity, group size and foraging behavior. Tamarins were less likely to harvest Leonia trees that were not or less completely covered by surrounding vegetation. This probably reflects a behavior to avoid predation by forest raptors. At high con-specific fruit abundance in the neighborhood, the proportion of fruits removed by tamarins was reduced. This suggests competition of trees for the disperser. My study revealed selection of the disperser on seasonal fruit crop size of L. cymosa. My results are consistent with the “fruit crop size hypothesis”. FCSH appears to constitute a valid framework also in the monkey-dispersed L. cymosa. My findings also show that factors beyond the tree’s control influenced fruit removal from Leonia trees. Disperser-mediated selection may be constrained (yet not impeded) by neighborhood conditions.
The bidirectional influence of parenchymal cells and cells of the immune system, especially of antigen-presenting and CD8\(^+\) T cells, in situations of putative auto- immune pathogenicity and degeneration was the main topic of this thesis. In the first part, the influence of human muscle cells on antigen-presenting cells was investigated. In inflammatory myopathies prominent infiltrates of immune cells containing T cells and antigen-presenting cells like macrophages and dendritic cells are present. The hypothesis was that human myoblasts have an inhibiting influence on these antigen-presenting cells under homeostatic conditions. A dysfunction or impairment under inflammatory circumstances might contribute to the development of myopathic conditions. The surface analysis of dendritic cells cocultured with myoblasts showed that immature dendritic cells could be driven into a reversible semi- mature state with significantly elevated levels of CD80. These dendritic cells were additionally characterized by their inhibiting function on T-cell proliferation. It was also shown that the lysates of healthy myoblasts could strongly enhance the phagocytic ability of macrophages, which could help with muscle regeneration and which might be disturbed in myositis patients. The second part of this thesis was about the clonal specificity of CD8\(^+\) T cells in a mouse model with genetically induced over-expression of PLP in oligodendrocytes. Here, we could show that the cytotoxic T lymphocytes, which had previously been shown to be pathogenic, were clonally expanded in the CNS of the transgenic mice. The amino acid sequences of the corresponding receptor chains were not identical, yet showed some similarities, which could mean that these clones recognize similar antigens (or epitopes of the same antigen). The knockout of PD-1 in this setting allowed for an analysis of the importance of tissue immune regulation. It became evident that the absence of PD-1 induced a larger number of clonal expansions in the CNS, hinting towards a reduced threshold for clonal disturbance and activation in these T cells. The expansions were, however, not pathogenic by themselves. Only in the presence of tissue damage and an antigenic stimulus (in our case the overexpression of PLP), the PD-1 limitation exacerbated the immune pathogenicity. Therefore, only in the presence of a “tissue damage signal”, the dyshomeostasis of T cells lacking PD-1 achieved high pathogenetic relevance. Finally, we investigated the pathogenetic role of CD8 T cells in Rasmussen encephalitis, a rare and chronic neurological disease mainly affecting children. The analysis of the T-cell receptor repertoire in Rasmussen encephalitis patients in the peripheral CD4\(^+\) and CD8\(^+\) T-cell compartments as well as the brain revealed the involvement of T cells in the pathogenicity of this disease. Many clonal expansions in the brain matched CD8\(^+\) T-cell expansions in the periphery on the sequence level. These putatively pathogenic clones could be visualized by immunohistochemistry in the brain and were found in close proximity to astrocytes and neurons. Additionally, the expanded clones could be found in the periphery of patients for at least one year.
The study investigates the water resources and aquifer dynamics of the igneous fractured aquifer-system of the Troodos Mountains in Cyprus, using a coupled, finite differences water balance and groundwater modelling approach. The numerical water balance modelling forms the quantitative framework by assessing groundwater recharge and evapotranspiration, which form input parameters for the groundwater flow models. High recharge areas are identified within the heavily fractured Gabbro and Sheeted Dyke formations in the upper Troodos Mountains, while the impervious Pillow Lava promontories - with low precipitation and high evapotranspiration - show unfavourable recharge conditions. Within the water balance studies, evapotranspiration is split into actual evapotranspiration and the so called secondary evapotranspiration, representing the water demand for open waters, moist and irrigated areas. By separating the evapotranspiration of open waters and moist areas from the one of irrigated areas, groundwater abstraction needs are quantified, allowing the simulation of single well abstraction rates in the groundwater flow models. Two sets of balanced groundwater models simulate the aquifer dynamics in the presented study: First, the basic groundwater percolation system is investigated using two-dimensional vertical flow models along geological cross-sections, depicting the entire Troodos Mountains up to a depth of several thousands of metres. The deeply percolating groundwater system starts in the high recharge areas of the upper Troodos, shows quasi stratiform flow in the Gabbro and Sheeted Dyke formations, and rises to the surface in the vicinity of the impervious Pillow Lava promontories. The residence times mostly yield less than 25 years, the ones of the deepest fluxes several hundreds of years. Moreover, inter basin flow and indirect recharge of the Circum Troodos Sedimentary Succession are identified. In a second step, the upper and most productive part of the fractured igneous aquifer-system is investigated in a regional, horizontal groundwater model, including management scenarios and inter catchment flow studies. In a natural scenario without groundwater abstractions, the recovery potential of the aquifer is tested. Predicted future water demand is simulated in an increased abstraction scenario. The results show a high sensitivity to well abstraction rate changes in the Pillow Lava and Basal Group promontories. The changes in groundwater heads range from a few tens of metres up to more than one hundred metres. The sensitivity in the more productive parts of the aquifer-system is lower. Inter-catchment flow studies indicate that - besides the dominant effluent conditions in the Troodos Mountains - single reaches show influent conditions and are sub-flown by groundwater. These fluxes influence the local water balance and generate inter catchment flow. The balanced groundwater models form thus a comprehensive modelling system, supplying future detail models with information concerning boundary conditions and inter-catchment flow, and allowing the simulation of impacts of landuse or climate change scenarios on the dynamics and water resources of the Troodos aquifer-system.
The widely used chemical acrylamide (AA) has been classified as a probable human carcinogen. This classification was based on positive results in rodent carcinogenicity studies as well as on a number of in vitro mutagenicity assays. In 2002, AA was discovered to be formed during the preparation of starch-containing foods. According to the latest FDA exposure assessment (2006), the average daily intake has been estimated from AA levels in foodstuffs and from nutritional habits to be around 0.4 µg/kg b.w. with a 90th percentile of 0.95 µg/kg b.w.. In children and adolescents however, the daily AA intake is about 1.5 times higher, due to lower body weight and differing consumption patterns. Apart from the diet, humans may be exposed to AA during the production or handling of monomeric AA, from AA residues in polyacrylamides, and from cigarette smoke. After oral administration, AA is readily absorbed and distributed throughout the organism. AA is metabolized to the reactive epoxide glycidamide (GA) via the CYP 450 isoenzyme CYP 2E1. Both, AA and GA are conjugated with glutathione. After enzymatic processing, the mercapturic acids N-Acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA) as well as the regioisomers N-Acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine (GAMA) and N-Acetyl-S-(1-carbamoyl-2-hydroxy-ethyl)-L-cysteine (iso-GAMA) are excreted with urine. An additional pathway for the metabolic conversion of GA is the epoxide hydrolase mediated hydrolysis to the diol compound glyceramide. Following administration of AA at doses exceeding the daily dietary intake by a factor of 1000 - 6000 to human subjects, a new urinary metabolite was found, which could be identified as the S-oxide of AAMA (AAMA-sulfoxide). In general, data from animal studies are used for risk assessment of (potential) human carcinogens. However, inter-species differences in toxicodynamics or toxicokinetics, e.g. in biotransformation may lead to under- or overestimation of human risk. The objective of this work was to establish a highly specific and sensitive analytical method to quantify the major urinary metabolites of AA. Other aims apart from measurements concerning the human background exposure were the evaluation of biotransformation and toxicokinetics of AA in humans and rats after oral administration of 13C3-AA. The obtained data was intended to help avoid linear extrapolation from animal models for future risk assessments of AA carcinogenicity.
Mechanisms and adaptive significance of interspecific associations between tropical ant species
(2009)
Aggression between ants from different colonies or species is ubiquitous. Exceptions to this rule exist in the form of supercolonies (within a species) and interspecific associations (between species). Probably the most intimate interspecific association is the parabiosis, where two ant species live together in a common nest. They keep their brood separate but jointly use trails and often share food resources. Parabioses are restricted to few species pairings and occur in South American and Southeast Asian rainforests. While the South American parabioses have been studied, albeit poorly, almost nothing is known about their Southeast Asian counterparts. My PhD project focuses on Southeast Asian parabioses between the myrmicine Crematogaster modiglianii Emery 1900 and the considerably larger formicine Camponotus rufifemur Emery 1900. The two species frequently nest together in hollow trees in the tropical lowland rainforest of Borneo. The basic question of my PhD project is why these two species live together. I investigated both proximate and ultimate aspects of this question. For comparative purposes, I included studies on a trail-sharing association in the same habitat. On the proximate level, I investigated which mechanisms facilitate tolerance towards hetero-spe¬ci¬fic nestmates. Ants generally discriminate nestmates from non-nestmates via cuticular hydro¬carbons that function as colony recognition cues. I studied the specificity of nestmate recognition within and between the two parabiotic species. Using gas chromatography-mass spectrometry (GC-MS), I analyzed the cuticular substances in both ant species to find potential differences to non-parabiotic species, and to estimate the substance overlap among the two species. A high substance overlap would e.g. suggest that interspecific tolerance is caused by chemical mimicry. Finally, bioassays were conducted to evaluate the function of different cuticular compounds. Interspecific tolerance in the two parabiotic species was species-specific but not colony-specific. Ca. rufifemur tolerated all Cr. modiglianii individuals, even those from foreign colonies, but strongly attacked workers of other Crematogaster species. Cr. modiglianii, in turn, tolerated Ca. rufifemur workers of certain foreign colonies but attacked those of others. Chemical analyses revealed two sympatric, chemically distinct Ca. rufifemur varieties (‘red’ and ‘black’) with almost no hydrocarbon overlap. Cr. modiglianii only tolerated foreign Ca. rufifemur workers if they belonged to the same chemical variety as their own Ca. rufifemur partner. It also attacked other, non-parabiotic Camponotus species. Thus, reciprocal interspecific tolerance was restricted to the species Cr. modiglianii and Ca. rufifemur. Ca. rufifemur frequently tolerated conspecific non-nestmates of the same chemical variety. Minor workers were more often tolerated than majors, possibly because they possess two to three times lower hydrocarbon quantities per body surface than majors. In contrast, Cr. modiglianii nearly always attacked conspecific non-nestmates. Both species possessed hydrocarbons with considerably higher chain lengths than congeneric, non-parabiotic ant species. Long-chain hydrocarbons are less volatile than shorter ones and thus harder to perceive. They may thus considerably facilitate interspecific tolerance. Moreover, up to 98% of the cuticular hydrocarbons in Ca. rufifemur were methylbranched alkenes, which are highly unusual among insect cuticular hydrocarbons. Cr. modiglianii and Ca. rufifemur had almost no hydrocarbons in common, refuting chemical mimicry as a possible cause of interspecific tolerance. The only hydrocarbons common to both species were two methylbranched alkenes, which constituted 89% of the ‘red’ Ca. rufifemur hydrocarbon profile and also occurred in those Cr. modiglianii colonies that lived together with this Ca. rufifemur variety. Cr. modiglianii presumably acquired these two compounds from its red Ca. rufifemur partner. Cr. modiglianii was significantly less aggressive towards foreign Cr. modiglianii workers that were associated with the same Ca. rufifemur variety than to those associated with the respective other one. Hence, this species seemed to use recognition cues acquired from its parabiotic partner. Apart from hydrocarbons, both species possessed a set of hitherto unknown substances on their cuticle. The quantitative composition of the unknown compounds varied between parabiotic nests but was similar among the two species of a nest. They are probably produced in the Dufour glanf of Cr. modiglianii and transferred to their Ca. rufifemur partner. Possible transfer mechanisms include interspecific trophallaxis and ‘mounting behaviour’, where Cr. modiglianii climbed onto Ca. rufifemur workers without being displaced. Although the composition of the unknown compounds greatly varied between nests, they did not function as nestmate recognition cues since both species used hydrocarbons for nestmate recognition. However, the unknown compounds significantly reduced aggression in Ca. rufifemur. The ultimate, i.e. ecological and evolutionary aspects of my PhD research deal with potential costs and benefits that Cr. modiglianii and Ca. rufifemur may derive from the parabiotic association, their interactions with other species, and population genetic analyses. Additional studies on a trail-sharing association between three other ant species deal with two possible mechanisms that may cause or facilitate trail-sharing. Whether parabioses are parasitic, commensalistic, or mutualistic, is largely unknown and depends on the costs and benefits each party derives from the association. I therefore investigated food competition (as one of the most probable costs), differentiation of foraging niches (which can reduce competition), and several potential benefits of the parabiotic way of life. Besides, I studied interactions between the ant species and the hemiepiphyte Poikilospermum cordifolium. The foraging niches of the two species differed regarding foraging range, daily activity pattern, and food preferences. None of the two species aggressively displaced its partner species from baits. Thus, interference competition for food seemed to be low or absent. For both ant species, a number of benefits from the parabiotic lifestyle seem possible. They include interspecific trail-following, joint nest defence, provision of nest space by the partner species, food exchange via trophallaxis, and mutual brood care. If an ant species follows another species’ pheromone trails, it can reach food resources found by the other species. As shown by artificial extract trails, Ca. rufifemur workers indeed followed trails of Cr. modiglianii but not vice versa. Thus, Ca. rufifemur benefited from Cr. modiglianii’s knowledge on food sources (informational parasitism). In turn, Cr. modiglianii seemed to profit from nest defence by Ca. rufifemur. Ca. rufifemur majors are substantially larger than Cr. modiglianii workers. Although Cr. modiglianii often effectively defended the nest as well, it seemed likely that this species derived a benefit from its partner’s defensive abilities. In neotropical parabioses (ant-gardens), mutualistic epiphytes play an important role in providing nest space. The neotropical Camponotus benefits its Crematogaster partner by planting epiphyte seeds, for which Crematogaster is too small. Similarly, the Bornean parabioses often were inhabited by the hemiepiphyte Poikilospermum cordifolium (Barg.-Petr.) Merr (Cecropiaceae). P. cordifolium seedlings, saplings and sometimes larger indivi¬duals abundantly grew at the entrances of parabiotic nests. However, P. cordifolium provides no additional nest space and, apart from nutritive elaiosomes, perianths, and extrafloral nectar probably plays a less important role for the ants than the neotropical epiphytes. In conclusion, the parabiosis is probably beneficial to both species. The main benefits seem to be nest defence (for Cr. modiglianii) and interspecific trail-following (for Ca. rufifemur). However, Ca. rufifemur seems to be more dependent on its partner than vice versa. For both parabiotic species, I analyzed mitochondrial DNA of ants from different regions in Borneo. My data suggest that there are four genetically and chemically distinct, but closely related varieties of Camponotus rufifemur. In contrast, Crematogaster modiglianii showed high genetic differentiation between distant populations but was not differentiated into genetic or chemical varieties. This argues against variety-specific cocladogenesis between Cr. modiglianii and Ca. rufifemur, although a less specific coevolution of the two species is highly likely. In Bornean rainforests, trail-sharing associations of Polyrhachis (Polyrhachis) ypsilon Emery 1887 and Camponotus (Colobopsis) saundersi Emery 1889 are common and often include further species such as Dolichoderus cuspidatus Smith 1857. I investigated a trail-sharing association between these three species and studied two mechanisms that may cause or facilitate these associations: interspecific trail-following, i.e. workers following another species’ pheromone trail, and differential inter¬specific aggression. In trail-following assays, D. cuspidatus regularly followed extract trails of the other two species, thus probably parasitizing on their information on food sources. In contrast, only few P. ypsilon and Ca. saundersi workers followed hetero¬speci¬fic extract trails. Hence, the association between P. ypsilon and Ca. saundersi cannot be ex¬plained by foragers following heterospecific trails. In this case, trail-sharing may originate from few scout ants that do follow heterospecific pheromone trails and then lay their own trails. Interspecific aggression among P. ypsilon, Ca. saundersi and D. cuspidatus was strongly asymmetric, Ca. saundersi being submissive to the other two species. All three species discriminated between heterospecific workers from the same and a distant trail-sharing site. Thus, it seems likely that the species of a given trail-sharing site habituate to one another. Differential tolerance by dominant ant species may be mediated by selective habituation towards submissive species, and thereby influence the assembly of trail-sharing associations.
The spectroscopic properties of molecular aggregates have been investigated by means of quantum dynamical calculations. Thereby both linear and nonlinear spectroscopic techniques have been taken into account. For the simulation of absorption and CD-spectra, coupling effects were regarded as well as the relative orientation of the monomer units in order to determine the parameters by reproducing measured spectra. For a more detailled description, results from quantum chemical calculations have also been included. Furthermore, investigations on nonlinear spectroscopy of molecular dimers have been performed.
The taphonomic and paleoecologic aspects of the Upper Hauterivian to Lower Barremian Agua de la Mula Member of the Agrio Formation (Neuquén Basin, Argentina) were studied in the frame of the sequence stratigraphic paradigm. The Agua de la Mula Member, a ca. 600 m thick succession of highly cyclic marine sediments was surveyed at two localities. Detailed bed-by-bed sedimentologic, stratigraphic, ichnologic, taphonomic and paleoecologic data collection allowed a precise paleoenvironmental, stratigraphic, taphonomic and synecologic interpretation, in a controlled sequence stratigraphic framework. The main architectural stratigraphic component is the Starvation-Dilution Sequence, interpreted as a the effect of a sixth-order, Milankovitch precession-driven cycle. Dilution hemisequences are siliciclastic-dominated and show evidence of depth changes. Starvation hemisequences show a diverse variation of mixed carbonate-siliciclastic facies that is linked to sequence stratigraphy. Ammonite-based biostratigraphy was revised and new knowledge proposed. The stratigraphic framework was improved by combining biostratigraphy, sequence stratigraphy and event stratigraphy. Nine main sequences were described, linked to other stratigraphic markers and correlated with other sequence stratigraphic charts. Several orders of cyclicity were inferred. Third- and fourth-order sequences are the major sequences, not subordinated to higher hierarchies (lower order). Precession, obliquity, and short and long eccentricity cycles of the Milankovitch band are proposed. Among the different sequence stratigraphic models the transgression-regression model fits the majority of the sequences described in this work. The depositional-sequence model could be applied only to the first third-order sequence, in which the true sequence boundary is identifiable. Starvation-dilution sequences, however, are composed by to components that are not completely explained by those models. Starvation hemisequences developed in intermediate to deep settings record the transgressive phase as well as the earLy regressive one without visible stratigraphic boundaries. 112 samples with 22,572 individuals were grouped into fifteen fossil associations and one assemblage that reflect the interaction of different factors: age, position in major, medium and starvation dilution sequences and, linked to sequence stratigraphy, depth, oxygen availability, rate of terrigenous input, water agitation, and substrate conditions. Temporary possible reduction in oxygen content is inferred based on all sources of available evidence. Organic buildups are briefly described and their development interpreted in terms of the sequence stratigraphic framework. Vertical patterns of replacement of fossil associations are described and related to sequence stratigraphy. Five types of skeletal concentrations represent the diversity of coquinas decribed in this study. Type 1, 2, 4 and 5 correspond to starvation hemisequences deposited in progressively shallower settings, from basin to inner ramp. Type 3 is embedded into dilution hemisequences and inferred to be linked to shell bed type I of Kidwell (1985). Types 1 and 2 correspond to transgression, maximum flooding and early regression without distinction. Type 4A as well as Type 5 are interpreted as onlap shell beds (Kidwell 1991a) or early TST shell beds (Fürsich and Pandey 2003). Type 4B corresponds to the MFZ shell bed (Fürsich and Pandey 2003) or mid-cycle shell bed (Abbott 1997), while Type 4C to the downlap shell bed (Kidwell 1991a). Time-averaging of shell beds was assessed with precision as the time involved in the deposition of the starvation hemisequences could be inferred. All shell beds comprise within-habitat assemblages forming within a few thousand years, with little environmental condensation. The fossilization of the marine calcareous shells is modelled as a series of steps called windows: environmental, destructional, burial and diagenetic. The “diagenetic window” is the most relevant. Connected to this it is proposed that carbonate dissolution is the primary control on the development of shell beds, as has been proposed before (Fürsich 1982; Fürsich and Pandey 2003). The interpretative power resulting from combining several lines of evidence, e.g., facies analysis, sequence stratigraphy, biostratigraphy, trace fossil analysis, paleoecology and taphonomy, and unravelling their multiple relationships, are the most relevant conclusions of this study.
It is well known, that the least squares estimator performs poorly in the presence of multicollinearity. One way to overcome this problem is using biased estimators, e.g. ridge regression estimators. In this study an estimation procedure is proposed based on adding a small quantity omega on some or each regressor. The resulting biased estimator is described in dependence of omega and furthermore it is shown that its mean squared error is smaller than the one corresponding to the least squares estimator in the case of highly correlated regressors.
Diverse roles of B cells in the pathophysiology of rheumatoid arthritis are now well established. B cells contribute to autoimmunity by producing autoantibodies, processing autoantigen and the production of different cytokines which are involved in the inflammatory cascade. Therefore approaches to target B lymphocytes directly or indirectly are developed for clinical practice to treat autoimmune diseases including rheumatoid arthritis. Transient B cell depletion by rituximab (anti-CD20 antibody) has gained prime importance in recent years. Meanwhile anti-CD20 mediated transient B cell depletion therapy is now used with clinical efficiency in the treatment of patients with rheumatoid arthritis. Rituximab induces noteworthy changes in the homeostasis of peripheral B cell subpopulations during the repletion phase with emerging immature B cells in peripheral blood followed by normalization of the naïve B cell pool and a longterm delay in memory B cell subsets in patients with rheumatoid arthritis. Particularly IgD+CD27+ memory B cells repopulate very slowly during B cell regeneration. In a prospective clinical study, our laboratory has shown that the overall number of memory B cells correlates well to the duration of clinical response to rituximab. Little is known about the particular molecular changes in the memory B cell repertoire after rituximab therapy. To better understand peripheral memory B cell subsets, we explored in detail the somatic mutational frequency and pattern of Ig-VH3 gene rearrangements by using a single B cell sorting technique followed by nested PCR before and up to 6 years after rituximab therapy in 18 RA patients. We compared rituximab inflicted dynamics of mutational acquisition to memory B cell repopulation in 4 healthy donors and 6 non RA patients undergoing high dose chemotherapy followed by autologous or allogeneic stem cell transplantation (SCT). Firstly we analyzed the peripheral composition of memory B cell subsets. The phenotypic analysis of peripheral pre-switch (IgD+CD27+) and post-switch (IgD-CD27+) memory B cells did not reveal any quantitative differences in RA patients prior to B cell depletion therapy compared to healthy donors. However extending those studies in directly analysing the B cell immunoglobulin receptor from individual B cells of RA patients and healthy controls brought interesting results. Pre-switched and post-switched memory B cells showed a highly significant difference in the amount of mutations/sequence. The population of IgD+CD27+ memory B cells is comprised of non-mutated, low and highly mutated (median= 9 mutations/ sequence) rearranged Ig receptors whereas the IgD-CD27+ memory B cell compartment shows quite uniformly highly mutated (median 18 mutations/ sequence) sequences indicating a significant difference between these two groups (mutational frequencies 3.83±0.19% vs. 7.1±0.53%; P=0.0001). Profound changes were noted in the re-emerging pre-switch memory B cells (IgD+/ CD27+) after transient B cell depletion with rituximab. These cells showed over a time period of 6 years after treatment with rituximab significantly delayed acquisition of mutations in Ig receptors on the single B cell level. One year after a single course of rituximab 84% of single repopulating IgD+/CD27+ B cells were unmutated and no highly mutated Ig-VH gene rearrangements were found(P=0.0001). Over time increasing numbers of mutations could be detected i-e 7.8% during 2nd year of regeneration (P=0.0001), 14% after 4 years (n=2). Nevertheless even 6 years after rituximab, VH mutations in IgD+ memory B cells were still reduced with 27% highly mutated sequences compared to 52% pre therapy(P=0.0001). Post-therapy analysis of CDR3 length of regenerated IgD+ memory B cells revealed increased CDR3 length which also correlates well with elevated number of non-mutated VH gene rearrangements observed during repletion phase. In comparison patients undergoing high dose chemotherapy followed by allogeneic stem cell transplantation repopulated IgD+ memory cells earlier with higher numbers of mutations in IgD+ memory B cells. One year after transplantation Ig receptors showed already 22% highly mutated and 42 % unmutated VH rearrangements. These findings indicated that anti-CD20 mediated B cell depletion seems not only to delay the production of pre-switch memory B cells but also significantly affects the acquisition of mutations in the IgD+ memory B cell pool. In contrary to the mutational pattern of IgD+ memory B cells after rituximab class switched memory B cells repopulate in the periphery with quantitatively normal mutations in their Ig receptors. Although the numeric replenishment of these recirculating class-switched memory B cells was also reduced after rituximab, we found no delay in quantitative acquisition of mutations also an increased proportion of IgA expressing B cells in this memory B cell subset was detected. Our data showed that post-therapy mutational targeting in RGYW/WRCY motifs were significantly increased as compared with that of pre-treatment (27% before rituximab vs. 43% after therapy, P=0.0003) indicating that affinity maturation may operate differently in class-switched memory B cells before and after B cell depletion. These results indicate a normal development process with an unimpaired mechanism of mutational acquisition in class-switched memory B cells. These data argue for different requirements to undergo somatic hypermutations in IgD+ memory B cells in comparison to class switched memory B cells. To conclude, our work has demonstrated for the first time a delayed acquisition of somatic hypermutations at single Ig receptor VH gene rearrangements of IgD+ memory B cells in comparison to class-switched memory B cells. These results demonstrate that IgD+ memory B cells are particularly susceptible to anti-CD20 treatment in patients with rheumatoid arthritis. In addition antigenic pressure and/or selection are substantially reduced by rituximab therapy which is basically not seen in the class-switched memory compartment. These data are in line with the hypothesis that IgD+ memory B cells have distinct requirements for activating their mutational machinery compared to class-switched memory B cells which recover normal mutations during regeneration phase. The results have implications in understanding the pathophysiology of memory B cell in rheumatoid arthritis and may be helpful in designing new targeted therapies.
In mammals, the RAF family of serine/threonine kinases consists of three members, A-, B- and C-RAF. Activation of RAF kinases involves a complex series of phosphorylations. Although the most prominent phosphorylation sites of B- and C-RAF are well characterized, little is known about regulatory phosphorylation of A-RAF. Using mass spectrometry, we identified here a number of novel in vivo phosphorylation sites in A-RAF. The physiological role and the function of these sites were investigated subsequently by amino acid exchange at the relevant positions. In particular, we found that S432 participates in MEK binding and is indispensable for A-RAF signaling. On the other hand, phosphorylation within the activation segment does not contribute to epidermal growth factor-mediated activation. Regarding regulation of A-RAF activity by 14-3-3 proteins, we show that A-RAF activity is regulated differentially by its C-terminal and internal 14-3-3 binding domain. Furthermore, by use of SPR technique, we found that 14-3-3 proteins associate with RAF in an isoform-specific manner. Of importance, we identified a novel regulatory domain in A-RAF (referred to as IH-segment) positioned between amino acids 248 and 267, which contains seven putative phosphorylation sites. Three of these sites, serines 257, 262 and 264, regulate A-RAF activation in a stimulatory manner. The spatial model of the A-RAF fragment including residues between S246 and E277 revealed a “switch of charge” at the molecular surface of the IH-region upon phosphorylation, suggesting a mechanism in which the high accumulation of negative charges may lead to an electrostatic destabilization of protein/membrane interaction resulting in depletion of A-RAF from the plasma membrane. Activation of B- and C-RAF is regulated by phosphorylation at conserved residues within the negative-charge regulatory region (N-region). Identification of phosphopeptides covering the sequence of the N-region led to the conclusion that, similar to B- and C-RAF, kinase activity of A-RAF is regulated by phosphorylation of the N-region. Abrogation of A-RAF activity by S299A substitution and elevated activity of the A-RAF-Y301D-Y302D mutant confirmed this conclusion. In addition, we studied the role of the non-conserved residues within the N-region in the activation process of RAF kinases. The non-conserved amino acids in positions –3 and +1 relative to the highly conserved S299 in A-RAF and S338 in C-RAF have so far not been considered as regulatory residues. Here, we demonstrate that Y296R substitution in A-RAF led to a constitutively active kinase. In contrast, G300S substitution (mimicking B- and C-RAF) acts in an inhibitory manner. These data were confirmed by analogous mutations in C-RAF. Based on the three-dimensional structure of the catalytic domain of B-RAF, a tight interaction between the N-region residue S339 and the catalytic domain residue R398 was identified in C-RAF and proposed to inhibit the kinase activity of RAF proteins. Furthermore, Y296 in A-RAF favors a spatial orientation of the N-region segment, which enables a tighter contact to the catalytic domain, whereas a glutamine residue at this position in C-RAF abrogates this interaction. Considering this observation, we suggest that Y296, which is unique for A-RAF, is a major determinant of the low activating potency of this RAF isoform. Finally, the residues R359 in A-RAF and R398 in C-RAF, which interact with the N-region, are also involved in binding of phosphatidic acid. Substitution of this conserved arginine by alanine resulted in accumulation of hyper-phosphorylated form of RAF, suggesting that this residue play a crucial role in phosphorylation-mediated feedback regulation of A- and C-RAF. Collectively, we provide here for the first time a detailed analysis of in vivo A-RAF phosphorylation status and demonstrate that regulation of A-RAF by phosphorylation exhibits unique features compared with B- and C-RAF.
The incidence of cardiovascular diseases including cardiac hypertrophy and failure in pre-menopausal women is lower compared to age-matched men but the risk of heart disease increases substantially after the onset of menopause. It has been postulated that female sex hormones play an important role in cardiovascular health in pre-menopausal women. In animal studies including spontaneously hypertensive (SHR) rats, the development of cardiac hypertrophy is attenuated by 17β-estradiol treatment. Cardiac energy metabolism is crucial for normal function of the heart. In cardiac hypertrophy and heart failure, the myocardium undergoes a metabolic shift from fatty acid as primary cardiac energy source to glucose, which re-introduces the fetal type of metabolism that representing the glucose as a major source of energy. Many studies have reported that the disruption of the balance between glucose and fatty acid metabolism plays an important role in cardiac pathologies including hypertrophy, heart failure, diabetes, dilative cardiomyopathy and myocardial infarction. Glucose enters cardiomyocytes via GLUT1 and GLUT4 glucose transporters and GLUT4 is the major glucose transporter which is insulin-dependent. Cardiac-selective GLUT4 deficiency leads to cardiac hypertrophy. This shows that the decrease in cardiac glucose uptake may play a direct role in the pathogenesis of cardiac hypertrophy. Estrogens modulate glucose homeostasis in the liver and the skeletal muscle. But it is not known whether estrogens affect also cardiac glucose uptake which could provide another mechanism to explain the prevention of cardiac hypertrophy by female sex hormones. In the present study, SHR Rats were ovariectomized (OVX), not ovariectomized (sham) or ovariectomized and treated with subcutaneous 17β-estradiol. After 6 weeks of treatment, body weight, the serum levels of estrogen, insulin, intra-peritoneal glucose tolerance test (IP-GTT), myocardial glucose uptake by FDG-PET (2-(18F)-fluoro-deoxyglucose (18FDG) and Positron Emission Tomography), cardiac glucose transporter expression and localization and cardiac hexokinase activity were analyzed. As results of this study, PET analysis of female SHR revealed decreased cardiac glucose uptake in OVX animals compared to intact that was normalized by estrogen supplementation. Interestingly, there was no change in global glucose tolerance among the treatment groups. Serum insulin levels and cardiac hexokinase activity were elevated by E2 substitution. The protein content of cardiac glucose transporters GLUT-4 and GLUT-1, and their translocation as determined by fractionation studies and immuno-staining did not show any significant change by ovariectomy and estrogen replacement. Also levels of insulin receptor substrate-1 (IRS-1) and its tyrosine phosphorylation, which is required for activation and translocation of GLUT4, was un-affected in all groups of SHR. Cardiac gene expression analysis in SHR heart showed that ei4Ebp1 and Frap1 genes which are involved in the mTOR signaling pathway, were differentially expressed upon estrogen treatment. These genes are known to be activated in presence of glucose in the heart. As a conclusion of this study, reduced myocardial FDG uptake in ovariectomized spontaneously hypertensive rat is normalized by 17β-estradiol treatment. Increased myocardial hexokinase appears as a potential mechanism to explain increased myocardial glucose uptake by 17β-estradiol. Increased cardiac glucose uptake in response to 17β-estradiol in ovariectomized SHR may provide a novel mechanism to explain the reduction of cardiac hypertrophy in E2 treated SHR. Therefore, 17β-estradiol improves cardiac glucose utilization in ovariectomized SHR which may give rise to possible mechanism for its protective effects against cardiac hypertrophy.
This thesis gives insights into the real-time dynamics of several free carbenes and radicals on a femtosecond and nanosecond time scale. The experiments were performed with radicals, singlet carbenes and triplet carbenes of various sizes. Several neutral excited states as well as the ionic ground state were characterized. Despite the relevance of such reactive intermediates in almost all chemical reactions, only relatively little experimental information on such systems is found in the literature. This is linked to the experimental challenge of producing such species under isolated conditions. The intermediates are formed from precursor molecules under interaction- free conditions by supersonic jet flash pyrolysis. The precursor molecules were synthetically designed to show clean thermal dissociation into one specific intermediate. A large variety of spectroscopic techniques was applied to study the intermediates. Each method augments the results of the other methods. This enabled to successfully approach the main goal of this thesis: to understand the excited-state dynamics of organic intermediates. The excited states were found to deactivate rapidly to the hot ground state. The observed fast decay is presumably linked to coupled electronically excited states and relaxation takes place by internal conversion or conical intersections. Further reactions then take place on the ground state surface. Absorption spectra, photodissociation dynamics, photoelectron spectra, ionization potentials, excited-state lifetimes and dissociative photoionization were elucidated by the measurements. Pulsed and continuous light sources were used over a large spectral range (UV, Vis, VUV). A well-defined amount of energy was deposited into the molecule. After internal conversion has taken place, a microcanonical ensemble of reactive intermediates can be studied. This data helps to understand the energetics and reaction channels of intermediates. Velocity map imaging enabled to monitor the pyrolysis efficiency in real time by analyzing photoion images. This observation facilitates clean intermediate generation. Experimental results were compared to quantum chemical calculations to aid the interpretation as well as to test the performance of theoretical approaches. Hydrocarbon radicals and carbenes are regarded as benchmark systems for computational methods due to their several low-lying electronic states and open-shell electronic configuration. The experimental data can help to identify and understand the contributions of the examined intermediates to the chemistry of high energy environments (e. g., hydrocarbon cracking reactors, interstellar space and combustion chambers). Here increased numbers of hydrocarbon intermediates are often present and usually have a strong impact on the overall reaction mechanism. Such environments contain in general a complex mixture of several different intermediates. The more spectroscopic and dynamic properties of each isolated intermediate are known, the easier it is to identify it among multiple components and to understand how it contributes to the overall reaction mechanism. Electronic excitation can take place by radiation, particle collisions or thermally at very high temperatures. How excited states influence the reaction mechanisms is still a matter of currant research.
Humans and other animals share choice preference for smaller-but-sooner over later-but-larger rewards, indicating that the subjective value of a reward is discounted as a function of time. This phenomenon referred to as delay discounting (DD), represents one facet of impulsivity which is inherently connected with reward processing and, within a certain range, adaptive. Maladaptive levels, however, can lead to suboptimal decision-making and represent important characteristics of psychopathologies such as attention-deficit/hyperactivity disorder (ADHD). In line with a proposed influence of dysregulated dopamine (DA) levels on impulsivity, neural structures involved in DD (the ventral-striatum [VS]; orbitofrontal cortex [OFC]) are highly innervated by dopaminergic neurons. However, studies explicitly testing the triadic interplay of dopaminergic neurotransmission, impulsivity and brain activation during intertemporal choice are missing. Therefore, the first study of the thesis examined the effect of different DA-bioavailability levels, indicated by a genetic polymorphism (Val158Met) in the gene of the catechol-O-methyltransferase, on the association of delay discounting and OFC activation. OFC response to monetary rewards that varied by delay-to-delivery was recorded with functional near-infrared spectroscopy (fNIRS) in a sample of 49 healthy human subjects. The results suggest a DA-related enhancement in OFC function from low (low DA level) to partial (intermediate DA level) and full (high DA level) reward delay sensitivity. Furthermore, DA-bioavailability was shown to moderate the association of neural reward delay sensitivity and impulsivity: OFC reward delay sensitivity was strongly correlated with impulsivity at intermediate DA-levels, but not at low or high DA-levels where impulsivity was related to delay-independent OFC amplitudes. It is concluded that DA-level should be considered as a crucial factor whenever impulsivity-related brain activation, in particular to reward delay, is examined in healthy subjects. Dysfunctional reward processing, accompanied by a limited ability to tolerate reward delays (delay aversion), has been proposed as an important feature in ADHD putatively caused by striatal hypo-dopaminergia. Therefore, the aim of the second study of this thesis was to examine subcortical processing of reward delays and to test for neural indicators of a negative emotional response to delay periods. Using functional magnetic resonance imaging (fMRI), brain activation in adult patients with ADHD (n=14) and healthy control subjects (n=12) was recorded during the processing of immediate and delayed rewards. Compared with healthy control subjects, hyporesponsiveness of the VS reward system was evident in patients with ADHD for both immediate and delayed rewards. In contrast, delayed rewards evoked hyperactivation in the dorsal caudate nucleus and the amygdala of ADHD patients, corroborating the central predictions of the delay aversion hypothesis. In combination both studies support the conception of a close link between delay discounting, brain activation and dopaminergic neurotransmission. The results implicate that studies on neural correlates of DD have to account for the DA-bioavailability level and for a negative emotional response to reward delays.
Gene and genome duplications are major mechanisms of eukaryotic genome evolution. Three rounds of genome duplication have occurred in the vertebrate lineage, two rounds (1R, 2R) during early vertebrate evolution and a third round, the fish-specific genome duplication (FSGD), in ray-finned fishes at the base of the teleost lineage. Whole genome duplications (WGDs) are considered to facilitate speciation processes and to provide the genetic raw material for major evolutionary transitions and increases in morphological complexity. In the present study, I have used comparative genomic approaches combining molecular phylogenetic reconstructions, synteny analyses as well as gene function studies (expression analyses and knockdown experiments) to investigate the evolutionary consequences and significance of the three vertebrate WGDs. First, the evolutionary history of the endothelin signaling system consisting of endothelin ligands and receptors was reconstructed. The endothelin system is a key component for the development of a major vertebrate innovation, the neural crest. This analysis shows that the endothelin system emerged in an ancestor of the vertebrate lineage and that its members in extant vertebrate genomes are derived from the vertebrate WGDs. Each round of WGD was followed by co-evolution of the expanding endothelin ligand and receptor repertoires. This supports the importance of genome duplications for the origin and diversification of the neural crest, but also underlines a major role for the co-option of new genes into the neural crest regulatory network. Next, I have studied the impact of the FSGD on the evolution of teleost pigment cell development and differentiation. The investigation of 128 genes showed that pigmentation genes have been preferentially retained in duplicate after the FSGD so that extant teleost genomes contain around 30% more putative pigmentation genes than tetrapods. Large parts of pigment cell regulatory pathways are present in duplicate being potentially involved in teleost pigmentary innovations. There are also important differences in the retention of duplicated pigmentation genes among divergent teleost lineages. Functional studies of pigment synthesis enzymes in zebrafish and medaka, particularly of the tyrosinase family, revealed lineage-specific functional evolution of duplicated pigmentation genes in teleosts, but also pointed to anciently conserved gene functions in vertebrates. These results suggest that the FSGD has facilitated the evolution of the teleost pigmentary system, which is the most complex and diverse among vertebrates. In conclusion, the present study supports a major role of WGDs for phenotypic evolution and biodiversity in vertebrates, particularly in fish.
Photosynthesis is the most fundamental process of life on earth. The biological production of oxygen in plant photosynthesis occurs in photosystem II (PSII). Here two water molecules are coupled in a four-electron oxidation to one O2 molecule, catalyzed by a tetranuclear manganese complex, known as the oxygen-evolving complex (OEC). In this thesis, density-functional theory (DFT) methods were validated and subsequently employed to study structures, spin-density distributions and EPR parameters of mono-, di-, and tetranuclear complexes with regard to the OEC. The goal was to draw conclusions on the molecular and electronic structure of the OEC.
The genus Borrelia belongs to the spirochete phylum, an ancient evolutionary branch of the domain bacteria that is only afar related to Gram-negative bacteria. Borreliae can be subdivided into the agents of the two borrelian-caused human diseases, Lyme disease and relapsing fever. Both disease patterns are closely related to the peculiar biology of Borrelia species and exhibit a wide spectrum of diverse clinical manifestations. Due to the small 0.91 Mb chromosome, borreliae have a lack of biosynthetic capacity. Thus, all Borrelia species are highly dependent on nutrients provided by their hosts. The transport of nutrients and other molecules across the outer membrane is enabled by pore-forming proteins, so-called porins. Porins are water-filled channels and can be subdivided into two different classes, general diffusion pores and substrate-specific porins. In terms of the Lyme disease agent Borrelia burgdorferi, three putative porins were characterized in previous studies: P13, Oms28 and P66. In contrast to Lyme disease species, the porin knowledge of relapsing fever Borrelia is low, which means that not any porin has actually been described for representatives of these agents. Thus, the general aim of this thesis was to provide insight into the porin content of both, Lyme disease and relapsing fever spirochetes. This aim could be achieved by isolating and identifying porins from Borrelia outer membranes and by biophysically characterizing them in artificial lipid membranes. In one chapter of this study, the first identification and characterization of a relapsing fever porin is presented. The pore-forming protein was isolated from outer membranes of Borrelia duttonii, Borrelia hermsii and Borrelia recurrentis and designated Oms38, for “outer membrane-spanning protein of 38 kDa”. Biophysical characterization of Oms38 was achieved by using the black lipid bilayer method and demonstrated that Oms38 forms small, water-filled channels with a single-channel conductance of 80 pS in 1 M KCl. The Oms38 channel did not exhibit voltage-dependent closure and is slightly selective for anions with a permeability ratio of cations over anions of 0.41 in KCl. Subsequently, a protein homologous to Oms38 was identified in the Lyme disease agents Borrelia burgdorferi, Borrelia garinii and Borrelia afzelii. The pore-forming protein of these species exhibits high sequence homology to Oms38 and similar biophysical properties, i.e. it forms pores of 50 pS in 1 M KCl. Interestingly, titration experiments revealed that this pore could be partly blocked by dicarboxylic anions, which means that this protein does not form a general diffusion pore but a channel with a binding-site specific for those compounds. Consequently, this porin was termed DipA, for “dicarboxylate-specific porin A”. In another set of experiments, it was shown that the porin P66 is present in both Lyme disease and relapsing fever species. Therefor, the outer membranes of the Lyme disease species Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii and the relapsing fever species Borrelia duttonii, Borrelia recurrentis and Borrelia hermsii were closer investigated. Except of the P66 homologue of Borrelia hermsii P66 of all species was highly active in artificial lipid membranes, forming pores with huge single-channel conductances between 9 and 11 nS in 1 M KCl. Moreover, the channel diameter and the constitution of Borrelia burgdorferi P66 were investigated in detail. Therefor, the P66 single-channel conductance in the presence of different nonelectrolytes with known hydrodynamic radii was analyzed in black lipid bilayers. The effective diameter of the P66 channel lumen was determined to be ~1.9 nm. Furthermore, as derived from multi-channel experiments the P66-induced membrane conductance could be blocked by certain nonelectrolytes, such as PEG 400, PEG 600 and maltohexaose. Additional blocking experiments on the single-channel level revealed seven subconducting states and indicated a heptameric constitution of the P66 channel. This indication could be confirmed by Blue native PAGE analysis which demonstrated that P66 units form a complex with a corresponding mass of approximately 440 kDa. Taking together, this thesis describes detailed biochemical and biophysical investigations of both Lyme disease and relapsing fever Borrelia porins and represents an important step forward in understanding the outer membrane pathways for nutrient uptake of these strictly host-dependent, pathogenic spirochetes. Furthermore, it provides some knowledge of the outer-membrane protein composition of Borrelia spirochetes. A profound knowledge of surface-exposed proteins, such as porins, is one precondition for the production of a successful vaccine and the drug design against the two borrelian-caused diseases.
Activation of mitogen-activated protein (MAP) kinases is a common reaction of plant cells in defense-related signal transduction pathways. Since the downstream events after the activation of MAP kinases are largely unknown in plants, the role of MAP kinases in the co-ordinate regulation of defense reactions and primary carbon metabolism by stress related stimuli has been analyzed in tomato. Thus, the relationship between mitogen activated protein kinases (LpMPK2 and LpMPK3) and extracellular invertases Lin6, as the key enzyme of an apoplasmic phloem unloading pathway, has been analyzed. The results showed that the mRNAs of LpMPK3 and Lin6 are sequentially induced by the same set of stress related stimuli (E-Fol, PGA,wounding, and KCl). The induction of the Lin6 promotor, as revealed by an increase in β-glucuronidase activity after 2 hours, was dependent both on the expression and activation of LpMPK3. These data suggest that the induction of extracellular invertase Lin6 by stress related stimuli requires LpMPK3. Glucose, metabolic molecule, was shown to result in the simultaneous induction of AtMPK4 and AtMPK6 activities that could be separated by anion-exchange chromatography, and characterized by differential cross-reaction with MAP kinase antibodies. Taken together, these data suggest that the activation of MAP inases play central roles in the regulation of sugar signaling. Stomatal movement is controlled by environmental signals including light intensity,humidity and atmospheric CO2 level. In Arabidopsis, a complete MAP kinase signaling cascade regulates stomatal development and patterning. However, the movement of stomata mediated by CO2 induced signaling pathways is not fully studied in higher plants. Here, we show that elevated levels of CO2 induce rapid and transient activation of SIPK and NtMPK4. The activation of both MAP kinases may regulate the anion channel activation for stomatal movement by the elevated level CO2. Up to now, the non-antioxidant function of tocopherol is not clear in higher plant,whereas the ability of tocopherol to modulate the stress tolerance mediated by function of antioxidant has been described in numerous studies. Thus, the function of α-tocopherol in stimuli-induced signal transduction pathways mediated by MAP kinase has been analyzed in tobacco. It has been shown that the activation of MAP kinase was induced by treatment of fungal elicitor and α-tocopherol phosphate but not α-tocopherol. Interestingly, α-tocopherol showed the transient inhibitory effect on the activation of stimuli-induced MAP Kinases in BY2 cells and tobacco plants, whereas ascorbate did not inhibit the activation of MAP kinases. The inhibitory activity test indicated that current application may indirectly affect the activity of MAP kinases. These results suggest that α-tocopherol can negatively regulate stimuliinduced signal transduction pathways via inactivation of MAP kinases. The purine-analogues have been tested and reported to be specific inhibitors of protein kinases mediated by structural-based selectivity in mammalian. Here, we tested C2, N6, N9-trisubstituted purines to determine basic relationship between their chemical structure and inhibitory activity using a particular plant MAP kinase. The modification of substitution in position C2 and N9 caused the increased inhibitory activity of 6-(benzylamino) purine analogue. In addition, 6-(isopentenylamino) purine analogues suggested that addition of a methyl group to position N9 caused at least 2-fold increased inhibitory activity compared with the addition of isopropyl group.Taken together, our study suggests that the selectivity and potency of inhibitors can be improved by structure modification. In addition, we have characterized the physiological function of Arabidopsis thaliana PLAT domain protein 1 (AtPDP1) in modulating the interaction of defense pathways mediated by biotic and abiotic factors. Interestingly, overexpression of AtPDP1 resulted in increasing susceptibility of virulent pathogens and necrotrophic fungus, and developing necrosis induced by unknown biotic factors. However, these overexperssion lines showed the significantly delayed senescence and higher level of phosystem II quantum yield compared with control plants against high salt stress. Our results strongly indicate that AtPDP1 positively regulate with salt tolerance, and enhances the sensitivity to biotic stresses. We propose that the AtPDP1 might be regulated with the complex pathways of interplay among various signaling during stress adaptation.
We discuss exceptional polynomials, i.e. polynomials over a finite field $k$ that induce bijections over infinitely many finite extensions of $k$. In the first chapters we give the theoretical background to characterize this class of polynomials with Galois theoretic means. This leads to the notion of arithmetic resp. geometric monodromy groups. In the remaining chapters we restrict our attention to polynomials with primitive affine arithmetic monodromy group. We first classify all exceptional polynomials with the fixed field of the affine kernel of the arithmetic monodromy group being of genus less or equal to 2. Next we show that every full affine group can be realized as the monodromy group of a polynomial. In the remaining chapters we classify affine polynomials of a given degree.