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The Transforming Growth Factor (TGF) superfamily of cytokines and their serine/threonine kinase receptors play an important role in the regulation of cell division, differentiation, adhesion, migration, organization, and death. Smad proteins are the major intracellular signal transducers for the TGF receptor superfamily that mediate the signal from the membrane into the nucleus. Bone Morphogenetic Protein-4 (BMP-4) is a representative of the TGF superfamily, which regulates the formation of teeth, limbs and bone, and also plays a role in fracture repair. Binding of BMP-4 to its receptor stimulates phosphorylation of Smad1, which subsequently recruits Smad4. A hetero-oligomeric complex consisting of Smad1 and Smad4 then translocates into the nucleus and regulates transcription of target genes by interacting with transcription factors. Although the individual steps of the signaling cascade from the receptor to the nucleus have been identified, the exact kinetics and the rate limiting step(s) have remained elusive. Standard biochemical techniques are not suitable for resolving these issues, as they do not offer sufficiently high sensitivity and temporal resolution. In this study, advanced optical techniques were used for direct visualization of Smad signaling in live mammalian cells. Novel fluorescent biosensors were developed by fusing cyan and yellow fluorescent proteins to the signaling molecules Smad1 and Smad4. By measuring Fluorescence Resonance Energy Transfer (FRET) between the two fluorescent proteins, the kinetics of BMP/Smad signaling was unraveled. A rate-limiting delay of 2 - 5 minutes occurred between BMP receptor stimulation and Smad1 activation. A similar delay was observed in the complex formation between Smad1 and Smad4. Further experimentation indicated that the delay is dependent on the Mad homology 1 (MH1) domain of Smad1. These results give new insights into the dynamics of the BMP receptor – Smad1/4 signaling process and provide a new tool for studying Smads and for testing inhibitory drugs.
The covalent linkage of the aryloxy-substituents through macrocyclisation was applied for the synthesis of perylene bisimide atropo-enantiomers. The synthesis of macrocyclic perylene bisimides was achieved by using a tetra(3-hydroxyphenoxy)-functionalized perylene bisimide with achiral 2,6-diisopropylphenyl as imide substituent through Williamson´s etherfication which could be realized for four different oligoethylene glycol bridging units. Two regioisomeric macrocycles, namely the diagonally bridged (1,7- and 6,12- linkage) and the laterally bridged (1,12- and 6,7-linkage) isomers, were obtained for each bridging unit. The structural assignment of the isolated regioisomeric macrocycles was unambiguously accomplished by X-ray analysis of two macrocycles and by 1H NMR spectroscopy for all isomers. The conformational influence of the aryloxy-substituents on the functional properties of this class of chromophores could be derived by comparison of the optical and electrochemical properties of all isolated macrocylces with those of an open-chained reference compound. It was shown that the aryloxy-substituents prefer a lateral conformation in solution. Furthermore, solvent dependent fluorescence studies indicated that a photoinduced electron transfer process is of importance for the fluorescence quenching of electron-rich aryloxy-substituted perylene bisimides. The resolution of the atropo-diastereomers of diagonally bridged macrocyclic perylene bisimides with chiral 2-(R)-octylamine as imide substituent and diethylene glycol bridging units could be accomplished by semi-preparative HPLC on a chiral column. The chiroptical properties of the isolated epimerically pure macrocycles were determined by CD spectroscopy. Based on the experimental CD spectra, the stereochemical assignment of the isolated epimers was accomplished by application of the excition chirality method and confirmed by quantum chemical calculation of the CD spectra. The synthetical concept was extended successfully to 1,7-diaryloxy-substituted perylene bisimides. The structure of the diagonally bridged macrocycle was unambiguously confirmed by X-ray analysis and NMR spectroscopy. The atropo-enantiomers of this macrocycle could be resolved by semi-preparative HPLC on a chiral column and the assignment of the absolute configuration was achieved by comparison of the CD spectra of the resolved enantiomers with those of epimerically pure bis(macrocycles) reported before. By comparison of the X-ray structures obtained for the racemic mixture as well as one enantiomer important information could be extracted for the formation of p-dimers of perylene bisimides. The dependence of the interconversion barrier on the bulkiness of the bay-substituents was investigated for four halogen-substituted perylene bisimides. The dynamic properties were investigated by temperature-dependent NMR spectroscopy and kinectic measurements using CD spectroscopy. By applying the concept of the “apparent overlap” a convincing linear relationship between the size of the substituents and the free enthalpy of activation could be derived. Furthermore, the resolution of the atropo-diastereomers or enantiomers of the tetrachloro and tetrabromo-substituted derivates was accomplished, whereupon especially the 1,6,7,12-tetrabromosubstituted perylene bisimide provided at room temperature stable enantiomers. Additionally, the derived structure-property relationship allows the design of conformationally stable perylene bisimide enantiomers by proper choice of the bay substituents. In order to utilize the reversibility of self-assembly for the quantitative formation of macrocyclic perylene bisimides, a tetrazinc porphyrin-functionalized perylene bisimide was synthesized. The self-assembly of the zinc porphyrin perylene bisimide bichromophoric building block and diazabicyclo-[2.2.2]-undecane into the desired 1:2 sandwich complex was investigated by UV/Vis and 1H NMR spectroscopy and the macrocyclic structure was unequivocally proven by diffusion-ordered NMR spectroscopy (DOSY NMR). Furthermore, the controlled deposition of these well-defined macrocycles on highly ordered pyrolitic graphite (HOPG) was demonstrated by atomic force microscopy (AFM) investigations. The alignment of a linear amino functionalised p-conjugated polymers upon addition of the bichromphoric tetrazinc porphyrin-perylene bisimide was investigated by UV/Vis spectroscopy and AFM measurement. The surface analysis by AFM investigations revealed that the bichromophoric system composed of perylene bisimide and zinc porphyrin is able to cross-link the linear p-conjugated polymer over a wide range of the graphite surface which provided a defined arrangement of three different functional p-systems.
The RS1 protein, a 67 kDa protein, encoded by an intronless single copy gene that was only detected in mammals, mediates transcriptional and post-transcriptional down-regulation of the sodium-D-glucose co-transporter SGLT1. The short-term post-transcriptional down-regulation of SGTL1 by RS1 has been shown to occur at the trans-Golgi network (TGN). In the present study, two tripeptides from the human RS1 protein (hRS1), GlnCysPro and GlnSerPro, that induce the post-transcriptional down-regulation of SGLT1 at the TGN, were identified. The application of the tripeptides led to 40-50% reduction of the amount of the SGLT1 protein in the plasma membrane, which correlated to the degree of decrease in SGLT1-mediated glucose transport. For the short-term down-regulation of SGLT1 by the tripeptides, the effective intracellular concentrations IC50 values of 2.0 nM (GlnCysPro, QCP) and 0.16 nM (GlnSerPro, QSP) were estimated. The observed down-regulation of SGLT1 by the tripeptides QCP and QSP, similar to hRS1 protein, was attenuated by different intracellular monosaccharides including nonmetabolized methyl-α-D-glucopyranoside and 2-deoxyglucose. On the contrary, the short-term inhibition of the hOCT2 by QCP could only be observed after rising of intracellular concentration of AMG. QCP and QSP are transported by H+-peptide cotransporter PEPT1 that is co-located with SGLT1 in the small intestinal enterocytes and thereafter effectively down-regulate hSGLT1-mediated transport of AMG. The data indicates that orally applied tripeptides QCP or QSP can be used to down-regulate D-glucose absorption in small intestine and used for treatment of obesity and diabetes mellitus.
Although known about and investigated since the late 1970’s, the picture of the basic principles governing inhibitor strengths and the structure-activity relationships of the cysteine protease inhibition mechanism is still very incomplete. Computational approaches can be a very useful tool for investigating such questions, as they allow the inspection of single, specific effects in isolation from all others, in a manner very difficult to achieve experimentally. The ab initio treatments of such large systems like proteins are still not feasible. However, there is a vast number of computational approaches capable of dealing with protein structures with reasonable accuracy. This work presents a summary of theoretical investigations into cysteine protease cathepsin B using a range of methods. We have concentrated on the investigation of cysteine protease inhibition by epoxide- and aziridine-based inhibitors in order to obtain better insight into these important topics. Various model systems are simulated by means of pure quantum mechanical methods and by hybrid (QM/MM) methods. Both approaches provide a static picture. Dynamical effects are then accounted for by additional molecular dynamics (MD) simulations, using both classical and QM/MM MD approaches. The quantum mechanical approach was used to study very small model systems consisting only of the electrophilic warhead of the inhibitor (both substitituted and not) and molecular moieties simulating a very simplified protein active site (methylthiolate instead of Cys29 and methylimidazolium instead of His199 residue) and solvent surroundings (two waters or two ammonium ions, in combination with a continuum solvent model). Although simple, such a system provides a good description of the most important interactions involved in the inhibition reaction. It also allows investigation of the influence of the properties of the electrophilic warhead on the reaction rate. Beside the properties of the electrophilic warhead, the protein and solvent environment is also an important factor in the irreversible deactivation of the enzyme active site by the inhibitor. The non-covalent interactions of the inhibitor with the oxyanion hole and other subsites of the enzyme, as well as its interaction with the solvent molecules, need to be explicitly taken into account in the calculations, because of their possible impact on the reaction profile. As molecular modeling methods allow the treatment of such large systems, but lack the possibility of describing covalent interactions, our method of choice was the combined quantum mechanics/molecular modeling approach. By splitting the system into a smaller part that undergoes the bond cleavage/formation process and must be treated quantum mechanically, and a larger part, comprised of the rest of the protein, which could be treated using force fields, we managed to simulate the system at the desired precision. Our investigations concentrated on the role of His199 in the inhibition mechanism as well as on the structure-reactivity relationships between cysteine protease and various inhibitors, yielding new insight into the kinetics, regio- and stereospecificity of the inhibition. In particular, our calculations provide the following insights: i.) an explanation for the regioselectivity of the reaction, and original insight into which interactions affect the stereoselectivity; ii.) a clear model which explains the known structure-activity relationships and connects these effects with the pH-dependency of the inhibition; iii.) our computations question the generally accepted two-step model by showing that substituent effects accelerate the irreversible step to such an extent that the achievement of an equilibrium in the first step is doubtful; iv.) by way of theoretical characterizations of aziridine models, the reasons for similarities and differences in the mode of action of epoxide- and aziridine-based inhibitors are elucidated; and finally, v.) combining our results with experimental knowledge will allow rational design of new inhibitors. To account for dynamical effects as well, molecular dynamics (MD) computations were also performed. In these calculations the potential energy was computed at the force field level. The results not only supported and clarified the QM/MM results, but comparison with previous X-ray structures helped correct existing errors in the available geometrical models and resolved inconsistencies in the weighting of various factors governing the inhibition. In the work the first QM/MM MD calculations on the active site of the cysteine proteases are presented. In contrast to the MD simulations, these calculations used potential energies computed at the QM/MM-level. With the help of these computations we sought to address strongly disputed questions about the reasons for the existence of the active site ion pair and its role in the high activity of the enzyme.
Neural crest cells and sensory neurons are two prominent cell populations which are induced at the border between neural and non-neural ectoderm during early vertebrate development. The neural crest cells are multipotent and highly migratory precursors that give rise to face cartilage, peripheral neurons, glia cells, pigment cells and many other cell types unique to vertebrates. Sensory neurons are located dorsally in the neural tube and are essential for sensing and converting environmental stimuli into electrical motor reflexes. In my PhD thesis, I obtained novel insights into the complex processes of cell induction at the neural plate border by investigating the regulation and function of mdkb in zebrafish. First, it was possible to demonstrate that mdkb expression is spatiotemporally correlated with the induction of neural crest cells and primary sensory neurons at the neural plate border. Second, it became evident that the expression of mdkb is activated by known neural crest cell inducing signals, like Wnts, FGFs and RA, but that it is independent of Delta-Notch signals essential for lateral inhibition. Knockdown experiments showed that mdkb function is necessary for induction of neural crest cells and sensory neurons at the neural plate border, probably through determination of a common pool of progenitor cells during gastrulation. The present study also used the advantages of the zebrafish model system to investigate the in vivo function of all midkine gene family members during early brain development. In contrast to the situation in mouse, all three zebrafish genes show distinct expression patterns throughout CNS development. mdka, mdkb and ptn expression is detected in mostly non-overlapping patterns during embryonic brain development in the telencephalon, the mid-hindbrain boundary and the rhombencephalon. The possibility of simultaneously knocking down two or even three mRNAs by injection of morpholino mixtures allowed the investigation of functional redundancy of midkine factors during brain formation. Knockdown of Midkine proteins revealed characteristic defects in brain patterning indicating their association with the establishment of prominent signaling centers such as the mid-hindbrain boundary and rhombomere 4. Interestingly, combined knockdown of mdka, mdkb and ptn or single knockdown of ptn alone prevented correct formation of somites, either by interfering with the shifting of the somite maturation front or interferance with cell adhesion in the PSM. Thus, Ptn was identified as a novel secreted regulator of segmentation in zebrafish.
During the last few years an increasing number of physiological processes in plants have been shown to be regulated by NO. NO plays important roles in growth and development, plant disease resistance, abiotic stress, and in above and underground plant organs. In recent years several enzymatic pathways and few non-enzymatic pathways were proposed for nitric oxide production in plants. The major goal of this work was to quantify NO production by plants and especially by roots, and to identify the enzymes responsible for NO production. As a major method, NO production by roots was followed through on-line measurement of NO emission into the gas phase by chemiluminescence (= direct chemiluminescence), and also by indirect chemiluminescence where trace amounts of oxidized products like NO2- and NO3- can be easily measured. Plants used were tobacco wild-type (N. tabacum cv Xanthi or cv Gatersleben), NR-free mutants grown on ammonium in order to prevent NR induction, plants grown on tungstate to inhibit synthesis of functional MoCo-enzymes, and a NO-overproducing nitrite reductase (NiR)-deficient transformant as well as barley, rice and pea. Induction of a hypersensitive response (HR) in tobacco leaves was achieved by using avirulent Pseudomonas syringae pv phaseolicola. At oxygen concentrations of <1%, even completely nitrate reductase (NR)-free root tissues reduced added nitrite to NO, indicating that in roots, NR was not the only source for nitrite-dependent NO formation. By contrast, NR-free leaf slices were not able to reduce nitrite to NO. Root NO formation was blocked by inhibitors of mitochondrial electron transport (Myxothiazol and SHAM), whereas NO formation by NR containing leaf slices was insensitive to the inhibitors. Consistent with that, mitochondria purified from roots, but not those from leaves, reduced nitrite to NO at the expense of NADH. The inhibitor studies suggest that, in root mitochondria, both terminal oxidases participate in NO formation, and they also suggest that even in NR-containing roots, a large part of the reduction of nitrite to NO was catalysed by mitochondria, and less by NR. The differential capacity of root and leaf mitochondria to reduce nitrite to NO appears to be common among higher plants, since it was observed with Arabidopsis, barley, pea, and tobacco. Nitrite and NADH consumption by mitochondria were also measured. Anaerobic, nitrite-dependent NO emission was exclusively associated with the membrane fraction, without participation of matrix components. It was also examined whether root mitochondria and mitochondrial membranes produce nitric oxide (NO) exclusively by reduction of nitrite or also via a nitric oxide synthase (NOS),- and to what extent direct NO measurements could be falsified by NO oxidation. In addition to chemiluminescence, Diaminofluoresceins (DAF) were used as an NO indicators for comparison. In air, mitochondria apparently produced no nitrite-dependent NO, and no NOS activity was detected by direct or indirect chemiluminescence. In contrast, with DAF-2 and DAR-4M an L-arginine-dependent fluorescence increase took place. However, the response of this apparent NOS activity to inhibitors, substrates and cofactors was untypical when compared with commercial iNOS and is considered an artefact. With iNOS, about 2/3 of the NO were oxidized to (nitrite + nitrate). Mitochondria also appear to consume NO without increasing oxidation to (nitrite+ nitrate). We therefore assume formation of NO to a volatile intermediate (eventually N2O3). It was recently shown that the hypersensitive response (HR) of tobacco triggered by the fungal elicitor cryptogein occurred independent of the presence or absence of nitrate reductase (NR). One conclusion was that NR-dependent NO formation played no role in the HR. Here we present evidence that the described scenario may be specific for cryptogein. Pseudomonas syringae pv. phaseolicola was infiltrated into tobacco leaves from WT plant and from the NiR-deficient NO-overproducing clone 271, grown either on nitrate or ammonium. Lesion development as well as bacterial growth and sugar concentrations in leaves and in the leaf apoplast was monitored. Lesion development was positively and bacterial growth was negatively correlated with nitrate nutrition and eventually with NO formation. Bacterial growth was positively correlated with ammonium nutrition and apoplastic sugar concentrations. Total (free and conjugated) SA content were always drastically increased by bacterial infection, but there was no clear correlation with NO production. In the presence of cryptogein, Pseudomonas growth was drastically reduced. This shows that the assumed interdependence of bacterial growth, NO production and the HR is complex and not unifactorial.
Adaptive Polarization Pulse Shaping and Modeling of Light-Matter Interactions with Neural Networks
(2007)
The technique of ultrafast polarization shaping is applied to a model quantum system, the potassium dimer. The polarization dependence of the multiphoton ionization dynamics in this molecule is first investigated in pump–probe experiments, and it is then more generally addressed and exploited in an adaptive quantum control experiment utilizing near–IR polarization–shaped laser pulses. The extension of these polarization shaping techniques to the UV spectral range is presented, and methods for the generation and characterization of polarization–shaped laser pulses in the UV are introduced. Systematic scans of double–pulse sequences are introduced for the investigation and interpretation of control mechanisms. This concept is first introduced and illustrated for an optical demonstration experiment, and it is then applied for the analysis of the intrapulse dumping mechanism that is observed in the excitation of a large dye molecule in solution with ultrashort laser pulses. Shaped laser pulses are employed as a means for obtaining copious amounts of data on light–matter interactions. Neural networks are introduced as a novel tool for generating computer–based models for these interactions from the accumulated data. The viability of this approach is first tested for second harmonic generation (SHG) and molecular fluorescence processes. Neural networks are then utilized for modeling the far more complex coherent strong–field dynamics of potassium atoms.
RS1, a gene product of RSC1A1, is critically involved in cell density-dependent transcriptional down-regulation of SGLT1 in LLC-PK1 cells and in the post-transcriptional down-regulation of SGLT1 in small intestine. RS1 inhibits the release of SGLT1 containing vesicles from the trans-Golgi network and migrates into the nucleus where it inhibits transcription of SGLT1. In the present work we identified a novel 21 amino acids-long nonconventional nuclear localization sequence (RS1 NLS) in porcine RS1 (pRS1) that is necessary and sufficient for nuclear targeting of pRS1. RS1 NLS is framed by two consensus sequences for phosphorylation which are responsible for confluence-dependent regulation of RS1 NLS: a casein kinase 2 (CK2) site in position 348 and a protein kinase C (PKC) site in position 370. Confluence-dependent nuclear targeting was observed with amino acids 342-374 (R-NLS-Reg). Mutation analysis suggested that nuclear targeting is blocked by phosphorylation of serine 370 (PKC) and that phosphorylation of serine 348 (CK2) prevents phosphorylation of serine 370. Because CK2 is down-regulated and PKC is up-regulated during confluence of LLC-PK1 cells, our data suggest that nuclear localization coordinates cell density-dependent changes in transcriptional and post-transcriptional inhibition of SGLT1 expression.
Brassicaceae and a few related plant families are characterized by possession of the glucosinolate-myrosinase system. Glucosinolates are amino-acid derived allelochemicals which are hydrolysed upon tissue damage by myrosinase enzymes to produce various degradation products which can be toxic for generalist insects. The larvae of the crucifer-specialist Athalia rosae, the turnip sawfly, sequester glucosinolates into their haemolymph. The role of the glucosinolate-myrosinase system for the interaction of the turnip sawfly with Brassicaceae was examined in this study from two different perspectives: variation within individual plants and between plant species. The plant responses to the feeding by herbivores and the short-term effects this induction had on insect behaviour were investigated in white mustard. Furthermore, plants can use multiple defences. Hence correlations of glucosinolates and myrosinase activities with other defences and nutritional quality and their long-term effects on the development of the insects were investigated in seven different plant species.
In the first part of this work a new approach to measure transient absorption spectra of fluorescent compounds by means of laser flash photolysis technique was presented. Generally, the recorded transient absorption signal consists of transient absorption, fluorescence and ground state bleaching. Thus, for fluorescent chromophores a fluorescence correction is indispensable in order to obtain undisturbed absorption decay curves as well as accurate transient absorption spectra. Due to time response characteristics of the PMT detector the fluorescence contribution cannot be corrected by recording the fluorescence separately. Measuring two transient absorption signals with probe light differing in intensity, compounds with quantum yields up to ~ 35 % can be investigated. This is a major improvement because transient absorption spectroscopy is a powerful method to gain insight into the kinetics and the energy of excited states and information in the time domain of fluorescence are no longer lost. In the second part the synthesis and the photophysical characterisation of redox cascades were reported. These cascades consist of an acridine acceptor and up to three triarylamine donor subunits. The redox potentials of the triarylamines were tuned by adequate substituents in the para-position of the phenyl ring to ensure a directed redox gradient. Upon photoexcitation a locally excited state or a CT state is populated which then injects a hole onto the adjacent donor and consequently results in a CS state. Fluorescence and transient absorption measurements revealed that HT depends strongly on donor strength and solvent polarity. Formation of a CS state was only observed in case of strong terminal donors or polar solvents. A low lying localised triplet state acts as an energy trap and quenches all CS states even in case of the cascade with the strongest terminal donor in very polar solvents. Furthermore, population of a CS state catalyses the formation of this triplet states which results in a shorter lifetime of the CS state compared to the lifetime of the CT state of the corresponding reference compound. Compared to redox cascades already reported in literature, the electronic coupling between the redox centres was decreased by sterical as well as electronic effects. To prolong the lifetime of the CS state saturated spacers on the one hand and a perpendicular orientation of the acceptor and the adjacent donor on the other hand were selected. The twisting of the subunits forming the CT state results in a higher degree of charge separation but its contribution to increase the lifetimes of the CS states is of minor importance. The longer lifetime of the CS states can be ascribed to the saturated spacers. Experimental data in combination with calculated values indicate that charge recombination takes place in the Marcus normal region by a superexchange mechanisms. Although charge recombination of the known cascades is located in the Marcus inverted region, these CS states decay faster than the CS states of the compounds investigated in this work.
A small percentage (1-5%) of the blood lymphocytes expresses alternative T-cell antigen receptor that uses g and d TCR rearranging genes. A subset of them expresses the Vg9Vd2 TCR. Those cells respond to self-nonpeptide and foreign antigens presented by unknown antigen-presenting molecules. Vg9Vd2 T cells also express Toll-like receptors and natural killer receptors that allow them to respond to other nonpeptide microbial components or to alterations in the expression of stress cell surface ligands such as NKG2D ligands. Vg9Vd2 T cells frequently are regulated by the expression of activating and/or inhibitory NKRs (iNKRs) that can fine-tune their activation threshold and the activating NKG2D receptor is one of the most studied until now. NKG2D, a C-type lectin receptor directed against MICA/MICB and UL16-binding protein (ULBP) molecules, have been reported a powerful co-stimulus for Ag-mediated activation of CD8 and Vg9Vd2 T cells. Indeed, NKG2D is recruited within the Vg9Vd2 TCR immunological synapse and enhances recognition by Vg9Vd2 T cells of Mycobacteria-infected DCs and various MICA/MICB or ULBP hemopoietic and non-hemopoietic tumors. The level of NKG2D is upregulated by inflammatory cytokines (e.g. IL-15), and NKG2D ligands are induced after a physical or genotoxic stress and/or along infection by intracellular pathogens. Therefore, NKG2D is a key stress sensor that strongly enhances recognition of altered or infected self by human gd T cells. Recent progress in the field supports the idea that gd T cells fulfill a role in the innate and adaptative immune response in different way of the conventional ab T cells. We demonstrated direct activation of Vg9Vd2 T cells by NKG2D ligation through the association with DAP10 adapter molecules and independently of TCR-Ag recognition, similar to the NKG2D-mediated activation of NK cells. Culture of peripherical blood mononuclear cells with immobilized NKG2D mAb or NKG2D ligand MICA induces up-regulation of CD69 and CD25 in NK and Vg9Vd2 T cells but not in CD8 T cells. Additionally, the ligation of NKG2D induces in Vg9Vd2 T cells the up-regulation of molecules typical for antigenpresenting cells, such as co-stimulator molecules (CD86) antigen presenting molecules (CD1a, HLA-DR), adhesion molecules (CD54), and activation molecules (CD69). Furthermore, NKG2D ligation in Vg9Vd2 T cells induces the production of cytokines such as TNF-a and chemokines such as, MIP-1a, but cannot induce the production of cytokines such as IL-6 or IFN-g and chemokines such as RANTES, MCP-1 and GM-CSF. In addition, NKG2D triggers the activation of the cytolytic machinery as efficient as CD3 stimulation as shown by measurement of the release of granules with esterase activity (BLT assay), perforin and the up-regulation of CD107a on the surface of Vg9Vd2 T cells. This NKG2D dependent cytolysis has been confirmed using purified Vg9Vd2 T cells, which kill MICA-transduced RMA cells but not the control cells. The TCR independence and NKG2D dependence of this killing is supported by mAb inhibition experiment. Finally, DAP 10, which mediates NKG2D signaling of human NK cells, is found in resting and activated Vg9Vd2 T cells. Moreover, data of intracellular signaling studies suggest an important role of Scr kinases in the NKG2D mediated killing and involvement of DAP-10-PI3K and PLCg 1 pathways as mayor proteins implicated in target cell lysis, and shows remarkable difference with the TCR signaling. The identification of these similarities in NKG2D function between NK and Vg9Vd2 T cells may be of interest for development of new strategies for Vg9Vd2 T cell-based immunotherapy in certain types of cancer and help to understand Vg9Vd2 T cell function in general.
B cells play diverse roles in the immunopathogensis of autoimmune diseases several approaches targeting B cell directly or indirectly are in clinical practice in the treatment of autoimmunity. In this regard, temporal B cell depletion by rituximab (anti CD20 antibody) is being appreciated and gaining more importance in recent years. To date, little is known about the regeneration profile of B cells following B cell depletion. We wanted to investigate the early replenishing B cells and examine the dynamic changes in the repertoire. we studied the immunoglobulin receptor (IgR) modulation of Ig-VH4 genes as representative of the heavy chain family. Five patients were included in the study and therapy induced alterations were assessed. Three time points namely before therapy, early regeneration phase (ERP- the early time point during regeneration where just above 1% B cells were found in the peripheral lymphocyte pool) and later regeneration phase (LRP- which commenced 2-3 months following ERP) were chosen. In three patients (A-C), Ig-VH4 genes were amplified from total genomic DNA during the above-mentioned all time points and in another two patients (D and E), Ig genes during ERP were studied by single cell amplification technique. Firstly, B cell regeneration followed the characteristic regeneration pattern as reported by several groups, with a predominant circulation of CD38hi expressing plasma cells and immature B cells in the ERP. During LRP, the proportion of these cells reduced relatively and the levels of naïve B cells rose gradually. On a molecular level, Ig-VH4 variable gene usage prior and post B cell depletion was determined and it was noticed that a diverse set of Ig-VH4 genes were employed in the repertoire before and after therapy. Mini gene segments such as VH4-34 and VH-4-39, which were reported to be connected with autoimmunity, were over expressed in the B cell repertoire before therapy. Profound changes were noticed in the early reemerging repertoire with a relatively increased population of intensely mutated B cells. These B cells acquired >=9 mutations in the Ig genes. Immunophenotyping with specific surface markers revealed that these highly mutated B cells evolve from the isotype-switched memory compartment especially the plasma cells. To support the hypothesis that the highly mutated B cells observed during ERP were plasma cells we carried out single cell amplification of individual plasma cells in another two patients during ERP and compared the mutational load, which remained similar. Actually plasma cells do not express CD20 on their surface and are not eliminated by rituximab therapy. However they were not observed in the peripheral blood following B cell depletion. The earliest time point when plasma cells are found again in peripheral circulation is the early recovery period (ERP). Therefore, it was intriguing to ascertain if the plasma cells were also modulated by rituximab therapy although they were not directly targeted by the therapy. We investigated if there is a therapy mediated mutational modulation of the plasma cells though these are not directly targeted by the therapy. We examined the confinement of mutations to the pre-defined RGYW/WRCY hotspot motifs (R=purine, Y=pyrimidine, W=A/T) in the plasma cells, which provides information on the involvement of T cells in B cell somatic hypermutation (SHM). Plasma cells before rituximab manifested the characteristics of active disease, which was revealed by restricted mutational targeting to the RGYW/WRCY motifs. The reemerging plasma cells during ERP had an increased targeting of the RGYW/WRCY motifs which indicated for a more pronounced T cell mediated B cell mutations which is the scenario observed in the healthy subjects. To further support the hypothesis of rituximab-mediated plasma cell modulation, we delineated the replacement to silent mutations ratio (R/S) in the hypervariable regions (CDRs) of the plasma cell Ig sequences. Within our study, the mean R/S ratio in the plasma cell CDRs of the patient group was relatively low (1.87) before rituximab treatment and interestingly this ratio increased significantly in the recirculating plasma cells to values of 2.67 and 3.60 in ERP and LRP status respectively. The increase in R/S ratios in reemerging plasma cells can be interpreted as a shaping of the Ig-repertoire by positive antigen selection as seen in healthy individuals. To conclude, our study demonstrates temporal B cell depletion by rituximab therapy seems to modulate also the plasma cell compartment, which is not directly targeted by the therapy. Modulation of plasma cells in RA could be also used as a potential biomarker in studying the effective response in RA treatment. This needs to be further explored to gain deeper insights into the underlying processes, which may be influenced by future therapies.
The process of sex-determination can be better understood through examinations of developing organs and cells, which are involved in the formation of undifferentiated gonad. This mechanisms show in fish a broad variety, ranging from hermaphroditism to gonochorism and environmental to genetic sex determination. Hormones and abiotic factors such as temperature and pH can influence teleost development and reproductive traits. These factors are vulnerable to pollutants and climate changes. Therefore, it is important to examine gonad development and sex-determination/differentiation in teleost fish. Teleost fish are the largest known group of vertebrates with approximately 25,000 species and are used for such kind of examinations as model organisms. Recently, in Oryzias latipes (medaka), dmrt1bY (or dmy), a member of the Dmrt gene family, has been described as testis-determining gene. However, this gene is not the universal master sex-determining gene in teleost fish. Although dmrt1bY is present in the most closely related species of the genus, namely Oryzias curvinotous, it is absent from other Oryzias species, like Oryzias celebensis, and other fish. During my thesis, I studied gonad development in medaka and in the closely related species Oryzias celebensis. Germ cell specification in medaka seems to be dependent on maternally provided cytoplasmatic determinants, so called germ plasm. Nanos and vasa are such germ cell specific genes. In zebrafish they are asymmetrically localized in the early embryo. I have shown that nanos mRNA is evenly distributed in the early embryo of medaka. A similar pattern has been already described for the medaka vasa homolog, olvas. This suggests differences in PGC specification in zebrafish and medaka. Further, the vasa homolog was isolated and the expression pattern examined in O. celebensis. The results show that it can be used as a germ cell specific marker. Additionally, the primordial germ cell migration in O. celebensis was followed, which is similar to medaka PGC migration. Primordial germ cell migration in vertebrates is dependent on the chemokine stromal cell-derived factor 1 (Sdf-1). Medaka has two different sdf-1 genes, sdf-1a and sdf-1b. Both genes are expressed in the lateral plate mesoderm (LPM). During late embryonic development, I could show that sdf-1a is expressed in newly formed somites and not longer in the LPM. Sdf-1b expression persisted in the posterior part of the lateral plate mesoderm in the developing gonad. In terms of early and late functions, this suggests subfunctionalization of sdf-1a and sdf-1b. In “higher” vertebrates, genes that are involved in the process of gonad development have been studied in detail, e.g. Wt1, Sox9, and Amh. I have analyzed the expression pattern of wt1 and sox9 co-orthologs and amh. In both, the medaka and O. celebensis, wt1a transcripts were localized in the LPM and its expression was similar to sdf-1a gene expression in medaka. Wt1b expression was restricted to the developing pronephric region. During later embryonic development, wt1a is specifically expressed in the somatic cells of the gonad primordium in both sexes. This is the first time that in fish wt1 gene expression in developing gonads has been described. Therefore, this result suggests that wt1a is involved in the formation of the bipotential gonad. Furthermore, I have analyzed the gonad specific function of the wt1 co-orthologs in medaka. I could show that a conditional co-regulation mechanism between Wt1a and Wt1b ensures PGC maintenance and/or survival. The expression of sox9 genes in medaka and sox9b in O. celebensis were detected in the somatic cells of the gonad primordium of both sexes. Additionally, I have shown that amh and amhrII in medaka are expressed in somatic cells of the gonad primordium of both sexes. This suggests that sox9b, amh and amhrII are involved in gonad development and have specific functions in the adult gonad. In O. celebensis I could detect an expression of dmrt1 already six days after fertilization in half of the embryos, which is similar to the dmrt1bY expression in medaka. Whether the expression of dmrt1 is male specific in O. celebensis is currently under investigation. Altogether, the obtained results provide new insights into gene expression patterns during the processes of gonad development. Furthermore, no differences in the expression pattern of wt1a and sox9b during gonad development between the medaka and O. celebensis could be detected. This might indicate that the genetic mechanisms during gonad development are similar in both species.
Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is an autosomal recessive neuronal disorder in infants. The disease is marked by early onset of respiratory distress and predominantly distal muscle weakness, as consequences of diaphragmatic paralysis and progressive degeneration of  motor neurons in the spinal cord, respectively. Genetically, SMARD1 is caused by mutations in the single gene encoding Immunoglobulin µ-Binding Protein 2 (IGHMBP2). Despite the tissue specific degeneration observed in SMARD1 patients, the disease gene product IGHMBP2 is ubiquitously expressed in human and mouse tissues. Therefore, SMARD1 appears to be a motor neuron disease caused by the malfunction of a “housekeeping” protein, rather than a neuron specific factor. IGHMBP2 harbors an N-terminal DEXDc-type helicase/ATPase domain and has been classified as a member of the Superfamily 1 (SF1) of helicases. This protein has been assigned to various cellular activities such as DNA replication, pre-mRNA splicing and transcription. However its precise function in either process has remained elusive. The study presented here aimed at the enzymatic characterization of IGHMBP2, the identification of a specific cellular process to which IGHMBP2 is connected and the role of this factor in the pathophysiology of SMARD1. As a first step toward this end, a two-step purification strategy was established, which enabled the large-scale purification of properly folded and enzymatically active IGHMBP2. In vitro enzymatic studies using this recombinant protein defined IGHMBP2 as an ATP-dependent helicase that catalyzes unwinding of duplices composed of either DNA or RNA in a 5’→3’ direction. In contrast to previous reports, indirect immunofluorescence studies revealed a predominantly cytoplasmic localization of IGHMBP2. Size-fractionation studies and affinity-purification experiments further showed that IGHMBP2 is part of an RNase-sensitive macromolecular complex, which was identified as the ribosome. Interestingly, IGHMBP2 was abundantly detected in both subunits as well as to 80S ribosomes but only in small amounts in actively translating polysomes. These data strongly point to a role of IGHMBP2 in ribosomes-associated gene regulation control, such as in mRNA stabilization or mRNA translation. However, its precise function in those pathways remains to be identified. The biochemical and enzymatic characterization of IGHMBP2 allowed for the first time insights into the pathomechanism of SMARD1. SMARD1-causing pathogenic IGHMBP2 variants were investigated for their enzymatic activities and interaction with ribosomal subunits. Interestingly, among all missense mutations that have been tested thus far, none obstructs association with ribosomal subunits. However, these mutants exhibit specific defects in either the ATPase or RNA helicase activity or both. The data suggest that defects in the enzymatic activity of IGHMBP2 directly correlate with the pathogenesis of SMARD1. Furthermore, these data also raise the possibility that the disease SMARD1 is caused by alterations in the cellular translation machinery.
Infrared photodissociation spectroscopy of ionic hydrocarbons : microsolvation and protonation sites
(2007)
This work has presented a spectroscopic analysis of three types of hydrocarbon cations: two ionized aromatic hydrocarbons, two protonated aromatic hydrocarbons and the cation of a fundamental radical hydrocarbon. The experiments were centered on the proton stretch vibrations of mass-selected complexes of these systems and polar (H2O) and non polar (Ar, N2, CO2) ligands. The experiments have been done in a tandem mass spectrometer coupled with an electron impact ionization ion source; an OPO laser system was used as tunable IR light source. All the proposed dimer structures have been also modeled using quantum chemical calculations (QCC). These calculations have consistently been matched with the experimental results and have enabled clear identification of the spectral features observed. This has enabled the evaluation of thermochemical properties which could not be extracted directly from experiment. The experiments done on complexes of 1-Np+ and Im+ have allowed for the acidity of their various groups to be probed: the shifts in the frequency as well as the enhancement in the intensity of the OH and NH stretch vibrations resulting from the complexation have yielded dependences on both the species (L) and the number (n) of the ligands. OH bound 1-Np+···Ar has been detected for the first time, showing that the REMPI-IRPD method is severely limited with respect to the production of the most stable isomer of a given cationic complex. The detection of c-1-Np+···(N2)n corresponds to the first observation of c-1-Np+ complexes and enables thus direct comparison of both 1-Np+ rotamers. The shift of the NH vibration of Im+···N2(H) yielded the first experimental estimate for the PA of the imidazyl radical. It was also found that the most stable 1-Np+···Ar and Im+···Ar structures differ qualitatively from that of the corresponding neutral dimers (H-bound vs pi-bound), emphasizing the large impact of ionization on the interaction potential and the preferred recognition motif between acidic aromatic molecules (A) and nonpolar ligands. The IRPD spectra of 1-Np+···Ln and Im+···Ln yielded spectroscopic information about the CH, NH and OH stretch vibrations of bare 1-Np+ and Im+. The dependence of the shifts in the frequency of the OH and NH stretch vibrations allows for creating microsolvation models. The spectroscopic results obtained on size-selected 1-NpH+···Ln show that, in the output of the presently used ion source, three classes of 1-NpH+ isomers can be identified: oxonium ions (1-Np protonated at the O atom); carbenium ions obtained by protonation in the para and ortho positions with respect to the OH functional group; carbenium ions obtained by the addition of a proton to well-defined sites on the second naphthalene ring. The spectral identification of these three classes of protonation sites is supported by their different photofragmentation patterns. It was demonstrated that the spectroscopic monitoring of the microsolvation of ImH+ in Ar and N2 together with the QCCs paint a very detailed picture of the microsolvation process, evidencing clear differences between the microsolvation models as function of the PA of the ligands. Important differences have also been identified between the various binding sites, enabling the creation of a clear scale of priorities for occupation of the binding sites during microsolvation. The application of IRPD to the study of microhydrated ImH+ provided for the first time direct spectroscopic information on the properties of the N-H bonds of this biomolecular building block under controlled microhydration. It was demonstrated that, as protonation enhances the acidity of the NH groups, the ability for proton conductivity of ImH+ increases. A very important result is derived from the IRPD spectroscopy of C2H5+···L (L = Ar, N2, CO2, CH4) dimers. The equilibrium geometry of the C2H5+ has long been debated. Now, IRPD spectra were recorded over the range of the CH stretch fundamentals (covering possible sp3 and sp2 hybridization of C). Depending on the ligand species, the spectra are found to be dominated by the fingerprint of two largely different dimer geometries. Using the experimental C2H5+···Ar spectrum and the corresponding QCCs, the structure of the (weakly perturbed) C2H5+ was found to be the nonclassical one, with one proton straddling across the C=C bond of the H2C=CH2. On the other hand, ligands like N2 and CH4 are strongly influencing the geometry, as seen in the spectral signatures of the C2H5+···N2 and C2H5+···CH4, which correspond to the classical [H2CCH3]+. It was thus demonstrated that while the nonclassical C2H5+ is the global minimum on the PES of the free [C2,H5]+, the structure of the C2H5+ can be strongly influenced by the chemical properties of the environment.
A torsion free abelian group of finite rank is called almost completely decomposable if it has a completely decomposable subgroup of finite index. A p-local, p-reduced almost completely decomposable group of type (1,2) is briefly called a (1,2)-group. Almost completely decomposable groups can be represented by matrices over the ring Z/hZ, where h is the exponent of the regulator quotient. This particular choice of representation allows for a better investigation of the decomposability of the group. Arnold and Dugas showed in several of their works that (1,2)-groups with regulator quotient of exponent at least p^7 allow infinitely many isomorphism types of indecomposable groups. It is not known if the exponent 7 is minimal. In this dissertation, this problem is addressed.
The present work deals with the synthesis and the investigation of the photophysical properties of covalently constructed calix[4]arene–perylene bisimide dye arrays containing various PBI units. The obtained conjugates are characterized with respect towards their application in a new, zigzag-type architecture of artificial light-harvesting systems. For this purpose, orange (core-unsubstituted), red (6,7,11,12-tert-butylphenoxy-functionalized) and green (1,7-pyrrolidino-substituted) perylene bisimide building blocks have been attached to the calix[4]arene scaffold. First, the monochromophoric reference systems have been studied, and second, the photophysical properties of a comprehensive series of newly synthesized, multichromophoric calix[4]arene–perylene bisimide conjugates showing efficient energy transfer processes between the individual dye subunits have been investigated. Furthermore, a series of bichromophoric compounds containing identical chromophoric units has been obtained. Towards this goal, a variety of spectroscopic techniques such as UV/vis absorption, steady state and time-resolved fluorescence emission, and femtosecond transient absorption spectroscopy as well as a spectrotemporal analysis of the obtained data has been applied. This work presents a new concept for an artificial light-harvesting system positioning the dye units by means of calix[4]arene spacers along a zigzag chain. The investigations start with the syntheses and optical properties of the monochromophoric building blocks and result in an elaborate study on the energy and electron transfer processes occurring after photoexcitation in a comprehensive series of multichromophoric calix[4]arene–perylene bisimide conjugates. Finally, the photophysical properties of a series of compounds containing each two identical PBI units are discussed.
Sumoylation of transcription factors modulate their activity (either upregulating or downregulating) by altering protein-protein interactions as well as subcelluar/subnuclear localization. The transcription factor family of NFAT (Nuclear Factor of Activated T cells) plays an important role in cytokine gene regulation in T cells. Due to alternative usage of two promoters (P1 & P2), two polyadenylation sites (pA1 and pA2) and alternative splicing events, NFATc1 is expressed in six isoforms which are NFATc1/alphaA, betaA, alphaB, betaB, alphaC and betaC, where alpha and beta refer to two different 1st exons and A, B, C to the differentially spliced and extended C-termini. The short isoforms of NFATc1 (NF-ATc1/A) contain a relatively short C terminus whereas, the longer isoforms, B and C, span the extra C-terminal peptides of 128 and 246 aa, respectively. To analyze the specific biological effects of NFATc1 isoform, a yeast two hybrid screening of a human spleen cDNA library with extra C-terminal peptide of NFATc1 as a bait, was performed. At the end of the assay, the proteins involved in the sumoylation pathway such as Ubc9, PIAS1 were detected with highest frequencies and subsequently were were able to demonstrate that NFATc1 is sumoylated. The extent of sumoylation is isoform specific. While NFATc1/A, harboring only one sumoylation site, shows very weak sumoylation, the two additional sites within NFATc1/C lead to efficient sumoylation. This modification directs NFATc1/C into SUMO-1 bodies, which in turn colocalize with PML-nbs. Furthermore, sumoylated NFATc1/C recruits the transcriptional co-repressors HDAC (both class I as well as class II HDACs) which results in a significant decrease of the level of histone acetylation on the IL-2 promoter, an important NFATc1 target gene. As a consequence of this, a decrease of IL-2 production was observed, while NFATc1/C, which can no longer be sumoylated due to mutating the target lysines, exhibited dramatic elevated transcriptional potential on the IL2 promoter. This supports our finding from IL-2 promoter-driven reporter gene assay, which shows downregulation of NFATc1/C transactivation upon sumoylation. Hence, sumoylation exerts a negative effect on NFATc1 transcriptioanl activity. Immunofluorescence studies showed SUMO modification to relocate NFATc1/C also into transcriptionally inactive heterochromatin regions, demonstrated by H3K9 m3 (tri-methylated histone lysine 9) colocalization studies. Interestingly, in the absence of sumoylation, NFATc1 was partially colocalized with transcriptional hotspots in the nucleus, which might contribute to the higher transcription potentiality of the non-sumoylated NFATc1. It is important to note that, the transcriptional activity of other NFATc1 target genes (IL-13, IFN-gamma etc.) was positively upregulated upon sumoylation of NFATc1, suggesting a non-universal effect of sumoylation on NFATc1/C function. In conclusion, sumoylation directs NFATc1 into nuclear bodies where it interacts with transcriptional co-repressors and relocalize itself with heterochromatin, leading to repression of NFATc1/C-mediated transcription. Most importantly, the effect of NFATc1/C sumoylation is promoter specific. Taken together, SUMO modification alters the function of NFATc1 from an activator to a site-specific transcriptional repressor. This study unraveled a novel regulatory mechanism, which controls isoform specific NFATc1 function.
In this work, the laser control of molecules was investigated theoretically. In doing so, emphasis was layed on entering vectorial properties and in particular the orientation in the laboratory frame. Therefore, the rotational degree of freedom had to be included in the quantum mechanical description. The coupled vibrational and rotational dynamics was examined, which is usually not done in coherent control theory. Local control theory was applied, where the field is determined from the dynamics of a system, which reacts with an instantaneous response to the perturbation and, in turn, determines the field again. Thus, the field is entangled with the quantum mechanical motion and the presented examples document, that this leads to an intuitive interpretation of the fields in terms of the underlying molecular dynamics. The limiting case of a classical treatment was shown to give similar results and hence, eases to understand the complicated structure of the control fields. In a different approach, the phase- and amplitude shaping of laser fields was systematically studied in the context of controlling population transfer in molecules.
Microtubules are a fascinating component of the cellular scaffold protein network, the cytoskeleton. These hollow tubular structures are assembled of laterally associated proto-filaments containing ab-tubulin heterodimers in a head to tail arrangement. Accordingly microtubules have a defined polarity, which sets the base for the polarity of the cell. The microtubule lattice can be arranged in two conformations: In the more abundant B-lattice conformation, where the protofilaments interact laterally through a- to a- and b- to b-tubulin contacts and in the less stable A-lattice conformation, where a-tubulin interacts laterally with b-tubulin. In cells the microtubules generally contain 13 protofilaments of which usually one pair interacts in the A-lattice conformation, forming the so-called lattice seam. Microtubule dynamics and interactions are strongly regulated by micro-tubule associate proteins (MAPs). Structural investigations on MAPs and microtubule associated motor proteins in complex with microtubules have become possible in combination with modern electron microscopy (EM) and image processing. We have used biochemistry and different advanced EM techniques to study the interaction between microtubules and the MAP Mal3p in vitro. Mal3p is the sole member of the end-binding protein 1 (EB1) protein family in the fission yeast Schizosaccharomyces pombe. Previous in vivo studies have shown that Mal3p promotes microtubule growth. Our studies with high-resolution unidirectional shadowing EM revealed that Mal3p interacts with the microtubule lattice in a novel way, using binding sites on the microtubule that are different from those reported for other MAPs or motor proteins. Full-length Mal3p preferentially binds between two protofilaments on the microtubule lattice, leaving the rest of the lattice free. A case where Mal3p was found in two adjacent protofilament, revealed an A-lattice conformation on the microtubules, surprisingly indicating specific binding of Mal3p to the microtubule seam. With a lattice enhancer, in form of a b-tubulin binding kinesin motor domain, it was demonstrated that Mal3p stabilizes the seam which is thought to be the weakest part of a microtubule. Further, the presence of Mal3p during microtubule polymerization enhances the closure of protofilament sheets into a tubular organization. Cryo-EM and 3-D helical reconstruction on a monomeric microtubule binding domain of Mal3p, confirm the localization in between the protofilament and result in an accurate localization on the microtubule lattice. The results also indicate Mal3p’s capacity to influence the microtubule lattice conformation. Together, studies approached in vitro demonstrate that an EB1-family homolog not only interacts with the microtubule plus end, but also with the microtubule lattice. The structure of Mal3p interacting with microtubules reveals a new mechanism for microtubule stabilization and further insight on how plus end binding proteins are able promote microtubule growth. These findings further suggest that microtubules exhibit two distinct reaction platforms on their surface that can independently interact with selected MAPs or motors.
The main focus of this work was to get a deeper understanding of the relationship between the structure of sol-gel films, their densification and their macroscopic cracking. First of all titania was chosen as model system. Therefore a synthesis route starting from the preparation of long-term stable amorphous redissoluble precursor powders based on acetylacetone as chelate ligand was utilized. The solubility and stability of the powders in various solvents can be determined by chemical synthesis and technological parameters. When dissolved in a solvent mixture of ethanol and 1,5-pentanediol, thin films can be easily prepared by dip-coating technique. Thereby the quality of the titania films enormously depends on the calcinations temperature and the solvent mixture is used. In order to investigate the influence of different solvents and solvent mixtures on the microstructure and densification of the precursors, the coating solutions were stripped off (sol powder) and analyzed as function of annealing temperature. It was pointed out that a high densification rate caused by the addition of 1,5-pentanediol, results in dense microstructure with trapped residual carbon. These impurities can retard the phase transformation of anatase to rutile. The analysis of so-called “film powders” scraped off multiple dip-coated substrates provides valuable information on the effect of air moisture and unidirectional densification during drying and aging on the structure of thin films. The high surface-to-volume ratio and access to air moisture determine the chemical composition of the as-prepared film, which controls shrinkage, crystallization and defect structure of the coatings. Further it was shown, that drying as a thin film results in the formation of closed pores and much denser microstructure than the respective sol powder. Without the addition of 1,5-pentanediol all –OEt moieties undergo hydrolysis reactions, which causes the formation of a rigid network. The presence of 1,5-pentanediol retards this hydrolysis reactions and provides some network plasticity. Generally the microstructure of thin films is comparatively close to the microstructure of the film powders. The addition of 1,5-pentandiol prevents hydrolysis and condensation reactions as like in the film powders. However even at 700 °C, thin films never transform to rutile, which was attributed to the tensile stresses in thin films. In thin films and in film powders as well a comparable amount of closed pores are formed during annealing. Further it was shown that most of the thin sol-gel films investigated form a dense crust on their tops during annealing. This explains why crack free films exhibit only closed pores. However, when cracks appear during thin film shrinkage in the coating, this crust is burst, which generates open porosity. The defect density in the coatings was determined by an automated analysis of surface images. The crack formation and quantity can be directly referred to tensile stresses in the coatings, which arise from hydrolysis and condensation during thin film drying and aging. Therefore when 1,5-pentanediol is added to the sol, thin film cracking was avoided, because hydrolysis and condensation reactions are retarded, which preserves a higher network flexibility. Furthermore the crack formation was significantly influenced by the atmospheric humidity that was used during the coating process, which was explained by different drying and condensation rates. Under certain chemical starting conditions water soluble precursor powders can be also obtained. In general the observations made with the water based coating solutions are mostly in agreement with the former results based on ethanol based coating solutions. For example the high surface-to-volume ratio of film powders compared to sol powders also significantly enhances film drying and densification. The addition of 1,5-pentanediol also clearly contributes to their densification behavior and phase evolution. As seen before in the case of ethanol based coatings, 1,5-pentanediol enhances the stability towards hydrolysis and condensation reactions and preserves some network plasticity. Therefore coatings prepared without the addition of 1,5-pentanediol already form cracks during film drying and aging because of tensile stresses. Thus, the addition of 1,5-pentanediol results in a reduction/prevention of crack formation. Nevertheless some differences were observed, i.e. the critical single coating film thickness of ethanol based coatings is nearly twice that of water based coatings. This was explained by the different surface tensions of the basis solvents, which during thin film drying causes significantly higher capillary forces and tensile stresses in water based coatings. When acetylacetone is replaced by triethanolamine as chelating ligand for titanium also re-dissolvable precursor powders can be synthesized. The film powders combine a high hydrolytic stability of the precursor with sufficient intermediate network flexibility. The different type of organics changes the drying and densification behavior: i.e. in contrast to film powders obtained from acetylacetone based precursor powders the structure of triethanolamine based film powders is unaffected by the thin film drying process. This high hydrolytic stability and plasticity of this precursor allows the preparation of defect free coatings up to single film thickness of 300 nm. However triethanolamine based thin films present at intermediary annealing temperatures a distinctively different microstructure compared to acetylacetone based films. The general validity of the conclusions was proved on the basis of zirconia coatings that were also prepared by the use of re-dissolvable precursor powders. In principle all conclusions concerning the interconnection of precursor chemistry, film formation, densification and structure were transferable to the respective zirconia coatings. Differences mainly arise only from differential material properties i.e. bulk density. Finally, it has been pointed out that the findings obtained on the densification behavior of thinsol-gel films are also a valuable tool for improved explanations of other important scientific questions concerning sol-gel films, i.e. scratch resistance of sol-gel coatings, fiber -bridging and – degradation of sol-gel coated fibers.
The effective binding of anions like carboxylates and phosphates in aqueous solutions is of particular interest for various reasons. The natural archetypes of effective anion receptors are enzymes that contain often arginine as relevant amino acid in the binding pocket. For this reason, one class of artificial anion receptors that emerged more than two decades ago mimics the anion binding with the guanidinium group present in the amino acid side chain. In 1999, Schmuck and coworkers developed a new class of guanidinium-based oxo anion receptor that binds carboxylates even in aqueous media. The binding modes of the 2-(guanidiniocarbonyl)-1H-pyrroles are based on individually weak non-covalent interaction between artificial host and substrate like ion pairing and multiple hydrogen bonds. The zwitterionic derivative with substitution of a carboxylate group in position 5 of the pyrrole ring system shows a strong self-assembly to discrete dimers (dimer 1) with an estimated association constant of 170 M-1 even in water. In order to further improve the structure motif for an effective oxo anion binding it is therefore of great interest to quantify the different intermolecular interactions between two monomeric units of 1. Against this background several theoretical ab initio studies were conducted in order to elucidate the influences of intrinsic properties as well as solvent effects on the stability of self-assembled dimers. In chapter 4.1 the molecular interactions in dimer 1 were investigated by comparison to various “knock-out” analogues. In these analogues single hydrogen bonds were switched off by substitution of hydrogen donor atoms with either methylene groups or ether bridges. The calculations were done for vacuum and solvation, as represented by a conductor-like polarizable continuum. It could be shown that the application of a simple continuum solvent model fails to predict the absolute energies of the knock-out analogues in strongly polar solvents. However, the calculated trends can explain the relative stabilities. In chapter 4.2 the structural similarity of arginine with structure 1 was used in order to examine the dependence of self-assembly from the flexibility of the molecular structure. In chapter 4.2.1 new global minimum structures of the canonical and zwitterionic arginine in gas phase were found by means of exhaustive force field based conformational searches in conjunction with ab initio structure optimizations of the lowest energy conformers. Most of the newly identified minimum conformers of both the zwitterionic and canonical tautomer revealed geometrical arrangements with hitherto unreported stacked orientations of the terminal groups. Finally a novel global minimum structure was detected that is more than 8 kJ mol-1 lower in energy than the previously published conformers. The same strategy for finding minimum energy conformers of the arginine monomer has also been employed for the arginine dimer structures. While previous theoretical studies favoured directed hydrogen bonds the new global minimum structure MMFF1 is about 60 kJ mol-1 more stable and exhibits a stacked orientation of the guanidinium and carboxylate groups. The importance of rigidity on the dimer stability was proven by calculations of an artificially stiffened arginine dimer system. The high binding affinity dimer 1 results by about 50% from the rigidity of the monomers which prevents any intramolecular stabilization. In chapter 4.3 novel structure motifs with varying ring systems have been examined on a DFT level of theory in order to make proposals for an improved carboxylate binding motif. The direct dependency of the dimerization energy on an increasing dipole moment was demonstrated by various anellated ring structures. The influence of the delocalization in the monomer on the dimerization energy was examined by variation of the electronic structure of electronically decoupled biphenylenes. With the aid of various substituted 7-guanidinioindole-2-carboxylate derivatives we could show that the carbonyl function is mainly responsible for the advantageous preorganisation, whereas the effect on the acidity seems to be only of minor importance. In the last chapter cooperativity effects in supramolecular assemblies have been investigated. This was achieved by NMR shift calculations of adenosine-carboxylic acid complexes as model systems and comparison to experimental low-temperature NMR studies. We could demonstrate that only by applying vibrational averaged NMR shifts the experimental proton shifts obtained at very low temperatures in the hydrogen bond exchange regime could be reproduced.
The continuously increase in resistance of human pathogenic microorganisms to the known antibiotics leads to the necessity for searching new sources for production of new active antimicrobial compounds from different natural sources especially plants, since many plants have been found to be able to produce antimicrobial compounds as a defense phenomenon against invading microorganisms. The aim of this work is to screen cultures for production of antimicrobial activity against representative of human pathogenic microorganisms and selection the most active cell culture producing antimicrobial protein(s) which are active against these pathogenic microorganisms and also isolation ,purification of the active protein(s) and cloning of its/their genes. Ten different plant suspension cultures have been screened in presence of nine elicitors for their antimicrobial activity against five selected human pathogenic microorganisms, and it has been found that the heterotrophic cultures are more active against the tester isolates than the autotrophic ones. The intracellular fraction of the mixotrophic Arabidopsis thaliana culture elicited with salicylic acid showed the highest antimicrobial activity against the tester isolates. The presence of proteinous antimicrobial activity has been elucidated by testing the activity of ammonium sulphate precipitate against Candida maltosa. High speed centrifugation technique has been used for partial purification of the active protein. The proteinous nature of the isolated compound has been confirmed by using bioautography technique and its molecular weight could be estimated to be around 26KDa. The active protein has been purified using gel filtration, and using mass spectrometry technique, for microsequencing of the active protein, it has been found that the function of the protein is unknown and we have termed it as AtPDP1 according to Arabidopsis thaliana Plat-Domain Protein1, since it contains a plant stress domain termed PLAT domain. It has been found that a second protein from the same plant with high homology level to AtPDP1 with the same domain, we termed it as AtPDP2. Genes for AtPDP1 and AtPDP2 have been cloned in E. coli using PGEM-T easy vector. The expression of both genes have been tested using Digital Northern program, and it has been observed that both genes are induced by different pathogens, chemicals known to induce defense in plant cells and also different hormones. We tried to clone the gene for AtPDP1 in PBI121 binary vector under the control of an elicitor inducible promoter of a proteinase inhibitor gene, to test its function in plant by overexpression, but we did not succeeded. Also the work aims to cloning the different known thaumatin genes from Arabidopsis thaliana for future work which represented by testing their expression under different stimuli, since most thaumatins have antimicrobial activity and some of them are active against Candida spp..Thirteen genes of known thaumatins from Arabidopsis thaliana have been cloned in PGEM-Teasy vector in DH5-alpha cells. coli cells. The expression of the thirteen genes has been done using Digital Northern program and it has been found that different genes show different expressions under different stimuli and the expression of At1g75800 gene was the maximum under all stimuli. The minimum expression of genes was for At1g75050. The rest of thaumatin genes showed moderate expressions under different stimuli.
Despite its precise agreement with the experiment, the validity of the standard model (SM) of elementary particle physics is ensured only up to a scale of several hundred GeV so far. Even more, the inclusion of gravity into an unifying theory poses a problem which cannot be solved by ordinary quantum field theory (QFT). String theory, which is the most popular ansatz for a unified theory, predicts QFT on noncommutative space-time as a low energy limit. Nevertheless, independently of the motivation given by string theory, the nonlocality inherent to noncommutative QFT opens up the possibility for the inclusion of gravity. There are no theoretical predictions for the energy scale Lambda_NC at which noncommutative effects arise and it can be assumed to lie in the TeV range, which is the energy range probed by the next generation of colliders. Within this work we study the phenomenological consequences of a possible realization of QFT on noncommutative space-time relying on this assumption. The motivation for this thesis was given by the gap in the range of phenomenological studies of noncommutative effects in collider experiments, due to the absence in the literature of Large Hadron Collider (LHC) studies regarding noncommutative QFTs. In the first part we thus performed a phenomenological analysis of the hadronic process pp -> Z gamma -> l^+l^- gamma at the LHC and of electron-positron pair annihilation into a Z boson and a photon at the International Linear Collider (ILC). The noncommutative extension of the SM considered within this work relies on two building blocks: the Moyal-Weyl star-product of functions on ordinary space-time and the Seiberg-Witten maps. The latter relate the ordinary fields and parameters to their noncommutative counterparts such that ordinary gauge transformations induce noncommutative gauge transformations. This requirement is expressed by a set of inhomogeneous differential equations (the gauge equivalence equations) which are solved by the Seiberg-Witten maps order by order in the noncommutative parameter Theta. Thus, by means of the Moyal-Weyl star-product and the Seiberg-Witten maps a noncommutative extension of the SM as an effective theory as expansion in powers of Theta can be achieved, providing the framework of our phenomenological studies. A consequence of the noncommutativity of space-time is the violation of rotational invariance with respect to the beam axis. This effect shows up in the azimuthal dependence of cross sections, which is absent in the SM as well as in other models beyond the SM. Thus, the azimuthal dependence of the cross section is a typical signature of noncommutativity and can be used in order to discriminate it against other new physics effects. We have found this dependence to be best suited for deriving the sensitivity bounds on the noncommutative scale Lambda_NC. By studying pp -> Z gamma -> l^+l^- gamma to first order in the noncommutative parameter Theta, we show in the first part of this work that measurements at the LHC are sensitive to noncommutative effects only in certain cases, giving bounds on the noncommutative scale of Lambda_NC > 1.2 TeV. Our result improved the bounds present in the literature coming from past and present collider experiments by one order of magnitude. In order to explore the whole parameter range of the noncommutativity, ILC studies are required. By means of e^+e^- -> Z gamma -> l^+l^- gamma to first order in Theta we have shown that ILC measurements are complementary to LHC measurements of the noncommutative parameters. In addition, the bounds on Lambda_NC derived from the ILC are significantly higher and reach Lambda_NC > 6 TeV. The second part of this work arose from the necessity to enlarge the range of validity of our model towards higher energies. Thus, we expand the neutral current sector of the noncommutative SM to second order in $\theta$. We found that, against the general expectation, the theory must be enlarged by additional parameters. The new parameters enter the theory as ambiguities of the Seiberg-Witten maps. The latter are not uniquely determined and differ by homogeneous solutions of the gauge equivalence equations. The expectation was that the ambiguities correspond to field redefinitions and therefore should vanish in scattering matrix elements. However, we proved that this is not the case, and the ambiguities do affect physical observables. Our conjecture is, that every order in Theta will introduce new parameters to the theory. However, only the experiment can decide to what extent efforts with still higher orders in Theta are reasonable and will also give directions for the development of theoretical models of noncommutative QFTs.
Oxylipins are important biological active compounds that play essential roles in defense, growth, development, and reproduction of plants and animals. Oxylipins are formed either by enzymatic pathways or radical catalyzed reaction from polyunsaturated fatty acids. Products of oxidation of arachidonic acid (C20:4) in animals by enzymatic and non-enzymatic pathways are prostaglandins and isoprostanes, respectively. In plants, radical catalyzed reaction of -linolenic acid (C18:3) forms phytoprostanes and enzymatic oxidation of this fatty acid produces OPDA and jasmonic acid. Like plants, cyanobacterial membranes contain a high ratio of polyunsaturated fatty acid, about 25% of total fatty acids. Oxylipin biosynthesis and function was studied in two model cyanobacteria, Anabaena PCC 7120 and Synechocystis PCC 6803, for the first time: 1. The filamentous cyanobaterium Anabaena PCC 7120 can naturally produce phytoprostanes type I and II as well as hydroxy fatty acids like in plants but lacks the enzymatic capacity to form jasmonates (12-oxo-phytodienoic acid and jasmonic acid) and prostaglandins. Data obtained provide the first evidence for the occurence of phytoprostanes in cyanobacteria as well as in the baterial kingdom. 2. By GC-MS analysis, the E1- and F1-phytoprostanes in Anabaena PCC 7120 were detected both in free and esterified form. Their levels are comparable with those in plants, in the range of ng/g DW. In one week old cultures, there was no evidence of PPF1 in the medium but its level accumulated up to 142 ng/l in six weeks old cultures. In contrast, PPE1 was stable over time, about 20 ng/g DW. Free cellular PPE1 was found about 4 times higher than that of PPF1, 80.5  23.6 and 24.1  10.9 ng/g DW, respectively. However, there was no significant difference in the total cellular levels of PPF1 and PPE1, ranging from 150 to about 200 ng/g DW. 3. Phytoprostanes are inducible in Anabaena. In the combination of oxidative stress (200 µM H2O2 or 10 µM CuSO4) with high light intensity (330 µE.m-2.s-1) for 8 h, levels of total cellular PPE1 and PPF1 were increased about 2 to 4 times. Interestingly, unlike in higher plants, application of oxidative stress or high light intensity alone showed no phytoprostaneous induction in this cyanobacterium. 4. When Anabaena cells were treated with phytoprostanes, Anabaena cells became remarkably resistant against subsequently applied – otherwise lethal – oxidative stress. All phytoprostanes displayed a high protective effect except for PPE1. The highest protection level was contributed by a mixture of PPA1 type I and II. After preincubation of Anabena cells with 100 µM PPA1–type I/II for 16 h followed by application of 1 mM H2O2 or 50 µM CuSO4 for 5 h, A1-phytoprostane pre-treatment protected 84.2% and 77.5% of the cells from cell death, respectively. Without oxylipins pre-treatment, about 98% of the cells were dead. Surprisingly, preincubation of Anabaena with other oxylipins derived from enzymatic pathway in plants and animals showed also an effect, however, the protection effect was low and ranged from 10 to 30%. In contrast, phytoprostanes did not protect Pseudomonas syringae and Escherichia coli from the toxicity of hydrogen peroxide. However, these bacteria do not synthesize polyunsaturated fatty acids and are therefore devoid of and not exposed to endogenously formed oxidized lipids. 5. Exogenous application of 100 µM PPF1 or 1.5 mM H2O2 for 90 min did not activate the expression of isiA in Anabaena. Oxylipins also displayed no effect on shinorine and tocopherol levels in Anabaena. However, application of 100 µM PPF1 for 6 h altered the protein expression in Anabaena. Most PPF1-modulated proteins are down-regulated and related to photosynthesis. Since oxidative stress only in combination with high light intensity increased lipid peroxidation, down-regulation of photosynthesis after recognition of oxidised lipids (phytoprostanes) may be a survival strategy of Anabaena to avoid damage by peroxidized lipids. 6. Dead plants may be the main source of (exogenous) phytoprostanes in the natural environment of Anabaena. Dry hay releases PPE1 and PPF1 (11 µg/g DW) into an aqueous environment. Anabaena is the typical cyanobacterium in paddy rice fields. After harvesting, most of uneconomical parts of rice plants are abundant on the field, which may release phytoprostanes that in turn might have an impact on cyanobacteria in the rice ecosystems. However, field research is needed to clarify this suspection. 7. A new class of oxylipins, phytoprostanes type III and IV, was identified and quantified in vitro. The two main phytoprostanes, PPE1 and PPF1 (type III and IV), can be obtained by autoxidation of -linolenic acid or Borage oil (containing 25% esterified -linolenic acid). After 12 days of autoxidation and subsequent hydrolysis, 1 g of Borage oil yielded 112.71 ± 1.93 µg of PPF1 and 3.80 ± 0.14 mg of PPE1. PPB1 and PPA1 (type III and IV) were prepared by isomerization and dehydration of PPE1 (type III and IV). The overall yield of PPB1 was 1.71 ± 0.04 mg/g oil (type III) and 2.09 ± 0.12 mg/g oil (type IV). Those of PPA1 were 8.38 ± 0.35 µg/g and 10.18 ± 0.30 µg/oil, respectively. 8. A rapid HPLC-MS/MS method for phytoprostane and phytohormone analysis has been developed. This method was applied to quantify free and esterified E1- and F1-phytoprostanes type III and IV in Synechocystis PCC 6803. The in vivo phytoprostanes type III and IV are present both in free and esterified form. The total cellular level of PPE1 type III and IV in Synechocystis is at least 2 times higher than that of PPF1. Unlike Anabaena, PPE1 and PPF1 were detectable in the medium of one week old Synechocystis cultures. Free levels of PPF1 in the medium (231.8 ± 36.2 ng/l) and in the cells (164.9 ± 15.2 ng/g DW) are lower than those of PPE1 (1003.3 ± 365.2 ng/l and 2331.0 ± 87.7 ng/g DW).
With ageing, the loss of bone mass correlates with the expansion of adipose tissue in human bone marrow thus facilitating bone-related diseases like osteopenia and osteoporosis. The molecular mechanisms underlying these events are still largely unknown. Reduced osteogenesis and concurrently enhanced adipogenesis might not only occur due to the impairment of conventional osteogenic differentiation originating from mesenchymal stem cells (MSCs). Additionally, transdifferentiation of (pre-)osteoblasts into adipocytes could contribute to the fatty conversion. Therefore, the aim of the present study was to prove the existence of transdifferentiation between the adipogenic and osteogenic lineage and to elucidate molecular mechanisms underlying this phenomenon. At first, a cell culture system of primary human MSCs was established that allowed for differentiation into the adipogenic and osteogenic lineage and proved that the MSC-derived adipocytes and pre-osteoblasts were capable of transdifferentiation (reprogramming) from one into the other lineage. Thereby, lineage-specific markers were completely reversed after reprogramming of pre-osteoblasts into adipocytes. The osteogenic transdifferentiation of adipocytes was slightly less efficient since osteogenic markers were present but the adipogenic ones partly persisted. Hence, plasticity also reached into the differentiation pathways of both lineages and the better performance of adipogenic reprogramming further supported the assumption of its occurrence in vivo. The subsequent examination of gene expression changes by microarray analyses that compared transdifferentiated cells with conventionally differentiated ones revealed high numbers of reproducibly regulated genes shortly after initiation of adipogenic and osteogenic reprogramming. Thereof, many genes were correlated with metabolism, transcription, and signal transduction as FGF, IGF, and Wnt signalling, but only few of the established adipogenesis- and none of the osteogenesis-associated marker genes were detected within 24 h after initiation of transdifferentiation. To find possible key control factors of transdifferentiation amongst the huge amount of regulated genes, a novel bioinformatic scoring scheme was developed that ranked genes due to their potential relevance for reprogramming. Besides the reproducibility and level of their regulation, also the possible reciprocity between the adipogenic and osteogenic transdifferentiation pathway was taken into account. Fibroblast growth factor 1 (FGF1) that ranked as one of the leading candidates to govern reprogramming was proven to inhibit adipogenic differentiation as well as adipogenic transdifferentiation in our cell culture system. Further examination of the FGF signalling pathway and other highly ranked genes could help to better understand the age-related fatty degeneration at the molecular level and therefore provide target molecules for therapeutic modulation of the plasticity of both lineages in order to inhibit adipogenic degeneration and to enhance osteogenesis.
The Nuclear Factors of Activated T cells (NFATs) are critical transcription factors that direct gene expression in immune and non-immune cells. Interaction of T cells with Ag-presenting cells results in the clustering of T-cell antigen receptor (TCR), co-receptors and integrins. Subsequent signal transduction resulting in NFAT activation leads to cytokine gene expression. Among the NFATs expressed in T cells, NFATc1 shows a unique induction property, which is essential for T cell differentiation and activation. It was revealed before that 3 major isoforms of NFATc1 are generated in activated T cells – the inducible short NFATc1/A, and the longer isoforms NFATc1/B and C. However, due to alternative splicing events and the existence of two different promoters and two alternative polyadenylation, we show here that 6 isoforms are synthesized in T cells which differ in their N-terminal and C-terminal peptides. In these experiments, we have identified these 6 isoforms by semi-quantitative long distance RT-PCR in several T cells subsets, and the inducible properties of 6 isoforms were investigated in those cells. The short NFATc1/A which is under control of the P1 promoter and the proximal pA1 polyadenylation site was the most prominent and inducible isoform in T effector cells. The transcription of the longer NFATc1/B and C isoforms is constitutive and even reduced in activated T lymphocytes. In addition to NFATc1 autoregulation, we tried to understand the NFATc1 gene regulation under the control of PKC pathways by microarray analysis. Compared to treatment of T cells with ionomycin alone (which enhances Ca++ flux), treatment of cells with the phorbolester TPA (leading to PKC activation) enhanced the induction of NFATc1. Microarray analysis revealed that PKC activation increased the transcription of NF-B1, Fos and JunB, which are important transcription factors binding to the regulatory regions of the NFATc1 gene. Besides the promoting effect of these transcription factors, we provided evidence that p53 and its targeting gene, Gadd45, exerted a negative effect on NFATc1 gene transcription. Summarizing all these results, we drew novel conclusions on NFATc1 expression, which provide a more detailed view on the regulatory mechanisms of NFATc1 transcription. Considering the high transcription and strong expression of NFATc1 in various human lymphomas, we propose that similar to NF-B, NFATc1/A plays a pivotal role in lymphomagenesis.
Cardiovascular disease is the major cause of mortality morbidity in both men and women in industrialized countries. The incidence of cardiovascular diseases in pre-menopausal women is lower compared to age-matched men but the risk of heart diseases increases dramatically after the onset of menopause.Therefore, it has been postulated that female sex hormones play an important role in cardiovascular health in pre-menopausal women. In contrast to clinical data, which failed to show positive estrogen effects on cardiovascular system of post- menopausal women, extensive experimental studies indicated cardioprotective effects of estrogens in laboratory animals. The majority of experimental estrogen substitution studies were performed with young individuals, thus the effects of ageing remain neglected and are poorly understood. The present project is the first attempt to study the cardiac effects of each estrogen receptor isoform (estrogen receptor alpha (ERa) and estrogen receptor beta (ERb)) in adult (“menopausal”) and senescent (“post- menopausal”) hypertensive rats. The female senescent spontaneously hypertensive rats (SHR) served as a model system for age- associated hypertension in females whereas young individuals were used for control experiments. Young and senescent SHR rats were treated with 17b- estradiol as well as new estrogen receptor isoform selective ligands 16a-LE2 (ERa agonist) and 8b-VE2 (ERb agonist). The results showed different functions of both estrogen receptor isoforms in cardiovascular system: ERa attenuated cardiac hypertrophy but not hypertension whereas ERb could significantly reduce both, blood pressure and cardiac hypertrophy. Surprisingly, both agonists and 17b- estradiol were effective in young animals but not in senescent SHR rats. These findings match with the clinical data and could be related to altered estrogen metabolism in senescent rats, since estrogen plasma levels did not increase to measurable extent in senescent animals receiving estrogen. Estrogen is metabolized by several 17b- hydroxysteroid dehydrogenase isoforms. In the current study, 17b- hydroxysteroid dehydrogenase type 10 (17b- HSD10) was identified as a novel protein- protein interaction partner of estrogen receptor alpha ligand binding domain (ERaLBD) in human heart. Cellular localization experiments of ERa in the cardiac myocytes showed nuclear and cytosolic localization pattern which overlapped partially with that of cardiac mitochondria. 17b-HSD10 is localized only in mitochondria. Direct interaction of both proteins was confirmed by pull- down experiments where 17b-HSD10 could be co-precipitated with ERa. Interestingly, protein interaction could be detected only under estrogen- free conditions whereas the presence of estrogen in the system blocked this interaction. Enzymatic assay which was developed in our laboratory, helped to define functional relevance of this interaction. The data obtained from enzymatic assays and protein- protein interaction studies strongly suggest that estrogen receptor could play an important role in the control of intracellular (or mitochondrial) estogen metabolism. The second potential ERa interaction partner in the heart- bladder cancer associated protein 10 (BLCAP10) - was initially identified in non- invasive bladder cancer cell lines. BLCAP10 protein expression in the heart as well as its localization pattern in cardiac myocytes is shown in the last part of the theses. Due to perinuclear localization similarity with ERb, we conclude that BLCAP10 could interact with ERb rather than with ERa. Poor BLCAP10 protein overexpression and toxicity in both, bacteria and eukaryotic cells, suggested that BLCAP10 could be involved in cell- cycle and/ or protein expression control. In summary, the results showed that isoform selective activation of estrogen receptors exert divergent effects in the cardiovascular system both by upregulation of aMHC expression or by lowering blood pressure. Hormones were effective in young animals but had only minor effects in senescent rats. The new ERa protein- protein interaction partners identified during the project provide new information about estrogen receptor function in the heart and its possible role in the regulation of estrogen homeostasis.
Participants trained aiming movements of the right hand to several targets with a prism-like virtual displacement of the location of one of the targets, receiving either terminal or continuous visual feedback. After training, the same targets were to be reached with the untrained left hand under manipulated feedback conditions. The right hand movements continuously adapted to the unnoticed visual displacement, significantly less with continuous than with terminal feedback. Under terminal but not under continuous feedback the adaptation to the manipulated target generalized to targets in the same horizontal direction but not to targets in the opposite direction. Finally, the movements of the untrained left hand showed the same qualitative changes to the targets as the movements of the trained right hand. The data are in line with the notion that the adaptation of the right hand movements is mainly based on a re-interpretation of target locations on which movement control of both hands draws.
This thesis describes the inclusion of dynamical effects in the theoretical calculation of Electron Paramagnetic Resonance (EPR) spectroscopic parameters. The studies were performed using Density Functional Theory (DFT) methodology and a perturbation-theoretical approach to g-tensor calculations. Hydrogen atoms trapped in octasilasesquioxane cages display unexpectly high, positive g-values. Computational simulation of these systems successfully reproduced the positive g-values and found them to arise from spin-orbit coupling around the oxygen nuclei. Dynamical effects were estimated by calculating the potential well in which the hydrogen atom moves. Semiquinone radical anions are important bioradicals that play a role in photosynthesis and respiration. The simplest and most prototypical, benzosemiquinone anion, was simulated both in the gas phase and in aqueous solution by Car-Parrinello Molecular Dynamics (CPMD). The neutral benzoquinone was also simulated for comparison. The solvation environments of both the anionic and neutral molecules were analysed and compared. EPR parameters were calculated for the semiquinone, providing the first example of full inclusion of dynamic effects in g-tensor calculation. The effects of different solvation interactions on the g-tensor and hyperfine interactions were extensively examined. Additionally, static calculations (i.e., calculations not incorporating any dynamical effects) were performed. Comparison between these (and prior computational studies) and the dynamical system allowed an assessment of the effects of dynamics on solvation and EPR parameters. Ubisemiquinone radical anion, one of the most widely-occurring semiquinone radicals, was simulated in the aqueous phase using CPMD. The solvation environment was analysed and EPR parameters were calculated. The motion of the side-chain, and its effects on solvation and EPR parameters, were examined.
The internal transcribed spacer 2 (ITS2) of the ribosomal gene repeat is an increasingly important phylogenetic marker whose RNA secondary structure is widely conserved across eukaryotic organisms. The ITS2 database aims to be a comprehensive resource on ITS2 sequence and secondary structure, based on direct thermodynamic as well as homology modelled RNA folds. Results: (a) A rebuild of the original ITS2 database generation scripts applied to a current NCBI dataset reveal more than 60,000 ITS2 structures. This more than doubles the contents of the original database and triples it when including partial structures. (b) The end-user interface was rewritten, extended and now features user-defined homology modelling. (c) Other possible RNA structure discovery methods (namely suboptimal and shape folding) prove helpful but are not able to replace homology modelling. (d) A use case of the ITS2 database in conjunction with other tools developed at the department gave insight into molecular phylogenetic analysis with ITS2.
This work studies the convergence of trajectories of gradient-like systems. In the first part of this work continuous-time gradient-like systems are examined. Results on the convergence of integral curves of gradient systems to single points of Lojasiewicz and Kurdyka are extended to a class of gradient-like vector fields and gradient-like differential inclusions. In the second part of this work discrete-time gradient-like optimization methods on manifolds are studied. Methods for smooth and for nonsmooth optimization problems are considered. For these methods some convergence results are proven. Additionally the optimization methods for nonsmooth cost functions are applied to sphere packing problems on adjoint orbits.
The live sciences currently undergo a paradigm shift to computer aided discoveries. Discoveries in the live sciences were historically made by either direct observation or as a result of chemical assays. Today we see a growing shift toward computer aided analysis and visualization. This gradual process happens in microscopy. Multidimensional laser scanning microscopy can acquire very complex multichannel data from fixed or live specimen. New probes such as visible fluorescent proteins let us observe the expression of genes and track protein localization. Ion sensitive dyes change intensity with the concentration of ions in the cell. The laser scanning confocal allows us to record these processes in three dimensions over time. This work demonstrates the application of software analysis to multidimensional microscopy data. We introduce methods for volume investigation, ion flux analysis and molecular modeling. The visualization methods are based on a multidimensional data model to accommodate complex datasets. The software uses vector processing and multiple processors to accelerate volume rendering and achieve interactive rendering. The algorithms are based on human visual perception and allow the observer a wide range of mixed render modes. The software was used to reconstruct the pituitary development in zebrafish and observe the degeneration of neurons after injury in a mouse model. Calicum indicator dyes have long been used to study calcium fluxes. We optimized the imaging method to minimize impact on the cell. Live cells were imaged continuously for 45 minutes and subjected to increasing does of a drug. We correlated the amplitude of calcium oscillations to increasing doses of a drug and obtain single cell dose response curves. Because this method is very sensitive and measures single cell responses it has potential in drug discovery and characterization. Microtubules form a dynamic cytoskeleton, which is responsible for cell shape, intracellular transport and has an integral role in mitosis. A hallmark of microtubule organization is lateral interactions. Microtubules are bundles by proteins into dense structures. To estimate the contribution of this bundling process, we created a fractal model of microtubule organization. This model demonstrates that morphology of complex microtubule arrays can be explained by bundling alone. In summary we showed that advances in software for visualization, data analysis and modeling lead to new discoveries.
Oxygen-centered radicals are important intermediates in photobiological, mechanistic and synthetic studies. The majority of precursors of reactive oxyl radicals are labile and thus delicate to handle. Therefore N-(alkoxy)-pyridinethiones and N-(Alkoxy)-thiazolethiones have attracted attention as "mild'' photochemical source of alkoxyl radicals, in the last few years. A disadvantage of the pyridine compounds, is their sensibility to daylight. Despite of their similarities, both molecules behave surprisingly different, if photolyzed in the absence of trapping reagents. The pyridinethione compounds undergo highly efficient radical chain reactions under such conditions while the corresponding thiazolethiones react surprisingly sluggish and give rise to several unwanted side products. The properties of both compounds should be understood and optimized in the frame of this work. Additionally new compounds should be suggested that can also be applied in the photochemical alkoxyl radical generation. Some background information about the generation and application of alkoxyl radicals is provided in chapter 2. Electronic excitations and UV/vis spectroscopy together with a description of quantum chemical approaches that are able to calculate such phenomena are outlined in chapter 3. Chapter 4 deals with the description of the vertical excitation spectra. During the validation CASSCF, CASPT2, TD-DFT and RI-CC2 were tested with respect to their ability to describe the vertical excitations in both compounds. The CASPT2 approach gives accurate descriptions of the electronic excitation spectra of all compounds. The time-dependent DFT results are very sensitive on the choice of the functional and a validation of the results should be always done. On the basis of these computations the spectroscopic visible absorption bands of both compounds were assigned to a pi-->pi* transition in the thiohydroxamic acid functionality. In chapter 5 the mechanism of the thermally and the photochemically induced N,O homolysis in both compounds is unveiled. The near UV-induced N,O homolysis will start from the S2 state. The expected relaxation from the S2- to the S1-state and the dissociation process is expected to be very fast in the case of the thiazolethione compound. The potential surfaces of the pyridine compound in contrast point to a slower N,O bond dissociation. Due to the resulting faster dissociation process the excess energy which results from the photochemical activation is quenched only to small amounts. The maximal possible excess energy of the fragments is lower and a quenching is much more likely in the case of the pyridinethione compounds. This explaines the different reactivities of both compounds. For the also already successfully applied precursor system N-(alkoxy)-pyridineones the computed dissociation paths show courses that clearly predict a slow bond dissociation process. Chapter 6 deals with the tuning of the initial excitation wave length of the known pyridinethiones und thiazolethiones. In the first part the effects of substituents on the thiazolethione heterocycle was examined. The UV/vis spectra of 4 and 5 substituted thiazolethiones can be interpreted like the spectrum of the parent compound. The second part of chapter 6 deals with the identification of a substitution pattern on the pyridine heterocycle which induces a blue shift of the photo active band. The computations showed that electron rich and electron poor substituents result the same effects on the electronic excitation spectra. These substituent effects are additive, but the steric orientation of the substituents has to be taken into account. Chapter 7 describes a computer aided design of new alkoxyl radical precursors. Combining the advantages of both compounds the radical formation should be initiated by an irradiation with light at about 350 nm, and the amount of side products during the radical formation process should be small. To achieve this 18 test candidates were obtained by a systematic variation of the parent compound of the thiazolethione precursor. To identify the promising new precursor systems a screening of the lower electronic excitations of all resulting 18 systems was performed with TD-DFT. For promising systems the N,O or P,O dissociation paths, respectively, were analyzed according to the developed model. N-(methoxy)-azaphospholethione and N-(methoxy)-pyrrolethione seem to be the most promising candidates. The computations predict a strong absorption at about 350 nm respectively 320 nm. Due to the amounts of maximal excess energy and the shapes of the potential surfaces of the N,O bond dissociation paths their reactivity should resemble more the behavior of the pyridinethiones.
Directed cortical actin assembly is the driving force for intercellular adhesion. Vasodilator-stimulated phosphoprotein (VASP) participates in actin-fiber formation and VASP activity is regulated by phosphorylations. We screened for endothelial cell proteins, which bind to VASP dependent on its phosphorylation status. Differential proteomics identified αII-spectrin as novel VASP-interacting protein. αII-spectrin binds to the triple GP5-motif in VASP via its SH3 domain. cAMP-dependent protein kinase-mediated VASP phosphorylation at Ser157 inhibits αII-spectrin/VASP complex formation. VASP becomes dephosphorylated upon formation of cell-cell contacts and in confluent but not in sparse endothelial cells αII-spectrin colocalizes with non-phosphorylated VASP at cell-cell junctions. Ectopic expression of the αII-spectrin SH3 domain fused to claudin-5 translocates VASP to cell-cell contacts and is sufficient to initiate the formation of cortical actin cytoskeletons. αII-spectrin SH3 domain overexpression stabilizes cell-cell contacts and decreases endothelial permeability. Conversely, permeability of VASP-deficient endothelial cells is elevated. In a skin edema model, microvascular leakage is increased in VASP-deficient over wild-type mice. We propose that αII-spectrin/VASP complexes regulate cortical actin cytoskeleton assembly with implications for formation of endothelial cell-cell contacts and regulation of vascular permeability.
Clonidine is an agonist at alpha2-adrenergic receptors that mediate a wide variety of the physiological responses to epinephrine and norepinephrine, such as inhibition of neurotransmitter release as well as sedation and analgesia. As with other therapeutically used alpha2-agonists such as moxonidine and rilmenidine, clonidine possesses an imidazoline structure and is believed to lower blood pressure not only via central and peripheral alpha2-receptors, but perhaps even more so by acting on central “imidazoline I1 receptors” in the brain stem. The molecular structure of these hypothetical “imidazoline I1 receptors” has not yet been identified. In order to test whether ligands with an imidazoline structure elicit pharmacological effects via alpha2-adrenergic receptors or via “imidazoline receptors”, mice were generated with a targeted deletion of all three alpha2-adrenergic receptor subtypes (alpha2ABC-KO). These alpha2ABC-KO mice were an ideal model in which to examine the pharmacological effects of the centrally acting antihypertensives clonidine, moxonidine and rilmenidine in the absence of alpha2-adrenergic receptors. As expected, sedative and analgesic actions of clonidine were completely absent in alpha2ABC-KO mice, confirming the sole role of alpha2-receptors in these properties of clonidine. Clonidine significantly lowered heart rate in anesthetized alpha2ABC-KO and wild-type mice by up to 150 beats/min. A similar bradycardic effect of clonidine was observed in isolated spontaneously beating right atria from alpha2ABC-KO mice. After treatment with the specific If inhibitor ZD 7288, clonidine was no longer able to lower spontaneous beating frequency, suggesting a common site of action. Furthermore, in HEK293 cells stably transfected with HCN2 and HCN4, it could be shown that clonidine inhibits the If current via blockade of pacemaker channels with similar affinity as in isolated alpha2ABC-KO and wild-type atria. This inhibition was demonstrated again in isolated sinoatrial node (SAN) cells from alpha2ABC-KO mice and was identical in potency and efficacy to clonidine inhibition observed in isolated wild-type SAN cells, confirming that inhibition of atrial HCN channels constitutes the alpha2-independent bradycardic action of clonidine. Direct inhibition of cardiac HCN pacemaker channels contributes to the bradycardic effects of clonidine in gene-targeted mice. Thus clonidine-like drugs represent novel structures for future HCN channel inhibitors.
In this thesis we have investigated the effect of NFAT (Nuclear Factor of Activated T Cell) transcription factors on the expression of Rag-(Recombination Activating Genes) genes in murine thymus. The protein products of Rag genes, RAG1 and RAG2, are critical for the recombination and generation of the TCR (T Cell Receptor) repertoire during thymocyte development, and their expression can be suppressed by the activity of NFAT factors. In thymus, the expression of Rag1 and Rag2 genes is induced at the double-negative (DN, CD4-8-) 3 stage, down-regulated at the DN4 stage, re-induced at the double-positive (DP, CD4+8+) stage, and suppressed again at the single-positive (SP, CD4+8- or CD4-8+) stage. Although it is known that TCR signaling suppresses the expression of Rag1 and Rag2 at the SP stage, the signals that mediate the Rag gene down-reulation remain elusive. Here we report that both the calcineurin-NFAT-signaling and MAPKinase signaling pathways, which are activated by TCR signaling during positive selection, mediate the Rag gene down-regulation in DP thymocytes. The calcineurin-NFAT pathway suppresses both the Rag1 and the Rag2 gene expression. This pathway has a stronger suppressive effect on the Rag1 than the Rag2 gene. A synergistic activity between the two NFAT factors NFATc2 and NFATc3 is essential for calcineurin-NFAT signaling to efficiently suppress the Rag gene expression in DP thymocytes. It is likely that the calcineurin-NFAT signaling down-regulates Rag gene expression by suppressing both the Rag anti-silencer element (ASE) activity and the Rag promoter activity. Similarly, MEK-ERK signaling of MAPK signaling pathway mediates the Rag gene suppression in DP thymocytes although the mechanism through which MEK-ERK mediates the Rag gene down-regulation has to be elucidated. In DN thymocytes, it appears that neither the calcineurin-NFAT signaling nor MAPK signaling is involved in the Rag gene down-regulation. However, a role for these two signaling pathways in the Rag gene up-regulation in DN thymocytes is not excluded. In DN thymocytes, pre-TCR signaling stimulates the expression both Nfatc1 and Nfatc2 genes but has no effect on Nfatc3 gene expression. In DN thymocytes, pre-TCR signaling activates Nfatc1α expression but not Nfatc1ß expression, i.e. the two promoters controling Nfatc1 gene xpression are differently controled by pre-TCR signals. Nfatc1α gene expression in DN thymocytes is mainly regulated by the MAPK signaling pathway because activation of Nfatc1α is mediated by MEK-ERK signaling but opposed by JNK signaling. Calcineuirn-NFAT and p38 signaling pathways are not involved in Nfatc1α promoter regulation in DN thymocytes. In DP thymocytes, TCR signaling up-regulates Nfatc1 and Nfatc2 expression but down-regulates Nfatc3 expression. In DP thymocytes, TCR signaling activates Nfatc1α expression. The activation of Nfatc1α in DP thymocytes is mediated by NFATc1, but not or to a less degree by NFATc2 and NFATc3. MEK-ERK, JNK, and p38 signaling pathways are involved in Nfatc1α gene activation in DP thymocytes, probably by activating NFAT trans-activation activity. All these findings illustrate that in thymocytes the expression of NFAT transcription factors – which are essential for thymic development - is controled at multiple levels.
A series of experiments was conducted in order to investigate motor contributions to learning highly skilled action sequences in contrast to sensory contributions. Experiments 1–4 made use of a bimanual-bisequential variant of the serial reaction time task: Presentation of imperative stimuli was arranged such that participants’ left-hand and right-hand responses followed different sequences independently of one another, thus establishing a compound sequence spanning both hands. At least partly independent learning of the two concurrently implemented hand-related sequences was demonstrated after extensive practice under condi-tions of both simultaneous (Experiments 1 & 2) and alternating (Experiments 3 & 4) stimulus presentation and responding. It persisted when there was only one imperative stimulus for presenting both hand-related sequences (Experiments 2–4) instead of two separate imperative stimuli (Experiments 1 & 2), one for each sequence, even when the hand-related sequences were correlated and massive integrated learning of the compound sequence occurred (Ex-periment 4). As for the nature of the independently acquired sequence representations, trans-ferable sequence knowledge was acquired only when there was a separate imperative stimulus for each sequence (Experiments 1 & 2) but not otherwise (Experiments 2–4). The most likely stimulus-based representations which allow for intermanual transfer can be regarded as sen-sory components of highly skilled action sequences, whereas motor components can be con-sidered as being reflected in effector-specific, non-transferable sequence knowledge. The same decomposition logic applies to transferable and non-transferable sequence knowledge observed under conditions of unimanual practice of a single sequence (Experiments 6 & 7). The advantage of practicing a key press sequence with fingers of one hand as opposed to practicing it with fingers of both hands (Experiment 5) also implicates a motor component as the two assignments were equivalent in all other respects. Moreover, Experiments 6 and 7 showed that hand-specific sequence knowledge can develop after relatively little practice (as little as approximately 120 sequence repetitions). Presumably, this occurs especially in tasks with particularly pronounced requirements for coarticulation between consecutive finger movements. In sum, the present series of experiments provides compelling evidence for an effector-specific component of sequence learning. Albeit relatively small in size, it emerged consistently under various conditions. By contributing to the refinement of sequential action execution it can play a role in attaining high levels of performance.
Bacteriosponges contain large amounts of morphologically and phylogenetically diverse microorganisms in their mesohyl. The association is permanent, stable and highly specific, however, little is known about the establishment and maintenance of this association. The first aim of this Ph.D. thesis was to examine cospeciation between eight Aplysina species from the Mediterranean and Caribbean and their cyanobacterial associates. Host phylogeny was constructed with 18S rDNA and ITS-2 sequences using an alignment based on the secondary structure of the molecular markers and five different algorithms each. The genus Aplysina appeared as monophyletic. Aplysina sponges could be distinguished into a Caribbean and a Mediterranean cluster and a possible Tethyan origin is suggested. Comparison of the host phylogeny to the 16S rDNA phylogeny of the cyanobacterial strains revealed the lack of a congruent pattern. Therefore it is proposed that Aplysina sponges have not cospeciated with their cyanobacterial phylotypes and probably also not with other sponge specific microbes. The second aim of this Ph.D. thesis was to examine vertical transmission of microorganisms through reproductive stages of sponges. A general transmission electron microscopy (TEM) suvey revealed a clear correlation in that bacteriosponges always contained many microorganisms in their reproductive stages whereas non-bacteriosponges were always devoid of microbes in their reproductive stages. The transmission of the microbial community via sponge reproductive stages is concluded. Based on the previous results Ircinia felix was chosen for a detailed documentation of vertical transmission. I. felix larvae contained large amounts of microorganisms extracellularly in the central region whereas the outer region was almost free of microbes as shown by TEM. In I. felix juveniles microorganisms were located between densely packed sponge cells. The microbial profiles of I. felix adult, larvae, and juveniles were compared using denaturing gradient gel electrophoresis (DGGE). Similar microbial community patterns were found in adult and the respective larvae indicating that a large subset of the adult microbial community was vertically transmitted. In contrast, microbial communities of larvae pools released by different adult individuals seemed to be more variable. Juvenile banding patterns were a mixture of sponge specific and seawater microbes due to DNA extraction artefacts but demonstrated that at least half of the adult microbial community is present in the next generation. Finally, a comprehensive phylogenetic analysis was conducted by sequencing excised DGGE bands from adult and offspring of the bacteriosponges Agelas wiedenmayeri, I. felix, and Smenospongia aurea and by taking additional 16S rDNA sequences of Ectyoplasia ferox and Xestospongia muta (unpublished data of the laboratory). The identification of 24 vertical transmission clusters in at least 8 eubacterial phyla demonstrates that a complex and uniform microbial community is transferred via sponge reproductive stages. Vertical transmission is specific in that the microorganisms of bacteriosponges, but not those from seawater, are passed on, but unselective in that there appears to be no differentiation between individual sponge-specific lineages. In conclusion, vertical transmission points to a mutualistic and long-term association of bacteriosponges and complex microbial consortia.
The steroid hormones corticosterone/cortisol and aldosterone are synthesized and secreted by the adrenal gland in response to stress or an altered salt-water balance. This is controlled by a negative feedback mechanism referred to as the HPA axis and the RAAS. Actions of these steroid hormones are mediated by the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR), which reside in the cytoplasm in a complex with heat-shock proteins. Both, the GR and the MR belong to the nuclear receptor superfamily and share a common protein structure consisting of three separate domains. However, they have different affinities for various ligands, their actions depend on hormone concentration, they are modulated by pre-receptor mechanisms such as the 11β-HSD2 and they are differently distributed in several tissues. Aldosterone acts via the MR in epithelial and in non-epithelial cells and regulates sodium-water homeostasis, cardiovascular function, neuronal excitability and adipocyte differentiation. So far the analysis of gene inactivation in vivo was limited to mice, but disease models in rats sometimes more closely reflect the situation encountered in humans. Since embryonic stem cells and thus gene targeting in rats is not available, we generated MR knock-down transgenic rats by lentiviral delivery of a shRNA. The F1 progeny of the founder rats showed a wide range of reduced MR mRNA and protein levels in kidney and hippocampus, the two major sites of MR expression. In contrast, expression of the highly homologous GR was unaltered, indicating specificity of gene inactivation. The two MR target genes, Sgk1 and ENaC, were up-regulated while the mRNA levels of other genes such as IK1 and SCD2 was reduced. Similar to the knock-out mice and human patients, the knock-down rats displayed typical signs of pseudohypoaldosteronism type I such as increased serum levels of aldosterone and renin as well as growth retardation. Importantly, we found a linear relationship between MR mRNA expression in kidney, serum aldosterone levels and body weight. Thus, our MR knock-down rats are amongst the first examples of RNAi in vivo and confirm that this technique allows to accomplish graded levels of gene inactivation that mimick human genetic diseases. Secondly, we investigated the role of the GR and the MR for the immunomodulatory activities of glucocorticoids (GCs) in peritoneal macrophages. GCs are involved in the modulation of macrophage function and thereby control the host’s immune responses to pathogens. Therefore, GCs are widely used for the treatment of inflammation and autoimmune diseases. However, concerning these GC activities neither the role of hormone concentration nor the differential contribution of the GR and the MR are known. At first we confirmed that both receptors but not 11β-HSD2 are expressed in peritoneal macrophages. Next, we showed that low levels of corticosterone enhance NO production as well as mRNA expression of pro-inflammatory cytokines, chemokines and enzymes required for mediator synthesis. In contrast, at high corticosterone concentrations macrophage function was strongly repressed. Importantly, inactivation of the GR by lentiviral delivery of siRNAs abrogated both the immunostimulatory and the immunosuppressive GC actions whereas inactivation of the MR had no effect. Furthermore, removal of endogenous GCs by adrenalectomy in vivo induced a pre-activated state in macrophages that could be modulated by corticosterone. We conclude that GCs exert distinct effects on macrophage function dependent on their concentration, and that they act through the GR despite concomitant expression of the MR. In summary, our results confirm that lentiviral delivery of shRNAs is an efficient means to down-regulation gene expression in primary cells and transgenic rats and thereby allows to perform functional studies on gene function that were previously limited to mice.
Inhibition of Nuclear Import of Calcineurin Prevents the Development of Myocardial Hypertrophy
(2007)
The Calcineurin/NFAT signaling cascade is a crucial transducer of cellular function. It has recently been emerged that in addition to the transcription factor NFAT, the phosphatase Calcineurin is also translocated to the nucleus. Our traditional understanding of Calcineurin activation via sustained high Ca2+-levels was also advanced by recent findings from this working group (AG Ritter), which showed that Calcineurin is activated by proteolysis of the C-terminal autoinhibitory domain. This leads to the constitutive activation and nuclear translocation of Calcineurin. Therefore, Calcineurin is not only responsible for dephosphorylating of NFAT in the cytosol thus enabling its nuclear import, its presence in the nucleus is also significant in ensuring the full transcriptional activity of NFAT. Formation of complexes between transcription factors and DNA regulates the transcriptional process. Therefore, the time that transcription factors remain nuclear is a major determinant of transcriptional activity. The movement of proteins over ~40 kDa into and out of the nucleus is governed by the nuclear pore complex (NPC). Transcription factors and enzymes that regulate the activity of these proteins are shuttled across the nuclear envelope by proteins that recognize nuclear localization signals (NLS) and nuclear export signals (NES) within the amino acid sequence of these transcription factors. In this study, the precise mechanisms of Calcineurin nuclear import and export were identified. Additionally to the nuclear localization sequence (NLS) and the nuclear export sequence (NES) within the sequence of Calcineurin, the respective nuclear cargo proteins, responsible for nuclear import, Importinβ1, and for nuclear export, CRM1, were identified. Inhibition of the Calcineurin/importin interaction by a competitive peptide, called Import Blocking Peptide (IBP), which mimicked the Calcineurin NLS, prevented nuclear entry of Calcineurin. A non-inhibitory control peptide showed no effect. Using this approach, it was able to prevent the development of myocardial hypertrophy. In Angiotensin II stimulated cardiomyocytes, both the transcriptional and the translational level was suppressed. Additionally, cell size and expression of Brain natriuretic peptide (as molecular marker for hypertrophy) were significantly reduced compared untreated controls. IBP worked dose-dependent, but did not affect the Calcineurin phosphatase activity. In conclusion, Calcineurin is not only capable of dephosphorylating NFAT, thus enabling its nuclear import, its presence in the nucleus is also important for full NFAT transcriptional activity. Using IBP to prevent the nuclear import of Calcineurin is a completely new approach to prevent the development of myocardial hypertrophy.
Neural networks can synchronize by learning from each other. For that purpose they receive common inputs and exchange their outputs. Adjusting discrete weights according to a suitable learning rule then leads to full synchronization in a finite number of steps. It is also possible to train additional neural networks by using the inputs and outputs generated during this process as examples. Several algorithms for both tasks are presented and analyzed. In the case of Tree Parity Machines the dynamics of both processes is driven by attractive and repulsive stochastic forces. Thus it can be described well by models based on random walks, which represent either the weights themselves or order parameters of their distribution. However, synchronization is much faster than learning. This effect is caused by different frequencies of attractive and repulsive steps, as only neural networks interacting with each other are able to skip unsuitable inputs. Scaling laws for the number of steps needed for full synchronization and successful learning are derived using analytical models. They indicate that the difference between both processes can be controlled by changing the synaptic depth. In the case of bidirectional interaction the synchronization time increases proportional to the square of this parameter, but it grows exponentially, if information is transmitted in one direction only. Because of this effect neural synchronization can be used to construct a cryptographic key-exchange protocol. Here the partners benefit from mutual interaction, so that a passive attacker is usually unable to learn the generated key in time. The success probabilities of different attack methods are determined by numerical simulations and scaling laws are derived from the data. If the synaptic depth is increased, the complexity of a successful attack grows exponentially, but there is only a polynomial increase of the effort needed to generate a key. Therefore the partners can reach any desired level of security by choosing suitable parameters. In addition, the entropy of the weight distribution is used to determine the effective number of keys, which are generated in different runs of the key-exchange protocol using the same sequence of input vectors. If the common random inputs are replaced with queries, synchronization is possible, too. However, the partners have more control over the difficulty of the key exchange and the attacks. Therefore they can improve the security without increasing the average synchronization time.
Ferromagnetic semiconductors (FS) promise the integration of magnetic memory functionalities and semiconductor information processing into the same material system. The prototypical FS (Ga,Mn)As has become the focus of semiconductor spintronics research over the past years. The spin-orbit mediated coupling of magnetic and semiconductor properties in this material gives rise to many novel transport-related phenomena which can be harnessed for device applications. In this thesis we address challenges faced in the development of an all-semiconductor memory architecture. A starting point for information storage in FS is the knowledge of their detailed magnetic anisotropy. The first part of this thesis concentrates on the investigation of the magnetization behaviour in compressively strained (Ga,Mn)As by electrical means. The angle between current and magnetization is monitored in magnetoresistance(MR) measurements along many in-plane directions using the Anisotropic MR(AMR) or Planar Hall effect(PHE). It is shown, that a full angular set of such measurements displayed in a color coded resistance polar plot can be used to identify and quantitatively determine the symmetry components of the magnetic anisotropy of (Ga,Mn)As at 4 K. We compile such "anisotropy fingerprints" for many (Ga,Mn)As layers from Wuerzburg and other laboratories and find the presence of three symmetry terms in all layers. The biaxial anisotropy term with easy axes along the [100] and [010] crystal direction dominates the magnetic behaviour. An additional uniaxial term with an anisotropy constant of ~10% of the biaxial one has its easy axis along either of the two <110> directions. A second contribution of uniaxial symmetry with easy axis along one of the biaxial easy axes has a strength of only ~1% of the biaxial anisotropy and is therefore barely visible in standard SQUID measurements. An all-electrical writing scheme would be desirable for commercialization. We report on a current assisted magnetization manipulation experiment in a lateral (Ga,Mn)As nanodevice at 4 K (far below Tc). Reading out the large resistance signal from DW that are confined in nanoconstrictions, we demonstrate the current assisted magnetization switching of a small central island through a hole mediated spin transfer from the adjacent leads. One possible non-perturbative read-out scheme for FS memory devices could be the recently discovered Tunneling Anisotropic MagnetoResistance (TAMR) effect. Here we clarify the origin of the large amplification of the TAMR amplitude in a device with an epitaxial GaAs tunnel barrier at low temperatures. We prove with the help of density of states spectroscopy that a thin (Ga,Mn)As injector layer undergoes a metal insulator transition upon a change of the magnetization direction in the layer plane. The two states can be distinguished by their typical power law behaviour in the measured conductance vs voltage tunneling spectra. While all hereto demonstrated (Ga,Mn)As devices inherited their anisotropic magnetic properties from their parent FS layer, more sophisticated FS architectures will require locally defined FS elements of different magnetic anisotropy on the same wafer. We show that shape anisotropy is not applicable in FS because of their low volume magnetization. We present a method to lithographically engineer the magnetic anisotropy of (Ga,Mn)As by submicron patterning. Anisotropic strain relaxation in submicron bar structures (nanobars) and the related deformation of the crystal lattice introduce a new uniaxial anisotropy term in the energy equation. We demonstrate by both SQUID and transport investigations that this lithographically induced uniaxial anisotropy overwrites the intrinsic biaxial anisotropy at all temperatures up to Tc. The final section of the thesis combines all the above into a novel device scheme. We use anisotropy engineering to fabricate two orthogonal, magnetically uniaxial, nanobars which are electrically connected through a constriction. We find that the constriction resistance depends on the relative orientation of the nanobar magnetizations, which can be written by an in-plane magnetic field. This effect can be explained with the AMR effect in connection with the field line patterns in the respective states. The device offers a novel non-volatile information storage scheme and a corresponding non-perturbative read-out method. The read out signal is shown to increase drastically in samples with partly depleted constriction region. This could be shown to originate in a magnetization direction driven metal insulator transition of the material in the constriction region.
Abiotic environmental stress, as evoked by short-term exposure of greenhousegrown plants to ambient ultraviolet radiation (UV), induces chemical and morphological adaptations of plants. Responses depend on the strength of stress and differ between species and tissues of variable age. In two Brassicaceae, Sinapis alba and Nasturtium officinale, stress responses towards short-term exposure to ambient radiation including or excluding UV reveal a high phenotypic plasticity, with strong differences their chemical composition compared to plants that remained in the greenhouse. The most pronounced defensive response against UV, the accumulation of flavonoid pigments, was strongest in young UV-exposed leaves, with an increase of the more effectice flavonol quercetin on the expense of less effectice kaempferol. Glucosinolates and myrosinase enzymes showed highly species-specific responses to UV-stress. Feeding behaviour and larval performance of the oligophagous Brassicaceae specialist, Phaedon cochleariae (Chrysomelidae; Coleoptera) were poorly affected by these differently UV-exposed host plants. Effects of plant stress on larval development were restricted to a minor variation in body mass due to variable food conversion of certain larval instars, which were compensated until pupation. Moreover, larval developmental times were unaffected by UV-exposure, but varied between species and leaves of different age. For P. cochleariae, this lack of variation in larval and pupal development towards UV-altered phytochemistry may suggest a strong genetic fixation of life history traits. In combination, the high plasticity towards variable food quality may correspond to the beetles’s specialisation on a narrow range of chemically highly variable host plants. Apart from being involved in plant defence against generalist herbivores, glucosinolates may also act as recognition cues and feeding stimulants for specialist insects. In earlier studies, glucosinolates were assumed to stimulate feeding by P. cochleariae, and they were suggested to be present on outermost leaf surfaces. However, since these findings were based on crude extraction methods, the presence of feeding stimulants in epicuticular waxes of Brassicaceae was re-investigated. In our study, glucosinolates were not detectable in mechanically removed waxes in Brassica napus and N. officinale, whereas substrate concentrations in solvent leaf extracts corresponded to densities and closure of leaf surface stomata. Therefore, glucosinolates that originate from the mesophyll may have been washed out through open stomata. Neither leaf waxes, nor leaf waxes combined with sinigrin or pure sinigrin evoked feeding. Moreover, in choice tests, these leaf beetles clearly preferred to feed on de-waxed surfaces. Finally, the presence of feeding stimulants in epicuticular waxes is highly unlikely considering the physico-chemical properties of the plant cuticle. The lack of stimulants on the outermost surface corresponds to the plant’s perspective, which should avoid easily accessible feeding stimulants. Nevertheless, the role of glucosinolates for feeding stimulation of P. cochleariae remained unclear. Therefore, S. alba leaf extracts of different polarities were tested in bioassays in order to identify which chemical leaf compounds act as stimulants. In bioassay-guided fractionations of methanol extracts by semi-preparative HPLC, two distinct fractions with stimulating activity were detected, whereas other fractions were not effective. Flavonoids were identified as main component in one stimulating fractions, the second fraction mainly contained glucosinolates, including sinalbin. The combination of both fractions was significantly more stimulating than each individual fraction, indicating additive effects of at least one compound of each fraction. However, since the combined fractions were less effective compared to the original extracts, other compounds may additionally be involved in the complex composition of leaf compounds acting as feeding stimulants for P. cochleariae. Finally, fractionated extracts of UV altered plants were used to test whether the strength of feeding responses depend on different ratios of glucosinolates and flavonoids. However, since the feeding behavior of this leaf beetle was not affected, such quantitative variations were concluded to be less important. The initiation of feeding behaviour may solely depend on the presence of stimulating compounds.
The basic question which drove our whole work was to find a meaningful noncommutative gauge theory even for the time-like case ($\theta^{0 i} \neq 0$). In order to be able to tackle questions regarding unitarity, it is not sufficient to consider theories which include the noncommutative parameter only up to a finite order. The reason is that in order to investigate tree-level unitarity or the optical theorem in loops one has to know the behavior of the noncommutative theory for center-of-mass energies much greater than the noncommutative scale. Therefore an effective theory, that is by construction only valid up to the noncommutative scale, isn't sufficient for our purpose. Our model is based on two fundamental assumptions. The first assumption is given by the commutation relations \eqref{eq:ncalg}. This led to the Moyal-Weyl star-product \eqref{eq:astproduct2} which replaces all point-like products between two fields. The second assumption is to assume that the model built this way is not only invariant under the noncommutative gauge transformation but also under the commutative one. In order to obtain an action of such a model one has to replace the fields by their appropriate \swms. We chose the gauge fixed action \eqref{eq:actioncgf} as the fundamental action of our model. After having constructed the action of the NCQED including the {\swms} we were confronted with the problem of calculating the {\swms} to all orders in $\tMN$. By means of \cite{bbg} we could calculate the {\swms} order by order in the gauge field, where each order in the gauge field contains all orders in the noncommutative parameter (\cf chapter \ref{chapter:swms}). By comparing the maps with the result we obtained from an alternative ansatz \cite{bcpvz}, we realized that already the simplest {\swm} for the gauge field is not unique. In chapter \ref{chapter:ambiguities} we examined this ambiguity, which we could parametrised by an arbitrary function $\astf$. The next step was to derive the Feynman rules for our NCQED. One finds that the propagators remain unchanged so that the free theory is equal to the commutative QED. The fermion-fermion-photon vertex contains not only a phase factor coming from the Moyal-Weyl star-product but also two additional terms which have their origin in the \swms. Beside the 3-photon vertex which is already present in NCQED without {\swms} and which has also additional terms coming from the \swms, too, one has a contact vertex which couples two fermions with two photons. After having derived all the vertices we calculated the pair annihilation scattering process $e^+ e^- \rightarrow \gamma \gamma$ at Born level. By choosing the parameter $\kggg = 1$ (\cf section \ref{sec:represent}), we found that the amplitude of the pair annihilation process becomes equal to the amplitude of the NCQED without \swms. This means that, at least for this process, the NCQED excluding {\swms} is only a special case of NCQED including \swms. On the basis of the pair annihilation process, we afterwards investigated tree-level unitarity. In order to satisfy the tree-level unitarity we had to constrain the arbitrary function $\astf$. We found that the series expansion of $\astf$ has to start with unity. In addition, the even part of the function must not increase faster than $s^{-1/2} \log(s)$ for $s \rightarrow \infty$, whereas the odd part of the $\astf$-function can't be constrained, at least by the process we considered. By assuming these constrains for the $\astf$-function, we could show that tree-level unitarity is satisfied if one incorporates the uncertainties present in the energy and the momenta of the scattered particles, \ie the uncertainties of the center-of-mass energy and the scattering angles. This uncertainties are not exclusively present due to the finite experimental resolution. A delta-like center-of-mass energy as well as delta-like momenta are in general not possible because the scattered particles are never exact plane waves.
Performance Evaluation of Efficient Resource Management Concepts for Next Generation IP Networks
(2007)
Next generation networks (NGNs) must integrate the services of current circuit-switched telephone networks and packet-switched data networks. This convergence towards a unified communication infrastructure necessitates from the high capital expenditures (CAPEX) and operational expenditures (OPEX) due to the coexistence of separate networks for voice and data. In the end, NGNs must offer the same services as these legacy networks and, therefore, they must provide a low-cost packet-switched solution with real-time transport capabilities for telephony and multimedia applications. In addition, NGNs must be fault-tolerant to guarantee user satisfaction and to support business-critical processes also in case of network failures. A key technology for the operation of NGNs is the Internet Protocol (IP) which evolved to a common and well accepted standard for networking in the Internet during the last 25 years. There are two basically different approaches to achieve QoS in IP networks. With capacity overprovisioning (CO), an IP network is equipped with sufficient bandwidth such that network congestion becomes very unlikely and QoS is maintained most of the time. The second option to achieve QoS in IP networks is admission control (AC). AC represents a network-inherent intelligence that admits real-time traffic flows to a single link or an entire network only if enough resources are available such that the requirements on packet loss and delay can be met. Otherwise, the request of a new flow is blocked. This work focuses on resource management and control mechanisms for NGNs, in particular on AC and associated bandwidth allocation methods. The first contribution consists of a new link-oriented AC method called experience-based admission control (EBAC) which is a hybrid approach dealing with the problems inherent to conventional AC mechanisms like parameter-based or measurement-based AC (PBAC/MBAC). PBAC provides good QoS but suffers from poor resource utilization and, vice versa, MBAC uses resources efficiently but is susceptible to QoS violations. Hence, EBAC aims at increasing the resource efficiency while maintaining the QoS which increases the revenues of ISPs and postpones their CAPEX for infrastructure upgrades. To show the advantages of EBAC, we first review today’s AC approaches and then develop the concept of EBAC. EBAC is a simple mechanism that safely overbooks the capacity of a single link to increase its resource utilization. We evaluate the performance of EBAC by its simulation under various traffic conditions. The second contribution concerns dynamic resource allocation in transport networks which implement a specific network admission control (NAC) architecture. In general, the performance of different NAC systems may be evaluated by conventional methods such as call blocking analysis which has often been applied in the context of multi-service asynchronous transfer mode (ATM) networks. However, to yield more practical results than abstract blocking probabilities, we propose a new method to compare different AC approaches by their respective bandwidth requirements. To present our new method for comparing different AC systems, we first give an overview of network resource management (NRM) in general. Then we present the concept of adaptive bandwidth allocation (ABA) in capacity tunnels and illustrate the analytical performance evaluation framework to compare different AC systems by their capacity requirements. Different network characteristics influence the performance of ABA. Therefore, the impact of various traffic demand models and tunnel implementations, and the influence of resilience requirements is investigated. In conclusion, the resources in NGNs must be exclusively dedicated to admitted traffic to guarantee QoS. For that purpose, robust and efficient concepts for NRM are required to control the requested bandwidth with regard to the available transmission capacity. Sophisticated AC will be a key function for NRM in NGNs and, therefore, efficient resource management concepts like experience-based admission control and adaptive bandwidth allocation for admission-controlled capacity tunnels, as presented in this work are appealing for NGN solutions.
In spite of David Lodge’s rejection of the theories labelled as poststructuralist, this thesis proves that his novels can be interpreted from a Foucauldian perspective. The concept of discourse, seen by the French philosopher as intricately linked with knowledge, power and truth, enables the distinction of four main discourses in Lodge’s novels, religious, gender, ethnic and literary. The analysis reveals that in David Lodge’s fiction there is a perpetual struggle for power illustrating Foucault’s idea of the interdependence between power, knowledge, truth and discourses circulated by institutions.
In 2001 the 433 m deep Messel 2001 borehole was drilled in the centre of the Messel Pit, 25 km south of Frankfurt (Germany). Geoscientific results from this drilling clarified the origin of the circular-shaped basin as a maar-diatreme-structure. Recovered deposits consist of lacustrine sediments (0-240 m) and volcaniclastic rocks such as lapilli tuffs (240-373 m) as well as rocks of the underlying diatreme breccia (373 433 m). The lapilli tuffs, as main interest here, show little differentiation on a macro- and microscopic scale and appear as a massive and unsorted volcaniclastic body with dominating juvenile lapilli and accidental clasts mostly in the range of (sub)millimetres to centimetres in diameter. This study presents rock magnetic properties measured on core samples of the volcaniclastic units and explains the origin of downhole magnetic anomalies detected during the drilling project in 2001. Magnetic behaviour of the erupted material is related to fine-grained, Fe-rich (titano)-magnetites, which are dispersed within the juvenile lapilli. Temperature-dependent susceptibility experiments, isothermal remanent magnetisation and hysteresis investigations demonstrate similar ferrimagnetic properties throughout the volcaniclastic material, in terms of composition, coercivity and grain size (pseudo-single-domain particles) of the ferrimagnetic minerals. Thus, during emplacement of the erupted material, the ferrimagnetic minerals had the same remanence acquisition potential. However, demagnetisation experiments show different magnetic stability behaviour of the acquired natural remanent magnetisation (NRM). Heating experiments prove the acquisition of thermal remanent magnetisation (TRM) dominated by temperature effects which could have been occurred during eruption and deposition of volcanic material, forming the Messel maar-diatreme. It is assumed that the upper half of the lapilli tuffs was deposited at relatively low depositional temperatures (<300 °C), whereas the material of the lower half took advantage of higher temperatures (>>300 °C). To understand the rock magnetic character within the Messel maar-diatreme-facies, particle grain sizes, the degree of the relative fraction dominance and the shape of the juvenile fragments have been studied in more detail. Image analytical methods as well as major and trace element analyses on the juvenile fraction support the clear subdivision of the lapilli tuffs. These findings in combination with rockmagnetic data indicate a separation into a relatively hot, geochemically undifferentiated eruption phase and a colder, differentiated phase. A two-condition eruption stage at the end of the Messel volcanic activity is suggested. The juvenile particles account for the temperature evolution and heat conditions during deposition of the Messel tuffs and contribute to the origin of magnetic field anomalies. Based on gravity parameters and the results of magnetisation properties, the potential field 3D-model of the Messel subsurface explains the negative ground anomalies, calculates the mass and volume parameters of the drilled lithozones and shows the asymmetric appearance of the diatreme-structure.
NO has been described as an important component involved in the development of the hypersensitive reaction (Delledonne et.al., 1998). Furthermore, NO induces expression of a set of defence gene, such as PR-1, PAL1 and chalcone synthase (CHS), and accumulation of SA (Durner et al., 1998). In this study, transgenic plants with altered NO levels were used to study the role of NO in plant defence. Arabidopsis plants which, due to expression of a bacterial NO dioxygenase, exhibit lower levels of NO than wild-type plants, show several weakened defence response, including the oxidative burst and expression of phenylpropanoid pathway genes. By contrast, constitutive expression of a bacterial NO synthase in Arabisopsis results in increased levels of endogenous NO. However, these plants do not show constitutively activated defence responses, but suffer from increased susceptibility to various strains of P. syringae. This might indicate that a gradient in NO production rather than constitutive elevation of NO is necessary to trigger plant defence responses. Nevertheless, NO seems to be important for regulation of the oxidative state in plant cells. This function of NO is important during leaf senescence. The data of the present work indicate that NO acts as senescence-delaying factor during plant development. The molecular action of NO in plants and signalling cascades in which NO is involved as second messenger are still poorly understood. Experiments addressing the selective quantification of NO in intact plant tissue, the identification of NO-target proteins as well as the function of NO-modified biomolecules might help to understand the role of NO in plants. Non-host resistance consists of several layers of defence that include preformed compounds existing in plants before pathogen infection and induced defences which the plant activates after recognition of a pathogen. The role of inducible defences in preventing multiplication of non-adapted bacteria is not clear. Our experiments suggest that to restrict non-adapted bacterial growth, pre-formed antimicrobial compounds and an early inducible cell wall-based defence might play an important role in Arabidopsis leaves. Upon inoculation with non-adapted bacteria, we have observed early, TTSS-independent up-regulation of PAL1 and BCB, two lignin biosynthesis genes which might be involved in papilla formation or other kinds of cell wall fortification. Moreover, Arabidopsis pal1 knockout lines permit significantly higher survival of non-adapted bacteria in leaves than wild-type plants, suggesting a functional importance of PAL1 up-regulation. Although non-host bacteria, like host bacteria, induce accumulation of SA and PR gene expression in a TTSS-dependent manner, SA-dependent or JA/ET-dependent defences do not directly contribute to non-host resistance. Moreover, non-adapted bacteria activate similar defence signalling pathways as do host bacteria. However, because of varieties in effector protein composition between different non-adapted bacterial strains, the activated signalling pathways might also include different compounds. The Arabidopsis ecotype Ler 0 is more susceptible to a non-adapted strain of P. syringae than ecotype Col-0. Although differences in glucosinolate content and composition between those ecotypes exist, they are probably not a major reason for the observed difference in non-host resistance. To further understand the mechanisms underlying non-host resistance, the generation of double or triple mutants with deficits in both cell wall-based defences and SA-dependent signal cascades is necessary. Moreover, the study of genome polymorphism and composition of secondary metabolites between Ler-0 and Col-0 can shed new light into the mechanisms of non-host resistance against bacterial pathogens. Additionally, experiments addressing papilla formation and callose biosynthesis in Ler-0 and Col-0 could help to further elucidate bacterial non-host resistance. Our data indicate that localized contact of Arabidopsis leaves with non-adapted bacteria, type III secretion-defective P. syringae strains and bacterial pathogen-associated molecular patterns (PAMPs) induce systemic acquired resistance (SAR) at the whole plant level. This finding contrasts the general belief that an HR or other leaf necroses are required for SAR induction. The observed symptomless systemic response was abolished in all SAR-deficient mutants tested in this study, but was intact in the jar1 mutant, which is compromised in induction of ISR, indicating that non-host bacteria and PAMPs induce SAR in a mechanistically similar way than host bacteria. In addition, our data show that the extent of SA accumulation or PR gene expression induced at sites of virulent or avirulent P. syringae inoculation rather than the amount of tissue necroses or jasmonate accumulation determine the magnitude of SAR. The fact that systemic responses were also triggered after local treatment with type III secretion-defective P. syringae strains and bacterial PAMPs indicate that induction of SAR is TTSS-independent. Instead, recognition of general elicitors like flagellin and LPS play an important role in activation of the SAR process. To broaden the concept of PAMP-based SAR initiation, further general elicitors from bacteria and fungal pathogens should be tested for their capability to induce SAR. Screens for mutants with deficiency in SAR activation by individual PAMPs can help to identify new components involved in the SAR signalling cascade. Possible functions of PAMPs as mobile systemic signals should be tested in future experiments. By selection of candidate genes whose expression is up-regulated in Arabidopsis leaves infected with avirulent and virulent P. syringae and pathophysiological analyses of corresponding T-DNA knockout lines, FLAVIN-DEPENDENT MONOOXYGENASE1 (FMO1) was identified as a key SAR regulator. SAR triggered by P. syringae is completely abolished in fmo1 mutant plants, and pathogen-induced expression of FMO1 in systemic leaves is closely correlated with the capability of different Arabidopsis lines to develop SAR. According to our findings, we have proposed that the FMO1 acts in signal amplification in non-inoculated, systemic leaves to trigger SAR. Experimental verification of the postulated potential amplification cycle underlying SAR should be tested in future experiments. The generation of transgenic lines expressing FMO1::GFP will provide useful information about the cellular localization of the FMO1 protein. Moreover, a comparative metabolomic analysis using SAR-induced wild-type, fmo1 knockout and FMO1 overexpressing lines can be used to identify substrates and reaction products of the FMO1 monooxygenase. As the single yeast FMO (yFMO) provides oxidizing equivalents at the ER for correct protein folding, expression of FMO1 in yfmo mutant yeast combined with protein activity assays might indicate whether FMO1 exhibits functional similarities with yeast FMO, e.g. in assuring proper folding of ER-targeted proteins essential for SAR establishment. Identification of further genes involved in activation of systemic resistance and biochemical characterization of the corresponding proteins can help to understand the SAR process in more detail.