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This thesis presents a model for the dynamical description of deconfined quark matter created in ultra-relativistic heavy ion collisions, treating quarks and antiquarks as classical point particles subject to a colour-dependent, Cornell-type potential interaction. The model provides a dynamical handle for hadronization via the recombination of quarks and antiquarks in colour neutral clusters. Gluons are not included explicitly in the model,but are described in an effective manner by the means of the potential interaction. The model includes four different quark flavours (up, down, strange and charm) and uses current masses for the quarks. The dynamical evolution of a system of colour charges subject to the Hamiltonian equations of motion of the model yields the formation of colour neutral clusters of quarks and antiquarks, which are subject only to a small remaining interaction, the strong interquark potential notwithstanding. These clusters can be mapped onto hadrons and hadronic resonances. Thus, the model allows a dynamical description of quarks degrees of freedom in heavy ion collisions, including a recombination scheme for hadronization. The thermal properties of the model turn pout to be very satisfying. The model shows a transition from a confining phase to a deconfined phase with rising temperature, going hand in hand with a softest point in the equation of state and a rise of energy density and pressure to the Stefan-Boltzmann limit of a gas of quarks and antiquarks. Moreover, the potential interaction is screened in the deconfined phase. For the dynamical description of ultra-relativistic heavy ion collision, the qMD model is coupled to UrQMD as a generator for its initial conditions. In this way, a fully dynamical description of the expansion and hadronization of the fireball created in such collisions can be achieved. Non-equilibrium aspects of the expansion dynamics and hadronization by recombination of quarks and antiquarks are discussed in detail, and a comparison with experimental data of collisions at the CERN-SPS is presented. The big advantage of the qMD model is the possibility to study cluster formation, including exotic clusters, and fluctuations in a dynamical manner. As an example, event-by-event fluctuations in electric charge are studied. Such fluctuations have been proposed as a clear criterion to distinguish a deconfined system from a hadrons gas. However, experimental data show hadron gas fluctuation measures even at RHIC, where deconfinement is taken for granted. We will see how the dynamics of quark recombination washes out the quark-gluon plasma signal in the fluctuation criterion. Moreover, we will discuss briefly the problem of entropy at recombination. In a second application, the formation of exotic hadronic clusters, larger than usual mesons and baryons, is studied. Such clusters could provide new measures for the thermalization and homogenization of a deconfined gas of colour charges. Moreover, number estimates for exotic clusters from recombination are considerably lower than corresponding predictions from thermal models, providing a clear difference between statistical hadronization and hadronization via quark recombination. A detailed analysis is provided for pentaquark candidates such as the Theta-Plus. It turns out that the distribution of exotic states over strangeness, isospin, and spin could provide a sensitive measure for thermalization and decorrelation in the deconfined quark phase, if it could be measured.
Nucleotide-binding domains (NBDs), roughly 27 kDa in size, are conservative components of the large family of ABC (ATP-binding cassette) transporters, which includes importers, exporters, and receptors. NBDs or ABC-ATPases supply energy for the translocation of a vast variety of substrates across biological membranes. Despite their hydrophilic sequence, many NBDs tend to aggregate and precipitate in solution upon isolation from the complete transporter. The conditions stabilizing an extremely labile NBD component of the E.coli HlyA transporter, HlyB-NBD, were developed. As a result, the pure highly concentrated enzyme was protected from precipitation for months that allowed screening of the unlimited crystallization conditions in the presence of different substrates and performance of the reproducible functional assays. HlyB-NBD was characterized in regard to its uncoupled ATPase activity, oligomeric state, and stability in solution. Comparative analysis of protein stability and ATPase activity in various buffers suggested an inverse relationship between the two. Kinetic analysis of ATPase activity revealed ATP-induced protein dimerization. Gel-filtration experiments with the wild type protein and H662A-mutant of HlyB-NBD provided further evidence of protein dimerization in the presence of ATP. The crystal structures in post- and pre-hydrolysis nucleotide-bound states of HlyB-NBD were determined at 1.6Å and 2.5Å resolution, respectively. While the hydrolytically deficient H662A mutant of HlyB-NBD was crystallized as a stable dimer in the presence of ATP or ATP-Mg2+, with two nucleotide molecules sandwiched between the two monomers, the same protein was shown to be a monomer in the ADP-loaded state. The wild type protein failed to develop crystals with bound ATP, yet formed ADP-bound crystals identical to those of the H662A-mutant. The X-ray structures of HlyB-NBD in various states of the hydrolytic cycle and the functional studies of the enzyme have provided an opportunity to characterize enzyme-substrate complexes and protein-protein interactions between the NBD subunits in great detail. Comparison of the nucleotide-free, the ADP-, and the ATP-loaded states revealed oligomeric and conformational changes of the protein upon substrate binding and resulted in a molecular picture of the catalytic cycle. The correlated results of the structural and functional investigations of HlyB-NBD are discussed with relation to the mechanism of action of ABC transporters.
The analysis of doxorubicin-loaded poly(butyl cyanoacrylate) nanoparticles in in vitro glioma models
(2005)
The use of doxorubicin for the treatment of glioma tumours would be an important approach in the chemotherapy treatment since doxorubicin is a very effective neoplastic agent. However, one problem faced by the use of doxorubicin for the treatment of brain tumours is the fact that doxorubicin is a substrate of an efflux pump protein, P-glycoprotein (P-gp), which is located on the luminal side of the brain capillary endothelium and in many tumour cells, which acts pumping out of the cell such substrate, and blocking its transport into the cell. A strategy to enhance the doxorubicin delivery into the brain would be the use of nanoparticles. This work showed, that the treatment of doxorubicin bound to poly(butyl cyanoacrylate) nanoparticles decreased the viability of the three glioma cell lines, the GS-9L, the RG-2, and the F-98 cell lines significantly in comparison to doxorubicin in solution, indicating an improvement of the nanoparticles-bound doxorubicin transport into the cells. The modification of the nanoparticles surface with different surfactants may even enhance the delivery of the drug into the cells. Searching for an improvement of the doxorubicin internalization, the nanoparticles surface was modified using polysorbate 80, poloxamer 188 and poloxamine 908 surfactants. The poloxamer 188 and polaxamine 908 surfactant modified nanoparticles did not show a significant enhancement of the doxorubicin internalization. Contrary, the treatment of polysorbate 80 surfactant modified nanoparticles led in some cases to a significant decrease of cancer cell viability. The use of doxorubicin in the three glioma cell lines allowed the measurement of different responses towards doxorubicin treatment. The different responses were due to the entry of various amounts of doxorubicin into the glioma cells, which express the P-glycoprotein in their cellular membrane. A higher level of the P-gp expression correlated with a weaker response towards the doxorubicin treatment. The GS-9L cell line showed a significant higher level of P-gp expression than the F-98, and RG-2 cell lines, and consequently, the GS-9L cell line presented the highest resistance to doxorubicin with the highest viability values after doxorubicin treatment. Due to the fact that the transport of doxorubicin is governed by the activity of the P-gp in the studied glioma cells, the use of poloxamer 185 as a P-gp inhibitor resulted in an enhancement of the uptake as well as of the accumulation of doxorubicin into the cells. The effect of poloxamer 185 on the doxorubicin uptake was significant marked in the case of doxorubicin-resistance cells, as the GS-9L cell line. In some cases, the presence of the nanoparticles formulation showed also an influence on such uptake improvement. The use of a P-gp inhibitor in combination with chemotherapeutic agents leads to encouraging results. Because of the wide spectrum of substances acting as P-gp inhibitors, the exact inhibitory mechanisms remain still unclear. For instance in our results the evaluation of a described P-gp inhibitor, polysorbate 80 did not show an important improvement in the doxorubicin uptake in the P-gp-expressing cell line, GS-9L. On the other hand, the Polysorbate 80-Dox-PBCA nanoparticles formulation decreased in greater extend the viability of the glioma cells than the poloxamer185-Dox-PBCA nanoparticles. Although, the P-gp inhibition was undoubtedly higher in the presence of poloxamer 185, polysorbate 80 showed a main effect on the disruption of the cellular membrane, resulting in an important cellular viability decrease. It seems that poloxamer 185 presents a direct effect on the functionality of the P-gp protein, which would be of great importance in the sensitization of resistant cancer cells. The range of concentration of poloxamer 185 is very important to yield an inhibitory effect on the P-gp-mediated transport mechanism. The accumulation of Rhodamine-123 (Rho-123), a known P-gp substrate, increased in a range of concentration from 0.001 % to 0.01, whereas at 0.1 % poloxamer 185 the accumulation significantly decreased. A maximal Rho-123 accumulation was reached at 0.01 % poloxamer 185.
Stem cells capable of self-renewal and differentiation into multiple tissues are important in medicine to reconstitute the hematopoietic system after myelo-ablative chemo- or radiotherapy. In the present situation, adult stem cells such as Mesenchymal stem cells (MSC) and Hematopoietic stem cells (HSC) are used for therapeutic purposes. For tissue regeneration and tissue constitution, engraftment of transplanted stem cells is a necessary feature. However, in many instances, the transplanted stem cells reach the tissues with low efficiency. Considering the three-step model of leukocyte extravasation by Springer et al, the rolling, adhesion and transmigration form the three major steps for the transplanted stem cells to enter the desired tissues. One of the molecular switches reported to be involved in these mechanisms are the Rho family GTPases. The present study investigates the role of Rho GTPases in adhesion and migration of stem and progenitor cells. Chemotactic and chemokinetic migration assays, transendothelial migration assays, migration of cells under shear stress, microinjection, retroviral and lentiviral gene transfer methods, oligonucleotide microarray analysis and pull down assays were employed in this study for the elucidation of Rho GTPase involvement in migration and adhesion of stem and progenitor cells. The transmigration assay used for the migration determination of the adherent cell type, MSC, was optimized for the efficient and effective assessment of the migrating cells. The involvement of Rho was found to be critical for stem and progenitor cell migration where inactivation of Rho by C2I-C3 transferase toxin and/or overexpression of C3 transferase cDNA increased the migration rate of Hematopoietic progenitor cells (HPC) and MSC. Moreover, modulation of Rho caused predictable cytoskeletal and morphological changes in MSC. Assessment of Rho GTPase involvement in the interacting partner, the endothelial cells during stem cell migration, revealed that active Rho expression induced E-selectin expression. The increased levels of E-selectin were functionally confirmed by the increased adhesion of progenitor cells (HPC) to the Human umbilical vein endothelial cell (HUVEC) layer. Moreover, inhibition of Rac in the migrating endothelial progenitor cells (eEPC) increased their adhesion to HUVEC correlating with the increased percentage expression of cell surface receptor, CD44 in Rac inactivated eEPC. In conclusion, this study shows that Rho GTPases control the adhesion and migration of stem and progenitor cells, HPC and MSC. Rho inhibition drives the cells to migrate in the blood vessels. The substantial increase in the level of active Rho in endothelial layer, manifested by the E-selectin surface expression assists the better adhesion of stem and progenitor cells to the endothelial layer. Serum factors and growth factors in the physiological system influence the Rho GTPase expression in both migrating stem cells and the barrier endothelial cells. Thus, specific modulation of Rho GTPases in the transplanted stem and progenitor cells could be an interesting tool to improve the migration and homing processes of stem cells for cellular therapy in future.
This work is dedicated to the investigation of nuclear matter at non-zero temperatures within an effective hadronic model based on the Walecka model. It includes fermions as well as a vector omega meson and a scalar sigma meson where for the latter a quartic self-interaction has been considered. The coupling constants have been adapted to the saturation properties of infinite nuclear matter. A set of self-consistent Schwinger-Dyson equations has been set up for all included particles within the Cornwall-Jackiw-Tomboulis formalism. This has been expanded to non-zero temperatures via the imaginary time formalism. Beside tree-level two different stages of approximations have been considered: the Hartree approximation which takes into account the double-bubble diagram for the scalar meson, and an improved approximation where in addition two-particle irreducible sunset diagrams for all fields were included. In the Hartree-approximation the Schwinger-Dyson equations can be solved by quasi-particle ansaetze, while in the improved approximation spectral functions with non-zero widths have to be introduced. The Schwinger-Dyson equations are solved by the fully dressed propagators. Comparing the two levels of approximation shows the influence of finite widths on the temperature dependence of the particle properties. The consideration of finite widths in fact has a significant influence on the transition from a phase of heavy nucleons to a transition of light nucleons, observed in the Walecka-model. The temperature dependence is weakend when finte widths are taken into account.
The present work was devised to address the systematic analysis of samples from a range of Roman non-ferrous metal artefacts from different archaeological contexts and sites in the Roman provinces of Germania Superior. One of the focal points of this study is the provenancing of different lead objects from five important Roman settlements between 15 BC and the beginning of fourth century AD. For this purpose, measurements were made on lead and copper ore samples from the Siegerland, Eifel, Hunsrück and Lahn-Dill area in Germany and supplemented with data from the literature to create a data bank of lead isotope ratios of European deposits. Compositional analysis of lead objects by Electron Microprobe analysis showed that Romans were able to purify lead from ore up to 99%. Multi-Collector Inductively Coupled Plasma Mass-Spectrometry was used to determine the source of lead, which played an important role in nearly all aspects of Roman life. Lead isotope ratios were measured for ore samples from German deposits from the eastern side of the Rhine (Siegerland, Lahn-Dill, Ems) and the western side of the Rhine (Eifel, Hunsrück), which contained enough ore reserves to answer the increasing local demand and are believed to have been mined during the Roman period. This data together with those from Mediterranean ore deposits from the literature was used to establish a data bank. The Mediterranean ore deposits range from Cambrian (high 207Pb/206Pb) to tertiary (lower 207Pb/206Pb) values. In particular, the Cypriot deposits are younger, while the Spanish deposits fall either with the younger Sardic ores or close to the older Cypriot ores. The lead isotope ratios of most German ore deposits fall in between the 208Pb/206Pb vs. 207Pb/206Pb ratios of Sardinia and Cyprus, where the lead isotope signature of ore deposits from France and Britain are also found. Over 240 lead objects were measured from Wallendorf (second century BC to first century AD) Dangstetten (15-8 BC), Waldgirmes (AD 1-10), Mainz (AD 1-300), Martberg (first to fourth centuries AD) & Trier (third to fourth centuries AD). Comparing the lead isotope ratios of lead objects and those from German ores shows that the source of over 85 percent of objects are Eifel ore deposits, but the Roman’s had also imported lead from the Southern Massif Central and from Great Britain. A further topic of this work was the systematic study of the variation of copper isotope ratios in different copper minerals and the mechanisms, which controls copper isotope fractionation in ores deposits. For this purpose, copper isotope analyses were made by Multi-Collector Inductively Coupled Plasma Mass-Spectrometry from a series of hydrothermal copper sulphides and their alteration products. Copper and lead isotope ratios were measured in coexisting phases of chalcopyrite and malachite and also coexisting malachite and azurite. No significant fractionation was observed in malachite-azurite phases, but in chalcopyrite-malachite coexisting phases, malachite always shows a positive fractionation to heavier isotope values. Zhu et al. and Larson et al. showed that isotopic variations in copper principally reflect mass fractionation in response to low temperature processes rather than source heterogeneity. The low temperature ore formation processes are mostly represented by weathering of primary sulphide ores to produce secondary carbonate phases and therefore are usually observed on the surface of ore deposits, which were probably removed during the early Bronze Age. Using this concept, copper isotope ratios were measured in some Early Bronze Age copper alloys and Roman copper alloys. However, no large copper isotope fractionation has been observed. Lead and copper isotope ratios were measured on samples from the Kupferschiefer. Two profiles were investigated; 1) Sangerhausen, which was not directly influenced by the oxidizing brines of Rote Fäule and 2) Oberkatz, where both Rote Fäule-controlled and structure-controlled mineralization were observed. Results from maturation studies of organic matter suggest the maximum temperature affecting the Kupferschiefer did not exceed 130°C. delta-65-Cu ranges between -0.78-+0.58‰, shows a positive correlation with copper concentration. Maximum temperature in the Kupferschiefer profile from Oberkatz is supposed to be around 150°C. delta-65Cu in this profile ranges between -0.71-+0.68‰. The pattern of copper isotope fractionation and copper concentration is same as the for profile of Sangerhausen. Origina lead isotope ratios are strongly overprinted by high concentrations of uranium in bottom of both profiles causing more radiogenic lead.
In order to investigate the role of neuronal synchronization in perceptual grouping, a new method was developed to record selectively from multiple cortical sites of known functional specificity as determined by optical imaging of intrinsic signals. To this end, a matrix of closely spaced guide tubes was developed in cooperation with a company providing the essential manufacturing technique RMPD® (Rapid Micro Product Development). The matrix was embedded into a framework of hard and software that allowed for the mapping of each guide tube onto the cortical site an electrode would be led to if inserted into that guide tube. With these developments, it was possible to determine the functional layout of the cortex by optical imaging and subsequently perform targeted recordings with multiple electrodes in parallel. The method was tested for its accuracy and found to target the electrodes with a precision of 100 µm to the desired cortical locations. Using the developed technique, neuronal activity was recorded from area 18 of anesthetized cats. For stimulation, Gabor-patches in different geometrical configurations were placed over the recorded receptive fields merging into visual objects appropriate for testing the hypothesis of feature binding by synchrony. Synchronization strength was measured by the height of the cross-correlation centre peaks. All pairwise synchronizations were summarized in a correlation index which determined the mean difference of the correlation strengths between conditions in which recording sites should or should not fire in synchrony according to the binding hypothesis. The correlation index deviated significantly from zero for several of these configurations, further supporting the hypothesis that synchronization plays an important role in the process of perceptual grouping. Furthermore, direct evidence was found for the independence of the synchronization strength from the neuronal firing rate and for neurons that change dynamically the ensemble they participate in. In parallel to the experimental approach, mechanisms of oscillatory long range synchronization were studied by network simulations. To this end, a biologically plausible model was implemented using pyramidal and basket cells with Hodgkin-Huxley like conductances. Several columns were built from these cells and intra- and inter-columnar connections were mimicked from physiological data. When activated by independent Poisson spike trains, the columns showed oscillatory activity in the gamma frequency range. Correlation analysis revealed the tendency to locally synchronize the oscillations among the columns, but a rapid phase transition occurred with increasing cortical distance. This finding suggests that the present view of the inter-columnar connectivity does not fully explain oscillatory long range synchronization and predicts that other processes such as top-down influences are necessary for long range synchronization phenomena.
Systematisch verabreichte Chemotherapeutika sind oft uneffektiv bei der Behandlung von Krankheiten des zentralen Nervensystems (ZNS). Eine der Ursachen hierfür ist der unzureichende Arzneistoff-Transport ins Gehirn aufgrund der Blut-Hirn-Schranke. Eine der Strategien für den nicht-invasiven Wirkstoff-Transport ins Gehirn ist die Verwendung von Nanopartikeln. Polybutylcyanoacrylat-Nanopartikel, die mit Polysorbat 80 (Tween® 80) überzogen wurden, können die Blut-Hirn-Schranke passieren und somit Wirkstoffe ins Gehirn transportieren. Wird die Blut-Hirn-Schranke durch einen Hirntumor partiell beschädigt und hierdurch ihre Permeabilität am Ort des Tumors erhöht, können Nanopartikel den Tumor zusätzlich durch den sogenannten EPR-Effekt erreichen. Im ersten Teil der vorliegenden Arbeit wurde die Beladung der Nanopartikel durch Variation der Formulierungparameter mit dem Ziel optimiert, eine Formulierung mit höherer Wirksamkeit für die Therapie von Glioblastom-tragenden Ratten zu entwickeln. Außerdem wurde das Potential von Doxorubicin, das an mit „Stealth Agents“ überzogenen Polybutylcyanoacrylat-Nanopartikel gebunden war, für die Chemotherapie von Hirntumoren untersucht. Im zweiten Teil dieser Studie wurden die Gehirn- und Körperverteilung in gesunden und in Glioblastom-101/8-tragenden Ratten nach i.v.-Gabe von Poly(butyl-2-cyano[3- 14C]acrylat)-Nanopartikeln, die mit Polysorbat 80 beschichtet wurden, und solchen, die noch zusätzlich mit Doxorubicin geladen waren (DOX-14C-PBCA + PS), untersucht. Die Standardformulierung von Doxubicin-Polybutylcyanoacrylat-Nanopartikeln (DOX-NP) wurde durch anionische Polymerisierung von Butylcyanoacrylat in Anwesenheit von DOX hergestellt. Zusätzlich wurden unterschiedliche DOX-NP Formulierungen durch Veränderung der Herstellung produziert. Das therapeutische Potential der Formulierungen wurde in Ratten mit ins Gehirn transplantieren Glioblastom 101/8 untersucht. Neben Polysorbat 80 wurden Poloxamer 188 und Poloxamin 908 als Überzugsmaterial verwendet. Die Resultate ergaben, dass die mit Polysorbat 80 überzogene Standardformulierung am effektivsten war. Die höhere Wirksamkeit von DOX-NP+PS 80 könnte durch die Fähigkeit dieser Träger erklärt werden, den Wirkstoff während eines frühen Stadiums der Tumorentwicklung durch einen Rezeptor-vermittelten Mechanismus, der durch den PS 80-Überzug aktiviert wurde über die intakte Blut-Hirn-Schranke, zu transportieren. Unsere Ergebnisse zeigen auch, dass Poloxamer 188 und Poloxamin 908 den antitumoralen Effekt von DOX-PBCA beträchtlich verbessern. Der anti-tumorale Effekt dieser Formulierungen könnte möglicherweise dem EPR-Effekt zugeschrieben werden. Es ist bekannt, dass die tumorale Arzneistoff-Aufnahme durch den EPR-Effektes für lang-zirkulierende Wirkstoffträger ausgeprägter ist und so mehr Wirkstoff durch die Tumor-geschädigte Blut-Hirn-Schranke gelangt. Unbeschichtete Nanopartikel, Polysorbat 80-beschichtete Nanopartikel oder mit Doxorubicin beladene und mit Polysorbat 80 beschichtete Nanopartikel wurden in gesunden und Tumor-tragenden Ratten injiziert. Diese Nanopartikel-Präparationen zeigten einer unterschiedliche Korpenverteilung in den Ratten. Unbeschichtete Nanopartikel sammelten sich in den RES-Organen an. Mit PS 80 beschichtete NP reduzierten die Aufnahme der NP in Leber und Milz, während sich die Konzentration der NP in der Lunge erhöhte. Diese Beobachtungen deuten darauf hin, dass die Änderung der Oberflächeneigenschaften der NP durch das Tensid, zu einer Interaktion mit unterschiedlichen Opsoninen führt, welches die Aufnahme der NP von verschiedenen phagozitierenden Zellen erleichtert. Hingegen war die Aufnahme der mit DOX beladenen, PS 80-beschichteten Nanopartikel den unbeschichteten Partikel ähnlich. Im Vergleich mit gesunden Ratten und mit Tumor-tragenden Ratten hingegen war die Konzentration der NP im Gehirn von Tumor tragenden Ratten 10 Tage nach der Tumor-implantation signifikant höher. In Anwesenheit des Glioblastoms ist der Transport von NP in das Gehirn das Resultat verschiedener Faktoren: zusätzlich zur Fähigkeit von PS 80-Nanopartikeln, die Blut-Hirn-Schranke zu passieren, extravasieren diese Träger wegen des EPR Effekts über das durch den Tumor undichte Endothelium. Die Konzentration von PS 80 [14C]-PBCA NP war im Glioblastom signifikant höher als mit DOX [14C]-PBCA NP. Dieses Phänomen kann durch die unterschiedliche Mikroumgebung von zerebralem intra-tumoralen und intaktem Gehirngewebe erklärt werde. Insbesondere können sich die positive Ladung der tumoralen Regionen und die positive Ladung der DOX [14C]-PBCA NP negativ beeinflussen. Dennoch waren die Doxorubicin-Konzentration in Glioblastom ausreichend, einen therapeutischen Effekt zu ermöglichen.
Group III presynaptic metabotropic glutamate receptors (mGluRs) play a central role in regulating presynaptic activity through G-protein effects on ion channels and signal transducing enzymes. Like all Class C G-protein coupled receptors, mGluR8 has an extended intracellular C-terminal domain (CTD) presumed to allow for modulation of downstream signaling. To elucidate the function and modulation of mGluR8, yeast two-hybrid screens of an adult rat brain cDNA library were performed with the CTDs of mGluR8a and 8b (mGluR8-C) as baits. Different components of the sumoylation cascade (ube2a, sumo-1, Pias1, Pias gamma and Pias xbeta) and some other proteins were identified as mGluR8 interacting proteins. Binding assays using recombinant GST-fusion proteins confirmed that Pias1 interacts not only with mGluR8-C, but all group III mGluR CTDs. Pias1 binding to mGluR8-C required a region N-terminally to a consensus sumoylation motif and was not affected by arginine substitution of the conserved lysine K882 within this motif. Co-transfection of fluorescently tagged mGluR8a-C, sumo-1 and enzymes of the sumoylation cascade into HEK 293 cells showed that mGluR8a-C can be sumoylated in cells. Arginine substitution of lysine K882 within the consensus sumoylation motif, but not of other conserved lysines within the CTD, abolished in vivo sumoylation. The results are consistent with post-translational sumoylation providing a novel mechanism of group III mGluR regulation.
Chemokines play a key role in the cellular infiltration of inflamed tissue. They are released by a wide variety of cell types during the initial phase of host response to injury, allergens, antigens, or invading microorganisms, and selectively attract leukocytes to inflammatory foci, inducing both migration and activation. Monocyte chemoattractant protein-1 (MCP-1), a member of the CC chemokine superfamily, functions in attracting monocytes, T lymphocytes, and basophils to sites of inflammation. MCP-1 is produced by monocytes, fibroblasts, vascular endothelial cells and smooth muscle cells in response to various stimuli such as tumour necrosis factor-a (TNF-a), interferon-g (IFN-g), and interleukin-1b (IL-1b). It also plays an important role in the pathogenesis of chronic inflammation, and overexpression of MCP-1 has been implicated in diseases including glomerulonephritis and rheumatoid arthritis. Oligonucleotide-directed triple helix formation offers a means to target specific sequences in DNA and interfere with gene expression at the transcriptional level. Triple helix-forming oligonucleotides (TFOs) bind to homopurine/homopyrimidine sequences, forming a stable, sequence-specific complex with the duplex DNA. Purine-rich sequences are frequent in gene regulatory regions and TFOs directed to promoter sequences have been shown to prevent binding of transcription factors and inhibit transcription initiation and elongation. Exogenous TFOs that bind homopurine/ homopyrimidine DNA sequences and form triple-helices can be rationally designed, while the intracellular delivery of single-stranded RNA TFOs has not been studied in detail before. In this study, expression vectors were constructed which directed transcription of either a 19 nt triplex-forming pyrimidine CU-TFO sequence targeting the human MCP-1 or two different 19 nt GU- or CA-control sequences, respectively, together with the vector encoded hygromycin resistance mRNA as one fusion transcript. HEK 293 cells were stable transfected with these vectors and several TFO and control cell lines were generated. Functional relevant triplex formation of a TFO with a corresponding 19 bp GC-rich AP-1/SP-1 site of the human MCP-1 promoter was shown. Binding of synthetic 19 nt CUTFO to the MCP-1 promoter duplex was verified by triplex blotting at pH 6.7. Underlining binding specificity, control sequences, including the GU- and CA-sequence, a TFO containing one single mismatch and a MCP-1 promoter duplex containing two mismatches, did not participate in triplex formation. Establishing a magnetic capture technique with streptavidin microbeads it was verified that at pH 7.0 the 19 nt TFO embedded in a 1.1 kb fusion transcript binds to a plasmid encoded MCP-1 promoter target duplex three times stronger than the controls. Finally, cell culture experiments revealed 76 ± 10.2% inhibition of MCP-1 protein secretion in TNF-a stimulated CU-TFO harboring cell lines and up to 88% after TNF-a and IFN-g costimulation in comparison to controls. Expression of interleukin-8 (IL-8) as one TNF-a inducible control gene was not affected by CU-TFO, demonstrating both highly specific and effective chemokine gene repression. Furthermore, another chemokine target, regulated upon activation normal T cell expressed and secreted (RANTES), which plays an essential role in inflammation by recruiting T lymphocytes, macrophages and eosinophils to inflammatory sites, was analysed using the triplex approach. A 28 nt TFO was designed targeting the murine RANTES gene promoter, and gel mobility shift assays demonstrated that the phosphodiester TFO formed a sequencespecific triplex with the double-stranded target DNA with a Kd of 2.5 x 10-7 M. It was analysed whether RANTES expression could be inhibited at the transcriptional level testing the TFO in two different cell lines, T helper-1 lymphocytes and brain microvascular endothelial cells (bend3 cells). Although there was a sequence-specific binding of the TFO detectable in the gel shift assays, there was no inhibitory effect of the exogenously added and phosphorothioate stabilised TFO on endogenous RANTES gene expression visible. Additionally, the small interfering RNA (siRNA) approach was tested as another strategy to inhibit expression of the pro-inflammatory chemokines MCP-1 and RANTES. Two different methods were pursuit, describing transient transfection with vector derived and synthetic siRNA. The vector pSUPER containing the siRNA coding sequence was used to suppress endogenous MCP-1 in HEK 293 cells. An empty vector without RNA sequence served as a control. Inhibition due to the siRNA was measured in stimulated and unstimulated cells. In TNF-a stimulated cells MCP-1 protein synthesis was decreased by 35 ± 11% after siRNA transfection. Using a synthetic double-stranded siRNA, the TNF-a induced MCP-1 protein secretion could be successfully inhibited about 62.3 ± 10.3% in HEK 293 cells, indicating that the siRNA is functional in these cells to suppress chemokine expression. The siRNA approach targeting murine RANTES in Th1 cells and b-end3 cells revealed no inhibition of endogenous gene expression. Gene therapy approaches rely on efficient transfer of genes to the desired target cells. A wide variety of viral and nonviral vectors have been developed and evaluated for their efficiency of transduction, sustained expression of the transgene, and safety. Among them, lentiviruses have been widely used for gene therapy applications. In order to improve the delivery of TFOs or siRNAs into the target cells, cloning of the lentiviral transfer vector SEW, the production of lentiviral particles by transient transfection were performed with the aim to generate lentiviral vector-derived TFOs in further experiments. Here, Th1 cells were transduced with infectious lentiviral particles and transduction efficacy was measured. Transduction efficacy higher than 82% could be achieved using the lentiviral vector SEW, opening optimal possibilities for the TFO or siRNA approach.
Lesion of the rat entorhinal cortex denervates the outer molecular layer of the fascia dentata followed by layer-specific axonal sprouting of uninjured fibers in the denervated zone. One of the candidate molecules regulating the laminar-specific sprouting response in the outer molecular layer is the transmembrane chondroitin sulfate proteoglycan NG2. NG2 is found in glial scars and has been suggested to impede axonal regeneration following injury of the spinal cord. The present study adressed the question whether NG2 could also regulate axonal growth in denervated areas of the brain. Therefore, (1) changes in NG2 mRNA and NG2 protein levels, (2) the cellular and the extracellular localisation of the molecule, (3) the identity of NG2 expressing cells, and (4) the generation of NG2-positive cells were studied in the rat fascia dentata before and following entorhinal deafferentation. Laser microdissection was employed to selectively harvest the denervated molecular layer and combined with quantitative reverse transcription-PCR to measure changes in NG2 mRNA amount (6h, 12h, 2d, 4d, 7d post lesion). The study revealed increases of NG2 mRNA at day 2 (2.5-fold) and day 4 (2-fold) post lesion. Immunocytochemistry was used to detect changes in NG2 protein distribution (1d, 4d, 7d, 10d, 14d, 30d, 6 months post lesion). NG2 staining was increased in the denervated outer molecular layer at 1 day post lesion, reached a maximum at 10 days post lesion, and returned to control levels within 6 month. Interestingly, the accumulation of NG2 protein was strongly restricted to the denervated outer molecular layer forming a border to the unaffected inner molecular layer. Using electron microscopy, NG2-immunoprecipitate was localized not only on glial surfaces and in the extracellular matrix but also in the vicinity of neuronal profiles indicating that NG2 is secreted following denervation. Double-labelings of NG2-immunopositive cells with markers for astrocytes, microglia/macrophages, and oligodendrocytes suggested that NG2-cells are a distinct glial subpopulation before and after entorhinal deafferentation. Bromodeoxyuridine-labeling revealed that some of the NG2-positive cells are postlesional generated. Taken together, the data revealed a layer-specific upregulation of NG2 in the denervated outer molecular layer of the fascia dentata that coincides with the sprouting response of uninjured fibers. This suggests that NG2 could regulate lesion-induced axonal growth in denervated areas of the brain.
In this dissertation a non-deterministic lambda-calculus with call-by-need evaluation is treated. Call-by-need means that subexpressions are evaluated at most once and only if their value must be known to compute the overall result. Also called "sharing", this technique is inevitable for an efficient implementation. In the lambda-ND calculus of chapter 3 sharing is represented explicitely by a let-construct. Above, the calculus has function application, lambda abstractions, sequential evaluation and pick for non-deterministic choice. Non-deterministic lambda calculi play a major role as a theoretical foundation for concurrent processes or side-effected input/output. In this work, non-determinism additionally makes visible when sharing is broken. Based on the bisimulation method this work develops a notion of equality which respects sharing. Using bisimulation to establish contextual equivalence requires substitutivity within contexts, i.e., the ability to "replace equals by equals" within every program or term. This property is called congruence or precongruence if it applies to a preorder. The open similarity of chapter 4 represents a new concept, insofar that the usual definition of a bisimulation is impossible in the lambda-ND calculus. So in section 3.2 a further calculus lambda-Approx has to be defined. Section 3.3 contains the proof of the so-called Approximation Theorem which states that the evaluation in lambda-ND and lambda-Approx agrees. The foundation for the non-trivial precongruence proof is set out in chapter 2 where the trailblazing method of Howe is extended to be capable with sharing. By the use of this (extended) method, the Precongruence Theorem proves open similarity to be a precongruence, involving the so-called precongruence candidate relation. Joining with the Approximation Theorem we obtain the Main Theorem which says that open similarity of the lambda-Approx calculus is contained within the contextual preorder of the lambda-ND calculus. However, this inclusion is strict, a property whose non-trivial proof involves the notion of syntactic continuity. Finally, chapter 6 discusses possible extensions of the base calculus such as recursive bindings or case and constructors. As a fundamental study the calculus lambda-ND provides neither of these concepts, since it was intentionally designed to keep the proofs as simple as possible. Section 6.1 illustrates that the addition case and constructors could be accomplished without big hurdles. However, recursive bindings cannot be represented simply by a fixed point combinator like Y, thus further investigations are necessary.
Jet physics in ALICE
(2005)
This work aims at the performance of the ALICE detector for the measurement of high-energy jets at mid-pseudo-rapidity in ultra-relativistic nucleus-nucleus collisions at LHC and their potential for the characterization of the partonic matter created in these collisions. In our approach, jets at high energy with E_{T}>50 GeV are reconstructed with a cone jet finder, as typically done for jet measurements in hadronic collisions. Within the ALICE framework we study its capabilities of measuring high-energy jets and quantify obtainable rates and the quality of reconstruction, both, in proton-proton and in lead-lead collisions at LHC conditions. In particular, we address whether modification of the jet fragmentation in the charged-particle sector can be detected within the high particle-multiplicity environment of the central lead-lead collisions. We comparatively treat these topics in view of an EMCAL proposed to complete the central ALICE tracking detectors. The main activities concerning the thesis are the following: a) Determination of the potential for exclusive jet measurements in ALICE. b) Determination of jet rates that can be acquired with the ALICE setup. c) Development of a parton-energy loss model. d) Simulation and study of the energy-loss effect on jet properties.
This thesis has explored how structural techniques can be applied to the problem of formal verification for sequential circuits. Algorithms for formal verification which operate on non-canonical gate netlist representations of digital circuits have certain advantages over the traditional techniques based on canonical representations as BDDs. They allow to exploit problem-specific knowledge because they can take into account structural properties of the designs being analyzed. This allows us to break the problem down into sub-problems which are (hopefully) easier to be solved. However, in the past, the main application of such structural techniques was in the field of combinational equivalence checking. One reason for this is that the behaviour of a sequential system does not only depend on its inputs but also on its internal states, and no concepts had been developed to-date allowing structural methods to deal with large sets of states. An important goal of this research was therefore to develop structural, non-canonical forms of representing the reachable states of a finite state machine and to develop methods for reachability analysis based on such representations. In order to reach this goal, two steps were taken. Firstly, a framework for manipulating Boolean functions represented as gate netlists has been established. Secondly, using this framework, a structural method for FSM traversal was developed serving as the basis for an equivalence checking algorithm for sequential circuits. The framework for manipulating Boolean functions represented as multi-level combinational networks is based on a new concept of an implicant in a multi-level network and on an AND/ORtype enumeration technique which allows us to derive such implicants. This concept extends the classical notion of an implicant in two-level circuits to the multi-level case. Using this notion, arbitrary transformations in multi-level combinational networks can be performed. The multi-level network implicants can be determined from AND/OR reasoning graphs, which are associated with an AND/OR reasoning technique operating directly on the gate netlist description of a multi-level circuit. This reasoning technique has the important property that it is complete, i.e. the associated AND/OR trees contain all prime implicants of a Boolean function at an arbitrary node in a combinational circuit. In other words, AND/OR graphs constructed for a network function serve as a representation of this function. A great advantage over BDDs is that AND/OR graphs, besides representing the logic function, also represent some structural properties of the analyzed circuitry. This permits to develop heuristics that are specially tailored for certain applications such as logic optimization or verification. Another advantage which is especially useful for logic optimization is the fact that the proposed AND/OR enumeration scheme is not restricted to the use of a specific logic alphabet such as B3 = {0, 1, X}. By using Roth’s D-calculus based on B5 = {0, 1, D, D-Komplement} permissible implicants can be determined. Transformations based on permissible implicants exploit observability don’t-care conditions in logic synthesis by creating permissible functions at internal network nodes. In order to evaluate the new structural framework for manipulating Boolean functions represented as gate netlists, several experiments with implicant-based optimization of multi-level circuits were performed. The results show that implicant-based circuit transformations lead to significantly better optimization results than traditional synthesis techniques. Next, based on the proposed structural methods for Boolean function manipulation, techniques for representing and manipulating the set of states of a sequential circuit have been developed. The concept of a “stub circuit” was introduced which implicitly represents a set of state vectors as the range of a multi-output function given as a gate netlist. The stub circuit is the result of an existential quantification operation which is obtained by functional decomposition using implicant-based netlist transformations and a network cutting procedure. Using this existential quantification operation, a new structural FSM traversal algorithm was formulated which performs a fixed point iteration on the set of reachable states represented by the stub circuit. The proposed approach performs a reachability analysis of the states of a sequential circuit. It operates on gate netlists and naturally allows to incorporate structural properties of a design under consideration into the reasoning. Therefore, structural FSM traversal is an interesting alternative to traditional symbolic FSM traversal, especially in those applications of formal verification, where structural properties can be exploited. Structural FSM traversal was applied to the problem of sequential equivalence checking. Here, structural similarities between the designs to be compared can effectively reduce the complexity of the verification task. The FSM to be traversed is a special product machine called sequential miter. The special structural properties of this product machine have made it possible to formulate an approximate algorithm for structural FSM traversal, called record and play(). This algorithm uses an approximation on the reachable state set represented by the stub circuit which is very beneficial for performance. Instead of calculating the stub circuit using the exact algorithm, implicant-based transformations directly using structural design similarities are performed. These transformations, together with existential quantification implemented by the cutting procedure, lead to an over-approximation of the reachable state set. By this overapproximation, only such unreachable product states are added to the set of states represented by the stub circuit which are unreachable at the current point in time but which are nevertheless equivalent. Therefore, more product states are added to the set of reachable states sometimes leading to drastic acceleration of the traversal, i.e. the fixed point is reached in much fewer steps. The algorithm record and play() was applied to the problem of checking the equivalence of a circuit with its optimized and retimed version. Retiming is a form of sequential circuit optimization which can radically alter the state encoding of a circuit. Traditional FSM traversal techniques often fail because the BDDs needed to represent the reachable state set and the transition relation of the product machine become too large. Experiments were conducted to evaluate the performance of record and play() on a standard set of sequential benchmark circuits. The algorithm was capable of proving the equivalence of optimized and retimed circuits with their original versions, some of which (to our knowledge) have never before been verified using traditional techniques like symbolic FSM traversal. The experimental results are very promising. Future research will therefore explore how structural FSM traversal can be applied to model checking.
The results presented here strongly indicate that ubiquitination of the recombinant human alpha1 GlyR at the plasma membrane of Xenopus oocytes is involved in receptor internalisation and degradation. Ubiquitination of the human alpha1 GlyR has been demonstrated by radio-iodination of plasma membrane-boundalpha1 GlyRs, whose subunits differed in molecular weight by additional 7, 14 or 21 kDa, corresponding to the molecular weights of one, two and three conjugated ubiquitin molecules, respectively, and by co-isolation of the non-tagged human alpha1 GlyR through hexahistidyl-tagged ubiquitin. Ubiquitin conjugated GlyRs where prominent at the plasma membrane, but could be hardly detected in total cell homogenates, indicating that ubiquitination takes place exclusively at the plasma membrane. Ubiquitination of the alpha1 GlyR at the plasma membrane was no longer detectable when the ten lysine residues of the cytoplasmic loop between transmembrane segments M3 and M4 were replaced by arginines. Despite this proteolytic cleavage continued to take place at the same extent as with the wild type alpha1 GlyR, suggesting that removal of GlyRs from the plasma membrane and routing to lysosomes for degradation were not dependent on ubiquitination. Also replacing a tyrosine in position 339, which was speculated to be part of an additional endocytosis motif, did not lead to a significant reduction of cleavage of the GlyR alpha1 subunits. However, a mutant lacking both, ubiquitination sites and 339Y, was significantly less processed. These results may suggest that the GlyR alpha1 subunit harbors at least two endocytosis motifs, which may act independently to regulate the density of alpha1 GlyR. Apparently, each of the two signals may be capable of compensating entirely the loss of the other. Part two of this Dissertation demonstrates that the correct topology of the glycine receptor alpha1 subunit depends critically on six positively charged residues within a basic cluster, RFRRKRR, located in the large cytoplasmic loop following the C-terminal end of M3. Neutralization of one or more charges of this cluster, but not of other charged residues in the M3-M4 loop, led to an aberrant translocation into the endoplasmic reticulum lumen of the M3-M4 loop. However, when two of the three basic charges located in the ectodomain linking M2 and M3 were neutralized, in addition to two charges of the basic cluster, endoplasmic reticulum disposition of the M3-M4 loop was prevented. We conclude that a high density of basic residues C-terminal to M3 is required to compensate for the presence of positively charged residues in the M2-M3 ectodomain, which otherwise impair correct membrane integration of the M3 segment. Part three of this Dissertation describes my contribution (blue native PAGE analysis of metabolically labeled alpha7 and 5HT3A receptors and the examination of the glycosylation state of metabolically labeled alpha7 subunits) to a work on the limited assembly capacity of Xenopus oocytes for nicotinic alpha7 subunits. While 5HT3A subunits combined efficiently to pentamers, alpha7 subunits existed in various assembly states including trimers, tetramers, pentamers, and aggregates. Only alpha7 subunits that completed the assembly process to homopentamers acquired complex-type carbohydrates and appeared at the cell surface. We conclude that Xenopus oocytes have a limited capacity to guide the assembly of alpha7 subunits, but not 5HT3A subunits to homopentamers. Accordingly, ER retention of imperfectly assembled alpha7 subunits rather than inefficient routing of fully assembled alpha7 receptors to the cell surface limits surface expression levels of alpha7 nicotinic acetylcholine receptors. Part four of this Dissertation describes my contribution (the biochemical analysis of the human P2X2 and P2X6 subtypes) to studies on the quaternary structure of P2X receptors. Armaz Aschrafi, the main author of the paper showed that subsequent to isolation under non-denaturing conditions from Xenopus oocytes the His-rP2X2 protein migrated on blue native PAGE predominantly in an aggregated form. The only discrete protein band detectable could be assigned to homotrimers of the His-rP2X2 subunit. Because of the exceptional assembly-behaviour of the rP2X2 protein compared to the rP2X1, rP2X3, rP2X4 and rP2X5 proteins, its human orthologue was investigated in the same manner. In contrast to rP2X2 subunits, hP2X2 subunits migrated under virtually identical conditions in a single defined assembly state, which could be clearly assigned to a trimer. P2X6 subunits represent the sole P2X subtype that is unable to form functional homomeric receptors in Xenopus oocytes. The blue native PAGE analysis of metabolically labeled hP2X6 receptors and the examination of the glycosylation state revealed that hP2X6 subunits form tetramers and aggregates that are not exported to the plasma membrane of Xenopus oocytes.
In the present work, the Heidelberg electron beam ion trap (EBIT) at the Max-Planck-Institute für Kernphysik (MPIK) has been used to produce, trap highly charged argon ions and study their magnetic dipole (M1) forbidden transitions. These transitions are of relativistic origin and, hence, provide unique possibilities to perform precise studies of relativistic effects in many electron systems. In this way, the transitions energies of the 1s22s22p for the 2P3/2 - 2P1/2 transition in Ar13+ and the 1s22s2p for the 3P1 - 3P2 transition in Ar14+, for 36Ar and 40Ar isotopes were compared. The observed isotopic effect has confirmed the relativistic nuclear recoil effect corrections due to the finite nuclear mass in a recent calculation made by Tupitsyn [TSC03], in which major inconsistencies of earlier theoretical methods have been corrected for the first time. The finite mass, or recoil effect, composed of the normal mass shift (NMS), and the specific mass shift (SMS) were corrected for relativistic contributions, RNMS and RSMS. The present experimental results have shown that the recoil effects on the Breit level are indeed very important, as well as the effects of the correlated relativistic dynamics in a many electron ion.
This thesis examines the spread and promotion of English on a global level, from a historical perspective in particular ‘Third World’ contexts. The globalization of English as an exclusive language of power is considered to be a trap, when accompanied by an ideology aiming to universalize monolingual and monocultural norms and standards. World-wide English diffusion is related - not to any mystical effects of some psycho-social mechanisms or transmuting alchemy - but to a global rise of military, political, economic, communicational and cultural Euro-American hegemony. The fact that the English language has become perhaps the primary medium of social control and power has not been given a prominent place in the analyses of established social scientists or political planners. On the contrary, the positively idealized dominance of English as a universal medium has become part of a collection of myths seeking to deny the global reality of multilingualism. Not allowing for the existence of any power besides itself, the perpetuation of this hegemony of English within a multilingual scenario has become a contradiction in terms. Centuries of colonialism, followed by neo-colonialism, are seen to have resulted in a world-wide consensus favouring centralization and homogenization of state and world economies, administrations, language, education and mass media systems, as prerequisites to local and global unity. The particular case of India as encountered by a colonizing Britain is used to illustrate the historical clash between differing language and educational traditions and cultures. It was on the strength of their own predominantly positive attitudes towards diversity - encoded in their promotion of complex social and religious philosophies, as well as varied economic and educational practices of pluralism and hierarchy-without-imposition, unity in diversity, etc. - that the people and their leaders finally achieved Indian independence from British colonialism. Contemporary Indian society, however, is still grappling with the legacy of a Eurocentric civilizational model - encoded in the neo-colonial system of English education - and in conflict with its own positively idealized and actively promoted traditions of pluralism. On national and international levels, the destabilization and destruction of diversity continues to threaten more than the linguistic and cultural uniqueness of numerous communities and individuals. For those majorities and minorities who refuse to give up their ‘differences’, political, economic and physical survival is at stake. A paradoxical reality, seldom acknowledged, is that while for the politically and economically already powerful language groups, the enormous resources spent on formal (language) education have become a means to maintain their material and political capital, whereas for the majority of modern societies' marginalized members, powerful linguistic barriers to full economic or political participation remain firmly in place. The justifications for perpetuating exclusionary policies and sustaining structural inequality have come from monocultural ideological assumptions in education and language policies as one of the key mechanisms for state control of labour. This thesis concludes that the trap of an ideologically exclusive status for English can be avoided by theoretically positivizing and institutionally promoting existing multilingual and multicultural peoples’ realities as an integral part of their human rights, in order to resist global Englishization.
Different numerical approaches and algorithms arising in the context of modelling of cellular tissue evolution are discussed in this thesis. Being suited in particular to off-lattice agent-based models, the numerical tool of three-dimensional weighted kinetic and dynamic Delaunay triangulations is introduced and discussed for its applicability to adjacency detection. As there exists no implementation of a code that incorporates all necessary features for tissue modelling, algorithms for incremental insertion or deletion of points in Delaunay triangulations and the restoration of the Delaunay property for triangulations of moving point sets are introduced. In addition, the numerical solution of reaction-diffusion equations and their connection to agent-based cell tissue simulations is discussed. In order to demonstrate the applicability of the numerical algorithms, biological problems are studied for different model systems: For multicellular tumour spheroids, the weighted Delaunay triangulation provides a great advantage for adjacency detection, but due to the large cell numbers the model used for the cell-cell interaction has to be simplified to allow for a numerical solution. The agent-based model reproduces macroscopic experimental signatures, but some parameters cannot be fixed with the data available. A much simpler, but in key properties analogous, continuum model based on reaction-diffusion equations is likewise capable of reproducing the experimental data. Both modelling approaches make differing predictions on non-quantified experimental signatures. In the case of the epidermis, a smaller system is considered which enables a more complete treatment of the equations of motion. In particular, a control mechanism of cell proliferation is analysed. Simple assumptions suffice to explain the flow equilibrium observed in the epidermis. In addition, the effect of adhesion on the survival chances of cancerous cells is studied. For some regions in parameter space, stochastic effects may completely alter the outcome. The findings stress the need of establishing a defined experimental model to fix the unknown model parameters and to rule out further models.
My graduate thesis is on the "Structural studies of membrane transport proteins". Transporters are membrane proteins that have multiple membrane-spanning a-helices. They are dynamic and diverse proteins, undergoing a large conformational change and transporting wide range of susbtrates. Based on their energy source they can be classified into primary and secondary transport systems. Primary transport systems are driven by the use of chemical (ATP) or light energy, while secondary transporters utilize ion gradients to transport substrates. I began my PhD dissertation on secondary transporters by two-dimensional crystallization and electron crystallographic analysis and recently my focus also has shifted towards 3D crystallization. The following projects constitute my PhD thesis: 1) 2D crystallization of MjNhaP1 and pH induced structural change: MjNhaP1, a Na+/H+ antiporter that is regulated by pH has been implicated in homeostasis of H+ and Na+ in Methanococcus jannaschii, a hyperthermophilic archaeon that grows optimally at 85°C. MjNhaP1 was cloned and expressed in E. coli. Two-dimensional crystals were obtained from purified protein at pH4. Electron cryo-microscopy yielded an 8Å projection map. The map of MjNhaP1 shows elongated densities in the centre of the dimer and a cluster of density peaks on either side of the dimer core, indicative of a bundle of 4-6 membrane-spanning helices. The effect of pH on the structure of MjNhaP1was studied in situ in 2D crystals revealing a major change in density within the helix bundle relative to the dimer interface. This change occurred at pH6 and above. The two conformations at low and high pH most likely represent the closed and open states of the antiporter, respectively. This is the first instance where a conformational change associated with the regulation of a secondary transporter appears to map structurally. Reconstruction of 3D map and high-resolution structure by x-ray crystallography would be necessary to understand the mechanism of ion transport and regulation by pH. 2) 2D crystallization of Proline transporter: Proline transporter (PutP) from E.coli belongs the sodium-solute symporter family that includes disease related sodium dependent glucose and iodide transporter in humans. Sodium and proline are co-transported with a stoichiometry of 1:1. Purified PutP was reconstituted to yield 2D crystals that were hexagonal in nature. The 2D crystals had tendency to stack indicating their willingness to form 3D crystals. A projection map of PutP from negatively stained crystals showed trimeric arrangement of protein. Other members of the SSF family have been shown to be monomers. My analysis of oligomeric state of PutP in detergent by blue native gel indicates a monomer in detergent solution. It is likely that PutP can function as a monomer but at higher concentration and in lipid bilayer it tends to form trimer. 3) Oligomeric state and crystallization of carnitine transporter from E.coli: E.coli carnitine transporter (CaiT) belongs to the BCCT (Betaine, Carnitine and Choline) superfamily that transports molecules with quaternary amine groups. CaiT is predicted to span the membrane 12 times and acts as a L-carnitine/g-butyrobetaine exchanger. Unlike other members in this transporter family, it does not require an ion gradient and does not respond to osmotic stress. Over-expression of the protein yielded ~2mg of protein/L of culture. The structure and oligomeric state of the protein were analyzed in detergent and lipid bilayers. Blue native gel electrophoresis indicated that CaiT was a trimer in detergent solution. Gel filtration and cross-linking studies further support this. Reconstitution of CaiT into lipid bilayers resulted in 2D crystals. Analysis of negatively stained 2D crystals confirmed that CaiT is a trimer in the membrane. Initial 3D crystallization trials have been successful and currently, the crystals diffract to 6Å and are being improved. 4) Monomeric porin OmpG: OmpG is a bacterial outer membrane b-barrel protein. It is monomeric and its size (33kDa) places it as a prime candidate for a structural solution, using the recently developed method of solid state NMR (work in collaboration with Prof.Hartmut Oskinat, FMP, Berlin). A long-term aim would be to study porins as templates for designing nanopores, for DNA sequencing and identification. I have expressed OmpG in inclusion bodies and refolded at an efficiency of >90% into a functional form using detergent. OmpG was then crystallized by 2D crystallization yielding an 8Å projection map whose structure was similar to native protein. In addition, these crystals were used for structure determination by solid state NMR. An initial spectrum of heavy isotopically labeled OmpG has allowed identification of specific amino acid residues including threonine and proline. Additionally, I obtained 3D crystals in detergent that diffract to 5.5Å and are being improved.
Protein-protein interactions within the plane of cellular membranes play a key role for many biological processes and in particular for transmembrane signaling. A prominent example is the ligand-induced crosslinking of cytokine receptors, where 3- dimensional cytokine binding followed by 2-dimensional interaction between the receptor subunits have been recognized to be important for regulating signaling specificity. The fundamental importance of such coupled interactions for cell-surface receptor activation has stimulated numerous theoretical studies, which have hardly been confirmed experimentally. An experimental approach to measure interactions and real time kinetics of type I interferon (IFN) induced assembly between interferon receptor subunits ifnar2 and ifnar1 on membrane was developed and determinants of the 2-dimensional interactions, such as dimensionality, size, valency, orientation, membrane fluidity and receptor density were quantitatively addressed The C-terminal decahistidine tagged extracellular domains (EC) of ifnar1 and ifnar2 were site- specifically tethered onto solid-supported fluid lipid membrane, which carried covalently attached chelator bis-nitrilotriacetic acid (bis-NTA) groups. Interactions on the lipid bilayer were detected with a novel solid phase detection technique, which allows simultaneous detection of ligand binding to a membrane anchored receptors and lateral interaction between them in the real time. This was achieved by combining two optical techniques: label-free reflectance interferometry (RIf) and total internal reflection fluorescence spectroscopy (TIRFS). Fluorescence signals, in the order of 10 fluorophores/µm2, were detected without substantial photobleaching. The sensitivity of the label-free interferometric detection was in the range of 10 pg/mm2. The crosstalk between the two signals was eliminated by means of spectral separation. Fluorescence was detected in the visible region and RIf was performed at 800 nm in the near infrared. Flow through conditions allowed to automate experiments and measure binding events as fast as ~ 5 s-1. Using this technique we have dissected the interactions involved in IFN-induced ifnar crosslinking. 2-dimensional association and dissociation rate constants were independently determined by tethering high stoichiometric excess of one of the receptor subunits and comparing dissociation of the labelled ligand away from the membrane in the absence and presence of the non-labelled high affinity competitor. Dissociation traces were fitted with the two-step dissociation model: the first step being the 2-dimensional separation of the ternary complex followed by the 3- dimensional ligand dissociation into solution. Label-free RIf detection allowed absolute parameterization of the 2-dimensional concentrations of the ifnar subunits on the membrane. The TIRFS signal provided high sensitivity of the ligand dissociation and was correlated against the RIf signal before fitting. These features of the detection system allowed us to parameterize the model, and the 2-dimensional association or dissociation rate constants were the only variables during the fitting. Another FRET based binding assay was developed to determine the 2- dimensional dissociation rate constant using a pulse-chase approach. The donor fluorescence from ifnar2-EC was quenched upon the ternary complex formation with the acceptor-labelled IFN and the nonlabelled ifnar1-EC. The equilibrium was perturbed by rapid tethering of substantial excess of the nonlabelled ifnar2-EC onto the membrane. The exchange of the labelled ifnar2-EC with the nonlabelled one was monitored as the decrease in the FRET signal with the 2-dimensional dissociation of ifnar2-EC from the ternary complex being the rate limiting step. Based on the several mutants and variants of the interacting proteins, the effect of different rate constants and receptor orientation on the 2-dimensional crosslinking dynamics was studied. We have identified several critical features of the 2- dimensional interactions on membranes, which cannot be readily concluded from the solution binding assays. The restricted rotation and the increased lifetime of the encounter complex due to high membrane viscosity are the main determinants of the 2-dimensional association. Tethering ifnar1-EC to the membrane via N-terminal decahistidine tag decreased the 2-dimensional association rate constant 4-5 fold. Electrostatic attraction and steering, the important mechanism to enhance association rate constant between the soluble proteins, are not pronounced for interactions on the membrane. Protein orientation due to membrane anchoring dominates over electrostatic effects and together with the increased lifetime of the encounter complex consequence that 2-dimensional association rate constants are quite similar and do not correlate with association rate constants in solution. The 2- dimensional dissociation rate constants were generally 2-5-fold lower compared to the corresponding 3-dimensional dissociation rate constants in solution. Possible explanations for this are that long lifetime of the encounter complex stabilizes the ternary complex or that membrane tethering affects the interaction diagram. In conclusion, combined TIRFS-RIf detection turn to be powerful and versatile technique to characterize protein-protein interactions on membranes.
Virtual screening of potential bioactive substances using the support vector machine approach
(2005)
Die vorliegende Dissertation stellt eine kumulative Arbeit dar, die in insgesamt acht wissenschaftlichen Publikationen (fünf publiziert, zwei eingerichtet und eine in Vorbereitung) dargelegt ist. In diesem Forschungsprojekt wurden Anwendungen von maschinellem Lernen für das virtuelle Screening von Moleküldatenbanken durchgeführt. Das Ziel war primär die Einführung und Überprüfung des Support-Vector-Machine (SVM) Ansatzes für das virtuelle Screening nach potentiellen Wirkstoffkandidaten. In der Einleitung der Arbeit ist die Rolle des virtuellen Screenings im Wirkstoffdesign beschrieben. Methoden des virtuellen Screenings können fast in jedem Bereich der gesamten pharmazeutischen Forschung angewendet werden. Maschinelles Lernen kann einen Einsatz finden von der Auswahl der ersten Moleküle, der Optimierung der Leitstrukturen bis hin zur Vorhersage von ADMET (Absorption, Distribution, Metabolism, Toxicity) Eigenschaften. In Abschnitt 4.2 werden möglichen Verfahren dargestellt, die zur Beschreibung von chemischen Strukturen eingesetzt werden können, um diese Strukturen in ein Format zu bringen (Deskriptoren), das man als Eingabe für maschinelle Lernverfahren wie Neuronale Netze oder SVM nutzen kann. Der Fokus ist dabei auf diejenigen Verfahren gerichtet, die in der vorliegenden Arbeit verwendet wurden. Die meisten Methoden berechnen Deskriptoren, die nur auf der zweidimensionalen (2D) Struktur basieren. Standard-Beispiele hierfür sind physikochemische Eigenschaften, Atom- und Bindungsanzahl etc. (Abschnitt 4.2.1). CATS Deskriptoren, ein topologisches Pharmakophorkonzept, sind ebenfalls 2D-basiert (Abschnitt 4.2.2). Ein anderer Typ von Deskriptoren beschreibt Eigenschaften, die aus einem dreidimensionalen (3D) Molekülmodell abgeleitet werden. Der Erfolg dieser Beschreibung hangt sehr stark davon ab, wie repräsentativ die 3D-Konformation ist, die für die Berechnung des Deskriptors angewendet wurde. Eine weitere Beschreibung, die wir in unserer Arbeit eingesetzt haben, waren Fingerprints. In unserem Fall waren die verwendeten Fingerprints ungeeignet zum Trainieren von Neuronale Netzen, da der Fingerprintvektor zu viele Dimensionen (~ 10 hoch 5) hatte. Im Gegensatz dazu hat das Training von SVM mit Fingerprints funktioniert. SVM hat den Vorteil im Vergleich zu anderen Methoden, dass sie in sehr hochdimensionalen Räumen gut klassifizieren kann. Dieser Zusammenhang zwischen SVM und Fingerprints war eine Neuheit, und wurde von uns erstmalig in die Chemieinformatik eingeführt. In Abschnitt 4.3 fokussiere ich mich auf die SVM-Methode. Für fast alle Klassifikationsaufgaben in dieser Arbeit wurde der SVM-Ansatz verwendet. Ein Schwerpunkt der Dissertation lag auf der SVM-Methode. Wegen Platzbeschränkungen wurde in den beigefügten Veröffentlichungen auf eine detaillierte Beschreibung der SVM verzichtet. Aus diesem Grund wird in Abschnitt 4.3 eine vollständige Einführung in SVM gegeben. Darin enthalten ist eine vollständige Diskussion der SVM Theorie: optimale Hyperfläche, Soft-Margin-Hyperfläche, quadratische Programmierung als Technik, um diese optimale Hyperfläche zu finden. Abschnitt 4.3 enthält auch eine Diskussion von Kernel-Funktionen, welche die genaue Form der optimalen Hyperfläche bestimmen. In Abschnitt 4.4 ist eine Einleitung in verschiede Methoden gegeben, die wir für die Auswahl von Deskriptoren genutzt haben. In diesem Abschnitt wird der Unterschied zwischen einer „Filter“- und der „Wrapper“-basierten Auswahl von Deskriptoren herausgearbeitet. In Veröffentlichung 3 (Abschnitt 7.3) haben wir die Vorteile und Nachteile von Filter- und Wrapper-basierten Methoden im virtuellen Screening vergleichend dargestellt. Abschnitt 7 besteht aus den Publikationen, die unsere Forschungsergebnisse enthalten. Unsere erste Publikation (Veröffentlichung 1) war ein Übersichtsartikel (Abschnitt 7.1). In diesem Artikel haben wir einen Gesamtüberblick der Anwendungen von SVM in der Bio- und Chemieinformatik gegeben. Wir diskutieren Anwendungen von SVM für die Gen-Chip-Analyse, die DNASequenzanalyse und die Vorhersage von Proteinstrukturen und Proteininteraktionen. Wir haben auch Beispiele beschrieben, wo SVM für die Vorhersage der Lokalisation von Proteinen in der Zelle genutzt wurden. Es wird dabei deutlich, dass SVM im Bereich des virtuellen Screenings noch nicht verbreitet war. Um den Einsatz von SVM als Hauptmethode unserer Forschung zu begründen, haben wir in unserer nächsten Publikation (Veröffentlichung 2) (Abschnitt 7.2) einen detaillierten Vergleich zwischen SVM und verschiedenen neuronalen Netzen, die sich als eine Standardmethode im virtuellen Screening etabliert haben, durchgeführt. Verglichen wurde die Trennung von wirstoffartigen und nicht-wirkstoffartigen Molekülen („Druglikeness“-Vorhersage). Die SVM konnte 82% aller Moleküle richtig klassifizieren. Die Klassifizierung war zudem robuster als mit dreilagigen feedforward-ANN bei der Verwendung verschiedener Anzahlen an Hidden-Neuronen. In diesem Projekt haben wir verschiedene Deskriptoren zur Beschreibung der Moleküle berechnet: Ghose-Crippen Fragmentdeskriptoren [86], physikochemische Eigenschaften [9] und topologische Pharmacophore (CATS) [10]. Die Entwicklung von weiteren Verfahren, die auf dem SVM-Konzept aufbauen, haben wir in den Publikationen in den Abschnitten 7.3 und 7.8 beschrieben. Veröffentlichung 3 stellt die Entwicklung einer neuen SVM-basierten Methode zur Auswahl von relevanten Deskriptoren für eine bestimmte Aktivität dar. Eingesetzt wurden die gleichen Deskriptoren wie in dem oben beschriebenen Projekt. Als charakteristische Molekülgruppen haben wir verschiedene Untermengen der COBRA Datenbank ausgewählt: 195 Thrombin Inhibitoren, 226 Kinase Inhibitoren und 227 Faktor Xa Inhibitoren. Es ist uns gelungen, die Anzahl der Deskriptoren von ursprünglich 407 auf ungefähr 50 zu verringern ohne signifikant an Klassifizierungsgenauigkeit zu verlieren. Unsere Methode haben wir mit einer Standardmethode für diese Anwendung verglichen, der Kolmogorov-Smirnov Statistik. Die SVM-basierte Methode erwies sich hierbei in jedem betrachteten Fall als besser als die Vergleichsmethoden hinsichtlich der Vorhersagegenauigkeit bei der gleichen Anzahl an Deskriptoren. Eine ausführliche Beschreibung ist in Abschnitt 4.4 gegeben. Dort sind auch verschiedene „Wrapper“ für die Deskriptoren-Auswahl beschrieben. Veröffentlichung 8 beschreibt die Anwendung von aktivem Lernen mit SVM. Die Idee des aktiven Lernens liegt in der Auswahl von Molekülen für das Lernverfahren aus dem Bereich an der Grenze der verschiedenen zu unterscheidenden Molekülklassen. Auf diese Weise kann die lokale Klassifikation verbessert werden. Die folgenden Gruppen von Moleküle wurden genutzt: ACE (Angiotensin converting enzyme), COX2 (Cyclooxygenase 2), CRF (Corticotropin releasing factor) Antagonisten, DPP (Dipeptidylpeptidase) IV, HIV (Human immunodeficiency virus) protease, Nuclear Receptors, NK (Neurokinin receptors), PPAR (peroxisome proliferator-activated receptor), Thrombin, GPCR und Matrix Metalloproteinasen. Aktives Lernen konnte die Leistungsfähigkeit des virtuellen Screenings verbessern, wie sich in dieser retrospektiven Studie zeigte. Es bleibt abzuwarten, ob sich das Verfahren durchsetzen wird, denn trotzt des Gewinns an Vorhersagegenauigkeit ist es aufgrund des mehrfachen SVMTrainings aufwändig. Die Publikationen aus den Abschnitten 7.5, 7.6 und 7.7 (Veröffentlichungen 5-7) zeigen praktische Anwendungen unserer SVM-Methoden im Wirkstoffdesign in Kombination mit anderen Verfahren, wie der Ähnlichkeitssuche und neuronalen Netzen zur Eigenschaftsvorhersage. In zwei Fällen haben wir mit dem Verfahren neuartige Liganden für COX-2 (cyclooxygenase 2) und dopamine D3/D2 Rezeptoren gefunden. Wir konnten somit klar zeigen, dass SVM-Methoden für das virtuelle Screening von Substanzdatensammlungen sinnvoll eingesetzt werden können. Es wurde im Rahmen der Arbeit auch ein schnelles Verfahren zur Erzeugung großer kombinatorischer Molekülbibliotheken entwickelt, welches auf der SMILES Notation aufbaut. Im frühen Stadium des Wirstoffdesigns ist es wichtig, eine möglichst „diverse“ Gruppe von Molekülen zu testen. Es gibt verschiedene etablierte Methoden, die eine solche Untermenge auswählen können. Wir haben eine neue Methode entwickelt, die genauer als die bekannte MaxMin-Methode sein sollte. Als erster Schritt wurde die „Probability Density Estimation“ (PDE) für die verfügbaren Moleküle berechnet. [78] Dafür haben wir jedes Molekül mit Deskriptoren beschrieben und die PDE im N-dimensionalen Deskriptorraum berechnet. Die Moleküle wurde mit dem Metropolis Algorithmus ausgewählt. [87] Die Idee liegt darin, wenige Moleküle aus den Bereichen mit hoher Dichte auszuwählen und mehr Moleküle aus den Bereichen mit niedriger Dichte. Die erhaltenen Ergebnisse wiesen jedoch auf zwei Nachteile hin. Erstens wurden Moleküle mit unrealistischen Deskriptorwerten ausgewählt und zweitens war unser Algorithmus zu langsam. Dieser Aspekt der Arbeit wurde daher nicht weiter verfolgt. In Veröffentlichung 6 (Abschnitt 7.6) haben wir in Zusammenarbeit mit der Molecular-Modeling Gruppe von Aventis-Pharma Deutschland (Frankfurt) einen SVM-basierten ADME Filter zur Früherkennung von CYP 2C9 Liganden entwickelt. Dieser nichtlineare SVM-Filter erreichte eine signifikant höhere Vorhersagegenauigkeit (q2 = 0.48) als ein auf den gleichen Daten entwickelten PLS-Modell (q2 = 0.34). Es wurden hierbei Dreipunkt-Pharmakophordeskriptoren eingesetzt, die auf einem dreidimensionalen Molekülmodell aufbauen. Eines der wichtigen Probleme im computerbasierten Wirkstoffdesign ist die Auswahl einer geeigneten Konformation für ein Molekül. Wir haben versucht, SVM auf dieses Problem anzuwenden. Der Trainingdatensatz wurde dazu mit jeweils mehreren Konformationen pro Molekül angereichert und ein SVM Modell gerechnet. Es wurden anschließend die Konformationen mit den am schlechtesten vorhergesagten IC50 Wert aussortiert. Die verbliebenen gemäß dem SVM-Modell bevorzugten Konformationen waren jedoch unrealistisch. Dieses Ergebnis zeigt Grenzen des SVM-Ansatzes auf. Wir glauben jedoch, dass weitere Forschung auf diesem Gebiet zu besseren Ergebnissen führen kann.
After a brief introduction on QCD and effective models in the first chapter, I analyze the dependence of the QCD transition temperature on the quark (or pion) mass in the second chapter. I found that a linear sigma model, which links the transition to chiral symmetry restoration, predicts a much stronger dependence of T_c on m_pi than seen in present lattice data for m_pi >~ 0.4 GeV. On the other hand, an effective Lagrangian for the Polyakov loop requires only small explicit symmetry breaking to describe T_c(m_pi) in the above mass range. In the third and fourth chapter, I study the linear sigma model with O(N) symmetry at nonzero temperature in the framework of the Cornwall-Jackiw-Tomboulis formalism. Extending the set of two-particle irreducible diagrams by adding sunset diagrams to the usual Hartree-Fock (or Hartree) contributions, I derive a new approximation scheme which extends the standard Hartree-Fock (or Hartree) approximation by the inclusion of nonzero decay widths.
In the present study possible sources and pathways of the gasoline additive methyl tertiary-butyl ether (MTBE) in the aquatic environment in Germany were investigated. The objective of the present study was to clarify some of the questions raised by a previous study on the MTBE situation in Germany. In the USA and Europe 12 million t and 3 million t of MTBE, respectively, are used as gasoline additive. The detection of MTBE in the aquatic environment and the potential risk for drinking water resources led to a phase-out of MTBE as gasoline additive in single states of the USA. Meanwhile there is also an ongoing discussion about the substitution of MTBE in Europe and Germany. The annual usage of MTBE in Germany is about 600,000 t. However, compared to the USA, significant less data exists on the occurrence of MTBE in the aquatic environment in Europe. Because of its physico-chemical properties, MTBE readily vaporizes from gasoline, is water soluble, adsorbs only weakly to the underground matrix and is largely persistent to biological degradation. The toxicity of MTBE remains to be completely investigated, but MTBE in drinking water has low taste- and odor thresholds of 20-40 microgram/L. The present study was conducted by collecting water samples and analyzing them for their MTBE concentrations through a combination of headspace-solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS). The detection limit was 10 ng/L. The method was successfully tested in the framework of an interlaboratory study and showed recoveries of reference values of 89% (74 ng/L) and 104% (256 ng/L). The relative standard deviations were 12% and 6%. The investigation of 83 water samples from 50 community water systems (CWSs) in Germany revealed a detection frequency of 40% and a concentration range of 17-712 ng/L. The detection of MTBE in the drinking water samples could be explained by a groundwater pollution and the pathway river - riverbank filtration - waterworks. Rivers are important drinking water sources. MTBE is emitted into rivers through a variety of sources. In the present study, potential point sources were investigated, i.e. MTBE production sites/refineries/tank farms and groundwater pollutions. For this purpose, the spatial distribution of MTBE in three German rivers with the named potential emission sources located close to the rivers was investigated by analyzing 49 corresponding river water samples. The influence of the potential emission sources groundwater pollution and refinery/tank farm was successfully demonstrated in certain parts of the River Saale and the River Rhine. Increasing MTBE concentrations from 24 ng/L to 379 ng/L and from 73 ng/L to 5 microgram/L, respectively, could be observed in the parts investigated in these two rivers. The identification of such emission sources is important for future modeling. Further sources of MTBE emission into surface water are industrial (non-petrochemical) and municipal sewage plant effluents. In the present study long-term monitoring of water from the River Main (n=67 samples), precipitation (n=89) and industrial (n=34) and municipal sewage plant effluents (n=66) was conducted. The comparison of the data sets revealed that maximum MTBE concentrations in the River Main of up to 1 microgram/L were most possibly due to single industrial effluents with MTBE concentrations of up to 28 microgram/L (measured in this study). The average MTBE content of 66 ng/L in the River Main most probably originated from municipal sewage plant effluents and further industrial effluents. Background concentrations of <30 ng/L could be related to the direct atmospheric input via precipitation. A certain aspect of the atmospheric MTBE input is represented by the input of MTBE into river water or groundwater through snow. In the present study 43 snow samples from 13 different locations were analyzed for their MTBE content. MTBE could be detected in 65% of the urban and rural samples. The concentrations ranged from 11-613 ng/L and were higher than the concentrations in rainwater samples formerly analyzed. Furthermore, a temperature dependency and wash-out effects could be observed. The atmospheric input of MTBE was in part also visible in the analyzed groundwater samples (n=170). The detection frequencies in non-urban and urban wells were 24% and 63%, respectively. The median concentrations were 177 ng/L and 57 ng/L. In wells located in the vicinity of sites with gasoline contaminated groundwater, MTBE concentrations of up to 42 mg/L could be observed. The MTBE emission sources and the different pathways of MTBE in the aquatic environment demonstrated in the present study and other works raise the question whether the use of MTBE in a bulk product like gasoline should be continued in the future. Currently, possible substitutes like ethyl tertiary-butyl ether (ETBE) or ethanol are being discussed.
Biophysical investigation of the ligand-induced assembling of the human type I interferon receptor
(2005)
Type I interferons (IFNs) elicit antiviral, antiproliferative and immunmodulatory responses through binding to a shared receptor consisting of the transmembrane proteins ifnar1 and ifnar2. Differential signaling by different interferons – in particular IFNalpha´s and IFNbeta – suggest different modes of receptor engagement. In this work either single ligand-receptor interactions or the formation of the extracellular part of a signaling complex were investigated referring to thermodynamics, kinetics, stoichiometry and structural organization. Initially an expression and purification strategy for the extracellular domain of ifnar1 (ifnar1-EC) using Sf9 insect cells yielding in mg amounts of glycosylated protein was established. Using reflectometric interference spectroscopy (RIfS) the interactions between IFNalpha2/beta and ifnar1-EC and ifnar2-EC was studied in order to understand the individual energetic contributions within the ternary complex. For IFNalpha2 a Kd of 5 µM for the interaction with ifnar1-EC was determined. Substantially tighter binding of IFNbeta with both ifnar2-EC and ifnar1-EC compared to IFNalpha2 was observed. For neither IFNalpha2 nor IFNbeta stabilization of the complex with ifnar1-EC in presence of soluble ifnar2-EC was detectable. In addition, no direct interaction between ifnar2 and ifnar1 was could be shown. Thus, stem-stem interactions between the extracellular domains of ifnar1 and ifnar2 do not seem to play a role for ternary complex formation. Furthermore, ligand-induced cross-talk between ifnar1-EC and ifnar2-EC being tethered onto solid-supported, fluid lipid bilayers was investigated by RIfS and total internal reflection fluorescence spectroscopy. A very stable binding of IFNalpha2 at high receptor surface concentrations was observed with an apparent kd approximately 200-times lower than for ifnar2-EC alone. This apparent kd was strongly dependent on the surface concentration of the receptor components, suggesting kinetic rather than static stabilization, which was corroborated by competition experiments. These results indicate that signaling is activated by transient cross-talk between ifnar1 and ifnar2, which is by several orders of magnitude more efficiently engaged by IFNbeta than by IFNalpha2. With respect to differential recognition of different IFNs ifnar1-EC was dissected into sub-fragments containing different of the four Ig-like domains. The appropriate folding and glycosylation of these proteins, also purified in mg amounts were confirmed by SDS-PAGE, size exclusion chromatography and CD-spectroscopy. Surprisingly, only one construct containing all three N-terminal Ig-like domains was active in terms of ligand binding, indicating that these domains were required. Competitive binding of IFNalpha2 and IFNbeta to both this fragment and ifnar1-EC was demonstrated. Cellular binding assays with different fragments, however, highlight the key role of the membrane-proximal Ig-like domain for the formation of an in situ IFN-receptor complex and the ensuing signal activation. Even substitution with Ig-like domains from homologous cytokine receptors did not restore high-affinity ligand binding. Receptor assembling analysis on supported lipid bilayer revealed that appropriate orientation of the receptor is required, which is controlled by the membrane-proximal Ig-domain. All results indicate that differential signalling is encoded by the efficiency of signalling complex formation, which is controlled by the binding affinity of IFNs to the extracellular domains of ifnar1 and 2.
Here I analyse 23 populations of D. galeata, a large-lake cladoceran, distributed mainly across the Palaearctic. I detected high levels of clonal diversity and population differentiation using variation at six microsatellite loci across Europe. Most populations were characterised by deviations from H-W equilibrium and significant heterozygote deficiencies. Observed heterozygote deficiencies might be a consequence of simultaneous hatching of individuals produced during different times of the year or of the coexistence of ecologically and genetically differentiated subpopulations. A significant isolation by distance was only found over large geographic distances (> 700 km). This pattern is mainly due to the high genetic differentiation among neighbouring populations. My results suggest that historic populations of Daphnia were once interconnected by gene flow but current populations are now largely isolated. Thus local ecological conditions which determine the level of biparental sexual reproduction and local adaptation are the main factors mediating population structure of D. galeata. The population genetic structure and diversity in D. galeata was investigated at a European scale using six microsatellite loci and 12S rDNA sequence data to infer and compare historical and contemporary patterns of gene flow. D. galeata has the potential for long-distance dispersal via ephippial resting eggs by wind and other dispersing vectors (waterfowl), but shows in general strong population differentiation even among neighbouring populations. A total of 427 individuals were analysed for microsatellite and 85 individuals for mitochondrial (mtDNA) sequence data from 12 populations across Europe. I detected genetic differentiation among populations across Europe and locations within sampling regions for both genetic marker systems (average values: mtDNA FST = 0.574; microsatellite FST = 0.389), resulting in a lack of isolation by distance. Furthermore, several microsatellite alleles and one haplotype were shared across populations. Partitioning of molecular variance was inconsistant for both marker systems. Microsatellite variation was higher within than among populations, whereas mtDNA data yielded an inverse pattern. Relative high levels of nuclear DNA diversity were found across Europe. The amount of mitochondrial diversity was low in Spain, Hungary and Denmark. Gene flow analysis at a European scale did not reveal typical pattern of population recolonization in the light of postglacial colonization hypotheses. Populations, which recently experienced an expansion or population-bottleneck were observed both in middle and northern Europe. Since these populations revealed high genetic diversity in both marker systems, I suggest these areas to represent postglacial zones of secondary contact among divergent lineages of D. galeata. In order to reveal the relationship between population genetic structure of D. galeata and the relative contribution of environmental factors, I used a statistical framework based on canonical correspondence analysis. Although I detected no single ecological gradient mediating the genetic differentiation in either lake regions, it is noteworthy that the same ecological factors were significantly correlated with intra- and interspecific genetic variation of D. galeata. For example, I found a relationship between genetic variation of D. galeata and differentiation with higher and lower trophic levels (phytoplankton, submerged macrophytes and fish) and a relationship between clonal variation and species diversity within Cladocera. Variance partitioning had only a minor contribution of each environmental category (abiotic, biomass/density and diversity) to genetic diversity of D. galeata, while the largest proportion of variation was explained by shared components. My work illustrates the important role of ecological differentiation and adaptation in structuring genetic variation, and it highlights the need for approaches incorporating a landscape context for population divergence.
Die vorliegende Arbeit beschäftigt sich mit der Charakterisierung des ALTRO Chips (ALICE TPC Readout), der ein integraler und wichtiger Bestandteil der Auslesekette des TPC (Time Projection Chamber) Detektors von ALICE (A Large Ion Collider Experiment) ist. ALICE ist ein Experiment am noch im Bau befindlichen LHC (Large Hadron Collider) am CERN mit der zentralen Ausrichtung, Schwerionenkollisionen zu untersuchen. Diese sind von besonderem Interesse, da durch sie ein experimenteller Zugriff zu dem QGP (Quark Gluon Plasma) existiert, dem einzigen vom Standardmodell vorhergesagten Phasenübergang, der unter Laborbedingungen erreichbar ist. Im Jahr 2004 wurden Messungen an einem Teststrahl am CERN PS (Proton Synchrotron) durchgeführt. Der Prototyp wurde voll mit FECs bestückt, was 5400 Kanälen entspricht und einer anderen Gasmixtur (Ne/N2/CO2 90%/5%/5%) befüllt. Für das optimale Leistungsverhalten der ALICE TPC muß der Digitalprozessor im ALTRO, bestehend aus vier Berechnungseinheiten, mit den passenden Werten konfiguriert werden. Der Datenfluss beginnt mit dem BCS1 (Baseline Correction and Subtraction 1) Modul, das systematische Störungen und die Grundlinie entfernt. Da der ALTRO kontinuierlich das anliegende Signal abtastet, entfernt es automatisch langsame Grundlinienveränderungen, die Beispielsweise durch Temperaturänderungen auftreten können. Gefolgt von dem TCF (Tail Cancellation Filter), der den Schweif des langsam fallenden, vom PASA generierten Signals entfernt. Um die nichtsystematischen Störungen der Grundlinie zu entfernen, folgt die BCS2 (Baseline Correction and Subtraction 2), die auf einer gleitenden Mittelwertsberechnung mit Ausschluß von Detektorsignalen über einen doppelten Schwellenwert basiert. Die finale Einheit für die Signalverarbeitung ist die ZSU (Zero Suppression Unit), die Meßpunkte unterhalb eines definierten Schwellwertes entfernt. Hier wird der weg beschrieben die TCF und BCS1 Parameter aus vorhandenen Detektordaten zu extrahieren. Während der Analyse der Daten von kosmischen Teilchen fiel bei Signalen mit hoher Amplitude (>700 ADC) eine zusätzliche Struktur in dem Schweif auf. Der Monitor wurde deswegen mit einem gleitenden Mittelwertfilter erweitert, worauf sich diese Struktur auch in kleineren Signalen (> 200 ADC) zeigte. Dieses Signal wird von Ionen erzeugt, die zur Kathode oder zu den Pads driften, bisher ist jedoch weder die Streuung der Elektronenlawine an der Anode, noch die Variationsbreite in den erzeugten Elektronlawinen verstanden oder gemessen worden. Eine erfolgreiche Messung, sowie Charakterisierung wird in dieser Arbeit beschrieben. Im Jahr 2005 im Sommer beginnt der Einbau der Gaskammern der TPC in ALICE, die Elektronik folgt am Ende dieses Jahres. Parallel hierzu wurde der Prototyp der TPC wieder in Betrieb genommen und im Frühling wird ein kompletter Sektor mit der Detektorelektronik ausgestattet. An diesen zwei Aufbauten wird die ALTRO Charakterisierung fortgeführt, verfeinert und komplettiert.
Mitochondial NADH:ubiquinone oxidoreductase (complex I) the largest multiprotein enzyme of the respiratory chain, catalyses the transfer of two electrons from NADH to ubiquinone, coupled to the translocation of four protons across the membrane. In addition to the 14 strictly conserved central subunits it contains a variable number of accessory subunits. At present, the best characterized enzyme is complex I from bovine heart with a molecular mass of about 980 kDa and 32 accessory proteins. In this study, the subunit composition of mitochondrial complex I from the aerobic yeast Y. lipolytica has been analysed by a combination of proteomic and genomic approaches. The sequences of 37 complex I subunits were identified. The sum of their individual molecular masses (about 930 kDa) was consistent with the native molecular weight of approximately 900 kDa for Y. lipolytica complex I obtained by BN-PAGE. A genomic analysis with Y. lipolytica and other eukaryotic databases to search for homologues of complex I subunits revealed 31 conserved proteins among the examined species. A novel protein named “X” was found in purified Y. lipolytica complex I by MALDI-MS. This protein exhibits homology to the thiosulfate sulfurtransferase enzyme referred to as rhodanese. The finding of a rhodanese-like protein in isolated complex I of Y. lipolytica allows to assume a special regulatory mechanism of complex I activity through control of the status of its iron-sulfur clusters. The second part of this study was aimed at investigating the possible role of one of these extra subunits, 39 kDa (NUEM) subunit which is related to the SDRs-enzyme family. The members of this family function in different redox and isomerization reactions and contain a conserved NAD(P)H-binding site. It was proposed that the 39 kDa subunit may be involved in a biosynthetic pathway, but the role of this subunit in complex I is unknown. In contrast to the situation in N. crassa, deletion of the 39 kDa encoding gene in Y. lipolytica led to the absence of fully assembled complex I. This result might indicate a different pathway of complex I assembly in both organisms. Several site-directed mutations were generated in the nucleotide binding motif. These had either no effect on enzyme activity and NADPH binding, or prevented complex I assembly. Mutations of arginine-65 that is located at the end of the second b-strand and responsible for selective interaction with the 2’-phosphate group of NADPH retained complex I activity in mitochondrial membranes but the affinity for the cofactor was markedly decreased. Purification of complex I from mutants resulted in decrease or loss of ubiquinone reductase activity. It is very likely that replacement of R65 not only led to a decrease in affinity for NADPH but also caused instability of the enzyme due to steric changes in the 39 kDa subunit. These data indicate that NADPH bound to the 39 kDa subunit (NUEM) is not essential for complex I activity, but probably involved in complex I assembly in Y. lipolytica.
The thesis entitled „Investigations on the significance of nucleo-cytoplasmic transport for the biological function of cellular proteins" aimed to unreveal molecular mechanisms in order to improve our understanding of the impact of nucleo-cytoplasmic transport on cellular functions. Within the scope of this work, it could be shown that regulated nucleo-cytoplasmic transport of a subfamily of homeobox transcription factors controlled their intra- and intercellular transport, and thereby influencing also their transcriptional activity. This study describes a novel regulatory mechanism, which could in general play an important role for the ordered differentiation of complex organisms. Besides cis-active transport Signals, also post-translational modifications can influence the localization and biological activity of proteins in trans. In addition to the known impact of phosphorylation on the transport and activity of STAT1, experimental evidence was provided demonstrating that acetylation affected the interaction of STAT1 with NF-kB p65, and subsequently modulated the expression of apoptosis-inducing NF-kB target genes. The impact of nucleo-cytoplasmic transport on the regulation of apoptosis was underlined by showing that the evolutionary conservation of a NES within the anti-apoptotic protein survivin plays an essential role for its dual function in the inhibition of apoptosis and ordered cell division. Since survivin is considered a bona fide cancer therapy target, these results strongly encourage future work to identify molecular decoys that specifically inhibit the nuclear export of survivin as novel therapeutics. In order to further dissect the regulation of nuclear transport and to efficiently identify transport inhibitors, cell-based assays are urgently required. Therefore, the cellular assay Systems developed in this work may not only serve to identify synthetic nuclear export and Import inhibitors but may also be applied in systematic RNAi-screening approaches to identify novel components of the transport machinery. In addition, the translocation based protease- and protein-interaction biosensors can be applied in various biological Systems, in particular to identify protein-protein interaction inhibitors of cancer relevant proteins. In summary, this work does not only underline the general significance of nucleo-cytoplasmic transport for cell biology, but also demonstrates its potential for the development of novel therapies against diseases like cancer and viral infections.
Calcium-activated potassium channels are fundamental regulators of neuron excitability. SK channels are activated by an intracellular increase of Ca++ (such as occurs during an action potential). They have a small single channel conductance (less than 20pS) and show no voltage dependence of activation. To date, there are only a few examples of high-resolution structures of eukaryotic membrane proteins. All of them were purified from natural sources. Since no abundant natural sources of eukaryotic K+ channels are available we overexpressed rSK2 in order to produce the quantities necessary for structural analysis. Unfortunately the Pichia pastoris expression system did not yield sufficient amount of pure protein, mainly because most of the protein was retained by in the ER and was only partially soluble. Subsequently, two constructs were expressed: SK2-FCYENE (containing a specific sequence that promotes surface expression), and SK2-q-CaM a concatamer of SK2 and calmodulin. Although these proved an improvement in terms of solubilisation, little improvement was found in terms of amounts of purified material obtained. For this reason we tested the Semliki Forest virus expression system, since the protein is expressed in a mammalian system where we hoped that it would be trafficked in the same way as in vivo. Using this system it was possible to express rSK2 and solubilise it with several detergents and to achieve much better purification. However, the levels were still not sufficient for high-resolution structural studies, although sufficient for single particle electron microscopy analysis.
Molecular dynamics (MD) simulation serves as an important and widely used computational tool to study molecular systems at an atomic resolution. No experimental technique is capable of generating a complete description of the dynamical structure of the biomolecules in their native solution environment. MD simulations allow us to study the dynamics and structure of the system and, moreover, helps in the interpretation of experimental observations. MD simulation was first introduced and applied by Alder and Wainwright in 1957 \cite{Alder57}. However, the first MD simulation of a macromolecule of biological interest was published 28 years ago \cite{McCammon77}. The simulation was concerned with the bovine pancreatic trypsin inhibitor (BPTI) protein, which has served as the hydrogen molecule'' of protein dynamics because of its small size, high stability, and relatively accurate X-ray structure available in 1977 \cite{Deisenhofer75}. This method is now widely used to tackle larger and more complex biological systems \cite{Groot01,Roux02} and has been facilitated by the development of fast and efficient methods for treating the long-range electrostatic interactions \cite{Essmann95}, the availability of faster parallel computers, and the continuous development of empirical molecular mechanical force fields \cite{Langley98,Cheatham99,Foloppe00}. It took several years until the first MD simulations of nucleic acid systems were performed \cite{Levitt83,Tidor83,Prabhakaran83,Nilsson86}. These investigations, which were also performed in vacuo, clearly demonstrated the importance of proper handling of electrostatics in a highly charged nucleic acid system, and different approaches, such as reduction of the phosphate charges and addition of hydrated counterions, have been applied to remedy this shortcoming and to maintain stable DNA structures. A few years later, the first MD simulation of a DNA molecule, including explicit water molecules and counterions was published \cite{Seibel85}. Various MD simulations on fully solvated RNA molecules with explicit inclusion of mobile ions indicated the importance of proper treatment of the environment of highly charged nucleic acids \cite{Lee95,Zichi95,Auffinger97,Auffinger99}. Given the central roles of RNA in the life of cells, it is important to understand the mechanism by which RNA forms three dimensional structures endowed with properties such as catalysis, ligand binding, and recognition of proteins. Furthermore, the increasing awareness of the essential role of RNA in controlling viral replication and in bacterial protein synthesis emphazises the potential of ribonucleicacids as targets for developing new antibacterial and new antiviral drugs. Driven by fruitful collaborations in the Sonderforschungsbereich RNA-Ligand interactions" the model RNA systems in this study include various RNA tetraloops and HIV-1 TAR RNA. For the latter system, the binding sites of heteroaromatic compounds have been studied employing automated docking calculations \cite{Goodsell90}. The results show that it is possible to use this tool to dock small rigid ligands to an RNA molecule, while large and flexible molecules are clearly problematic. The main part of this work is focused on MD simulations of RNA tetraloops.
The quinol:fumarate reductase (QFR) is the terminal reductase of anaerobic fumarate respiration, the most commonly occurring type of anaerobic respiration. This membrane protein complex couples the oxidation of menaquinol to menaquinone to the reduction of fumarate to succinate. The three-dimensional crystal structure of the QFR from Wolinella succinogenes has previoulsy been solved at 2.2 Å resolution. Although the diheme-containing QFR from W. succinogenes is known to catalyze an electroneutral process, structural and functional characterization of parental and variant enzymes has revealed active site locations which indicate electrogenic catalysis across the membrane. A solution to this apparent controversy was proposed with the so-called “Epathway hypothesis”. According to this, transmembrane electron transfer via the heme groups is strictly coupled to a parallel, compensatory transfer of protons via a transiently established pathway, which is inactive in the oxidized state of the enzyme. Proposed constituents of the E-pathway are the side chain of Glu C180, and the ring C propionate of the distal heme. Previous experimental evidence strongly supports such a role for the former constituent. One aim of this thesis is to investigate by a combination of specific 13C-heme propionate labeling and FTIR difference spectroscopy whether the ring C propionate of the distal heme is involved in redox-coupled proton transfer in the QFR from W. succinogenes. In addition to W. succinogenes, the primary structures of the QFR enzymes of two other e- proteobacteria are known. These are Campylobacter jejuni and Helicobacter pylori, which unlike W. succinogenes are human pathogens. The QFR from H. pylori has previously been established to be a potential drug target, and the same is likely for the QFR from C. jejuni. The two pathogenic species colonize mucosal surfaces causing several diseases. The possibility of studying these QFRs from these bacteria and creating more efficient drugs specifically active for this enzyme depends substantially on the availability of large amounts of high-quality protein. Further, biochemical and structural studies on QFR enzymes from e- proteobacteria species other than W. succinogenes can be valuable to enlighten new aspects or corroborate the current understanding of this class of membrane proteins.
A strong interest is currently going on in the physics of high intensity and high energy beams: intense proton or deuteron beams are required in various fields of science and industry, including sources of neutrons for research experiments and material processing, nuclear physics experiments, tritium production and nuclear waste transmutation. High current heavy ion beams are envisaged for power production facilities (inertial fusion). Several projects presently under study are based on rf linacs as driver, sometimes followed by accumulation and/or compressor rings [Acc98]. The critical issue for all of them is to be operated in a low loss regime, because of activation problems in the structure. For this reason careful investigations have to be performed in order to understand and control the beam behaviour, aiming at conserving the beam quality, reducing the emittance growth and filamentation and avoiding the formation of halo. The beam current to be accelerated is actually limited by the amount of beam losses, which depends upon the beam halo: in order to reduce induced radioactivity and to allow for hands-on maintenance, normally losses <1 W/m are considered as acceptable [Sto96]. One of the major facilities under study is the European Spallation Source (ESS), a project based on a H- linac accelerating a 107 mA peak current beam (360 ns pulse in the DTL) and on two compressor rings, producing 5 MW average beam power [ESS]. Also the USA are developing a proposal for a Spallation Neutron Source (SNS), providing a short pulse H- beam with average power of 1÷2 MW; a 30 mA linac is required [SNS]. The Accelerator for Production of Tritium (APT), studied at Los Alamos, requires a 100 mA proton beam current (cw) to produce a power of 130÷170 MW [APT]. A similar but smaller accelerator (40 mA, 40 MW beam power) would serve as driver for the Accelerator Driven Transmutation of Waste (ADTW) system [ATW]. The accelerator system for the International Fusion Material Irradiation Facility (IFMIF) will test the behaviour of materials to be used for magnetic fusion (e.g. ITER); it consists of two 125 mA deuteron beams in parallel, to generate a fusion-like neutron spectrum with 10 MW cw [IFM]. In the field of heavy ions, for about 20 years scientists have been working on inertial confinement fusion, as an alternative to magnetic confinement one, to find a practical and cleaner method for producing energy. Nuclear fusion occurs when the nuclei of lighter elements (in a state of matter called "plasma") merge to form heavier elements; the extremely high temperatures and densities needed to get the nuclei to collide in the proper way and release big amounts of energy are obtained in a small "pellet" of fusion fuel, which receives energy from laser or ion beams, implodes and its inertia compresses it hard enough to hold together the plasma until it reaches ignition. Both laser and accelerator facilities have been investigated as drivers, since a demonstration of ignition at low gains is more easily accessible by lasers, whereas the intrinsic properties of accelerators -efficiency and repetition rate- will be essential for a medium-gain power plant. One study for a fusion power system driven by heavy ion beams (HIBALL) was completed in Europe already in 1982 [Bad81]. When the USA declassified essential information on pellet design, "indirect drive" targets have been considered openly, where the pellet is hit by X-rays generated from laser or ion beams rather than directly from the beams. Main progress has been achieved during the latest years in the understanding of pellet dynamics after ignition, i.e. in plasma physics [Sym1][Sym2][Sym3][Bas97][Lut97], imposing also new requirements on the layout of the driver accelerator facilities. In 1994-95 Frankfurt University and several other European laboratories (leaded by GSI) started a new collaboration called HIDIF (Heavy Ion Driven Ignition Facility) in order to simplify the accelerator plant design owing to the new technique of indirectly driven targets and to some technological improvements. First studies were oriented towards the conceptual goal of a facility providing just enough beam energy for the ignition of fusion reactions at very low gain (a "proof of principle") [Hof98]. In a recent phase of the study, it was realized that the proposed concept would make this scheme a more appropriate choice for energy production rather than for ignition; the acronym HIDIF was therefore intended as Heavy Ion Driven Inertial Fusion, and the parameters are going to be modified accordingly [Hof96][Hof97][Hof98]. The scenario presently discussed by this group proposes the formation and acceleration of an intense beam (400 mA) of singly charged heavy ions of three different atomic species, with mass differences of about 10% (the reference one is 209Bi+) in a main rf linac; they are then injected into some storage rings at an energy of 50 MeV/u, bunched in induction linacs and finally transported to a target with different velocities in such a way that the three species merge on the pellet ("telescoping") at 500 TW peak power. In this thesis the main linac of the HIDIF proposal is extensively investigated as an example of a high intensity heavy ion linac. Results are presented from numerical simulations of multi-particle beam dynamics carried out for the first time in this context. After a short presentation of the HIDIF reference scenario (Ignition Facility), including a discussion of the motivations for a high current heavy ion linac, some elements of the theory of beam transport and acceleration are recalled [Con91][Hof82][Kap85] [Lap87][Law88][Mit78][Rei94][Str83]. Then the used simulation programs are described, and a particle dynamics layout of a conventional 200 MHz Alvarez DTL is discussed with respect to low emittance growth at high transmission, including large space-charge effects, taking into account the influence of different kinds of statistical errors and of input mismatch on the beam dynamics. The modifications needed for "telescoping" are investigated with simulations for the nominal mass difference (10%) and for a smaller one (5%); finally the transfer line between DTL and rings is discussed and studied both analytically and by numerical calculations. The large mass number (A= 209) helps to reduce the space-charge effects with respect to protons, therefore the behaviour of the beam is not space-charge dominated. Nevertheless the tune depression values (similar to those of the ESS linac e.g.) indicate that these effects cannot be neglected. For a linac with low duty cycle, as in the case of an ignition facility, the results from particle dynamics calculations can be considered as a reliable guideline for the DTL layout, since they indicate that such a high intensity linac can fulfill the requirements on smooth beam behaviour and low losses.
The challenging intricacies of strongly correlated electronic systems necessitate the use of a variety of complementary theoretical approaches. In this thesis, we analyze two distinct aspects of strong correlations and develop further or adapt suitable techniques. First, we discuss magnetization transport in insulating one-dimensional spin rings described by a Heisenberg model in an inhomogeneous magnetic field. Due to quantum mechanical interference of magnon wave functions, persistent magnetization currents are shown to exist in such a geometry in analogy to persistent charge currents in mesoscopic normal metal rings. The second, longer part is dedicated to a new aspect of the functional renormalization group technique for fermions. By decoupling the interaction via a Hubbard-Stratonovich transformation, we introduce collective bosonic variables from the beginning and analyze the hierarchy of flow equations for the coupled field theory. The possibility of a cutoff in the momentum transfer of the interaction leads to a new flow scheme, which we will refer to as the interaction cutoff scheme. Within this approach, Ward identities for forward scattering problems are conserved at every instant of the flow leading to an exact solution of a whole hierarchy of flow equations. This way the known exact result for the single-particle Green's function of the Tomonaga-Luttinger model is recovered.
Cancer has become one of the most fatal diseases. The Heidelberg Heavy Ion Cancer Therapy (HICAT) has the potential to become an important and efficient treatment method because of its excellent “Bragg peak” characteristics and on-line irradiation control by the PET diagnostics. The dedicated Heidelberg Heavy Ion Cancer Therapy Project includes two ECR ion sources, a RF linear injector, a synchrotron and three treatment rooms. It will deliver 4*10 high 10 protons, or 1*10 high 10 He, or 1*10 high 9 Carbons, or 5*10 high 8 Oxygens per synchrotron cycle with the beam energy 50-430AMeV for the treatments. The RF linear injector consists of a 400AkeV RFQ and of a very compact 7AMeV IH-DTL accelerator operated at 216.816MHz. The development of the IH-DTL within the HICAT project is a great challenge with respect to the present state of the DTL art because of the following reasons: • The highest operating frequency (216.816MHz) of all IH-DTL cavities; • Extremely large cavity length to diameter ratio of about 11; • IH-DTL with three internal triplets; • The highest effective voltage gain per meter (5.5MV/m); • Very short MEBT design for the beam matching. The following achievements have been reached during the development of the IH-DTL injector for HICAT : The KONUS beam dynamics design with LORASR code fulfills the beam requirement of the HICAT synchrotron at the injection point. The simulations for the IH-DTL injector have been performed not only with a homogeneous input beam, but also with the actual particle distribution from the exit of the HICAT RFQ accelerator as delivered by the PARMTEQ code. The output longitudinal normalized emittance for 95% of all particles is 2.00AkeVns, the emittance growth is less than 24%, while the X-X’ and Y-Y’ normalized emittance are 0.77mmmrad and 0.62mmmrad, respectively. The emittance growth in X-X’ is less than 18%, and the emittance growth in Y-Y’ is less than 5%. Based on the transverse envelopes of the transported particles, the redesign of the buncher drift tubes at the RFQ high energy end has been made to get a higher transit time factor for this novel RFQ internal buncher. An optimized effective buncher gap voltage of 45.4KV has been calculated to deliver a minimized longitudinal beam emittance, while the influence of the effective buncher voltage on the transverse emittance can be neglected. Six different tuning concepts were investigated in detail while tuning the 1:2 scaled HICAT IH model cavity. ‘Volume Tuning’ by a variation of the cavity cross sectional area can compensate the unbalanced capacitance distribution in case of an extreme beta-lambda-variation along an IH cavity. ‘Additional Capacitance Plates’ or copper sheets clamped on drift tube stems are a fast way for checking the tuning sensitivity, but they will be replaced by massive copper blocks mounted on the drift tube girders finally. ‘Lens Coupling’ is an important tuning to stabilize the operation mode and to increase or decrease the coupling between neighboring sections. ‘Tube Tuning’ is the fine tuning concept and also the standard tuning method to reach the needed field distributions as well as the gap voltage distributions. ‘Undercut Tuning’ is a very sensitive tuning for the end sections and with respect to the voltage distribution balance along the structure. The different types of ‘plungers’ in the 3rd and 4th sections have different effects on the resonance frequency and on the field distribution. The different triplet stems and the geometry of the cavity end have been also investigated to reach the design field and voltage distributions. Finally, the needed uniform field distribution along the IH-DTL cavity and the corresponding effective voltage distribution were realized, the remaining maximum gap voltage difference was less than 5% for the model cavity. The several important higher order modes were also measured. The RF tuning of the IH-DTL model cavity delivers the final geometry parameters of the IH-DTL power cavity. A rectangular cavity cross section was adopted for the first time for this IH-DTL cavity. This eases the realization of the volume tuning concept in the 1st and 2nd sections. Lens coupling determines the final distance between the triplet and the girder. The triplets are mounted on the lower cavity half shell. The Microwave Studio simulations have been carried out not only for the HICAT model cavity, but also for the final geometry of the IH-DTL power cavity. The field distribution for the operation mode H110 fits to the model cavity measurement as well as the Higher Order Modes. The simulations prove the IH-DTL geometrical design. On the other hand, the precision of one simulation with 2.3 million mesh points for full cross section area and the CPU time more than 15hours on a DELL PC with Intel Pentium 4 of 2.4GHz and 2.096GRAM were exploited to their limit when calculating the real parameters for the two final machining iterations during production. The shunt impedance of the IH-DTL power cavity is estimated by comparison with the existing tanks to about 195.8MOmega/m, which fits to the simulation result of 200.3MOmega/m with reducing the conductivity to the 5.0*10 high 7 Omega-1m-1. The effective shunt impedance is 153 MOmega/m. The needed RF power is 755kW. The expected quality factor of the IH-DTL cavity is about 15600. The IH-DTL power cavity tuning measurements before cavity copper plating have been performed. The results are within the specifications. There is no doubt that the needed accuracy of the voltage distribution will be reached with the foreseen fine tuning concepts in the last steps.
Prion diseases, also called transmissible spongiform encephalopathies, are a group of fatal neurodegenerative conditions that affect humans and a wide variety of animals. To date there is no therapeutic or prophylactic approach against prion diseases available. The causative infectious agent is the prion, also termed PrPSc, which is a pathological conformer of a cellular protein named prion protein PrPc. Prions are thought to multiply upon conversion of PrPc to PrPSc in a self-propagating manner. Immunotherapeutic strategies directed against PrPc represent a possible approach in preventing or curing prion diseases. Accordingly, it was already shown in animal models, that passive immunization delays the onset of prion diseases. The present thesis aimed at the development of a candidate vaccine towards the active immunization against prion diseases, an immune response, which has to be accompanied by the circumvention of host tolerance to the self-antigen PrPc. The vaccine development was approached using virus-like particles (retroparticles) derived from either the murine leukemia (MLV) or the human immunodeficiency virus (HIV). The display of PrP on the surface of such particles was addressed for both the cellular and the pathogenic form of PrP. The display of PrPc was achieved by either fusion to the transmembrane domain of the platelet derived growth factor receptor (PDGFR) or to the N-terminal part of the viral envelope protein (Env). In both cases, the corresponding PrPD- and PrPE-retroparticles were successfully produced and analyzed via immune fluorescence, Western Blot analysis, immunogold electron microscopy as well as by ELISA methods. Both, PrPD- and PrPE-retroparticles showed effective incorporation of N-terminally truncated forms of PrPc but not for the complete protein. PrPc at this revealed the typical glycosylation pattern, which was specifically removed by a glycosidase enzyme. Upon display of PrPc on retroparticles the protein remained detectable by PrP-specific antibodies under native conditions. Electron microscopy analysis of PrPc-variants revealed no alteration of the characteristic retroviral morphology of the generated particles. MLV-derived PrPD-retroparticles were successfully used in immunization studies. Contrary to approaches using bacterially expressed PrPc, the immunization of mice resulted in a specific antibody response. The display of the pathogenic isoform was aimed by two different strategies. The first one was directed at the conversion of the proteinase K (PK) sensitive from of PrP on the surface of PrPD-retroparticles into the PK resistant form. Albeit specific adaption of the PK digestion assay detecting resistant PrP, no PrP conversion was observed for PrPD-retroparticles. The second approach utilized a replication competent variant of the ecotropic MLV displaying PrPc on the viral Env protein. This MLV variant was stable in cell culture for six passages but did not replicate on scrapie-infected, PrPSc-propagating neuroblastoma cells. Thus, besides PrPc-displaying virus-like particles a replication competent MLV variant was obtained, which stably incorporated PrPc at the N-terminus of the viral Env protein. The incorporation of the cell-surface located PrPc into particles was expected from previously obtained data on protein display in the context of retrovirus-derived particles. Thus, the lack of incorporation observed for the complete PrPc sequence was rather unexpected and was found to be inhibited at both, fusion to PDGFR and the viral Env. In contrast to N-terminally truncated PrPc, the complete PrPc was shown to exhibit increased cell surface internalization rates and half-life times eventually contributing to the observed results. The PrP-vaccination approach described in this work represents the first successful system inducing PrP-specific antibody responses against the prion protein in wt mice. Explanations at this are based on the induction of specific T cell help or effects of the innate immunity, respectively. MLV-and HIV-derived particles bearing the PrP-coding sequence or being replication competent variants generated during this thesis might help to further improve the PrP-specific immune response.
This study analyses the labour market effects of fixed-term contracts (FTCs) in West Germany by microeconometric methods using individual and establishment level data. In the first part of the study the role of FTCs in firms’ labour demand is analysed. An econometric investigation of the firms’ reasons for using FTCs focussing on the identification of the link between dismissal protection for permanent contract workers and the firms’ use of FTCs is presented. Furthermore, a descriptive analysis of the role of FTCs in worker and job flows at the firm level is provided. The second part of the study evaluates the short-run effects of being employed on an FTC on working conditions and wages using a large cross-sectional dataset of employees. The final part of the study analyses whether taking up an FTC increases the (permanent contract) employment opportunities in the long-run (stepping stone effect) and whether FTCs affect job finding behaviour of unemployed job searchers. Firstly, an econometric unemployment duration analysis distinguishing between both types of contracts as destination states is performed. Secondly, the effects of entering into FTCs from unemployment on future (permanent contract) employment opportunities are evaluated attempting to account for the sequential decision problem of job searchers.
Drug target 5-lipoxygenase : a link between cellular enzyme regulation and molecular pharmacology
(2005)
Leukotriene (LT) sind bioaktive Lipidmediatoren, die in einer Vielzahl von Entzündungskrankheiten wie z.B. Asthma, Psoriasis, Arthritis oder allergische Rhinitis involviert sind. Des Weiteren spielen LT in der Pathogenese von Erkrankungen wie Krebs, Osteoarthritis oder Atherosklerose eine Rolle. Die 5-Lipoxygenase (5-LO) ist das Enzym, das für die Bildung von LT verantwortlich ist. Aufgrund der physiologischen Eigenschaften der LT, ist die Entwicklung von potentiellen Arzneistoffen, welche die 5-LO als Zielstruktur besitzen, von erheblichem Interesse. Die Aktivität der 5-LO wird in vitro durch Ca2+, ATP, Phosphatidylcholin und Lipidhydroperoxide (LOOH) und durch die p38-abhängige MK-2/3 5-LO bestimmt. Inhibitorstudien weisen darauf hin, dass der MEK1/2-Signalweg ebenfalls in vivo an der 5-LO Aktivierung beteiligt ist. Hauptziel dieser Arbeit war es zu untersuchen, welche Rolle der MEK1/2-Signalweg bei der Aktivierung der 5-LO besitzt und welchen Einfluss der 5-LO Aktivierungsweg auf die Wirksamkeit potentieller Inhibitoren hat. „In gel kinase“ und „In vitro kinase“ Untersuchungen zeigten, dass die 5-LO ein Substrat für die Extracellular signal-regulated kinase (ERK) und MK-2/3 darstellt. Der Zusatz von mehrfach ungesättigten Fettsäuren (UFA), wie AA oder Ölsäure, verstärkte den Phosphorylierungsgrad der 5-LO sowohl durch ERK1/2 als auch durch MK-2/3. Die genannten Kinasen sind demnach auch für die 5-LO Aktivierung durch natürliche Stimuli verantwortlich, die den zellulären Ca2+-Spiegel kaum beeinflussen. Daraus ist ersichtlich, dass die Phosphorylierung der 5-LO durch ERK1/2 und/oder MK-2/3 einen alternativen Aktivierungsmechanismus neben Ca2+ darstellt. Ursprünglich wurden Nonredox-5-LO-Inhibitoren als kompetitive Wirkstoffe entwickelt, die mit AA um die Bindung an die katalytische Domäne der 5-LO konkurrieren. Vertreter dieser Inhibitoren, wie ZM230487 und L-739,010, zeigen eine potente Hemmung der LT-Biosynthese in verschiedenen Testsystemen. Sie scheiterten jedoch in klinischen Studien. In dieser Arbeit konnten wir zeigen, dass die Wirksamkeit dieser Inhibitoren vom Aktivierungsweg der 5-LO abhängig ist. Verglichen mit 5-LO Aktivität, die durch den unphysiologischen Stimulus Ca2+-Ionophor induziert wird, erfordert die Hemmung zellstress-induzierter Aktivität eine 10- bis 100-fach höhere Konzentration der Nonredox-5-LO-Inhibitoren. Die nicht-phosphorylierbare 5-LO Mutante (Ser271Ala/Ser663Ala) war wesentlich sensitiver gegenüber Nonredox-Inhibitoren als der Wildtyp, wenn das Enzym durch 5-LO Kinasen aktiviert wurde. Somit zeigen diese Ergebnisse, dass, im Gegensatz zu Ca2+, die 5-LO Aktivierung mittels Phosphorylierung die Wirksamkeit der Nonredox-Inhibitoren deutlich verringert. Des Weiteren wurde das pharmakologische Profil des neuen 5-LO Inhibitors CJ-13,610 mittels verschiedener in vitro-Testsysteme charakterisiert. In intakten PMNL, die durch Ca2+-Ionophor stimuliert wurden, hemmte die Substanz die 5-LO Produktbildung mit einem IC50 von 70 nM. Durch Zugabe von exogener AA, wird die Wirkung vermindert und der IC50 des Inhibitors steigt an. Dies deutet auf eine kompetitive Wirkweise hin. Wie die bekannten Nonredox-Inhibitoren, verliert auch CJ-13,610 seine Wirkung bei erhöhtem zellulärem Peroxidspiegel. Der Inhibitor CJ-13,610 zeigt jedoch keine Abhängigkeit vom Aktivierungsweg der 5-LO. Grundsätzlich ist es also von fundamentaler Bedeutung bei der Entwicklung von neuen Arzneistoffen, die zellulären Zusammenhänge, insbesondere die Regulierung der Aktivität von Enzymen, zu kennen. Wie in dieser Arbeit gezeigt, hat die Phosphorylierung der 5-LO einen starken Einfluss auf die Regulation der 5-LO Aktivität und eine elementare Wirkung auf die Hemmung des Enzyms durch verschiedene Wirkstoffe.
A fundamental work on THz measurement techniques for application to steel manufacturing processes
(2004)
The terahertz (THz) waves had not been obtained except by a huge system, such as a free electron laser, until an invention of a photo-mixing technique at Bell laboratory in 1984 [1]. The first method using the Auston switch could generate up to 1 THz [2]. After then, as a result of some efforts for extending the frequency limit, a combination of antennas for the generation and the detection reached several THz [3, 4]. This technique has developed, so far, with taking a form of filling up the so-called THz gap . At the same time, a lot of researches have been trying to increase the output power as well [5-7]. In the 1990s, a big advantage in the frequency band was brought by non-linear optical methods [8-11]. The technique led to drastically expand the frequency region and recently to realize a measurement up to 41 THz [12]. On the other hand, some efforts have yielded new generation and detection methods from other approaches, a CW-THz as well as the pulse generation [13-19]. Especially, a THz luminescence and a laser, originated in a research on the Bloch oscillator, are recently generated from a quantum cascade structure, even at an only low temperature of 60 K [20-22]. This research attracts a lot of attention, because it would be a breakthrough for the THz technique to become widespread into industrial area as well as research, in a point of low costs and easier operations. It is naturally thought that a technology of short pulse lasers has helped the THz field to be developed. As a background of an appearance of a stable Ti:sapphire laser and a high power chirped pulse amplification (CPA) laser, instead of a dye laser, a lot of concentration on the techniques of a pulse compression and amplification have been done. [23] Viewed from an application side, the THz technique has come into the limelight as a promising measurement method. A discovery of absorption peaks of a protein and a DNA in the THz region is promoting to put the technique into practice in the field of medicine and pharmaceutical science from several years ago [24-27]. It is also known that some absorption of light polar-molecules exist in the region, therefore, some ideas of gas and water content monitoring in the chemical and the food industries are proposed [28-32]. Furthermore, a lot of reports, such as measurements of carrier distribution in semiconductors, refractive index of a thin film and an object shape as radar, indicate that this technique would have a wide range of application [33-37]. I believe that it is worth challenging to apply it into the steel-making industry, due to its unique advantages. The THz wavelength of 30-300 ¼m can cope with both independence of a surface roughness of steel products and a detection with a sub-millimeter precision, for a remote surface inspection. There is also a possibility that it can measure thickness or dielectric constants of relatively high conductive materials, because of a high permeability against non-polar dielectric materials, short pulse detection and with a high signal-to-noise ratio of 103-5. Furthermore, there is a possibility that it could be applicable to a measurement at high temperature, for less influence by a thermal radiation, compared with the visible and infrared light. These ideas have motivated me to start this THz work.
Das Ziel der Untersuchung von ultra-relativistischen Schwerionenkollisionen ist die Suche nach dem Quark Gluon Plasma (QGP), einem Zustand hochdichter stark wechselwirkender Materie in dem der Einschluss von Quarks und Gluonen in Hadronen aufgehoben ist. Die bisher gewonnenen experimentellen Hinweise deuten daraufhin,daß in Schwerionenkollisionen bei den derzeit höchsten zur Verfügung stehenden Energien von 158 GeV/Nukleon in Pb+Pb Reaktionen am CERN-SPS die Rahmenbedingungen für einen Phasenübergang von hadronischer Materie zu einer partonischen Phaseerfüllt sind. Die exakte Phasenstruktur stark wechselwirkender Materie hingegen ist derzeit noch nicht vollständig verstanden. Da inklusive hadronische Observablen und "penetrierende Proben" nicht direkt sensitiv auf die Existenz und Natur des Phasenübergangs sind, wurde die Analyse von Einzelereignis-"event-by-event"-Fluktuationenvorgeschlagen. Das Fluktuationsverhalten von Einzelereignis-Observablen sollte direkt sensitiv auf die Natur des zu beobachtenden Phasenübergangssein. In dieser Arbeit wurden Fluktuationen in der "chemischen" Zusammensetzung der Teilchenquelle untersucht und erste Ergebnisse werden präsentiert.
The mammalian retina contains around 30 morphological varieties of amacrine cell types. These interneurons receive excitatory glutamatergic input from bipolar cells and provide GABA- and glycinergic inhibition to other cells in the retina. Amacrine cells exhibit widely varying light evoked responses, in large part defined by their presynaptic partners. We wondered whether amacrine functional diversity is based on a differential expression of glutamate receptors among cell populations and types. In whole cell patch-clamp experiments on mouse retinal slices, we used selective agonists and antagonists to discriminate responses mediated by NMDA/ non-NMDA (NBQX) and AMPA/ KA receptors (cyclothiazide, GYKI 52466, GYKI 53655, SYM 2081). We sampled a large variety of individual cell types, which were classified by their dendritic field size into either narrow-field or wide-field cells after filling with Lucifer yellow or neurobiotin. In addition, we used transgenic GlyT2-EGFP mice, whose glycinergic neurons express EGFP. This allowed us to classify amacrines on basis of their neurotransmitter into either glycinergic or GABAergic cells. All cells (n = 300) had good responses to non-NMDA agonists. Specific AMPA receptor responses could be obtained from almost all cells recorded: 94% of the AII (n = 17), 87% of the narrow-field (n = 45), 81% of the wide-field (n = 21), 85% of the glycinergic (n = 20) and 78% of the GABAergic cells (n = 9). KA receptor selective drugs were also effective on the majority of the AII (79%, n = 14), narrow-field (93%, n = 43), wide-field (85%, n = 26), glycinergic (94%, n = 16) and GABAergic amacrine cells (100%, n = 6). Among the cells tested for the two receptors (n = 65), we encountered both exclusive expression of AMPA or KA receptors and co-expression of the two types. Most narrow-field (70%, n = 27), glycinergic (81%, n = 16) and GABAergic cells (67%, n = 6) were found to have both AMPA and KA receptors. In contrast, only less than half of the wide-field cells (43%, n = 14) were found to co-express AMPA and KA receptors, most of them expressing exclusively AMPA (36%) or KA receptors (21%). We could elicit small NMDA responses from most of the wide-field (75%, n = 13) and GABAergic cells (67%, n = 3), whereas only 47% of the narrow-field (n = 15), 14% of the AII (n = 22) and no glycinergic cell (n = 2) reacted to NMDA. Abstract 83 Our data suggest that AMPA, KA and NMDA receptors are differentially expressed among different types of amacrine cells rather than among populations with different neurotransmitters or different dendritic coverage of the retina. Selective expression of kinetically different glutamate receptors among amacrine types may be involved in generating transient and sustained inhibitory pathways in the retina. Since AMPA and KA receptors are not generally clustered at the same postsynaptic sites, a single amacrine cell expressing both AMPA and KA receptors may provide inhibition with different temporal characteristics to individual synaptic partners.
Life of Varroa destructor, Anderson and Trueman, an ectoparasitic mite of honeybees, is divided into a reproductive phase in the bee brood and a phoretic phase during which the mite is attached to the adult bee. Phoretic mites leave the colony with workers involved in foraging tasks. Little information is available on the mortality of mites outside the colony. Mites may or not return to the colony as a result of death of the infested foragers, host change by drifting of foragers, or removal of mites outside the colony. That mites do not return to the colony was indicated by substantially higher infestation of outflying workers compared to the infestation of returning workers (Kutschker, 1999). The main objective of the study was to provide information whether V. destructor influences flight behaviour of foragers and consequently returning frequency of foragers to the colony. I first repeated the experiment of Kutschker (1999) examining the infestation of outflying and returning workers. Further, I registered flight duration of foragers using a video method. In this experiment I compared also the infestation and flight duration of bees of different genetic origin, Carnica from Oberursel and bees from Primorsky region. I investigated returning time of workers, returning frequency until evening, drifting to other colonies and orientation toward the nest entrance in the experiments in which workers were released in close vicinity of the colony. At last, I measured the loss of foragers in relation to colony infestation using a Bee Scan. Results from this study, listed below, showed considerable influence of V. destructor on flight behavior of foragers translating into loss of mites. Loss of mites with foragers add substantial component to mite mortality and was underestimated in previous studies. Such loss might be viewed as a mechanism of resistance against V. destructor. a) The mean infestation of outflying workers (0.019±0.018) was twice as the mean infestation of returning workers (0.009±0.018). The difference in the infestation between outflying and returning workers was more marked in highly infested colonies. b) Investigation of individually tagged workers by use of a two camera video recording device showed significantly higher infestation of outflying workers compared to returning workers. Mites were lost by the non returning of infested foragers (22%) and by loss of mites from foragers that returned to the colony without the mite (20%). A small portion of mites (1.8%) was gained. Loss of mites significantly exceeded mite gain. c) The flight duration of infested workers determined by using the same two camera video system was significantly higher in infested compared to uninfested workers of the same age that flew closest at time. The median flight duration of infested workers was 1.7 higher (214s) than the median duration of unifested workers (128s). d) Infested workers took 2.3 times longer to return to the colony than uninfested workers of the same age when released from the same locations, closest at time. The returning time increased with the distance of release. In a group of bees released simultaneously the infestation was higher in bees returning later and in those that did not return in the observation period of 15 min. e) Released workers did not return to the colony 1.5 more frequently than uninfested workers in evening. The difference in returning was significant for locations of 20 and 50m from the colony. No difference in returning between infested and uninfested workers were observed for the most distant location of 400m. f) No significant difference was found in returning time and/or in the returning frequency until evening between workers artificially infested overnight and naturally infested workers. Artificially infested workers returned later and less frequently than a control group indicating rapid influence of V. destructor on flight behavior of foragers. g) The orientation ability of infested workers toward the nest entrance was impaired. Infested workers compared to uninfested workers twice as often approached a dummy entrance before finding the nest entrance. h) No significant differences were found in drifting between infested and uninfested workers. Drifting in the neighboring nucleus colony occurred in about 1% occasions after release of marked workers. Similarly, more infested, but not significantly more infested workers (2.6%) entered a different colored hive than the same colored hive (1.9%). However, the number of drifting bees were to low to make results conclusive. i) The comparison between Carnica and Primorsky workers revealed higher infestation in Carnica compared to Primorsky. Further, Primorsky workers lost more mites during foraging due to mite loss from foragers and non returning of infested workers. No significant differences in flight duration were observed between the two bee stocks. j) Loss of foragers, as determined by the Bee Scan counts of outflying and returning foragers, and the infestation of outflying bees increased significantly over a period of 70 days. A colony with 7.7. higher infestation of outflying foragers lost 2.2. time more bees per flight per day compared to a low infested colony. k) The estimates of mite loss with foragers from mite population per day up to 3.1% exceeds approximately mite mortality of 1% within the colony as represented by counting dead mites on bottom board inserts.
Cold target recoil ion momentum spectroscopy (COLTRIM) has been employed to image the momentum distributions of continuum electrons liberated in the impact of slow He2+ on He and H2. The distributions were measured for fully determined motion of the nuclei that is as a function of the impact parameter and in a well de ned scattering plane The single ionization (SI) of H2 leading to H2+ recoil ions in nondissociative states (He2+ + H+ -> He2+ + H+ + e-) and the transfer ionization (TI) of H2 leading to H2 dissociation into two free protons (He2+ H2 -> He+ + H+ + H+ + e-) were investigated. Similar measurements have been carried out for He target, the corresponding atomic two electron system, i.e. the single ionization (SI) (He2+ + He -> He+ + He2+ e- and the transfer ionization (TI) (He2+ + He -> He+ + He2+ + e-). These measurements have been exploited to understand the results obtained for H target. In comparing the continuum electron momentum distributions for H2 with that for He, a high degree of similarity is observed. In the case of transfer ionization of H2, the electron momentum distributions generated for parallel and perpendicular molecular orientations revealed no orientation dependence. The in scattering plane electron momentum distributions for the transfer ionization of H2 by He2+ and for the transfer ionization of He by He2e showed that the salient feature of these distributions for both collisions systems consists in the appearance of two groups of electrons with difeerent structures. In addition to the group of the saddle electrons forming two jets separated by a valley along the projectile axis we nd a new group of electrons moving with a velocity higher than the projectile velocity These new fast forward electrons result from a narrow range of impact parameters and appear as image saddle in the projectile frame. In contrast to the transfer ionization of He, the fast forward electrons group disappears in the in scattering plane electron momentum distribution generated for the single ionization of He. Instead of this group another new group of electrons appear These electrons exhibit an amount of backscattering These backward elec trons appear as image saddle in the target frame The structures that the saddle electrons show are owing to the quasi molecular nature of the collision process For the TI of H2, the TI of He and the SI of He, a pi-orbital shape of the electron momentum distribution is observed This indicates the importance of the rotational coupling 2-p-theta -> 2p-pi in the initial promotion of the ground state followed by further promotions to the continuum The backward electrons as well as the fast forward electrons are not discussed in the theoretical literature at all. However, a number of obvious indications of the existence of the backward and fast forward electrons could be seen in the experimental works of Abdallah et al. as well as in the theoretical calculations of Sidky et al One might speculate that electrons which are promoted on the saddle for some time during the collision could finally swing around the He+ ion in the way out of the collision, i.e. either around the projectile in the forward direction as in the TI case forming the fast forward electrons or around the recoil ion in the backward direction as in the SI case forming the backward electrons. This might be a result of the strong gradient, and hence the large acceleration of the screened He+ potential.
Alzheimer’s disease (AD) is the most common neurodegenerative disorder world wide, causing presenile dementia and death of millions of people. During AD damage and massive loss of brain cells occur. Alzheimer’s disease is genetically heterogeneous and may therefore represent a common phenotype that results from various genetic and environmental influences and risk factors. In approximately 10% of patients, changes of the genetic information were detected (gene mutations). In these cases, Alzheimer’s disease is inherited as an autosomal dominant trait (familial Alzheimer’s disease, FAD). In rare cases of familial Alzheimer’s disease (about 1-3%), mutations have been detected in genes on chromosomes 14 and 1 (encoding for Presenilin 1 and 2, respectively), and on chromosome 21 encoding for the amyloid precursor protein (APP), which is responsible for the release of the cell-damaging protein amyloid-beta (ß-amyloid, Aß). Familial forms of early-onset Alzheimer’s disease are rare; however, their importance extends far beyond their frequency, because they allow to identify some of the critical pathogenetic pathways of the disease. All familial Alzheimer mutations share a common feature: they lead to an enhanced production of the Aß, which is the major constituent of senile plaques in brains of AD patients. New data indicates that Aß promotes neuronal degeneration. Therefore, one aim of these thesis was to elucidate the neurotoxic biochemical pathways induced by Aß, investigating the effect of the FAD Swedish APP double mutation (APPsw) on oxidative stress-induced cell death mechanisms. This mutation results in a three- to sixfold increased Aß production compared to wild-type APP (APPwt). As cell models, the neuronal PC12 (rat pheochromocytoma) and the HEK (human embryonic kidney 293) cell lines were used, which have been transfected with human wiltyp APP or human APP containing the Swedish double mutation. The used cell models offer two important advantages. First, compared to experiments using high concentrations of Aß at micromolar levels applied extracellularly to cells, PC12 APPsw cells secret low Aß levels similar to the situation in FAD brains. Thus, this cell model represents a very suitable approach to elucidate the AD-specific cell death pathways mimicking physiological conditions. Second, these two cell lines (PC12 and HEK APPwt and APPsw) with different production levels of Aß may additionally allow to study dose-dependent effects of Aß. The here obtained results provide evidence for the enhanced cell vulnerability caused by the Swedish APP mutation and elucidate the cell death mechanism probably initiated by intracellulary produced Aß. Here it seems likely that increased production of Aß at physiological levels primes APPsw PC12 cells to undergo cell death only after additional stress, while chronic high levels in HEK cells already lead to enhanced basal apoptotic levels. Crucial effects of the Swedish APP mutation include the impairments of cellular energy metabolism affecting mitochondrial membrane potential and ATP levels as well as the additional activation of caspase 2, caspase 8 and JNK in response to oxidative stress. Thereby ,the following model can be proposed: PC12 cells harboring the Swedish APP mutation have a reduced energy metabolism compared to APPwt or control cells. However, this effect does not leads to enhanced basal apoptotic levels of cultured cells. An exposure of PC12 cells to oxidative stress leads to mitochondrial dysfunction, e.g., decrease in mitochondrial membrane potential and depletion in ATP. The consequence is the activation of the intrinsic apoptotic pathway releasing cytochrome c and Smac resulting in the activation of caspase 9. This effect is amplified by the overexpression of APP, since both APPsw and APPwt PC12 cells show enhanced cytochrome c and Smac release as well as enhanced caspase 9 activity as vector transfected control. In APPsw PC12 cells a parallel pathway is additionally emphased. Due to reduced ATP levels or enhanced Aß production JNK is activated. Furthermore, the extrinsic apoptotic pathway is enhanced, since caspase 8 and caspase 2 activation was clearly enhanced by the Swedish APP mutation. Both pathways may then converge by activating the effector enzyme, caspase 3, and the execution of cell death. In addition, caspase independent effects also needs to be considered. One possibility could be the implication of AIF since AIF expression was found to be induced by the Swedish APP mutation. In APPsw HEK cells high chronic Aß levels leads to enhanced apoptotic levels, reduce mitochondrial membrane potential and ATP levels even under basal conditions. Summarizing, a hypothetical sequence of events is proposed linking FAD, Aß production, JNK-activation, mitochondrial dysfunction with caspase pathway and neuronal loss for our cell model. The brain has a high metabolic rate and is exposured to gradually rising levels of oxidative stress during life. In Swedish FAD patients the levels of oxidative stress are increased in the temporal inferior cortex. This study using a cell model mimicking the in vivo situation in AD brains indicates that probably both, increased Aß production and the gradual rise of oxidative stress throughout life converge at a final common pathway of an increased vulnerability of neurons to apoptotic cell death from FAD patients. Presenilin (PS) 1 is an aspartyl protease, involved in the gamma-secretase mediated proteolysis of Amyloid-ß-protein (Aß), the major constituent of senile plaques in brains of Alzheimer’s disease (AD) patients. Recent studies have suggested an additional role for presenilin proteins in apoptotic cell death observed in AD. Since PS 1 is proteolytic cleaved by caspase 3, it has been prosposed that the resulting C-terminal fragment of PS1 (PSCas) could play a role in signal transduction during apoptosis. Moreover, it was shown that mutant presenilins causing early-onset of familial Alzheimer's disease (FAD) may render cells vulnerable to apoptosis. The mechanism by which PS1 regulates apoptotic cell death is yet not understood. Therefore one aim of our present study was to clarify the involvement of PS1 in the proteolytic cascade of apoptosis and if the cleavage of PS1 by caspase 3 has an regulatory function. Here it is demonstrated that both, PS1 and PS1Cas lead to a reduced vulnerability of PC12 and Jurkat cells to different apoptotic stimuli. However a mutation at the caspase 3 recognition site (D345A/ PSmut), which inhibits cleavage of PS1 by caspase 3, show no differences in the effect of PS1 or PSCas towards apoptotic stimuli. This suggest that proteolysis of PS1 by caspase 3 is not a determinant, but only a secondary effect during apoptosis. Since several FAD mutation distributed through the whole PS1 gene lead to enhanced apoptosis, an abolishment of the antiapoptotic effect of PS1 might contribute to the massive neurodegeneration in early age of FAD patients. Here, the regulate properties of PS1 in apoptosis may not be through an caspase 3 dependent cleavage and generation of PSCas, but rather through interaction of PS1 with other proteins involved in apoptosis.
In contrast to the class A heat stress transcription factors (Hsfs) of plants, a considerable number of Hsfs assigned to classes B and C have no evident function as transcription activators on their own. In the course of my PhD work I showed that tomato HsfB1, a heat stress induced member of class B Hsf family, is a novel type of transcriptional coactivator in plants. Together with class A Hsfs, e.g. tomato HsfA1, it plays an important role in efficient transcrition initiation during heat stress by forming a type of enhanceosome on fragments of Hsp promoter. Characterization of promoter architecture of hsp promoters led to the identification of novel, complex heat stress element (HSE) clusters, which are required for optimal synergistic interactions of HsfA1 and HsfB1. In addition, HsfB1 showed synergistic activation of the expression of a subset of viral and house keeping promoters. CaMV35S promoter, the most widely expressed constitutive promoter turned out to be the the most interesting candidate to study this effect in detail. Because, for most house-keeping promoters tested during this study, the activators responsible for constitutive expression are not known, but in case of CaMV35S promoter they are quite well known (the bZip proteins, TGA1/2). These proteins belong to the acidic activators, similar to class A Hsfs. Actually, on heat stress inducible promoters HsfA1 or other class A Hsfs are the synergistic partners of HsfB1, whereas on house-keeping or viral promoters, HsfB1 shows synergistic transcriptional activation in cooperation with the promoter specific acidic activators, e.g. with TGA proteins on 35S promoter. In agreement with this the binding sites for HsfB1 were identified in both house-keeping and 35S promoter. It has been suggested during this study that HsfB1 acts in the maintenance of transcription of a sub-set of house-keeping and viral genes during heat stress. The coactivator function of HsfB1 depends on a single lysine residue in the GRGK motif in its CTD. Since, this motif is highly conserved among histones as the acetylation motif, especially in histones H2A and H4,. It was suggested that the GRGK motif acts as a recruitment motif, and together with the other acidic activator is responsible for corecruitment of a histone acetyl transferase (HAT). So, the effect of mammalian CBP (a well known HAT) and its plant orthologs (HAC1) was tested on the stimulation of synergistic reporter gene activation obtained with HsfA1 and HsfB1. Both in plant and mammalian cells, CBP/HAC1 further stimulated the HsfA1/B1 synergistic effect. Corecruitment of HAC1 was proven by in vitro pull down assays, where the NTD of HAC1 interacted specifically both with HsfA1 and HsfB1. Formation of a ternary complex between HsfA1, HsfB1 and CBP/HAC1 was shown via coimmunoprecipitation and electrophoretic mobility shift assays (EMSA). In conclusion, the work presented in my thesis presents a new model for transcriptional regulation during an ongoing heat stress.
In an attempt to search for potential candidate molecules involved in the pathogenesis of endometriosis, a novel 2910 bp cDNA encoding a putative 411 amino acid protein, shrew-1 was discovered. By computational analysis it was predicted to be an integral membrane protein with an outside-in transmembrane domain but no homology with any known protein or domain could be identified. Antibodies raised against the putative open-reading frame peptide of shrew-1 labelled a protein of ca. 48 kDa in extracts of shrew-1 mRNA positive tissues and also detected ectopically expressed shrew-1. In the course of my PhD work, I confirmed the prediction that shrew-1 is indeed a transmembrane protein, by expressing epitope-tagged shrew-1 in epithelial cells and analysing the transfected cells by surface biotinylation and immunoblots. Additionally, I could show that shrew-1 is able to target to E-cadherin-mediated adherens junctions and interacts with the E-cadherin-catenin complex in polarised MCF7 and MDCK cells, but not with the N-cadherin-catenin complex in non-polarised epithelial cells. A direct interaction of shrew-1 with beta-catenin could be shown in an in vitro pull-down assay. From this data, it could be assumed that shrew-1 might play a role in the function and/or regulation of the dynamics of E-cadherin-mediated junctional complexes. In the next part of my thesis, I showed that stable overexpression of shrew-1 in normal MDCK cells. causes changes in morphology of the cells and turns them invasive. Furthermore, transcription by ²-catenin was activated in these MDCK cells stably overexpressing shrew-1. It was probably the imbalance of shrew-1 protein at the adherens junctions that led to the misregulation of adherens junctions associated proteins, i.e. E-cadherin and beta-catenin. Caveolin-1 is another integral membrane protein that forms complexes with Ecadherin- beta-catenin complexes and also plays a role in the endocytosis of E-cadherin during junctional disruption. By immunofluorescence and biochemical studies, caveolin-1 was identified as another interacting partner of shrew-1. However, the functional relevance of this interaction is still not clear. In conclusion, it can be said that shrew-1 interacts with the key players of invasion and metastasis, E-cadherin and caveolin-1, suggesting its possible role in these processes and making it an interesting candidate to unravel other unknown mechanisms involved in the complex process of invasion.