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Altersabhängige Veränderungen von Immunsystem und Verhalten weiblicher Tapjas (Tupaia belangeri) (2006)

Christian Münkel

In der vorliegenden Arbeit wurde untersucht, ob es bei Tupaia belangeri (Spitzhörnchen) Veränderungen von Verhalten und im Immunsystem im Alter gibt. In einem ersten Versuch wurden unter Standard Laborbedingungen (Heimatkäfig) gehaltene, zwischen 3 Monaten und 7 Jahren alte, Tiere untersucht. Hierfür wurden Verhaltensaufzeichnungen der Hellphase (12h) per Video für eine spätere Auswertung erstellt. Für die Bestimmung von Hormonen und Immunparametern wurde den Tieren zwei Stunden vor Beginn der Aktivitätsphase Blut entnommen. In einem weiteren Versuch wurden dieselben Individuen für 2 Stunden in eine ihnen unbekannte Umgebung gebracht, um zu erforschen, ob die Tiere verschiedenen Alters unterschiedlich in Verhalten und Physiologie auf diese leichte Belastung reagieren. Während der Zeit in der neuen Umgebung (Versuchsräume) wurden wiederum Verhaltensaufzeichnungen angefertigt und im Anschluss eine Blutabnahme durchgeführt. Zusätzlich wurden 2 Wochen vor diesem Versuch Vorwerte zu dieser Tageszeit bestimmt, um über individuelle Veränderungen Aussagen treffen zu können. Die wichtigsten Befunde sind: -Die Aktivität nimmt mit zunehmendem Alter ab. -Körpermasse und Nahrungsaufnahme ändern sich nicht im Alter. -Die Funktionalität und Anteile der Zellen des spezifischen Immunsystems nehmen bei Tupajas mit zunehmendem Alter ab. -Die morgendlichen Glucokortikoidkonzentrationen im Serum nehmen mit dem Alter ab. -Auf eine leichte Belastung reagieren alte und junge Tiere mit vergleichbarem Verhalten; ältere Tiere explorieren im Unterschied weniger das neue Umfeld. -Die Glucokortikoidausschüttung auf eine Belastung hin nimmt mit zunehmendem Alter der Tiere ab.

Study of relaxation processes in simple glass formers by means of 2H NMR spectroscopy (2006)

Sorin A. Lusceac

In the present work molecular glass formers were investigated by means of multidimensional 2H NMR (solid echo and 2D spectra, stimulated echo decays, spin-lattice and spin-spin relaxation). We focused our attention on glass forming liquids that were composed of simple, rigid molecules in order to study the properties of the secondary relaxation processes (beta-processes) in the glassy state (0.5Tg<T<Tg). For several systems we also acquired NMR data at temperatures corresponding to the supercooled liquid state (T>Tg) providing information on the main relaxation process (alpha-process). In addition, extensive random walk simulations were performed to gain a deeper understanding of the experimental findings. As the molecular motion involved in the beta-process displays a highly restricted angular displacement, its effects on the line shape of the solid echo are subtle. The spectral changes due to molecular motion are quantified by the relative spectral intensity at zero 2H NMR frequency R(tp,T) (tp is the interpulse delay). Random walk simulations employing several simple models for the beta-process were performed in order to estimate the evolution of R(tp,T). They proved that 2H NMR can provide a time constant <ln(taubeta)> at a certain temperature. In addition, it was demonstrated that the R(tp,T) traces contain information about the angular amplitude and the distribution of correlation times G[ln(taubeta)]. The available data of ethanol-d1 and ethanol-d2 were successfully analyzed in the view of the simulation results, and the time constant of ethanol-d1 agrees well with dielectric spectroscopy (DS) data. We studied in detail m-fluoroaniline (m-FAN) by 2H NMR as it is a glass former that displays a relatively peculiar beta-process that is faster and shows a much smaller relative relaxation strength than most of the previously studied materials (e.g. toluene, polybutadiene). The geometry of molecular motion involved in the beta-process in m-FAN was found to be similar with the one in toluene with the difference that in the case of m-FAN a smaller angular amplitude suffices to explain the experimental findings. However, a large discrepancy between the NMR and DS time scale of the beta-process was found. The results support the claim of several authors that m-FAN forms hydrogen-bond-induced-clusters. In the quest of further understanding the secondary relaxation processes, toluene and o-terphenyl (OTP) confined in a nanoporous SBA-15 matrix were investigated. The temperature dependence of spin-lattice relaxation time allows us to differentiate type A (without a secondary dielectric beta-peak) from type B glass formers (with a beta-peak). We find that the alpha-process in both OTP (type A) and toluene (type B) in confinement is governed by a broad heterogeneous distribution of correlation times, not seen in the bulk. Additionally there is evidence that the beta-process in toluene changes under confinement conditions, where it exhibits a distribution of spin-lattice relaxation times that is substantially broadened towards faster times. In contrast, the corresponding low temperature relaxation time for OTP is not altered in confinement (T<Tg). Polybutadiene (PBD) is a widely investigated polymer without side groups. At low temperatures (T<Tg) we identified a further secondary process in addition to the beta-process that was designated as the gamma-process. In addition, a mixture of roughly 10% deuterated benzene in PBD was also measured by 2H NMR. The results proved that the benzene molecules display features very similar to those of neat PBD, thus being sensitive to both beta and gamma-process of its host. In the end we established several constraints for any model to explain the gamma-process ruling out a large angle reorientation of the C- 2H bonds at low temperatures (T<Tg). The supercooled plastically crystalline phase of cyanoadamantane was investigated in order to study the alpha-process (T>Tg). Although the orientationally disordered crystalline phase always coexisted with the orientationally ordered crystalline phase, we were able to single out the signal from the former by selective excitation and it was possible to carry out line shape measurements and 2D experiments. The latter directly reveal 6-fold reorientation of the molecular C3 axis via 90° angles, thus reflecting the symmetry of the lattice. We can reproduce the line shape by random walk simulations properly taking into account the molecular motion. Both line shape and 2D experiments yield time constants which agree with those reported by other techniques. We did not find any indication of a small angle motion as usually found for the alpha-process in structural glasses. Thus, the motional process in the glassy crystal appears to be simple and quite different from that in the structural glasses.

Crystal Structures of Perovskite-related Can(Nb,Ti)nO3n+2 (n=5 and 6) (2006)

Jonathan Guevarra

In this thesis, the crystal structures at room temperature of perovskite-related materials belonging to the homologous series Can(Nb,Ti)nO3n+2 with n = 5 and 6 have been accurately determined by single-crystal X-ray diffraction. The three compounds Ca5Nb5O17 (n = 5), Ca5Nb4TiO17 (n = 5), and Ca6(Nb,Ti)6O20 (n = 6) have been investigated. Their general structural features are the same, independent of the composition n. The crystal structures are composed of slabs of corner-sharing (Nb,Ti)O6 octahedra stacked along the [110] direction of the cubic perovskite structure. Neighboring slabs are separated by additional layers of oxygen atoms and are shifted with respect to each other by half the body diagonal of an octahedron along a. The number of layers of octahedra in a slab, indicated by the parameter n, determines the structure type, which varies therefore with composition. The crystal structures are all monoclinic (b unique) with a centrosymmetric P21/c symmetry for the n = 5 type compounds and an acentric P21 symmetry for the n = 6 type compound (in the latter, pseudo-inversion symmetries have been observed). The distortion of the (Nb,Ti)O6 octahedra increases from the center to the borders of the slabs. For the Ti-doped compounds, an ordering of Nb and Ti ions is observed with Ti ions preferentially occupying the central region of the slabs where the octahedral is least distorted. Nb and Ti ions have computed valences close to their formal valences of 5+ and 4+, respectively, for all sites in the structure. This indicates that Nb and Ti ions are equally well accommodated by the different oxygen environments in the slabs and that the observed ordering of Nb and Ti ions can only be the result of the different octahedral distortions. For the undoped Ca5Nb5O17 (n = 5), computed valences of Nb ions are close to 5+ at the borders and smaller fractional valences are obtained in the middle of the slabs. This suggests that the electrical conduction in this compound is most likely accommodated by the least distorted octahedra. Owing to the similarity in the structural features, a common model was successfully employed in the structural descriptions of the compounds investigated. Applying the superspace approach to the Ti-doped compounds, the crystal structures were characterized as commensurately modulated structures with a common basic structure and a centrosymmetric superspace group valid for both compounds, while the modulation wave vectors and the modulation functions depend on the composition n. Structure refinements in superspace demonstrate the efficiency of the superspace approach. The different occupation factors of Nb and Ti ions in the slabs as obtained in the three-dimensional structure model have been reproduced in the superspace refinements. The higher quality of the structure model obtained from the superspace refinements is illustrated by the computed valences of Nb and Ti ions which are much closer to their formal values of 5+ and 4+, respectively, for all sites in the structure compared to the three-dimensional or supercell model. In the case of n = 6 compound, the application of a centrosymmetric superspace group leads to an equal Nb/Ti ratio on sites that are related by superspace inversion, but that are independent sites in the supercell. In this way, the superspace approach provides a natural explanation for the pseudo-inversion symmetries as they have been observed in the supercell structure, while removing the interdependencies between parameters. The displacive modulation functions for the two compounds exhibit similarities, but in general are not identical. The modulation functions in n = 6 may not be used to describe exactly the atomic displacements in n = 5. Thus it is concluded that for this homologous series, the idea of a unified superspace model does not extend towards the precise atomic positions of the two compounds which are characterized by the displacive modulation functions.

Gating of water channels (aquaporins) in plants: effects of osmotic and oxidative stresses and the role of unstirred layers (2006)

Qing Ye

There is accumulating evidence that water channels (aquaporins; AQPs) play an important role in plant water relations at the levels of cells, tissues, organs, and whole plants. AQPs facilitate the rapid, passive exchange of water across cell membranes. Most of the water permeability of plasma membranes is due to AQP activity, i.e. the open/closed state of AQPs can be ‘gated’ in order to regulate water movement. Many internal (metabolic) factors or external (environmental) stresses have been found to cause a gating of AQPs, but the precise gating mechanisms are not well understood. Using pressure probe techniques, water and solute flows across cell membranes (internodes of giant green alga Chara and of cortical cells of corn root) and across an entire organ (roots of young corn seedlings) were studied. Based on detailed information on the function of AQPs, two new gating mechanisms of AQPs have been proposed. One is the ‘cohesion/tension (C/T) mechanism’ for the osmotic gating of AQPs, which is used to interpret the effect of high concentration on cell hydraulic conductivity (Lp) during osmotic stress. The other one is the ‘oxidative gating mechanism’ of AQPs in the presence of hydroxyl radicals (*OH) or of hydrogen peroxide (H2O2). Evidence for an osmotic gating of AQPs was provided from measurements of effects of high concentration on cell Lp in Chara internodes. The osmotic dehydration is thought to be caused by the fact that the exclusion of solutes from AQPs creates tensions (negative pressures) within the water channel pores, when the osmolyte is present on both sides of the membrane. This should have affected the open/closed state by changing the free energy between states favouring a reversible distorted/collapsed state rather than the open. As expected from the C/T model, effects increased with increasing both the concentration and size of osmolytes. Pore volumes of AQPs in the plasma membrane of Chara internodes were estimated from exponential ‘dehydration curves’. The analysis of osmotic responses showed that there were narrow pores with a volume of 2.3 ± 0.2 nm3 and bigger ones with a volume of between 5.5 ± 0.8 and 6.1 ± 0.8 nm3. Alternatively, pore volumes were estimated from ratios between osmotic ( ) and diffusional (Pd) water flow as derived from measurements of hydraulic and isotopic water flows, which represent the number of water molecules (N) in a single-file pore transporting water. Values of N ranged between 35 and 60, which referred to volumes of 0.51 and 0.88 nm3/pore. This value was substantially smaller than that obtained during osmotic dehydration, but bigger than values from literature. The difference may be due to an underestimation caused by unstirred layers (USLs), which would affect Pd rather than Pf (Lp). Based on analytical solutions from diffusion kinetics and measured experimental results, even for the most rapidly permeating solute heavy water (HDO), USLs should have resulted in an underestimation of Pd by less than 30 %. Evidence for an ‘oxidative gating’ of AQPs was derived from experiments with Chara internodes, which was then tested for higher plant tissue cells as well (root cortical cells of corn). In the presence of hydroxyl radicals (*OH) as produced during the Fenton reaction (Fe2+ + H2O2 = Fe3+ + OH- + *OH), cell Lp of Chara internodes was dramatically but reversibly reduced. In experiments with young corn roots, in the presence of hydrogen peroxide (H2O2), half times of water flows increased at the level of both entire roots and individual cortical cells by factors of 3 and 9, respectively, i.e., hydraulic conductivity decreased by the same factors. Results indicated that there might be a common interaction between the redox state (oxidative stress) and water relations (water stress) in plants. For solutes rapidly permeating through cell membranes, closure of water channel resulted in anomalous osmosis (negative reflection coefficients), i.e., in the striking situation that a cell did not shrink but swelled in a hypertonic medium. For the first time, anomalous osmosis has been demonstrated in roots as well, i.e., for an entire organ and in the presence of a rather complicated osmotic barrier. The phenomenon has been interpreted in terms of the composite transport structure of both the cell membrane and the root. In the presence of a rapidly permeating solute like acetone, channel closure resulted in a situation that the solute moved faster than the water, and the reflection coefficient (ss) reversed its sign.

Immobilisation von Enzymen auf sphärischen Polyelektrolytbürsten (2006)

Björn Haupt

In dieser Arbeit wurde die adsorptive Immobilisation von Enzymen auf kolloidalen Partikeln untersucht. Diese Partikel bestehen aus Polystyrolkernen auf denen Polymerketten durch eine grafting from-Technik angeknüpft wurden. Sie werden als sphärische Polyelektrolytbürsten bezeichnet. Die Parameter dieser Partikel lassen sich gezielt über die Synthese einstellen. Durch die hohe Oberflächendichte wird ein so genannter brush erhalten, d.h. eine Schicht von Polymerketten, die fest an eine Oberfläche geknüpft sind und deren benachbarten Ketten sich deutlich überlappen. Der Dissoziationsgrad der geladenen Gruppen ist für annealed brushes, die aus schwachen Polyelektrolyten aufgebaut sind, vom pH-Wert abhängig. Für quenched brushes, deren Ketten aus starken Elektrolyten aufgebaut sind, ist der Dissoziationsgrad unabhängig vom pH-Wert. In Abwesenheit von Fremdsalzionen sind die Gegenionen der Polyelektrolytketten innerhalb des brush gefangen und die Ketten werden durch den resultierenden hohen osmotischen Druck bis fast an ihre Konturlänge gestreckt. Es wird hier von einem osmotic brush gesprochen. Für die hier durchgeführten Adsorptionsexperimente wurden drei verschiedene Enzyme verwendet: Glucoamylase, alpha-D-Glucosidase und beta-D-Glucosidase. Als Triebkraft der Adsorption trotz gleichnamiger Ladung der Trägerpartikel und der Enzyme wurde die counterion release force diskutiert: Die positiv geladenen Bereiche eines überwiegend negativ geladenen Enzyms wechselwirken mit negativ geladenen Ketten eines brush. Das zu adsorbierende Protein besitzt N+ positive und N- negative Ladungen. Durch die Adsorption werden nun je N+ Gegenionen des Enzyms und der Polymerketten frei gesetzt und N- Gegenionen in den brush aufgenommen. Somit werden 2 N+ - N- Gegenionen frei gesetzt und erhöhen die Entropie. Für die Beschreibung der Adsorptionskurven wurde ein Modell entwickelt, das als Grenzfälle die Langmuir-, Langmuir-Freundlich- und Brunnauer-Emmet-Teller-Theorie enthält. Allerdings wurde hier nicht von reversiblen Gleichgewichtszuständen ausgegangen. Die Adsorption von flexiblen Enzymen folgte einem BET-Verlauf. Ein Langmuir-Freundlich Verlauf wurde für globuläre Proteine beobachtet. Die Immobilisation von Glucoamylase lieferte sowohl für ein annealed- als auch für ein quenched brush-System übereinstimmende Ergebnisse. Dies kann mit der im Vergleich zur Adsorption von BSA geringeren Wechselwirkung zwischen Glucoamylase und brush erklärt werden. Die Aktivität der adsorbierten Enzyme wurde untersucht und die Ergebnisse mittels Michaelis-Menten-Kinetik ausgewertet. Diese liefert die Parameter Michaelis-Konstante KM und die Wechselzahl kcat. Als Untersuchungsmethoden standen UV/VIS-Spektroskopie und isotherme Titrationskalorimetrie (ITC) zur Verfügung. Aktivitätsuntersuchungen von gelösten Enzymen unter gleichen Bedingungen dienten als Vergleich für die immobilisierten Enzyme. Für ein annealed und ein quenched brush-System konnte durch Untersuchungen von adsorbierter Glucoamylase gezeigt werden, dass die KM-Werte der immobiliserten Enzyme sich nicht signifikant im Vergleich zur Michaelis-Konstante des nativen Enzyms ändern. Untersuchungen von alpha- und beta-D-Glucosidase wurden mittels UV/VIS-Spektroskopie und ITC durchgeführt. Es wurde hier ebenfalls ein Erhalt der enzymatischen Aktivität gefunden. Für beta-D-Glucosidase konnte beobachtet werden, dass die KM-Werte mit steigender Beladung sich dem des nativen Enzyms annähern und für höchste Beladungen identisch sind. UV/VIS-Spektroskopie und ITC liefern vergleichbare Daten. ITC eignet sich somit als Untersuchungsmethode für die Enzymaktivität. Die Wechselzahl der immobilisierten Enzyme war in allen Messungen herabgesetzt. Besonders ausgeprägt war dieser Abfall für alpha-D-Glucosidase. Da KM erhalten bleibt und Konformationsuntersuchungen anderer adsorbierter Proteine zeigten, dass Adsorption nicht zu starken Konformationsänderungen führt, kann dieser Aktivitätsverlust nicht durch Störung der Konformation auftreten. Die verringerte Aktivität ist auf zur Vergleichsmessung unterschiedliche Bedingungen zurückzuführen. So ist der pH-Wert innerhalb des brush abgesenkt und die Ionenstärke erhöht. Es herrschen für innerhalb des brush adsorbierte Enzyme veränderte Bedingungen, die sich nach außen hin denen in Lösung angleichen. Enzyme, deren Aktivität für niedrige pH-Werte abgesenkt ist, erniedrigen somit kcat. Alpha-D-Glucosidase ist für pH-Werte kleiner als fünf nicht mehr im verwendeten Test aktiv. Zudem zeigten Untersuchungen, dass kcat abhängig von der Vorbehandlung des Enzyms ist. Es konnte in dieser Arbeit gezeigt werden, dass durch Adsorption von Enzymen eine hohe Beladung sphärischer Polyelektrolytbürsten möglich ist. Durch diese einfache Vorgehensweise konnten mit Enzym beladene Trägerpartikel erhalten werden, die unter Erhalt der KM-Werte enzymatisch aktiv waren. Somit eignen sich sphärische Polyelektrolytbürsten ideal als Trägerpartikel für die Immobilisation von Enzymen.

Low Density 2D-Electron Sheets on Liquid 3He Surface (2006)

Sergiy Putselyk

In this work, experiments with electron mobility on the surface of 3He were carried out. Mobility measurements were done in the temperature range 0.6-600mK, the operating frequencies 1Hz-100kHz, the excitation voltage 4mV - 1V and pressing electrical fields 0-255V/cm on the top electrode and 0-47.5V/cm for three bottom electrodes. Films with different thicknesses 0.2, 0.5 and 1.0mm were also under investigations. In the temperature region above 300mK, electron scattering on 3He vapor atoms was observed. In our measurements we failed to observe Wigner solid crystallization. Possible explanations could be: Interplay of two conductivities, namely conductivity through the electron layer in the center ("bulk") and conductivity through the boundary layer of electrons ("boundary"). Another explanation could be overheating effect or sliding mode of Surface State Electrons, when electron layer was near decoupled from helium surface. In our opinion, it is most probable explanation. We also did measurements below temperature of the superfluid point transition, though we did not observed any change in the signal. Possible explanation could be overheating of electrons by excitation voltage or sliding mode.

Quality Assurance for Eddy Covariance Measurements of Turbulent Fluxes and its Influence on the Energy Balance Closure Problem (2006)

Matthias Mauder

The eddy covariance method enables direct measurements of turbulent fluxes at the earth’s surface. Such measurements are required to study the surface energy balance and the exchange of gaseous air constituents. Due to improvements in the construction of adequate sensors and the progress in computer technology during the last decades this method is now well-established. However, a general failure to close the energy balance equation has been reported for many micrometeorological field experiments. This unresolved problem motivated this dissertation, which aims at the quality assurance for eddy covariance measurements. The presented concept for quality assurance comprises investigations on the accuracy of the deployed sensors and on the impact of the data analysis for such measurements. A specific focus is set on possible implications for the determination of reliable CO2 flux estimates, since this issue gained importance during the last years for studies on the global carbon cycle related to global warming due to the green house effect. Data from several field experiments in Germany, California and Nigeria form the experimental basis for these investigations. A software package was developed to perform the necessary post processing for all eddy covariance measurements presented in this thesis. The results of sensor intercomparison experiments show a typical random error of eddy covariance measurements of 5% for the sensible heat flux and 10% for the latent heat flux, if sensors are well-calibrated and maintained and the assumptions for this method are fulfilled. The applicability of an objective quality assessment scheme of flux data was demonstrated for large datasets from a field campaign comprising 14 measuring systems. The energy balance closure problem has been studied at two exemplary sites. The energy balance could not be closed for measurements over an agricultural area in Germany. For this experiment the sum of turbulent heat fluxes was 30% smaller than the available energy at the surface. In contrast, no systematic bias of the energy balance could be found for measurements over fallow bush-land in Nigeria, although the measures of quality assurance were similar. Neither differences in instrumentation nor in the post-field data processing between both experiments can explain these findings. A further analysis of the dataset from the agricultural area in Germany showed that additional flux contributions can be found when extending the averaging time of covariances beyond the conventional 30 minute interval. The energy balance can even be closed for this site when applying an averaging time of 24 hours. Longwave flux contributions seem to be generated here by the much stronger heterogeneity of the surrounding terrain compared to the more or less homogeneous environment of the Nigerian site. The filtering of heterogeneity induced flux contributions from very low frequency covariances through the commonly used averaging times of less than 30 minutes is identified as major reason of the energy balance closure problem. To improve the understanding of the processes leading to low frequency flux contributions a more detailed analysis of further experiments in combination with large eddy simulation modelling are required. The impact of post-field data processing was not only evaluated for energy flux estimates but also for fluxes of CO2, which showed similar additional flux contributions for extended averaging times. Finally, it was demonstrated that the quality assessment scheme presented in this thesis provides a fundamental and robust rejection criterion for a successful gap-filling strategy to determine annual sums of CO2 net ecosystem exchange.

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