Untersuchung der DNA-bindenden Eigenschaften neuer Nukleobasen-gekoppelter Pyrrolcarboxamide, Combilexine und Hetaren[a]carbazol-Derivate: Validierung und Etablierung von in-vitro- und in-silico-Methoden

Da maligne Neoplasien durch Mutationen in Proto-Onko- und/oder Tumorsuppressorgenen ausgelöst werden, stellt die DNA eines der wichtigsten Targets für die Entwicklung neuer Zytostatika dar. Auch bei den im Arbeitskreis Pindur designten und synthetisier-ten Verbindungen der Nukleobasen-gekoppelten Pyrrolcarboxamid-, der Hetaren[a]carbazol- und der Combilexin-Reihe handelt es sich um DNA-Liganden mit potentiell antitumoraktiven Eigenschaf-ten. Die einen dualen Bindemodus aufweisenden Combilexine bestehen aus einem Interkalator (u. a. Naphthalimid, Acridon), der über einen Linker variabler Kettenlänge mit einer rinnenbin-denden, von Netropsin abgeleiteten Bispyrrol-, oder einer bioisosteren Imidazol-, Thiazol- oder Thiophen-pyrrolcarboxamid-struktur verknüpft ist. Das N-terminale Ende der Combilexine wird von einer N,N-Dimethylaminopropyl- oder -ethyl-Seitenkette gebildet. Die DNA-Affinitäten der Liganden wurden mittels Tm-Wert-Messung-en bestimmt. Diese Denaturierungsexperimente wurden sowohl mit poly(dAdT)2- als auch mit Thymus-DNA (~42% GC-Anteil) durchge-führt, um Aussagen zur Stärke und zur Sequenzselektivität der DNA-Bindung machen zu können. Des Weiteren wurden die Bindekon-stanten einiger ausgewählter Vertreter mit Hilfe des Ethidium-bromid-Verdrängungsassays ermittelt; einige Testverbindungen wurden zudem auf potentiell vorhandene, TOPO I-inhibierende Eigenschaften untersucht. Diese biochemischen und biophysika-lischen Tests wurden durch Molecular Modelling-Studien ergänzt, die die Berechnung von molekularen Eigenschaften, die Durch-führung von Konformerenanalysen und die Simulation von DNA-Ligand-Komplexen (Docking) umfassten. Durch Korrelation der in vitro-Befunde mit den in silico-Daten gelang es, vor allem für die Substanzklasse der Combilexine einige richtungweisende Struktur-Wirkungsbeziehungen aufzustellen. So konnte gezeigt werden, dass die Einführung eines Imidazol-Rings in die rinnen-bindende Hetaren-pyrrolcarboxamid-Struktur der Combilexine aufgrund der H-Brücken-Akzeptor-Funktion des sp2-hybridisierten N-Atoms eine Verschiebung der Sequenzselektivität der DNA-Bindung von AT- zu GC-reichen Arealen der DNA bedingt. Zudem erwies sich ein C3-Linker für die Verknüpfung des Naphthalimids mit dem rinnenbindenden Strukturelement als am besten geeignet, während bei den Acridon-Derivaten die Verbindungen mit einem N-terminalen Buttersäure-Linker die höchste DNA-Affinität aufwiesen. Dies ist sehr wahrscheinlich auf die im Vergleich zum Naphthalimid-Molekül geringere y-Achsen-Ausdehnung (bzgl. eines x/y-Koordinatensystems) des Acridons zurückzuführen. Die ermittelten Struktur-Wirkungsbeziehungen können dazu herangezogen werden, das rationale Design neuer DNA-Liganden mit potentiell stärkerer DNA-Bindung zu optimieren.

Multifunctional water-soluble phosphorus dendrimers

Dendrimers are polymeric macromolecules with a regularly branched structure and are synthesised in an iterative fashion. Due to their monodispersity, well-defined shape and extremely high functionality, dendrimers are ideal nano-sized objects for functional and biocompatible surface coatings, biosensing and biomedicine. This dissertation describes the synthesis of ten novel water-soluble phosphorus containing dendrimers and their application in different biological and biomimetic systems. The dendrimers can be divided into two classes; the first type contains either a ferrocene at the core or 24 ferrocenes in the branches. They showed reversible reduction-oxidation behaviour and might be applied in electronic multilayered architectures. Dendrimers of the second class carry a dithiolane functionalised core that can strongly bind to noble metals, like gold substrates. Although such dendrimer coated substrates were unable to tether defect-free lipid bilayer membranes, the coatings were successfully applied for culturing Human Osteoblast cells. The cell adhesion to a coating of polycationic dendrimers was so strong that cell division could not take place, specifically evoking apoptosis. The polyanionic dendrimers, however, promoted excellent cell adhesion and proliferation. Therefore, the practical application of such macromolecular architectures can be envisioned, such as in dendrimer coatings for tissue engineering and or medical implants.

The C-terminal domain of coilin interacts with Sm proteins and U snRNPs

Coilin is the signature protein of the Cajal body (CB), a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). Newly imported Sm-class snRNPs are thought to traffic through CBs before proceeding to their final nuclear destinations. Loss of coilin function in mice leads to significant viability and fertility problems. Coilin interacts directly with the spinal muscular atrophy (SMA) protein via dimethylarginine residues in its C-terminal domain. Although coilin hypomethylation results in delocalization of survival of motor neurons (SMN) from CBs, high concentrations of snRNPs remain within these structures. Thus, CBs appear to be involved in snRNP maturation, but factors that tether snRNPs to CBs have not been described. In this report, we demonstrate that the coilin C-terminal domain binds directly to various Sm and Lsm proteins via their Sm motifs. We show that the region of coilin responsible for this binding activity is separable from that which binds to SMN. Interestingly, U2, U4, U5, and U6 snRNPs interact with the coilin C-terminal domain in a glutathione S-transferase (GST)-pulldown assay, whereas U1 and U7 snRNPs do not. Thus, the ability to interact with free Sm (and Lsm) proteins as well as with intact snRNPs, indicates that coilin and CBs may facilitate the modification of newly formed snRNPs, the regeneration of 'mature' snRNPs, or the reclamation of unassembled snRNP components.

THE FUNCTIONAL ORGANIZATION OF SYNAPTIC VESICLE POOLS IN A RETINAL BIPOLAR NEURON

Ribbon synapses are found in sensory systems and are characterized by ‘ribbon-like’ organelles that tether synaptic vesicles. The synaptic ribbons co-localize with sites of calcium entry and vesicle fusion, forming ribbon-style active zones. The ability of ribbon synapses to maintain rapid and sustained neurotransmission is critical for vision, hearing and balance. At retinal ribbon synapses, three vesicle pools have been proposed. A rapid pool of vesicles that are docked at the plasma membrane, and whose fusion is limited only by calcium entry, a releasable pool of ATP-primed vesicles whose size also correlates with the number of ribbon-tethered vesicles, and a reserve pool of non-ribbon-tethered cytoplasmic vesicles. However evidence of vesicle fusion at sites away from ribbon-style active zones questions this organization. Another fundamental question underlying the mechanism of vesicle fusion at these synapses is the role of SNARE (Soluble N-ethylmaleimide sensitive factor Attachment Protein Receptor) proteins. Vesicles at conventional neurons undergo SNARE complex-mediated fusion. However a recent study has suggested that ribbon synapses involved in hearing can operate independently of neuronal SNAREs. We used the well-characterized goldfish bipolar neuron to investigate the organization of vesicle pools and the role of SNARE proteins at a retinal ribbon synapse. We blocked functional refilling of the releasable pool and then stimulated bipolar terminals with brief depolarizations that triggered the fusion of the rapid pool of vesicles. We found that the rapid pool draws vesicles from the releasable pool and that both pools undergo release at ribbon-style active zones. To assess the functional role of SNARE proteins at retinal ribbon synapses, we used peptides derived from SNARE proteins that compete with endogenous proteins for SNARE complex formation. The SNARE peptides blocked fusion of reserve vesicles but not vesicles in the rapid and releasable pools, possibly because both rapid and releasable vesicles were associated with preformed SNARE complexes. However, an activity-dependent block in refilling of the releasable pool was seen, suggesting that new SNARE complexes must be formed before vesicles can join a fusion-competent pool. Taken together, our results suggest that SNARE complex-mediated exocytosis of serially-organized vesicle pools at ribbon-style active zones is important in the neurotransmission of vision.

Sea-floor videos and photographs (benthos) along 6 shelf profiles from the eastern Weddell Sea, Antarctica taken with remote operated vehicel CHEROKEE during Polarstern cruise ANT-XXI/2

The ROV operations had three objectives: (1) to check, whether the "Cherokee" system is suited for advanced benthological work in the high latitude Antarctic shelf areas; (2) to support the disturbance experiment, providing immediate visual Information; (3) to continue ecological work that started in 1989 at the hilltop situated at the northern margin of the Norsel Bank off the 4-Seasons Inlet (Weddell Sea). The "Cherokee" is was equipped with 3 video cameras, 2 of which support the operation. A high resolution Tritech Typhoon camera is used for scientific observations to be recorded. In addition, the ROV has a manipulator, a still camera, lights and strobe, compass, 2 lasers, a Posidonia transponder and an obstacle avoidance Sonar. The size of the vehicle is 160 X 90 X 90cm. In the present configuration without TMS (tether management system) the deployment has to start with paying out the full cable length, lay it in loops on deck and connect the glass fibres at the tether's spool winch. After a final technical check the vehicle is deployed into the water, actively driven perpendicular to the ship's axis and floatings are fixed to the tether. At a cable length of approx. 50 m, the tether is tightened to the depressor by several cable ties and both components are lowered towards the sea floor, the vehicle by the thruster's propulsion and the depressor by the ship's winch. At 5 m intervals the tether has to be tied to the single conductor cable. In good weather conditions the instruments supporting the navigation of the ROV, especially the Posidonia system, allow an operation mode to follow the ship's course if the ship's speed is slow. Together with the lasers which act as a scale in the images they also allow a reproducible scientific analysis since the transect can be plotted in a GIS system. Consequently, the area observed can be easily calculated. An operation as a predominantly drifting system, especially in areas with bottom near currents, is also possible, however, the connection of the tether at the rear of the vehicle is unsuitable for such conditions. The recovery of the system corresponds to that of the deployment. Most important is to reach the surface of the sea at a safe distance perpendicular to the ship's axis in order not to interfere with the ship's propellers. During this phase the Posidonia transponder system is of high relevance although it has to be switched off at a water depth of approx. 40 m. The minimum personal needed is 4 persons to handle the tether on deck, one person to operate the ship's winch, one pilot and one additional technician for the ROV's operation itself, one scientist, and one person on the ship's bridge in addition to one on deck for whale watching when the Posidonia system is in use. The time for the deployment of the ROV until it reaches the sea floor depends on the water depth and consequently on the length of the cable to be paid out beforehand and to be tightened to the single conductor cable. Deployment and recovery at intermediate water depths can last up to 2 hours each. A reasonable time for benthological observations close to the sea floor is 1 to 3 hours but can be extended if scientifically justified. Preliminary results: after a first test station, the ROV was deployed 3 times for observations related to the disturbance experiment. A first attempt to Cross the hilltop at the northern margin of the Norsel Bank close to the 4- Seasons Inlet was successful only for the first hundreds of metres transect length. The benthic community was dominated in biomass by the demosponge Cinachyra barbata. Due to the strong current of approx. 1 nm/h, the design of the system, and an expected more difficult current regime between grounded icebergs and the top of the hilltop the operation was stopped before the hilltop was reached. In a second attempt the hilltop was successfully crossed because the current and wind situation was much more suitable. In contrast to earlier expeditions with the "sprint" ROV it was the first time that both slopes, the smoother in the northeast and the steeper in the southwest were continuously observed during one cast. A coarse classification of the hilltop fauna shows patches dominated by single taxa: cnidarians, hydrozoans, holothurians, sea urchins and stalked sponges. Approximately 20 % of the north-eastern slope was devastated by grounding icebergs. Here the sediments consisted of large boulders, gravel or blocks of finer sediment looking like an irregularly ploughed field. On the Norsel Bank the Cinachyra concentrations were locally associated with high abundances of sea anemones. Total observation time amounted to 11.5 hours corresponding to almost 6-9 km transect length.

Energy Analysis of Bare Electrodynamic Tethers

The design of an electrodynamic tether is a complex task that involves the control of dynamic instabilities, optimization of the generated power (or the descent time in deorbiting missions), and minimization of the tether mass. The electrodynamic forces on an electrodynamic tether are responsible for variations in the mechanical energy of the tethered system and can also drive the system to dynamic instability. Energy sources and sinks in this system include the following: 1) ionospheric impedance, 2) the potential drop at the cathodic contactor, 3) ohmic losses in the tether, 4) the corotational plasma electric field, and 5) generated power and/or 6) input power. The analysis of each of these energy components, or bricks, establishes parameters that are useful tools for tether design. In this study, the nondimensional parameters that govern the orbital energy variation, dynamic instability, and power generation were characterized, and their mutual interdependence was established. A space-debris mitigation mission was taken as an example of this approach for the assessment of tether performance. Numerical simulations using a dumbbell model for tether dynamics, the International Geomagnetic Reference Field for the geomagnetic field, and the International Reference Ionosphere for the ionosphere were performed to test the analytical approach. The results obtained herein stress the close relationships that exist among the velocity of descent, dynamic stability, and generated power. An optimal tether design requires a detailed tradeoff among these performances in a real-world scenario.

Orbital debris mitigation through deorbiting with passive electrodynamic drag

The increase of orbital debris and the consequent proliferation of smaller objects through fragmentation are driving the need for mitigation strategies. The issue is how to deorbit the satellite with an efficient system that does not impair drastically the propellant budget of the satellite and, consequently, reduces its operating life. We have been investigating, in the framework of a European-Community-funded project, a passive system that makes use of an electrodynamics tether to deorbit a satellite through Lorentz forces. The deorbiting system will be carried by the satellite itself at launch and deployed from the satellite at the end of its life. From that moment onward the system operates passively without requiring any intervention from the satellite itself. The paper summarizes the results of the analysis carried out to show the deorbiting performance of the system starting from different orbital altitudes and inclinations for a reference satellite mass. Results can be easily scaled to other satellite masses. The results have been obtained by using a high-fidelity computer model that uses the latest environmental routines for magnetic field, ionospheric density, atmospheric density and a gravity field model. The tether dynamics is modelled by considering all the main aspects of a real system as the tether flexibility and its temperature-dependent electrical conductivity. Temperature variations are computed by including all the major external and internal input fluxes and the thermal flux emitted from the tether. The results shows that a relatively compact and light system can carry out the complete deorbit of a relatively large satellite in a time ranging from a month to less than a year starting from high LEO with the best performance occurring at low orbital inclinations.

La electrodinámica de cables espaciales

Se discuten los fundamentos que subyacen en la concepción y en las aplicaciones de un cable espacial, o tether. Si el cable es metálico y órbita en un planeta con ionosfera y campo magnético propio —la Tierra misma, o uno de los grandes planetas exteriores-, su movimiento genera una fuerza electromotriz por el mismo proceso de inducción magnética de un simple generador eléctrico, y el circuito conductor se "cierra" a través del plasma ionosférico. Se analiza el problema técnico central al que se enfrentan los tethers electrodinámicos de potencia: cómo establecer un buen contacto eléctrico entre el cable y la rarificada ionosfera. Se discute una reciente solución al problema.

The radiation impedance of orbiting conductors

The dispersion relation for waves in a cold, magnetized plasma is discussed using the potential for the longitudinal part of the electric field. This clarifies wave emission from a conductor in low Earth orbit and should be useful in considering the far field and both hot plasma and nonlinear, near-field effects. General formulas for radiation impedance are directly obtained. For tethers a fundamental dependence on contactor size is discussed. Spherical and ellipsoidal contactors and an (anodcless) bare tether are considered. Simple arguments on nonlinear contactor effects lead to a surprisingly simple result for impedances off the Alfven branch.

The orbital-motion-limited regime of cylindrical Langmuir probes

An asymptotic analysis of electron collection at high bias Fp serves to determine the domain of validity of the orbital-motion-limited regime of cylindrical Langmuir probes, which is basic for the workings of conductive bare tethers. The radius of a wire collecting OML current in an unmagnetized plasma at rest cannot exceed a value, Rmax , which is found to exhibit a minimum as a function of Fp ; atFp values of interest, Rmax is already increasing and is larger than the electron Debye length lDe . The breakdown of the regime relates to conditions far fromthe probe, at electron energies comparable to the ion thermal energy, kTi ; Rmax is found to increase with Ti . It is also found that ~1! the maximumwidth of a thin tape, if used instead of a wire, is 4Rmax ; ~2! the electron thermal gyroradius must be larger than both R and lDe for magnetic effects to be negligible; and ~3! conditions applying to the tether case are such that trapped-orbit effects are negligible.

Cylindrical Langmuir probes beyond the orbital-motion-limited regime

The current I to a cylindrical probe at rest in an unmagnetized plasma, with probe bias highly positive, is determined. The way I lags behind the orbital-motion-limited OMLcurrent, 1 OML R, as the radius R exceeds the maximum radius for the OML regime to hold, is of interest for space-tether applications. The ratio I/I OML is roughly a decreasing function of R/lD R max /lDe , which is independent of bias, with lDe the electron Debye length and Rmax /l De roughly an increasing function of the temperature ratio, Ti /Te. The dependence of current on ion energy is used to discuss the effect of probe motion through the plasma, a case applying to tethers in low orbit.

E-beam propagation and interaction with atmosphere

This study purports to investigate whether a conductive tether left uninsulated and electrically floating in LEO could serve as an effective e-beam source to produce artificial auroras. An electrically floating tether comes out biased highly negative over most of its length. Ambient ions impacting it with KeV energies liberate secondary electrons, which are locally accelerated through the 2D tether voltage-bias, race down magnetic lines, and result in peak auroral emissions at about 120-160 km altitude. Since no current flows at either tether end, a bare-tether e-beam is fully free of spacecraft charging problems. Beam propagation and beam-atmosphere interactions need be modelled in a simple but quantitative way so as to allow a satisfactory discussion of observational options and their feasibility. The evolution in the energy spectrum of secondary electrons, their pitch distribution, and beam broadening due to collisions with neutrals, which would result in a broader but weaker tether footprint in the E-layer, need be modelled. Relations between particle/energy flux values, and ionization and accompanying emission rates, are considered.

Spherical collectors versus bare tethers for drag, thrust, and power generation

Deorbit, power generation, and thrusting performances of a bare thin-tape tether and an insulated tether with a spherical electron collector are compared for typical conditions in low-Earth orbit and common values of length L = 4−20 km and cross-sectional area of the tether A = 1−5 mm2. The relative performance of moderately large spheres, as compared with bare tapes, improves but still lags as one moves from deorbiting to power generation and to thrusting: Maximum drag in deorbiting requires maximum current and, thus, fully reflects on anodic collection capability, whereas extracting power at a load or using a supply to push current against the motional field requires reduced currents. The relative performance also improves as one moves to smaller A, which makes the sphere approach the limiting short-circuit current, and at greater L, with the higher bias only affecting moderately the already large bare-tape current. For a 4-m-diameter sphere, relative performances range from 0.09 sphere-to-bare tether drag ratio for L = 4 km and A = 5 mm2 to 0.82 thrust–efficiency ratio for L = 20 km and A = 1 mm2. Extremely large spheres collecting the short-circuit current at zero bias at daytime (diameters being about 14 m for A = 1 mm2 and 31 m for A = 5 mm2) barely outperform the bare tape for L = 4 km and are still outperformed by the bare tape for L = 20 km in both deorbiting and power generation; these large spheres perform like the bare tape in thrusting. In no case was sphere or sphere-related hardware taken into account in evaluating system mass, which would have reduced the sphere performances even further.

Fast magnetosonic wave excitation by an array of wires with time-modulated currents

The excitation of Fast Magnetosonic (FMS)waves by a cylindrical array of parallel tethers carrying timemodulated current is discussed. The tethers would fly vertical in the equatorial plane, which is perpendicular to the geomagnetic field when its tilt is ignored, and would be stabilized by the gravity gradient. The tether array would radiate a single FMS wave. In the time-dependent background made of geomagnetic field plus radiated wave, plasma FMS perturbations are excited in the array vicinity through a parametric instability. The growth rate is estimated by truncating the evolution equation for FMS perturbations to the two azimuthal modes of lowest order. Design parameters such as tether length and number, required power and mass are discussed for Low Earth Orbit conditions. The array-attached wave structure would have the radiated wave controlled by the intensity and modulation frequency of the currents, making an active experiment on non-linear low frequency waves possible in real space plasma conditions.

Frozen orbits for scientific missions using rotating tethers

We derive a semi-analytic formulation that permits to study the long-term dynamics of fast-rotating inert tethers around planetary satellites. Since space tethers are extensive bodies they generate non-keplerian gravitational forces which depend solely on their mass geometry and attitude, that can be exploited for controlling science orbits. We conclude that rotating tethers modify the geometry of frozen orbits, allowing for lower eccentricity frozen orbits for a wide range of orbital inclination, where the length of the tether becomes a new parameter that the mission analyst may use to shape frozen orbits to tighter operational constraints.