Mimicking the pre-hairpin intermediate of gp41

A novel screening platform for potential retroviral fusion inhibitors on the basis of fully functional membrane‐anchored coiled coil lipopeptide receptors has been established. The work comprises the scrutiny of lateral organization of functional lipids in phase separated bilayers and an in‐depth investigation of the biophysical properties of lipopeptide‐based receptors. Lateral sorting of lipids was detected by the recognition of streptavidin of biotinylated lipids in phase separated bilayers and by nanoscopic patterns in mixed fluorocarbon / hydrocarbon lipid bilayers, employing temperature controlled atomic force microscopy (AFM) as a versatile characterization method. Particular features of fluorocarbon bilayers were additionally investigated in great detail by means of ellipsometry and ATR‐IR spectroscopy. Lipopeptide‐receptors were synthesized on the basis of a robust and reliable in situ coupling reaction by coupling terminal cysteine modified receptor‐peptides to a maleimide functionalized lipid bilayer. Receptor functionality of the lipopeptides was visualized by specific binding of vesicles and nanoparticles tracked by a multiplicity of characterization methods, such as AFM, ellipsometry, CLSM and fluorescence spectroscopy. Finally, in situ coupling of viral peptides, originating from the fusion protein of HIV resulted in a mimic of the pre‐hairpin intermediate of gp41. Structural analysis of N36‐lipopepides by means of CD‐spectroscopy in combination with FT‐IR spectroscopy revealed a coiled coil assembly of lipopeptides, which render the aggregates fully functional receptors for potent fusion inhibitors. Thereby, reversible inhibitor binding of T20 and the corresponding C‐ peptides was detected by AFM and ellipsometry, rendering coiled coil lipopeptides a new promising technique for screening of retroviral fusion inhibitors.

Studien zur physiologischen Funktion löslicher Zytokinrezeptoren und zur Wirkungsweise viral kodierter Zytokine

Interleukin-6 (IL-6) aktiviert Zielzellen durch Bindung an den Interleukin-6-Rezeptor (IL-6R) und anschließende Homodimerisierung von gp130. IL-6 alleine kann nur Zellen aktivieren, die IL-6R exprimieren, der Komplex aus IL-6 und löslichem IL-6R (sIL-6R) kann gp130 auf Zellen aktivieren, die keinen IL-6R exprimieren. Von gp130 gibt es eine lösliche Form (sgp130), die in Komplexen mit sIL-6R und IL-6 vorliegen kann.Es wurden rekombinante Versionen von sgp130 konstruiert, exprimiert und aufgereinigt. Die sgp130 Proteine inhibieren die sIL-6R-abhängige Stimulation von Zellen, nicht jedoch über membrangebundenen IL-6R vermittelte IL-6-Aktivitäten. sgp130 inhibiert also selektiv sIL-6R-abhängige Antworten und hat keinen Einfluß auf IL-6-Antworten über membrangebundenen IL-6R.Das Genom von Humanem Herpesvirus-8 kodiert für ein virales IL-6 (vIL-6). Um zu klären, ob vIL-6 direkt an IL-6R oder gp130 bindet, wurden Immunpräzipitationen mit radioaktiv markiertem vIL-6 durchgeführt. Dabei zeigte vIL-6 eine direkte Interaktion mit gp130, nicht jedoch mit IL-6R.Die biologische Aktivität von vIL-6 ist IL-6R-unabhängig. Es gibt keinen Unterschied in der Effektivität von vIL-6 bei der Stimulation von Zellen die nur gp130 oder gp130 und IL-6R exprimieren. Die Ergebnisse demonstrieren, daß vIL-6 das erste bekannte Zytokin ist, welches direkt gp130 binden und aktivieren kann.

A single chain antibody against a viral RNA polymerase (TBSV-BS3-Statice)

Expression of antibodies in plant against essential viral proteins could provide an alternative approach to engineered viral resistance. Engineered single chain Fv antibodies scFV are particularly suitable for expression in plant because of their small size and the lack of assembly requirements. RNA-dependent RNA polymerases (RdRps) function as the catalytic subunit of viral replicases required for the replication of all positive strand RNA viruses. By using Phage technology we selected scFvs from a phage library using purified E.coli expressed TBSV(Tomato bushy stunt virus) replicase as antigen. The scFvs mediated-inhibition of RdRp activity was studied in vitro and in planta. In vitro experiments showed the inhibition of CNV(Cucumber necrosis virus) and TCV(Turnip crinkle virus) RdRp. Transient in planta assays based on agroinfiltration and an infectious clone of TBSV demonstrated the inhibition of the replication of TBSV(Tomato bushy stunt virus). Epitope mapping showed that the selected scFvs target the motif E of RdRp which is involved in template binding.Moreover T1 plants of transgenic lines of N. benthamiana expressing different scFvs either in the cytoplasm or the ER (endoplasmic reticulum) showed a high level of resistance against infection with TBSV and RCNMV(Red clover necrotic mosaic virus) upon inoculation with virus particles. This is the first report that scFvs against a RdRp of a plant viruses can inhibit viral replication in vivo. The resistance is even efficient against viruses belonging to different virus families.

Evaluation of a viral vector system, based on a defective interfering RNA of tomato bushy stunt virus, for protein expression and the induction of gene silencing

A viral vector system was developed based on a DI-RNA, a sub-viral particle derived from TBSV-BS3-statice. This newly designed vector system was tested for its applicability in protein expression and induction of gene silencing. Two strategies were pursued. The first strategy was replication of the DI-RNA by a transgenically expressed TBSV replicase and the second was the replication by a so called helper virus. It could be demonstrated by northern blot analysis that the replicase, expressed by the transgenic N. benthamiana plant line TR4 or supplied by the helper virus, is able to replicate DI-RNA introduced into the plant cells. Various genes were inserted into different DI constructs in order to study the vector system with regard to protein expression. However, independent of how the replicase was provided no detectable amounts of protein were produced in the plants. Possible reasons for this failure are identified: the lack of systemic movement of the DI-RNA in the transgenic TR4 plants and the occurrence of deletions in the inserted genes in both systems. As a consequence the two strategies were considered unsuitable for protein expression. The DI-RNA vector system was able to induce silencing of transgenes as well as endogenous genes. Several different p19 deficient helper virus constructs were made to evaluate their silencing efficiency in combination with our DI-RNA constructs. However, it was found that our vector system can not compete with other existing VIGS (virus induced gene silencing) systems in this field. Finally, the influence of DI sequences on mRNA stability on transient GUS expression experiments in GUS silenced plants was evaluated. The GUS reporter gene system was found to be unsuitable for distinguishing between expression levels of wild type plants and GUS silenced transgenic plants. The results indicate a positive effect of the DI sequences on the level of protein expression and therefore further research into this area is recommended.

On the geometry of the spin-statistics connection in quantum mechanics

The Spin-Statistics theorem states that the statistics of a system of identical particles is determined by their spin: Particles of integer spin are Bosons (i.e. obey Bose-Einstein statistics), whereas particles of half-integer spin are Fermions (i.e. obey Fermi-Dirac statistics). Since the original proof by Fierz and Pauli, it has been known that the connection between Spin and Statistics follows from the general principles of relativistic Quantum Field Theory. In spite of this, there are different approaches to Spin-Statistics and it is not clear whether the theorem holds under assumptions that are different, and even less restrictive, than the usual ones (e.g. Lorentz-covariance). Additionally, in Quantum Mechanics there is a deep relation between indistinguishabilty and the geometry of the configuration space. This is clearly illustrated by Gibbs' paradox. Therefore, for many years efforts have been made in order to find a geometric proof of the connection between Spin and Statistics. Recently, various proposals have been put forward, in which an attempt is made to derive the Spin-Statistics connection from assumptions different from the ones used in the relativistic, quantum field theoretic proofs. Among these, there is the one due to Berry and Robbins (BR), based on the postulation of a certain single-valuedness condition, that has caused a renewed interest in the problem. In the present thesis, we consider the problem of indistinguishability in Quantum Mechanics from a geometric-algebraic point of view. An approach is developed to study configuration spaces Q having a finite fundamental group, that allows us to describe different geometric structures of Q in terms of spaces of functions on the universal cover of Q. In particular, it is shown that the space of complex continuous functions over the universal cover of Q admits a decomposition into C(Q)-submodules, labelled by the irreducible representations of the fundamental group of Q, that can be interpreted as the spaces of sections of certain flat vector bundles over Q. With this technique, various results pertaining to the problem of quantum indistinguishability are reproduced in a clear and systematic way. Our method is also used in order to give a global formulation of the BR construction. As a result of this analysis, it is found that the single-valuedness condition of BR is inconsistent. Additionally, a proposal aiming at establishing the Fermi-Bose alternative, within our approach, is made.

The geometry of Lagrangian fibres

If the generic fibre f−1(c) of a Lagrangian fibration f : X → B on a complex Poisson– variety X is smooth, compact, and connected, it is isomorphic to the compactification of a complex abelian Lie–group. For affine Lagrangian fibres it is not clear what the structure of the fibre is. Adler and van Moerbeke developed a strategy to prove that the generic fibre of a Lagrangian fibration is isomorphic to the affine part of an abelian variety.rnWe extend their strategy to verify that the generic fibre of a given Lagrangian fibration is the affine part of a (C∗)r–extension of an abelian variety. This strategy turned out to be successful for all examples we studied. Additionally we studied examples of Lagrangian fibrations that have the affine part of a ramified cyclic cover of an abelian variety as generic fibre. We obtained an embedding in a Lagrangian fibration that has the affine part of a C∗–extension of an abelian variety as generic fibre. This embedding is not an embedding in the category of Lagrangian fibrations. The C∗–quotient of the new Lagrangian fibration defines in a natural way a deformation of the cyclic quotient of the original Lagrangian fibration.

New enhancement strategies for plasmon-enhanced fluorescence biosensors

This thesis investigates metallic nanostructures exhibiting surface plasmon resonance for the amplification of fluorescence signal in sandwich immunoassays. In this approach, an analyte is captured by an antibody immobilized on a plasmonic structure and detected by a subsequently bound fluorophore labeled detection antibody. The highly confined field of surface plasmons originates from collective charge oscillations which are associated with high electromagnetic field enhancements at the metal surface and allow for greatly increased fluorescence signal from the attached fluorophores. This feature allows for improving the signal-to-noise ratio in fluorescence measurements and thus advancing the sensitivity of the sensor platform. In particular, the thesis presents two plasmonic nanostructures that amplify fluorescence signal in devices that rely on epifluorescence geometry, in which the fluorophore absorbs and emits light from the same direction perpendicular to the substrate surface.rnThe first is a crossed relief gold grating that supports propagating surface plasmon polaritons (SPPs) and second, gold nanoparticles embedded in refractive index symmetric environment exhibiting collective localized surface plasmons (cLSPs). Finite-difference time-domain simulations are performed in order to design structures for the optimum amplification of established Cy5 and Alexa Fluor 647 fluorophore labels with the absorption and emission wavelengths in the red region of spectrum. The design takes into account combined effect of surface plasmon-enhanced excitation rate, directional surface plasmon-driven emission and modified quantum yield for characteristic distances in immunoassays. Homebuilt optical instruments are developed for the experimental observation of the surface plasmon mode spectrum, measurements of the angular distribution of surface plasmon-coupled fluorescence light and a setup mimicking commercial fluorescence reading systems in epifluorescence geometry.rnCrossed relief grating structures are prepared by interference lithography and multiple copies are made by UV nanoimprint lithography. The fabricated crossed diffraction gratings were utilized for sandwich immunoassay-based detection of the clinically relevant inflammation marker interleukin 6 (IL-6). The enhancement factor of the crossed grating reached EF=100 when compared to a flat gold substrate. This result is comparable to the highest reported enhancements to date, for fluorophores with relatively high intrinsic quantum yield. The measured enhancement factor excellently agrees with the predictions of the simulations and the mechanisms of the enhancement are explained in detail. Main contributions were the high electric field intensity enhancement (30-fold increase) and the directional fluorescence emission at (4-fold increase) compared to a flat gold substrate.rnCollective localized surface plasmons (cLSPs) hold potential for even stronger fluorescence enhancement of EF=1000, due to higher electric field intensity confinement. cLSPs are established by diffractive coupling of the localized surface plasmon resonance (LSPR) of metallic nanoparticles and result in a narrow resonance. Due to the narrow resonance, it is hard to overlap the cLSPs mode with the absorption and emission bands of the used fluorophore, simultaneously. Therefore, a novel two resonance structure that supports SPP and cLSP modes was proposed. It consists of a 2D array of cylindrical gold nanoparticles above a low refractive index polymer and a silver film. A structure that supports the proposed SPP and cLSP modes was prepared by employing laser interference lithography and the measured mode spectrum was compared to simulation results.rn

Stratospheric aerosol extinction retrieved from SCIAMACHY measurements in limb geometry

Stratosphärische Partikel sind typischerweise mit dem bloßen Auge nicht wahrnehmbar. Dennoch haben sie einen signifikanten Einfluss auf die Strahlungsbilanz der Erde und die heteorogene Chemie in der Stratosphäre. Kontinuierliche, vertikal aufgelöste, globale Datensätze sind daher essenziell für das Verständnis physikalischer und chemischer Prozesse in diesem Teil der Atmosphäre. Beginnend mit den Messungen des zweiten Stratospheric Aerosol Measurement (SAM II) Instruments im Jahre 1978 existiert eine kontinuierliche Zeitreihe für stratosphärische Aerosol-Extinktionsprofile, welche von Messinstrumenten wie dem zweiten Stratospheric Aerosol and Gas Experiment (SAGE II), dem SCIAMACHY, dem OSIRIS und dem OMPS bis heute fortgeführt wird. rnrnIn dieser Arbeit wird ein neu entwickelter Algorithmus vorgestellt, der das sogenannte ,,Zwiebel-Schäl Prinzip'' verwendet, um Extinktionsprofile zwischen 12 und 33 km zu berechnen. Dafür wird der Algorithmus auf Radianzprofile einzelner Wellenlängen angewandt, die von SCIAMACHY in der Limb-Geometrie gemessen wurden. SCIAMACHY's einzigartige Methode abwechselnder Limb- und Nadir-Messungen bietet den Vorteil, hochaufgelöste vertikale und horizontale Messungen mit zeitlicher und räumlicher Koinzidenz durchführen zu können. Die dadurch erlangten Zusatzinformationen können verwendet werden, um die Effekte von horizontalen Gradienten entlang der Sichtlinie des Messinstruments zu korrigieren, welche vor allem kurz nach Vulkanausbrüchen und für polare Stratosphärenwolken beobachtet werden. Wenn diese Gradienten für die Berechnung von Extinktionsprofilen nicht beachtet werden, so kann dies dazu führen, dass sowohl die optischen Dicke als auch die Höhe von Vulkanfahnen oder polarer Stratosphärenwolken unterschätzt werden. In dieser Arbeit wird ein Verfahren vorgestellt, welches mit Hilfe von dreidimensionalen Strahlungstransportsimulationen und horizontal aufgelösten Datensätzen die berechneten Extinktionsprofile korrigiert.rnrnVergleichsstudien mit den Ergebnissen von Satelliten- (SAGE II) und Ballonmessungen zeigen, dass Extinktionsprofile von stratosphärischen Partikeln mit Hilfe des neu entwickelten Algorithmus berechnet werden können und gut mit bestehenden Datensätzen übereinstimmen. Untersuchungen des Nabro Vulkanausbruchs 2011 und des Auftretens von polaren Stratosphärenwolken in der südlichen Hemisphäre zeigen, dass das Korrekturverfahren für horizontale Gradienten die berechneten Extinktionsprofile deutlich verbessert.