Parallele und miniaturisierte Analyse von Protein-Protein-Interaktionen in der T-Zell-Signaltransduktion mittels Fluoreszenz-Kreuzkorrelations-Spektroskopie und Peptidmikroarrays

The aim of this thesis was to develop methods for the parallel analysis of complexes of endogenous proteins in T-cell signal transduction. We opted for detection in cell lysates, as the labelling of proteins in lysates is more versatile and more amenable to parallelisation compared to the labelling of proteins in life cells. Two approaches were developed, based on peptide microarrays and on fluorescence correlation and cross correlation spectroscopy (FCS / FCCS). It is shown that peptide microarrays provide a robust and quantitative means for detecting signalling-dependent changes of protein interactions. Recruitment of a protein into a complex leads to the masking of an otherwise exposed binding site. In cell lysates this masking can be detected by reduced binding to a microarray carrying a peptide corresponding to the binding motif of the respective interaction domain. This approach provides a significant shortcut to the detection of molecular interactions. It was first exemplified for the binding of ZAP70 to a bis-pY-motif of the activated T-cell receptor via its tandem SH2 domain. The activation-dependent recruitment of ZAP70 into signalling complexes reduced the microarray-bound signal, providing quantitative information on the change of the fraction of available binding sites. The concept was extended with microarrays comprising 24 peptides representing binding motifs for SH2, SH3, PTB WH1 and PDZ domains. Lysates of resting or stimulated Jurkat cells were incubated on these arrays and the binding of two signalling proteins per array was detected by two-colour indirect immunofluorescence. With 16 arrays per slide incubated in parallel, this approach yields 768 data points on protein-protein interactions in cellular signalling per slide. In addition to changes caused by the masking of binding sites, recruitment of proteins into complexes also lead to signal changes. This endows the arrays with properties of co-purification methods, but in a highly parallel and miniaturised fashion. Competition with peptides corresponding to interaction motifs dismantled complexes on the arrays in a stepwise and dose dependent manner and provided detailed information on the architecture of signalling complexes. For stimulation of Jurkat cells through receptor crosslinking with anti-CD3 and/or anti-CD28 antibodies, the timecourse of the changes in the pattern of molecular interactions could be monitored. Experiments with Jurkat cell derivatives lacking key signalling components demonstrated that the peptide arrays provide a means to dissect functional interdependences in cellular signalling networks. FCCS and FCS were used to detect interactions of endogenous signalling proteins using simple “mix, incubate, and measure” protocols in microliter volumes of crude cell lysates. Both binding partners of interest were targeted by indirect immunolabelling. Using 384-well plates, measurements were conducted in 20 microlitres crude cell lysate per sample, corresponding to 0.2 - 1 * 10^6 cells. Matched pairs of primary antibodies against the interaction partners and labelled secondary reagents were all added to the crude cell lysates in one step. FCCS, using two spectrally different fluorophores as labels, proved superior to FCS, using one fluorophore and one mass-tag. The one-step addition of reagents and separation-free detection minimise the number of manipulations required for complex detection and make the protocol readily compatible with automation. The method was applied for detecting the signalling-dependent formation of molecular complexes in T-cell signal transduction and for determination of IC50 values for substances interfering with protein-protein interactions. Competition experiments with peptides disrupting interactions constituting a central protein complex in T-cell signalling, the LAT-signalosome, revealed their relative contributions to stabilising the complex. In conclusion, the FCCS-based approach for protein complex detection is well suited for the parallel detection of a set of protein-protein interactions within a cellular signalling network. In applications relating to systems biology, data sets on the evolution of interactions over time in response to different stimuli may be generated with comparably little effort. Here, the ability to work endogenous proteins is especially advantageous, as overexpression of fusion-proteins with detection tags may distort signalling by driving the formation of protein complexes. Other applications are the screening for inhibitory substances of protein-protein interactions and ranking of inhibitors according to their IC50 values. Especially for interactions depending on signalling-dependent posttranslational modifications, an approach based on unmodified cellular systems is preferable over reconstituted systems. Future applications may also be in the screening for new interactions and the generation of interaction maps using libraries of antibodies.Ziel dieser Doktorarbeit war, im Kontext der T-Zell-Signaltransduktion Methoden zur parallelen Analyse von Komplexen endogener Proteine zu entwickeln. Dabei wurde auf die Markierung von Proteinen im Zelllysat gesetzt, da diese flexibler einsetzbar und daher einfacher parallelisierbar ist als die Markierung von Proteinen in lebenden Zellen. Zwei Methoden wurden entwickelt, basierend auf Peptidmikroarrays und auf Fluoreszenz-Korrelations- und Kreuzkorrelations-Spektroskopie (FCS / FCCS). Die Arbeit zeigt, dass Peptidmikroarrays einen einfachen Weg zur quantitativen Detektion signalabhängiger Protein-Protein-Interaktionen eröffnen. Die Komplexierung eines Proteins maskiert eine andernfalls frei zugängliche Bindungsstelle. In Zelllysaten lässt sich so eine Maskierung detektieren, indem sie die Bindung des Proteins an einen Array vermindert, der ein zu der maskierten Bindungsstelle passendes Peptid-Motiv trägt. Dieser Effekt wurde validiert anhand der Bindung der SH2-Domänen von ZAP70 an ein bis-pY-Motiv des aktivierten T-Zell-Rezeptors. Die aktivierungsabhängige Einbeziehung von ZAP70 in Signalproteinkomplexe reduzierte das Fluoreszenzsignal auf dem Mikroarray. Aus der Signalreduktion ließ sich Änderung des Anteils verfügbarer Bindungsstellen ableiten. Der Ansatz wurde ausgeweitet, indem Mikroarrays mit 24 Peptiden, Repräsentanten für Bindemotive von SH2-, SH3-, PTB-, WH1- und PDZ-Domänen, eingesetzt wurden. Lysate ruhender oder stimulierter Jurkat-Zellen wurden auf den Arrays inkubiert und die Bindung von zwei Signaltransduktionsproteinen pro Array durch indirekte Immunfluoreszenz detektiert. Durch parallele Inkubation von 16 Mikroarrays pro Objektträger lieferte ein Experiment 768 Datenpunkte zu Interaktionen in der zellulären Signaltransduktion. Neben Maskierungseffekten führte auch die Einbeziehung von Proteinen in Komplexe zu Signaländerungen, analog zu Ko-Aufreinigungsmethoden, aber in paralleler und miniaturisierter Weise. Kompetitionen mit Peptiden, die den Bindemotiven innerhalb der Komplexe entsprachen, ermöglichten die schritt¬weise und konzentrationsabhängige Zerlegung der Komplexe auf den Arrays und lieferten somit detaillierte Informationen zur Komplexarchitektur. Nach Rezeptor-Kreuzvernetzung durch anti-CD3 und/oder anti-CD28 ließ sich der Zeitverlauf von Änderungen im Muster molekularer Interaktionen beobachten. In Experimenten mit Jurkat-Zellen, denen Schlüsselkomponenten der Signaltransduktion fehlten, zeigten die Peptidarrays funktionale Zusammen¬hänge im zellulären Signalnetzwerk. FCS und FCCS wurden genutzt, um Interaktionen endogener Signaltransduktions-Proteine in einfachen „Misch, inkubier und miss“-Protokollen in Mikrolitervolumina von Zelllysat zu detektieren. Beide hinsichtlich ihrer Bindung zu untersuchenden Proteine wurden indirekt immunmarkiert. Die Messungen wurden in Multititerplatten mit 384 Probenkammern in jeweils 20 micro-l Lysat pro Ansatz (entsprechend 0,2 – 1,0 * 10^6 Zellen) durchgeführt. Abgestimmte Paare von Primärantikörpern und markierten Sekundärreagenzien wurden in einem Schritt zu den Lysaten gegeben. FCCS mit zwei spektral unterschiedlichen Fluorophoren als Markierung erwies sich als sensitiver und robuster als FCS mit einem Fluorophor und einem Nanopartikel als Massenmarkierung. Die gleichzeitige Zugabe aller Reagenzien minimiert den Handhabungsaufwand und macht die Methode kompatibel für die Automatisierung. Bisher wurde sie für die Detektion signalabhängiger Proteinkomplexe in der T-Zell-Signaltransduktion und für die Bestimmung von IC50-Werten für Inhibitoren der Komplexbildung eingesetzt. Titrationsexperimente mit Peptiden, die die individuellen Interaktionen innerhalb des LAT-Signalosoms kompetieren, zeigten die relative Bedeutung der einzelnen Interaktionen für die Stabilisierung des gesamten Komplexes. Zusammenfassend ist der FCCS-basierte Ansatz gut geeignet, um mehrere Interaktionen innerhalb eines zellulären Signalnetzwerkes parallel zu verfolgen, beispielsweise in systembiologischen Fragestellungen. Datensätze zur Entwicklung von Interaktionen über die Zeit und in Antwort auf unterschiedliche Stimuli können so mit vergleichsweise wenig Aufwand gewonnen werden. Vorteilhaft ist, in Systemen mit ausschließlich endogenen Proteinen arbeiten zu können, da die Überexpression von Fusionsproteinen mit Detektions¬markierungen die Bildung von Proteinkomplexen favorisieren und das zelluläre Signalgeschehen verzerren könnte. Eine weitere Anwendung kann die Reihenuntersuchung potentieller Inhibitoren von Interaktionen sein. Inhibitorkandidaten ließen sich dabei anhand ihres IC50-Wertes vergleichend beurteilen. Da viele Interaktionen auf posttranslationalen Modifikationen beruhen, ist ein auf endogenen Proteinen in ihrem natürlichen zellulären Kontext basierender Ansatz einem rekonstitutierten System vorzuziehen. Zukünftig könnte die Methode auch für die Neuidentifikation von Interaktionen mit Hilfe von Antikörper-Bibliotheken eingesetzt werden

Erfahrungen bei der bakteriolgischen Untersuchung Meningitisverdächtigen Materials

ERFAHRUNGEN BEI DER BAKTERIOLGISCHEN UNTERSUCHUNG MENINGITISVERDÄCHTIGEN MATERIALS Erfahrungen bei der bakteriolgischen Untersuchung Meningitisverdächtigen Materials ( - ) Einband ( - ) Titelseite ( - ) Kapitel ([1]) Danksagung (15) Literatur. (15) Lebenslauf. ( - ) Einband ( -

Final results from the EU project AVATAR: aerodynamic modelling of 10 MW wind turbines

This paper presents final results from the EU project AVATAR in which aerodynamic models are improved and validated for wind turbines on a scale of 10 MW and more. Special attention is paid to the improvement of low fidelity engineering (BEM based) models with higher fidelity (CFD) models but also with intermediate fidelity free vortex wake (FVW) models. The latter methods were found to be a good basis for improvement of induction modelling in engineering methods amongst others for the prediction of yawed cases, which in AVATAR was found to be one of the most challenging subjects to model. FVW methods also helped to improve the prediction of tip losses. Aero-elastic calculations with BEM based and FVW based models showed that fatigue loads for normal production cases were over predicted with approximately 15% or even more. It should then be realised that the outcome of BEM based models does not only depend on the choice of engineering add-ons (as is often assumed) but it is also heavily dependent on the way the induced velocities are solved. To this end an annulus and element approach are discussed which are assessed with the aid of FVW methods. For the prediction of fatigue loads the so-called element approach is recommended but the derived yaw models rely on an annulus approach which pleads for a generalised solution method for the induced velocities

Risk factors and indicators of severe systemic insect sting reactions

Hymenoptera venom allergy ranks among the top three causes of anaphylaxis worldwide, and approximately one-quarter of sting-induced reactions are classified as severe. Fatal sting reactions are exceedingly rare, but certain factors may entail a considerably higher risk. Delayed administration of epinephrine and upright posture are situational risk factors which may determine an unfavorable outcome of the acute anaphylactic episode and should be addressed during individual patient education. Systemic mastocytosis and senior age are major, unmodifiable long-term risk factors and thus reinforce the indication for venom immunotherapy. Vespid venom allergy and male sex likewise augment the risk of severe or even fatal reactions. Further studies are required to assess the impact of specific cardiovascular comorbidities. Available data regarding potential effects of beta-blockers and/or ACE inhibitors in coexisting venom allergy are inconclusive and do not justify recommendations to discontinue guideline-directed antihypertensive treatment. The absence of urticaria/angioedema during sting-induced anaphylaxis is indicative of a severe reaction, serum tryptase elevation, and mast cell clonality. Determination of basal serum tryptase levels is an established diagnostic tool for risk assessment in Hymenoptera venom-allergic patients. Measurement of platelet-activating factor acetylhydrolase activity represents a complementary approach but is not available for routine diagnostic use

Computational Simulation of the Turbulent Flow around an Airfoil using a spectral/hp Method

The precise calculation of loads in turbulent flow is still problematic for most Computational fluid dynamics (CFD) approaches. The spectral/hp approach allows to solve the incompressible NavierStokes equation with spectral accuracy on unstructured grids by means of Direct Numerical simulation (DNS) or Large Eddy Simulation (LES). Here we present first results we obtained from DNS compared to LES and measurements at a low Reynolds number

Combined Structural Optimization and Aeroelastic Analysis of a Vertical Axis Wind Turbine

Floating offshore wind energy poses challenges on the turbine design. A possible solution is vertical axis wind turbines, which are possibly easier to scale-up and require less components (lower maintenance) and a smaller floating structure than horizontal axis wind turbines. This paper presents a structural optimization and aeroelastic analysis of an optimized Troposkein vertical axis wind turbine to minimize the relation between the rotor mass and the swept area. The aeroelastic behavior of the different designs has been analyzed using a modified version of the HAWC2 code with the Actuator Cylinder model to compute the aerodynamics of the vertical axis wind turbine. The combined shape and topology optimization of a vertical axis wind turbine show a minimum mass to area ratio of 1.82 kg/m2 for blades with varying blade sections from a NACA 0040 at the attachment points to a NACA 0015 in the equatorial region. During an aeroelastic analysis of the wind turbine a maximum flapwise deflection of 0.45 m and a maximum edgewise deflection of 0.47 m were found. While the turbine is aeroelastically stable, an oscillation as a result of resonance reduces the fatigue life

Comparison of 3D transitional CFD simulations for rotating wind turbine wings with measurements:Paper

Since the investigation of van Ingen et al., attempts were undertaken to search for laminar parts within the boundary layer of wind turbines operating in the lower atmosphere with much higher turbulence levels than seen in wind tunnels or at higher altitudes where airplanes usually fly. Based on the results of the DAN-Aero experiment and the Aerodynamic Glove project, a special work package Boundary Layer Transition was embedded in IAEwind Task 29 MexNext 3rd phase (MN3). Here, we report on the results of the application of various CFD tools to predict transition on the MEXICO blade. In addition, recent results from a comparison of thermographic pictures (aimed at detecting transition) with 3D transitional CFD are included as well. The MEXICO (2006) and NEW MEXICO (2014) wind tunnel experiments on a turbine equipped with three 2.5 m blades have been described extensively in the literature. In addition, during MN3, high-frequency Kulite data from experiments were used to detect traces of transitional effects. Complementary, the following set of codes were applied to cases 1.1 and 1.2 (axial inflow with 10 m/s and 15 m/s respectively) – elsA, CFX, OpenFOAM (with 2 different turbulence/transitional models), Ellipsys, (with 2 different turbulence models and eN transition prediction tool), FLOWer and TAU – to search for detection of laminar parts by means of simulation. Obviously, the flow around a rotating blade is much more complicated than around a simple 2D section. Therefore, results for even integrated quantities like thrust and torque are varying strongly. Nevertheless, visible differences between fully turbulent and transitional set-ups are present. We discuss our findings, especially with respect to turbulence and transition models used

Detailed analysis of the blade root flow of a horizontal axis wind turbine

The root flow of wind turbine blades is subjected to complex physical mechanisms that influence significantly the rotor aerodynamic performance. Spanwise flows, the Himmelskamp effect, and the formation of the root vortex are examples of interrelated aerodynamic phenomena that take place in the blade root region. In this study we address those phenomena by means of particle image velocimetry (PIV) measurements and Reynolds-averaged Navier–Stokes (RANS) simulations. The numerical results obtained in this study are in very good agreement with the experiments and unveil the details of the intricate root flow. The Himmelskamp effect is shown to delay the stall onset and to enhance the lift force coefficient Cl even at moderate angles of attack. This improvement in the aerodynamic performance occurs in spite of the negative influence of the mentioned effect on the suction peak of the involved blade sections. The results also show that the vortex emanating from the spanwise position of maximum chord length rotates in the opposite direction to the root vortex, which affects the wake evolution. Furthermore, the aerodynamic losses in the root region are demonstrated to take place much more gradually than at the tip

Argonaute Autoantibodies as Biomarkers in Autoimmune Neurologic Diseases

OBJECTIVE: To identify and characterize autoantibodies (Abs) as novel biomarkers for an autoimmune context in patients with central and peripheral neurologic diseases. METHODS: Two distinct approaches (immunoprecipitation/mass spectrometry-based proteomics and protein microarrays) and patients' sera and CSF were used. The specificity of the identified target was confirmed by cell-based assay (CBA) in 856 control samples. RESULTS: Using the 2 methods as well as sera and CSF of patients with central and peripheral neurologic involvement, we identified Abs against the family of Argonaute proteins (mainly AGO1 and AGO2), which were already reported in systemic autoimmunity. AGO-Abs were mostly of immunoglobulin G 1 subclass and conformation dependent. Using CBA, AGO-Abs were detected in 21 patients with a high suspicion of autoimmune neurologic diseases (71.4% were women; median age 57 years) and only in 4/856 (0.5%) controls analyzed by CBA (1 diagnosed with small-cell lung cancer and the other 3 with Sjögren syndrome). Among the 21 neurologic patients identified, the main clinical presentations were sensory neuronopathy (8/21, 38.1%) and limbic encephalitis (6/21, 28.6%). Fourteen patients (66.7%) had autoimmune comorbidities and/or co-occurring Abs, whereas AGO-Abs were the only autoimmune biomarker for the remaining 7/21 (33.3%). Thirteen (61.9%) patients were treated with immunotherapy; 8/13 (61.5%) improved, and 3/13 (23.1%) remained stable, suggesting an efficacy of these treatments. CONCLUSIONS: AGO-Abs might be potential biomarkers of autoimmunity in patients with central and peripheral nonparaneoplastic neurologic diseases. In 7 patients, AGO-Abs were the only biomarkers; thus, their identification may be useful to suspect the autoimmune character of the neurologic disorder. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that AGO-Abs are more frequent in patients with autoimmune neurologic diseases than controls