Assessing the functional structure of molecular transporters by EPR spectroscopy

In this thesis, the self-assembled functional structure of a broad range of amphiphilic molecular transporters is studied. By employing paramagnetic probe molecules and ions, continuous-wave and pulse electron paramagnetic resonance spectroscopy reveal information about the local structure of these materials from the perspective of incorporated guest molecules. First, the transport function of human serum albumin for fatty acids is in the focus. As suggested by the crystal structure, the anchor points for the fatty acids are distributed asymmetrically in the protein. In contrast to the crystallographic findings, a remarkably symmetric entry point distribution of the fatty acid binding channels is found, which may facilitate the uptake and release of the guest molecules. Further, the metal binding of 1,2,3-triazole modified star-shaped cholic acid oligomers is studied. These biomimetic molecules are able to include and transport molecules in solvents of different polarity. A pre-arrangement of the triazole groups induces a strong chelate-like binding and close contact between guest molecule and metal ion. In absence of a preordering, each triazole moiety acts as a single entity and the binding affinity for metal ions is strongly decreased. Hydrogels based on N-isopropylacrylamide phase separate from water above a certain temperature. The macroscopic thermal collapse of these hydrogels is utilized as a tool for dynamic nuclear polarization. It is shown that a radical-free hyperpolarized solution can be achieved with a spin-labeled gel as separable matrix. On the nanoscale, these hydrogels form static heterogeneities in both structure and function. Collapsed regions protect the spin probes from a chemical decay while open, water-swollen regions act as catalytic centers. Similarly, thermoresponsive dendronized polymers form structural heterogeneities, which are, however, highly dynamic. At the critical temperature, they trigger the aggregation of the polymer into mesoglobules. The dehydration of these aggregates is a molecularly controlled non-equilibrium process that is facilitated by a hydrophobic dendritic core. Further, a slow heating rate results in a kinetically entrapped non-equilibrium state due to the formation of an impermeable dense polymeric layer at the periphery of the mesoglobule.

Measurement of the elastic electron-proton cross section and separation of the electric and magnetic form factor in the Q 2 range from 0.004 to 1 (GeV/c) 2

The electromagnetic form factors of the proton are fundamental quantities sensitive to the distribution of charge and magnetization inside the proton. Precise knowledge of the form factors, in particular of the charge and magnetization radii provide strong tests for theory in the non-perturbative regime of QCD. However, the existing data at Q^2 below 1 (GeV/c)^2 are not precise enough for a hard test of theoretical predictions.rnrnFor a more precise determination of the form factors, within this work more than 1400 cross sections of the reaction H(e,e′)p were measured at the Mainz Microtron MAMI using the 3-spectrometer-facility of the A1-collaboration. The data were taken in three periods in the years 2006 and 2007 using beam energies of 180, 315, 450, 585, 720 and 855 MeV. They cover the Q^2 region from 0.004 to 1 (GeV/c)^2 with counting rate uncertainties below 0.2% for most of the data points. The relative luminosity of the measurements was determined using one of the spectrometers as a luminosity monitor. The overlapping acceptances of the measurements maximize the internal redundancy of the data and allow, together with several additions to the standard experimental setup, for tight control of systematic uncertainties.rnTo account for the radiative processes, an event generator was developed and implemented in the simulation package of the analysis software which works without peaking approximation by explicitly calculating the Bethe-Heitler and Born Feynman diagrams for each event.rnTo separate the form factors and to determine the radii, the data were analyzed by fitting a wide selection of form factor models directly to the measured cross sections. These fits also determined the absolute normalization of the different data subsets. The validity of this method was tested with extensive simulations. The results were compared to an extraction via the standard Rosenbluth technique.rnrnThe dip structure in G_E that was seen in the analysis of the previous world data shows up in a modified form. When compared to the standard-dipole form factor as a smooth curve, the extracted G_E exhibits a strong change of the slope around 0.1 (GeV/c)^2, and in the magnetic form factor a dip around 0.2 (GeV/c)^2 is found. This may be taken as indications for a pion cloud. For higher Q^2, the fits yield larger values for G_M than previous measurements, in agreement with form factor ratios from recent precise polarized measurements in the Q2 region up to 0.6 (GeV/c)^2.rnrnThe charge and magnetic rms radii are determined as rn⟨r_e⟩=0.879 ± 0.005(stat.) ± 0.004(syst.) ± 0.002(model) ± 0.004(group) fm,rn⟨r_m⟩=0.777 ± 0.013(stat.) ± 0.009(syst.) ± 0.005(model) ± 0.002(group) fm.rnThis charge radius is significantly larger than theoretical predictions and than the radius of the standard dipole. However, it is in agreement with earlier results measured at the Mainz linear accelerator and with determinations from Hydrogen Lamb shift measurements. The extracted magnetic radius is smaller than previous determinations and than the standard-dipole value.

Laser shock peening treatment to control and moderate fatigue crack growth in aircraft structure based on residual stress engineering approach

Laser Shock Peening (LSP) is a surface enhancement treatment which induces a significant layer of beneficial compressive residual stresses of up to several mm underneath the surface of metal components in order to improve the detrimental effects of the crack growth behavior rate in it. The aim of this thesis is to predict the crack growth behavior in metallic specimens with one or more stripes which define the compressive residual stress area induced by the Laser Shock Peening treatment. The process was applied as crack retardation stripes perpendicular to the crack propagation direction with the object of slowing down the crack when approaching the peened stripes. The finite element method has been applied to simulate the redistribution of stresses in a cracked model when it is subjected to a tension load and to a compressive residual stress field, and to evaluate the Stress Intensity Factor (SIF) in this condition. Finally, the Afgrow software is used to predict the crack growth behavior of the component following the Laser Shock Peening treatment and to detect the improvement in the fatigue life comparing it to the baseline specimen. An educational internship at the “Research & Technologies Germany – Hamburg” department of AIRBUS helped to achieve knowledge and experience to write this thesis. The main tasks of the thesis are the following: •To up to date Literature Survey related to “Laser Shock Peening in Metallic Structures” •To validate the FE model developed against experimental measurements at coupon level •To develop design of crack growth slowdown in Centered Cracked Tension specimens based on residual stress engineering approach using laser peened strip transversal to the crack path •To evaluate the Stress Intensity Factor values for Centered Cracked Tension specimens after the Laser Shock Peening treatment via Finite Element Analysis •To predict the crack growth behavior in Centered Cracked Tension specimens using as input the SIF values evaluated with the FE simulations •To validate the results by means of experimental tests

Computersimulationen zum Einfluss topologischer Beschränkungen auf Polymere

Topologische Beschränkungen beeinflussen die Eigenschaften von Polymeren. Im Rahmen dieser Arbeit wird mit Hilfe von Computersimulationen im Detail untersucht, inwieweit sich die statischen Eigenschaften von kollabierten Polymerringen, Polymerringen in konzentrierten Lösungen und aus Polymerringen aufgebauten Bürsten mit topologischen Beschränkungen von solchen ohne topologische Beschränkungen unterscheiden. Des Weiteren wird analysiert, welchen Einfluss geometrische Beschränkungen auf die topologischen Eigenschaften von einzelnen Polymerketten besitzen. Im ersten Teil der Arbeit geht es um den Einfluss der Topologie auf die Eigenschaften einzelner Polymerketten in verschiedenen Situationen. Da allerdings gerade die effiziente Durchführung von Monte-Carlo-Simulationen von kollabierten Polymerketten eine große Herausforderung darstellt, werden zunächst drei Bridging-Monte-Carlo-Schritte für Gitter- auf Kontinuumsmodelle übertragen. Eine Messung der Effizienz dieser Schritte ergibt einen Beschleunigungsfaktor von bis zu 100 im Vergleich zum herkömmlichen Slithering-Snake-Algorithmus. Darauf folgt die Analyse einer einzelnen, vergröberten Polystyrolkette in sphärischer Geometrie hinsichtlich Verschlaufungen und Knoten. Es wird gezeigt, dass eine signifikante Verknotung der Polystrolkette erst eintritt, wenn der Radius des umgebenden Kapsids kleiner als der Gyrationsradius der Kette ist. Des Weiteren werden sowohl Monte-Carlo- als auch Molekulardynamiksimulationen sehr großer Ringe mit bis zu einer Million Monomeren im kollabierten Zustand durchgeführt. Während die Konfigurationen aus den Monte-Carlo-Simulationen aufgrund der Verwendung der Bridging-Schritte sehr stark verknotet sind, bleiben die Konfigurationen aus den Molekulardynamiksimulationen unverknotet. Hierbei zeigen sich signifikante Unterschiede sowohl in der lokalen als auch in der globalen Struktur der Ringpolymere. Im zweiten Teil der Arbeit wird das Skalierungsverhalten des Gyrationsradius der einzelnen Polymerringe in einer konzentrierten Lösung aus völlig flexiblen Polymerringen im Kontinuum untersucht. Dabei wird der Anfang des asymptotischen Skalierungsverhaltens, welches mit dem Modell des “fractal globules“ konsistent ist, erreicht. Im abschließenden, dritten Teil dieser Arbeit wird das Verhalten von Bürsten aus linearen Polymeren mit dem von Ringpolymerbürsten verglichen. Dabei zeigt sich, dass die Struktur und das Skalierungsverhalten beider Systeme mit identischem Dichteprofil parallel zum Substrat deutlich voneinander abweichen, obwohl die Eigenschaften beider Systeme in Richtung senkrecht zum Substrat übereinstimmen. Der Vergleich des Relaxationsverhaltens einzelner Ketten in herkömmlichen Polymerbürsten und Ringbürsten liefert keine gravierenden Unterschiede. Es stellt sich aber auch heraus, dass die bisher verwendeten Erklärungen zur Relaxationsverhalten von herkömmlichen Bürsten nicht ausreichen, da diese lediglich den anfänglichen Zerfall der Korrelationsfunktion berücksichtigen. Bei der Untersuchung der Dynamik einzelner Monomere in einer herkömmlichen Bürste aus offenen Ketten vom Substrat hin zum offenen Ende zeigt sich, dass die Monomere in der Mitte der Kette die langsamste Relaxation besitzen, obwohl ihre mittlere Verrückung deutlich kleiner als die der freien Endmonomere ist.

Feasibility studies for accessing nucleon structure observables with the PANDA experiment at the future FAIR facility

The availability of a high-intensity antiproton beam with momentum up to 15,GeV/c at the future FAIR will open a unique opportunity to investigate wide areas of nuclear physics with the $overline{P}$ANDA (anti{$overline{P}$}roton ANnihilations at DArmstadt) detector. Part of these investigations concern the Electromagnetic Form Factors of the proton in the time-like region and the study of the Transition Distribution Amplitudes, for which feasibility studies have been performed in this Thesis. rnMoreover, simulations to study the efficiency and the energy resolution of the backward endcap of the electromagnetic calorimeter of $overline{P}$ANDA are presented. This detector is crucial especially for the reconstruction of processes like $bar pprightarrow e^+ e^- pi^0$, investigated in this work. Different arrangements of dead material were studied. The results show that both, the efficiency and the energy resolution of the backward endcap of the electromagnetic calorimeter fullfill the requirements for the detection of backward particles, and that this detector is necessary for the reconstruction of the channels of interest. rnrnThe study of the annihilation channel $bar pprightarrow e^+ e^-$ will improve the knowledge of the Electromagnetic Form Factors in the time-like region, and will help to understand their connection with the Electromagnetic Form Factors in the space-like region. In this Thesis the feasibility of a measurement of the $bar pprightarrow e^+ e^-$ cross section with $overline{P}$ANDA is studied using Monte-Carlo simulations. The major background channel $bar pprightarrow pi^+ pi^-$ is taken into account. The results show a $10^9$ background suppression factor, which assure a sufficiently clean signal with less than 0.1% background contamination. The signal can be measured with an efficiency greater than 30% up to $s=14$,(GeV/c)$^2$. The Electromagnetic Form Factors are extracted from the reconstructed signal and corrected angular distribution. Above this $s$ limit, the low cross section will not allow the direct extraction of the Electromagnetic Form Factors. However, the total cross section can still be measured and an extraction of the Electromagnetic Form Factors is possible considering certain assumptions on the ratio between the electric and magnetic contributions.rnrnThe Transition Distribution Amplitudes are new non-perturbative objects describing the transition between a baryon and a meson. They are accessible in hard exclusive processes like $bar pprightarrow e^+ e^- pi^0$. The study of this process with $overline{P}$ANDA will test the Transition Distribution Amplitudes approach. This work includes a feasibility study for measuring this channel with $overline{P}$ANDA. The main background reaction is here $bar pprightarrow pi^+ pi^- pi^0$. A background suppression factor of $10^8$ has been achieved while keeping a signal efficiency above 20%.rnrnrnPart of this work has been published in the European Physics Journal A 44, 373-384 (2010).rn

Nucleon electromagnetic structure studies in the spacelike and timelike regions

The thesis investigates the nucleon structure probed by the electromagnetic interaction. One of the most basic observables, reflecting the electromagnetic structure of the nucleon, are the form factors, which have been studied by means of elastic electron-proton scattering with ever increasing precision for several decades. In the timelike region, corresponding with the proton-antiproton annihilation into a electron-positron pair, the present experimental information is much less accurate. However, in the near future high-precision form factor measurements are planned. About 50 years after the first pioneering measurements of the electromagnetic form factors, polarization experiments stirred up the field since the results were found to be in striking contradiction to the findings of previous form factor investigations from unpolarized measurements. Triggered by the conflicting results, a whole new field studying the influence of two-photon exchange corrections to elastic electron-proton scattering emerged, which appeared as the most likely explanation of the discrepancy. The main part of this thesis deals with theoretical studies of two-photon exchange, which is investigated particularly with regard to form factor measurements in the spacelike as well as in the timelike region. An extraction of the two-photon amplitudes in the spacelike region through a combined analysis using the results of unpolarized cross section measurements and polarization experiments is presented. Furthermore, predictions of the two-photon exchange effects on the e+p/e-p cross section ratio are given for several new experiments, which are currently ongoing. The two-photon exchange corrections are also investigated in the timelike region in the process pbar{p} -> e+ e- by means of two factorization approaches. These corrections are found to be smaller than those obtained for the spacelike scattering process. The influence of the two-photon exchange corrections on cross section measurements as well as asymmetries, which allow a direct access of the two-photon exchange contribution, is discussed. Furthermore, one of the factorization approaches is applied for investigating the two-boson exchange effects in parity-violating electron-proton scattering. In the last part of the underlying work, the process pbar{p} -> pi0 e+e- is analyzed with the aim of determining the form factors in the so-called unphysical, timelike region below the two-nucleon production threshold. For this purpose, a phenomenological model is used, which provides a good description of the available data of the real photoproduction process pbar{p} -> pi0 gamma.

Crystal structure of a CAP-DNA complex: the DNA is bent by 90 degrees

The 3 angstrom resolution crystal structure of the Escherichia coli catabolite gene activator protein (CAP) complexed with a 30-base pair DNA sequence shows that the DNA is bent by 900. This bend results almost entirely from two 400 kinks that occur between TG/CA base pairs at positions 5 and 6 on each side of the dyad axis of the complex. DNA sequence discrimination by CAP derives both from sequence-dependent distortion of the DNA helix and from direct hydrogen-bonding interactions between three protein side chains and the exposed edges of three base pairs in the major groove of the DNA. The structure of this transcription factor-DNA complex provides insights into possible mechanisms of transcription activation

Using the Natural Period of a Structure as an Indicator of the Significance of Second-Order Effects

This study investigates the feasibility of predicting the momentamplification in beam-column elements of steel moment-resisting frames using the structure's natural period. Unlike previous methods, which perform moment-amplification on a story-by-story basis, this study develops and tests two models that aim to predict a global amplification factor indicative of the largest relevant instance of local moment amplification in the structure. To thisend, a variety of two-dimensional frames is investigated using first and secondorder finite element analysis. The observed moment amplification is then compared with the predicted amplification based on the structure's natural period, which is calculated by first-order finite element analysis. As a benchmark, design moment amplification factors are calculated for each story using the story stiffness approach, and serve to demonstrate the relativeconservatism and accuracy of the proposed models with respect to current practice in design. The study finds that the observed moment amplification factors may vastly exceed expectations when internal member stresses are initially very small. Where the internal stresses are small relative to the member capacities, thesecases are inconsequential for design. To qualify the significance of the observed amplification factors, two parameters are used: the second-order moment normalized to the plastic moment capacity, and the combined flexural and axial stress interaction equations developed by AISC

Conformationally Controlled Electron Delocalization in n-Type Rods: Synthesis, Structure, and Optical, Electrochemical, and Spectroelectrochemical Properties of Dicyanocyclophanes

A series of dicyanobiphenyl-cyclophanes 1-6 with various pi-backbone conformations and characteristic n-type semiconductor properties is presented. Their synthesis, optical, structural, electrochemical, spectroelectrochemical, and packing properties are investigated. The X-ray crystal structures of all n-type rods allow the systematic correlation of structural features with physical properties. In addition, the results are supported by quantum mechanical calculations based on density functional theory. A two-step reduction process is observed for all n-type rods, in which the first step is reversible. The potential gap between the reduction processes depends linearly on the cos(2) value of the torsion angle phi between the pi-systems. Similarly, optical absorption spectroscopy shows that the vertical excitation energy of the conjugation band correlates with the cos(2) value of the torsion angle phi. These correlations demonstrate that the fixed intramolecular torsion angle phi is the dominant factor determining the extent of electron delocalization in these model compounds, and that the angle phi measured in the solid-state structure is a good proxy for the molecular conformation in solution. Spectroelectrochemical investigations demonstrate that conformational rigidity is maintained even in the radical anion form. In particular, the absorption bands corresponding to the SOMO-LUMO+i transitions are shifted bathochromically, whereas the absorption bands corresponding to the HOMO-SOMO transition are shifted hypsochromically with increasing torsion angle phi.

Characterization of Molecular Determinants of the Conformational Stability of Macrophage Migration Inhibitory Factor: Leucine 46 Hydrophobic Pocket

Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of its interactions and biological activities, including its binding to its receptor CD74 and activation of certain signaling pathways. Therefore, understanding the molecular factors governing the oligomerization of MIF and the role of quaternary structure in modulating its structural stability and multifunctional properties is crucial for understanding the function of MIF in health and disease. Herein, we describe highly conserved intersubunit interactions involving the hydrophobic packing of the side chain of Leu46 onto the β-strand β3 of one monomer within a hydrophobic pocket from the adjacent monomer constituted by residues Arg11, Val14, Phe18, Leu19, Val39, His40, Val41, Val42, and Pro43. To elucidate the structural significance of these intersubunit interactions and their relative contribution to MIF’s trimerization, structural stability and catalytic activity, we generated three point mutations where Leu46 was replaced by glycine (L46G), alanine (L46A) and phenylalanine (L46F), and their structural properties, stability, oligomerization state, and catalytic activity were characterized using a battery of biophysical methods and X-ray crystallography. Our findings provide new insights into the role of the Leu46 hydrophobic pocket in stabilizing the conformational state of MIF in solution. Disrupting the Leu46 hydrophobic interaction perturbs the secondary and tertiary structure of the protein but has no effect on its oligomerization state.

Static and Dynamic Error Correction of a Computer-Aided Mechanical Navigation Linkage for Arthroscopic Hip Surgery

While beneficially decreasing the necessary incision size, arthroscopic hip surgery increases the surgical complexity due to loss of joint visibility. To ease such difficulty, a computer-aided mechanical navigation system was developed to present the location of the surgical tool relative to the patient¿s hip joint. A preliminary study reduced the position error of the tracking linkage with limited static testing trials. In this study, a correction method, including a rotational correction factor and a length correction function, was developed through more in-depth static testing. The developed correction method was then applied to additional static and dynamic testing trials to evaluate its effectiveness. For static testing, the position error decreased from an average of 0.384 inches to 0.153 inches, with an error reduction of 60.5%. Three parameters utilized to quantify error reduction of dynamic testing did not show consistent results. The vertex coordinates achieved 29.4% of error reduction, yet with large variation in the upper vertex. The triangular area error was reduced by 5.37%, however inconsistent among all five dynamic trials. Error of vertex angles increased, indicating a shape torsion using the developed correction method. While the established correction method effectively and consistently reduced position error in static testing, it did not present consistent results in dynamic trials. More dynamic paramters should be explored to quantify error reduction of dynamic testing, and more in-depth dynamic testing methodology should be conducted to further improve the accuracy of the computer-aided nagivation system.

An in vivo study of a growth-factor enhanced, cell free, two-layered collagen-tricalcium phosphate in deep osteochondral defects

Focal osteochondral defects are still a challenging problem in joint surgery. We have developed a two-layered implant consisting of a basal porous beta-tricalcium phosphate (TCP) for bone reconstruction and a superficial fibrous collagen type I/III layer for cartilage regeneration. Fifty-four osteochondral defects in the trochlear groove of 27 Göttinger Minipigs were created and either left untreated, treated with the implant alone, or the implant augmented with an additional growth factor mixture, which was assumed to stimulate cell and tissue differentiation. Follow-up was 6, 12 and 52 weeks with n=6 for each group. The repair tissue was evaluated for its gross appearance and biomechanical properties. Histological sections were semi-quantitatively scored for their histomorphological structure. Treatment with the two-layered implant improved defect filling and subchondral bone repair at 6 and 12 weeks follow-up. The TCP was replaced by cancellous bone at 52 weeks. Cartilage repair tissue mainly consisted of fibrocartilage and showed a moderate cell density up to the joint surface. Growth factor treatment improved the mechanical and histomorphological properties of the cartilage repair tissue at 12, but not at 52 weeks postoperatively. In conclusion, the two-layered collagen-TCP implant augmented with chondroinductive growth factors seems a promising new option for the treatment of deep osteochondral defects in joint surgery.

Snake venom C-type lectins interacting with platelet receptors. Structure-function relationships and effects on haemostasis

Snake venoms contain components that affect the prey either by neurotoxic or haemorrhagic effects. The latter category affect haemostasis either by inhibiting or activating platelets or coagulation factors. They fall into several types based upon structure and mode of action. A major class is the snake C-type lectins or C-type lectin-like family which shows a typical folding like that in classic C-type lectins such as the selectins and mannose-binding proteins. Those in snake venoms are mostly based on a heterodimeric structure with two subunits alpha and beta, which are often oligomerized to form larger molecules. Simple heterodimeric members of this family have been shown to inhibit platelet functions by binding to GPIb but others activate platelets via the same receptor. Some that act via GPIb do so by inducing von Willebrand factor to bind to it. Another series of snake C-type lectins activate platelets by binding to GPVI while yet another series uses the integrin alpha(2)beta(1) to affect platelet function. The structure of more and more of these C-type lectins have now been, and are being, determined, often together with their ligands, casting light on binding sites and mechanisms. In addition, it is relatively easy to model the structure of the C-type lectins if the primary structure is known. These studies have shown that these proteins are quite a complex group, often with more than one platelet receptor as ligand and although superficially some appear to act as inhibitors, in fact most function by inducing thrombocytopenia by various routes. The relationship between structure and function in this group of venom proteins will be discussed.

Crystal structure of the platelet glycoprotein Ib(alpha) N-terminal domain reveals an unmasking mechanism for receptor activation

Glycoprotein Ib (GPIb) is a platelet receptor with a critical role in mediating the arrest of platelets at sites of vascular damage. GPIb binds to the A1 domain of von Willebrand factor (vWF-A1) at high blood shear, initiating platelet adhesion and contributing to the formation of a thrombus. To investigate the molecular basis of GPIb regulation and ligand binding, we have determined the structure of the N-terminal domain of the GPIb(alpha) chain (residues 1-279). This structure is the first determined from the cell adhesion/signaling class of leucine-rich repeat (LRR) proteins and reveals the topology of the characteristic disulfide-bonded flanking regions. The fold consists of an N-terminal beta-hairpin, eight leucine-rich repeats, a disulfide-bonded loop, and a C-terminal anionic region. The structure also demonstrates a novel LRR motif in the form of an M-shaped arrangement of three tandem beta-turns. Negatively charged binding surfaces on the LRR concave face and anionic region indicate two-step binding kinetics to vWF-A1, which can be regulated by an unmasking mechanism involving conformational change of a key loop. Using molecular docking of the GPIb and vWF-A1 crystal structures, we were also able to model the GPIb.vWF-A1 complex.