Erstellung eines computergestützten Lernprogramms zum Ausdrucksverhalten des Hundes

Beißvorfälle mit zum Teil tödlichem Ausgang sowie die zunehmende Bevölkerungs- und damit auch Hundedichte vor allem in Ballungsräumen führten in den letzten Jahren zu einer starken Medienpräsenz des Themas Hundehaltung und Verantwortlichkeit der Besitzer. In der Folge wurden eine Reihe von Rechtsvorschriften erlassen, die, basierend auf dem Tierschutzgesetz, stets einen Sachkundenachweis des Hundehalters fordern. Ein äußerst wichtiger Themenkomplex innerhalb dieser Sachkundeprüfungen ist Hundeverhalten, insbesondere das Ausdrucksverhalten des Hundes. Durch Kenntnisse auf diesem Gebiet könnten gefährliche Situationen rechtzeitig erkannt und umgangen werden. Ziel der vorliegenden Arbeit war es, in zeitgemäßer und fundierter Form Kenntnisse über das Ausdrucksverhalten des Hundes zu vermitteln. Es wurde hierfür die Umsetzung in einem interaktiven multimedialen Lernprogramm auf CD-Rom gewählt, um mit Hilfe von Videos in bewegten Bildern den zeitlichen Ablauf der komplexen Körpersprache und Mimik von Hunden darzustellen. Das verwendete Videomaterial stammte sowohl aus eigenen Aufnahmen als auch aus Fremdaufnahmen. Der Inhalt des Programms gliedert sich in zwei große Themenkomplexe. Der geschichtliche Teil, der die Domestikation des Hundes beleuchtet, soll Hintergrundwissen über den Vorfahren des Hundes, den Wolf, vermitteln und die Umstände der Domestikation erläutern, die den Grundstock für die einzigartige Beziehung zwischen Menschen und Hunden legt. Im zweiten Themenkomplex folgen Ausführungen über die rassespezifischen Besonderheiten in der Ausdrucksmöglichkeit bestimmter Hundetypen. Ein weiteres Kapitel befasst sich mit der Individualentwicklung des Hundes. Anschließend folgt der Bereich verschiedener Kommunikationsformen des Hundes, wobei schwerpunktmäßig das optische Ausdrucksverhalten dargestellt und mit 107 Videos und 296 Fotos und Graphiken unterlegt wird. Dem Benutzer bleibt freigestellt, ob er sich Seite für Seite durch das Programm arbeiten möchte oder ob er gezielt einzelne Kapitel anwählt, je nach Kenntnisstand und Interessenslage. Insgesamt bietet das Programm einen breit gefächerten Überblick über die Verhaltensweise der Spezies Hund, was dem Benutzer zusätzlich zum eigentlichen Thema die Möglichkeit geben soll, sich Wissen über die Hintergründe der Verhaltensweisen anzueignen. Es soll sowohl Tierärzten wie auch Studenten der Tiermedizin, aber auch Hundebesitzern die Möglichkeit geben, sich Sachkunde im Hinblick auf das Ausdrucksverhalten des Hundes anzueignen, um insbesondere Gefahrensituationen rechtzeitig zu erkennen und zu umgehen

Multiple interactions and complex viscosity: The impact of frequency rheology for the development of high concentration protein formulations

Clinical doses of therapeutic proteins range up to 2 mg/kg bodyweigth per patient and even higher. For patient convenience and competitiveness, subcutaneous (s.c.) applications are required. Therefore, liquid formulations for s.c. applications can reach concentrations of up to 200 mg/ml. One key parameter for the development of biotherapeutics as high concentrated liquid formulations (HCLF) is viscosity. Consequently, high solution viscosity is challenging due to e.g. impeded syringeablitiy and injectability that directly link to patient inconvenience, and high shear stress that potentially impair protein inherent stability. Following Jezek et al. 2011, we consider protein concentrations of \u3e100 mg/ml as “highly concentrated”. During early phases in development of biopharmaceutics only limited material is available. Therefore, prediction of the solution viscosity at higher concentrations (e.g. for HCLF conditions), if required, will be of great benefit. In this study, we applied different approaches comprehensively investigating parameters describing protein-protein interaction, protein hydration, protein conformation at different concentrations, and the volume fraction of the protein molecule in solution. At a molecular level, Protein-Protein Interaction (PPI) are a result of electrostatic-interaction, van-der-Waal (vdW)-forces and hydrophobic forces of bi- or multimodal interaction as well as protein-excipients interaction. At the macroscopic level, these parameters describe a crucial influence on the protein-stability and its rheological behavior in solution. However, during formulation development commonly evaluated PPI parameter such as the second virial coefficient (B22), and/or the concentration- dependent diffusion coefficient (kD). These parameters only describe interactions in dilute conditions, which poses limitations in predicting interactions at high protein concentrations. At dilute conditions, mostly electrostatic double layer repulsion and charge-shielding effects of buffer and excipients components dominate. In contrast, at high concentration, distances between individual molecules are narrowed, and thus attractive forces such as vdW interactions are predominantly present. Therefore, a direct correlation of PPI parameter obtained from dilute to crowded conditions is only a shaky compromise. The mechanisms and principles driving the formation of highly viscose systems are not fully understood, especially at the molecular level. As a consequence, the attempts to reduce viscosity are often left to chance. In a case study, we evaluated the behaviour of concentrated protein formulations under high-frequency shear excitation in the MHz range. As a result, we propose an explanation for interaction potentials between individual protein molecules linked to high solution viscosity by extending the complex colloid theory. Multiple attraction forces result in a complex viscous behavior, the formation of a transient micro-rheological network of multiple interacting protein molecules, and the formation of an elastic modulus. In order to lower the viscosity, such multiple interactions have to be disrupted and disordered by different excipients. However, the interaction potential is correlated to the characteristics of each mAb molecule and can be altered by excipients in a defined way. By relating low concentration PPI measurements and wet-lab determined molecular characteristics (e.g. effective surface charge, dipole moment) it is possible to predicts the potency for describing a high viscosity for each mAb. Knowing the effects of pH and different buffer and excipients, a guided development for decreasing the viscosity by different excipients and formulation conditions is possible. High frequency rheology allows a rapid and early evaluation of the viscosity properties of early candidates and thus support subsequent formulation development

First principles calculation of inhomogeneous broadening in solid-state cw-EPR spectroscopy

We present a scheme for the first-principles calculation of EPR lineshapes for continuous-wave-EPR spectroscopy (cw-EPR) of spin centers in complex chemical environments. We specifically focus on poorly characterized systems, e.g. powders and frozen glasses with variable microsolvation structures. Our approach is based on ab initio molecular dynamics simulations and ab initio calculations of the ensemble of g- and A-tensors along the trajectory. The method incorporates temperature effects as well as the full anharmonicity of the intra- and intermolecular degrees of freedom of the system. We apply this scheme to compute the lineshape of a prototypical spin probe, the nitrosodisulfonate dianionic radical (Fremy's salt), dissolved in a 50 : 50 mixture of water and methanol. We are able to determine the specific effect of variations of local solvent composition and microsolvation structure on the cw-EPR lineshape. Our molecular dynamics reveal a highly anisotropic solvation structure with distinct spatial preferences for water and methanol around Fremy's salt that can be traced back to a combination of steric and polar influences. The overall solvation structure and conformational preferences of Fremy's salt as found in our MD simulations agree very well with the results obtained from EPR and orientation-selective ENDOR spectroscopy performed on the frozen glass. The simulated EPR lineshapes show good agreement with the experimental spectra. When combined with our MD results, they characterize the lineshape dependence on local morphological fluctuations

Two sub-states of the red2 state of methyl-coenzyme M reductase revealed by high-field EPR spectroscopy

Methyl-coenzyme M reductase (MCR) catalyzes the formation of methane from methyl-coenzyme M and coenzyme B in methanogenic archaea. The enzyme has two structurally interlinked active sites embedded in an α2β2γ2 subunit structure. Each active site has the nickel porphyrinoid F430 as a prosthetic group. In the active state, F430 contains the transition metal in the Ni(I) oxidation state. The active enzyme exhibits an axial Ni(I)-based continuous wave (CW) electron paramagnetic resonance (EPR) signal, called red1a in the absence of substrates or red1c in the presence of coenzyme M. Addition of coenzyme B to the MCR-red1 state can partially and reversibly convert it into the MCR-red2 form, which shows a rhombic Ni(I)-based EPR signal (at X-band microwave frequencies of approximately 9.4GHz). In this report we present evidence from high-field/high-frequency CW EPR spectroscopy (W-band, microwave frequency of approximately 94GHz) that the red2 state consists of two substates that could not be resolved by EPR spectroscopy at X-band frequencies. At W-band it becomes apparent that upon addition of coenzyme B to MCR in the red1c state, two red2 EPR signals are induced, not one as was previously believed. The first signal is the well-characterized (ortho)rhombic EPR signal, thus far called red2, while the second previously unidentified signal is axial. We have named the two substates MCR-red2r and MCR-red2a after their rhombic and axial signals, respectivel

Coordination and binding geometry of methyl-coenzyme M in the red1m state of methyl-coenzyme M reductase

Methane formation in methanogenic Archaea is catalyzed by methyl-coenzyme M reductase (MCR) and takes place via the reduction of methyl-coenzyme M (CH3-S-CoM) with coenzyme B (HS-CoB) to methane and the heterodisulfide CoM-S-S-CoB. MCR harbors the nickel porphyrinoid coenzyme F430 as a prosthetic group, which has to be in the Ni(I) oxidation state for the enzyme to be active. To date no intermediates in the catalytic cycle of MCRred1 (red for reduced Ni) have been identified. Here, we report a detailed characterization of MCRred1m ("m” for methyl-coenzyme M), which is the complex of MCRred1a ("a” for absence of substrate) with CH3-S-CoM. Using continuous-wave and pulse electron paramagnetic resonance spectroscopy in combination with selective isotope labeling (13C and 2H) of CH3-S-CoM, it is shown that CH3-S-CoM binds in the active site of MCR such that its thioether sulfur is weakly coordinated to the Ni(I) of F430. The complex is stable until the addition of the second substrate, HS-CoB. Results from EPR spectroscopy, along with quantum mechanical calculations, are used to characterize the electronic and geometric structure of this complex, which can be regarded as the first intermediate in the catalytic mechanis

Probing the Y2 Receptor on Transmembrane, Intra- and Extra-Cellular Sites for EPR Measurements

The function of G protein-coupled receptors is intrinsically linked to their conformational dynamics. In conjugation with site-directed spin labeling, electron paramagnetic resonance (EPR) spectroscopy provides powerful tools to study the highly dynamic conformational states of these proteins. Here, we explored positions for nitroxide spin labeling coupled to single cysteines, introduced at transmembrane, intra- and extra-cellular sites of the human neuropeptide Y2 receptor. Receptor mutants were functionally analyzed in cell culture system, expressed in Escherichia coli fermentation with yields of up to 10 mg of purified protein per liter expression medium and functionally reconstituted into a lipid bicelle environment. Successful spin labeling was confirmed by a fluorescence assay and continuous wave EPR measurements. EPR spectra revealed mobile and immobile populations, indicating multiple dynamic conformational states of the receptor. We found that the singly mutated positions by MTSL ((1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate) have a water exposed immobilized conformation as their main conformation, while in case of the IDSL (bis(1-oxyl-2,2,5,5-tetramethyl-3-imidazolin-4-yl) disulfide) labeled positions, the main conformation are mainly of hydrophobic nature. Further, double cysteine mutants were generated and examined for potential applications of distance measurements by double electron–electron resonance (DEER) pulsed EPR technique on the receptor