Hybrid Surgery for Severe Mitral Valve Calcification: Limitations and Caveats for an Open Transcatheter Approach

Background and Objectives: Mitral stenosis with extensive mitral annular calcification (MAC) remains surgically challenging in respect to clinical outcome. Prolonged surgery time with imminent ventricular rupture and systolic anterior motion can be considered as a complex of causal factors. The aim of our alternative hybrid approach was to reduce the risk of annual rupture and paravalvular leaks and to avoid obstruction of the outflow tract. A review of the current literature was also carried out. Materials and Methods: Six female patients (mean age 76 9 years) with severe mitral valve stenosis and severely calcified annulus underwent an open implantation of an Edwards Sapien 3 prosthesis on cardiopulmonary bypass. Our hybrid approach involved resection of the anterior mitral leaflet, placement of anchor sutures and the deployment of a balloon expanded prosthesis under visual control. Concomitant procedures were carried out in three patients. Results: The mean duration of cross-clamping was 95 31 min and cardiopulmonary bypass was 137 60 min. The perioperative TEE showed in three patients an inconspicuous, heart valve-typical gradient on all implanted prostheses and a clinically irrelevant paravalvular leakage occurred in the anterior annulus. In the left ventricular outflow tract, mild to moderately elevated gradients were recorded. No adverse cerebrovascular events and pacemaker implantations were observed. All but one patient survived to discharge. Survival at one year was 83.3%. Conclusions: This “off label” implantation of the Edwards Sapien 3 prosthesis may be considered as a suitable bail-out approach for patients at high-risk for mitral valve surgery or deemed inoperable due to extensive MAC

Numerische und experimentelle Untersuchungen zu den Spannungsumlagerungen von ermüdungsbeanspruchten Betonbauteilen im Very-High-Cycle-Fatigue Bereich

Ein zentraler Baustein zur Reduktion von CO2-Emissionen ist der Ausbau der erneuerbaren Energien, insbesondere der Windenergie. Forschungsbedarf besteht dabei bei der ressourceneffizienten Herstellung der Turmstrukturen. Bei Nabenhöhen von über 100 Metern sind Hybridtürme aus vorgespannten Stahlbetonsegmenten die geeignetste Konstruktion. Hierfür ist jedoch eine genaue Kenntnis des Ermüdungsverhaltens von Beton erforderlich. In der Literatur existieren überwiegend Untersuchungen an kleinformatigen zylindrischen Probekörpern, deren Ergebnisse nur bedingt auf die großmaßstäblichen Bauteile übertragen werden können. Im Rahmen dieses Vorhabens wurden daher zum einen Großversuche mit zyklisch biegebeanspruchten, vorgespannten Betonbalken sowie Begleitversuche an zylindrischen Probekörpern und zum anderen numerische Simulationen der Balkenversuche durchgeführt. Das numerische Materialmodell wurde aufbauend auf einem additiven Dehnungsmodell im Finite-Elemente-Programm ANSYS Mechanical in einem iterativen Berechnungsablauf implementiert. Die Betondehnungen setzen sich hierbei aus vier Anteilen zusammen, einem elastischen, einem plastischen, einem viskosen und einem Temperaturdehnungsanteil. Somit konnte der kombinierte Einfluss der Anteile auf das Ermüdungsverhalten von Beton dargestellt werden. In den Großversuchen konnte bei den Balkenprobekörpern ein Ermüdungsversagen der Betondruckzone erzeugt werden, das sich an dieser Stelle durch Risse parallel zur Drucknormalspannung sowie teilweises Abplatzen der Betondruckzone, die der größten Spannungsschwingbreite ausgesetzt war, einstellte. Es zeigte sich, dass dies erst nach deutlich mehr Lastwechseln eintrat als bei den axial beanspruchten Betonzylindern in den zyklischen Begleitversuchen mit derselben Spannungsschwingbreite. Dies ist auf die Spannungsumlagerung zurückzuführen, die im Querschnitt aufgrund der ermüdungsbedingten Materialdegradation und Steifigkeitsverringerung der stark beanspruchten Randbereiche stattfand. In den Begleitversuchen wurden die Materialparameter für das numerische Modell ermittelt, mit dem im Anschluss die Balkenversuche nachgerechnet wurden. Es konnten die in den Versuchen beobachteten Effekte der Steifigkeitsdegradation und Spannungsumlagerung und die daraus resultierende Lebensdauerverlängerung nachgebildet werden. Das Modell kann somit für weitergehende Lebensdaueruntersuchungen von ermüdungsbeanspruchten Betonbauteilen verwendet werden.:ABSCHLUSSBERICHT 1 Allgemeine Angaben 2 Zusammenfassung / Summary 3 Wissenschaftlicher Arbeits- und Ergebnisbericht 4 Veröffentlichte Projektergebniss

Self-Assembly and Structure Formation of Spider Silk Based Proteins in (Ultra)thin Films

Spider silk is one of the most fascinating materials found in nature. Besides its properties like biodegradability, low immunoreactivity, and biocompatibility, especially the mechanical properties outperforming today’s artificial high-tech materials like Kevlar® are of great interest in biomedicine or material science. Spider silk comprises highly repetitive amino acid sequence motives, whose structure is accepted to be responsible for the extraordinary properties of spider silk. Typically, hydrophilic sequence motives alternate with hydrophobic ones making spider silk proteins resemble block copolymers. Additionally, the simple amino acid sequence and the possibility to form fibrillar structures are common characteristics of spider silk proteins as well as intrinsically disordered proteins (IDP) or protein regions (IDR). Both are suspected of being involved in the development of certain neurodegenerative diseases like Alzheimer´s disease. These aspects open promising possibilities of the use of spider silk proteins in nanotechnology, but also as model systems for the fibrillization processes of IDPs and IDRs, which are still unresolved today. Currently, most of the research and application is focused on 1-dimensional spider silk protein fibrils and fibers or 0-dimensional spider silk particles. However, 2-dimensional spider silk protein films or porous 3-dimensional objects are highly relevant platforms with the potential for cell-supporting scaffolds, biodegradable electrolyte materials in transistors, or e.g., planar drug-eluting implant coatings. Generally, the effects of sequence-based and external influences on the self-assembly and folding of spider silk proteins have not yet been fully elucidated in all of these various dimensional spider silk materials, even concerning IDP and IDR models. Thus, basic research regarding assembly and folding processes is still needed, especially in films. Particularly, 2-dimensional films allow a broad spectrum of (surface) analytical techniques, from whose outcome general structure-property relations of spider silk materials across all material dimensions can be obtained. In this work, engineered spider silk proteins, which are based on the consensus sequence motives in the spider silk fibroin (spidroin) 3 and 4 of the European garden spider Araneus diadematus (eADF4(Cx), eADF3(AQ)x, eADF3(QAQ)x) as well as blends of two short peptides with the respective aa sequence of the hydrophobic (pep-c) and hydrophilic (pep-a) part of eADF4(Cx) proteins were used. Spider silk-related proteins and peptides were dissolved in 1,1,1,3,3,3-hexafluoroisopropanol or formic acid, processed as thin films, and post-treated with methanol vapor to induce β-sheet formation. Dichroic FTIR-spectroscopy was used, a powerful tool for studying protein secondary structure formation and orientation. Proteins reveal characteristic amide bands, which are highly sensitive to the conformation of the protein backbone. In the course of this work, a set of components for the line shape analysis (LSA) of the Amide I band was developed. Therby, each component was assigned to a typical secondary structure allowing a quantitative determination of the respective portions and their structural orientation. Quantitative secondary structure portions and their orientation could be determined on this basis. Furthermore, a comprehensive study of folding and self-assembly-influencing parameters like hydrophobic and hydrophilic sequences, molecular weight, the repeating sequence motive order, the film thickness, surface topography, and the surface chemistry in engineered spider silk protein and spider silk protein-based films was carried out. In general, methanol vapor post-treatment induced the formation of β-sheet structures in all films, causing phase separation and the formation of spherical and filamentous structures. The phase separation upon post-treatment was influenced by the covalent connectivity between hydrophobic and hydrophilic sequence parts as well as the repeating sequence motives. In thin films, the increased flexibility of shorter peptides enabled the formation of multipack filaments instead of spherical structures, which were formed by higher molecular weight proteins with several inter-connected repeating sequence motives. Stamping wrinkled structures using poly(dimethylsiloxane) substrates was possible. Filamentous structures were successfully assigned to β-sheet rich structures using infrared nanospectroscopy for the first time. Further, enhanced surface hydrophobicity led to the clustering of β-sheet filaments. The β-sheet content could be controlled by the amount of hydrophobic sequences in thin films. With a higher amount of hydrophobic sequences in the proteins or blends, the β-sheet content increased until a maximum β-sheet content of around 60% was reached. Additionally, β-sheet formation could be suppressed by increasing substrate hydrophobicity or by decreasing the number of repeating sequence motives by going from protein-like folding to peptide-like self-assembly. The backfolding of proteins with covalently linked repeating sequence motives further promoted the formation of more antiparallel β-sheets. Antiparallel β-sheet formation was also favored when the portion of the hydrophilic, amorphous phase was increased. Micrometer thick films did not reveal any preferred alignment of β-sheets, while a general out-of-plane orientation of β-sheets could be obtained in all thin protein, peptide, and blend films. Z-axial orientation in films was increased by using short pep-c and pep-a peptides, higher molecular weight proteins or the deposition of monolayered films instead of thin multilayered films. Also, increased hydrophilicity of the substrate promoted the alignment of β-sheets perpendicular to the substrate surface. The folding kinetics and final domain size were found to be directly correlated. The amount of hydrophobic phase, backfolding, and increased flexibility due to low chain lengths increased the folding kinetics and led to smaller domain sizes. Thus, competing effects of backfolding and flexibility of the protein/peptide backbone could be rationalized. The film integrity and water contact angle were directly related to the β-sheet content and the molecular weight. Beyond the classical protein conformation and orientation analysis, the possibilities and limits of orientation analysis using dichroic attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy were elaborated on the seemingly ideal oriented polymer model system of end-grafted poly(N,N-dimethylaminoethylmethacrylate) chains. Such a system featured a polymer brush regime in the swollen state with z-axial orientation expected similarly high as thin spider silk films after ptm. Moreover, dichroic ATR-FTIR spectroscopy is a promising analytical method for closing gaps in the defined assignment of brush regimes. In summary, general models of the structure formation and self-assembly of spider silk protein in films depending on the parameters mentioned above could be developed and set in relation to IDP/IDR self-assembly by using dichroic FTIR spectroscopy as the basic analysis method. The herein postulated models on the molecular level contribute to the understanding and development of future industrial applications of spider silk protein-based materials and the clarification of unresolved questions regarding IDP and IDR systems.:Abstract V Kurzfassung IX List of Publications XIII Publications in Trade Journals XIII Presentations and Posters XIII Contribution to Joint Publications XV List of Abbrevations XVII List of Symbols XIX List of Figures XXV List of Tables XXXIII 1 Introduction and Motivation 1 2 Theory 5 2.1 Proteins and Peptides 5 2.1.1 General Definition of Proteins and Peptides 5 2.1.2 Structure of Globular Proteins 7 2.1.3 Protein Folding 10 2.1.4 Intrinsically Disordered Proteins and Protein Regions 11 2.2 Block Copolymers 14 2.3 Spiders and Spider Silks 17 2.3.1 Classification of Spiders 17 2.3.2 The Natural Spider Silk Spinning Process 18 2.3.3 Structure of Spider Silk and Spider Silk Proteins 19 2.3.4 Structure-Property Relationships of Spider Silk 21 2.4 Infrared Spectroscopy 23 2.4.1 Basic Principles of Infrared Spectroscopy 23 2.4.2 Basic Equipment and IR-Technologies 27 2.4.3 Orientation Analysis using Dichroic FTIR Spectroscopy 32 2.4.4 Infrared Spectroscopy of Proteins and Peptides 38 2.4.5 Quantitative Analysis of TRANS- and ATR-FTIR Protein Spectra 43 2.5 Electronic Circular Dichroism 46 2.5.1 Basics Principles of Circular Dichroism 46 2.5.2 Circular Dichroism of Proteins and Polypeptides 48 2.5.3 Spectra Analysis 50 2.6 Atomic Force Microscopy 51 2.6.1 Setup of Atomic Force Microscopes 51 2.6.2 Basic Principles of Atomic Force Microscopy 52 2.6.3 AFM Operation Modes 55 3 Experimental Section 57 3.1 Materials 57 3.1.1 Chemicals 57 3.1.2 Substrates 57 3.1.3 Film Preparation 58 3.2 Analytical Methods 60 3.2.1 Dichroic FTIR Spectroscopy 60 3.2.2 Atomic Force Microscopy 64 3.2.3 Electronic Circular Dichroism 64 3.2.4 Spectroscopic Ellipsometry 64 3.2.5 Infrared Nanospectroscopy 65 3.2.6 Grazing Incident Small Angle X-Ray Scattering 66 4 Results 67 4.1 Self-Assembly of eADF4(C16) Films 67 4.1.1 Motivation 67 4.1.2 Dichroic FTIR Spectroscopy Characterization of ß-sheet Orientation in Spider Silk Films on Silicon Substrates 68 4.2 Influence of the Hydrophilic and Hydrophobic Blocks on Peptide Self-Assembly 90 4.2.1 Motivation 90 4.2.2 β-Sheet Structure Formation within Binary Blends of Two Spider Silk Related Peptides 90 4.2.3 Influence of the Hydrophilic and Hydrophobic Blocks on the Inner Morphology in Spider Silk Protein Based Blend Films 122 4.3 Influence of the Sequence Motive Repeating Number on Spider Silk Protein Folding 123 4.3.1 Motivation 123 4.3.2 Influence of Sequence Motive Repeating Number on Protein Folding in Spider Silk Protein Films 124 4.4 Influence of the Module Order on Spider Silk Protein Self-Assembly 152 4.4.1 Motivation 152 4.4.2 Secondary Structure upon Post-treatment 153 4.4.3 β-Sheet Orientation after Post-treatment 157 4.4.4 Morphology and Surface Properties 158 4.4.5 Conclusion 160 4.5 Surface Induced Changes of Spider Silk Protein Self-Assembly 161 4.5.1 Motivation 161 4.5.2 Variation of the Substrate Surface Chemistry and Topography 161 4.5.3 Influence of the Surface Topography on Protein Self-Assembly 162 4.5.4 Influence of the Surface Chemistry on Protein Self-Assembly 164 4.5.5 Conclusion 169 4.6 Chances and Limits of Dichroic ATR-FTIR Spectroscopy 170 4.6.1 Motivation 170 4.6.2 Novel Insights into Swelling and Orientation of End-Grafted PDMAEMA Chains by In-Situ ATR-FTIR Complementing In-Situ Ellipsometry 171 5 Conclusion and Outlook 197 6 References 203 7 Appendix 219 8 Danksagung 227 9 Eidesstattliche Versicherung 22

Construction and Characterization of T7 Bacteriophages Harboring Apidaecin-Derived Sequences

The global spread of multi- and pan-resistant bacteria has triggered research to identify novel strategies to fight these pathogens, such as antimicrobial peptides and, more recently, bacteriophages. In a proof-of-concept study, we have genetically modified lytic T7Select phages targeting Escherichia coli Rosetta by integrating DNA sequences derived from the proline-rich antimicrobial peptide, apidaecin. This allowed testing of our hypothesis that apidaecins and bacteriophages can synergistically act on phage-sensitive and phage-resistant E. coli cells and overcome the excessive cost of peptide drugs by using infected cells to express apidaecins before cell lysis. Indeed, the addition of the highly active synthetic apidaecin analogs, Api802 and Api806, to T7Select phage-infected E. coli Rosetta cultures prevented or delayed the growth of potentially phage-resistant E. coli Rosetta strains. However, high concentrations of Api802 also reduced the T7Select phage fitness. Additionally, plasmids encoding Api802, Api806, and Api810 sequences transformed into E. coli Rosetta allowed the production of satisfactory peptide quantities. When these sequences were integrated into the T7Select phage genome carrying an N-terminal green fluorescent protein (GFP-) tag to monitor the expression in infected E. coli Rosetta cells, the GFP–apidaecin analogs were produced in reasonable quantities. However, when Api802, Api806 and Api810 sequences were integrated into the T7Select phage genome, expression was below detection limits and an effect on the growth of potentially phage-resistant E. coli Rosetta strains was not observed for Api802 and Api806. In conclusion, we were able to show that apidaecins can be integrated into the T7Select phage genome to induce their expression in host cells, but further research is required to optimize the engineered T7Select phages for higher expression levels of apidaecins to achieve the expected synergistic effects that were visible when the T7Select phages and synthetic Api802 and Api806 were added to E. coli Rosetta cultures

A Rat Model of Post-Traumatic Stress Syndrome Causes Phenotype-Associated Morphological Changes and Hypofunction of the Adrenal Gland

Background: Rats exposed to chronic predator scent stress mimic the phenotype of complex post-traumatic stress disorder (PTSD) in humans, including altered adrenal morphology and function. High- and low-anxiety phenotypes have been described in rats exposed to predator scent stress (PSS). This study aimed to determine whether these high- and low-anxiety phenotypes correlate with changes in adrenal histomorphology and corticosteroid production. Methods: Rats were exposed to PSS for ten days. Thirty days later, the rats’ anxiety index (AI) was assessed with an elevated plus-maze test. Based on differences in AI, the rats were segregated into low- (AI ≤ 0.8, n = 9) and high- (AI > 0.8, n = 10) anxiety phenotypes. Plasma corticosterone (CORT) concentrations were measured by ELISA. Adrenal CORT, desoxyCORT, and 11-dehydroCORT were measured by high-performance liquid chromatography. After staining with hematoxylin and eosin, adrenal histomorphometric changes were evaluated by measuring the thickness of the functional zones of the adrenal cortex. Results: Decreased plasma CORT concentrations, as well as decreased adrenal CORT, desoxyCORT and 11-dehydroCORT concentrations, were observed in high- but not in low-anxietyphenotypes. These decreases were associated with increases in AI. PSS led to a significant decrease in the thickness of the zona fasciculata and an increase in the thickness of the zona intermedia. The increase in the thickness of the zona intermedia was more pronounced in low-anxiety than in high-anxiety rats. A decrease in the adrenal capsule thickness was observed only in low-anxiety rats. The nucleus diameter of cells in the zona fasciculata of high-anxiety rats was significantly smaller than that of control or low-anxiety rats. Conclusion: Phenotype-associated changes in adrenal function and histomorphology were observed in a rat model of complex post-traumatic stress disorder

Momentum work and the energetic foundations of physics: I. Newton’s laws of motion tailored to processes

Modern physics is based on Newton’s laws of motion, which describe interaction via forces. In this paper, we argue that interaction needs to be described in terms of processes. By introducing the momentum work and the associated momentum energy in mechanics, we present a coherent formulation of the process equations for mechanics and thermodynamics. This naturally leads to a simple derivation of the Lorentz-transformed mass, according to which any object changes its mass in real terms when its velocity is changed. Momentum work requires a revision of Newton’s laws of motion. For the first time in the history of physics, the elastic collision between objects, such as particles, can be described as a temporal process, not as interaction via force = counter-force. The mechanism of energy conversion during the elastic collision and other mechanical processes, such as free fall, becomes clear and demonstrates the validity of the principle of energy conservation on microscale at any point in time. The results suggest that physics can be rebuilt on a more coherent footing of dynamic processes up to quantum-process thermodynamics