Biophysical characterization of reactions associated with reverse cholesterol transport

This thesis aimed at improving our understanding of reactions relevant in the reverse cholesterol transport (RCT). RCT facilitates cholesterol homeostasis and is the most important pathway involved in cardiovascular disease. For this purpose three different projects were chosen. Thermodynamics of protein self-association and unfolding was characterized in detail at the example of Apolipoprotein A-1 (Apo A-1). Lipid binding was characterized by means of small peptides that mimic Apo A-1 function. The third project gained insight about the molecular mechanisms of ABCA1`s allocrite flopping. Apo A-1 is the main protein constituent of high density lipoprotein (HDL) and is together with ABCA1 a key player of the RCT. Apolipoprotein A-1 Protein self-association and unfolding are two processes whose understanding is of utmost importance for the development of biological phamaceuticals as oligomerisation may alter functional properties of proteins. Apo A-1 is a perfect candidate for these investigations as it undergoes a concentration dependent self-association process and has high physiological relevance. Even though Apo A-1 is a highly investigated macromolecule, self-association was not investigated in such a comprehensive approach. Additionally, we used highly purified recombinant human Apo A-1, which was generously provided by H.-J. Schönfeld. For analyzing thermodynamics of self-association and thermal unfolding we introduced new theoretical and experimental methods Self-association data was obtained by a combination of high sensitivity micro calorimetry and analytical ultracentrifugation. The dissociation reaction of highly concentrated and thus oligomeric Apo A-1 was followed by injection into buffer in an isothermal titration calorimeter (ITC). Dilution of the sample moved the chemical equilibrium towards monomers. Complementary, this equilibrium was analyzed by data obtained from analytical ultracentrifugation in a sedimentation equilibrium mode. If any, self-association was described in former studies as equilibrium between distinct species, for example between monomers and dimers. We introduced a cooperative self-association model that describes the equilibrium of the protein between each possible oligomer in a concentration dependent manner. Furthermore, we introduced a “binding partition function” that represents the sum of all concentrations found in the system. Together with a dissociation degree of the protein we found a link between thermodynamic data and theory of self-association. The binding partition function describes the statistical properties of the system in thermodynamic equilibrium. Hence, it is independent of the theoretical model that is utilized to describe the reaction. Thermal unfolding of Apo A-1 was followed by circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). We found that melting of Apo A-1 caused a transition of α–helix to β–sheet and random coiled secondary structure and appeared to be highly reversibly up to 75 °C. Thermal unfolding of Apo A-1 and in general of proteins is analyzed almost exclusively with an all-or-none model. As a powerful alternative for higly α–helical proteins such as Apo A 1, we introduced the cooperative Zimm-Bragg theory. Zimm-Bragg theory is commonly used for thermal unfolding of peptides, but fits well to our data and to data of other proteins obtained from literature. Apo A-1 mimetic peptides Apo A-1 was proposed as drug against cardiovascular disease. However, Apo A-1 mimetic peptides are more promising as they have to be administered in much lower dosage and are produced more easily. Understanding their lipid binding properties is essential for the estimation of in vivo effects as well as for formulation and dosage of possible drugs with these peptides. Apo A-1 structure is featured by several amphiphatic class A motif α-helices. Even though it is the main protein component of HDL, thermodynamic characterization of its lipid binding has not been achieved in detail. As a model of Apo A-1 we used two peptides (4F and P), which are featured by class A amphipathic α-helical sequences. 4F showed Apo A-1 mimetic properties in animal models and clinical studies. We used isothermal titration calorimetry to determine thermodynamic parameters of binding to POPC lipid vesicles. In order to understand this reaction several other experimental methods were used. Static and dynamic light scattering illustrated the ability of the peptides to rupture unilamellar vesicles and form micellar-like particles. In contrast, many other peptides such as cell penetrating peptides (CPPs) only partition into the membrane. This finding is in agreement with a 1:1 lipid-to-peptide stochiometry yielded from ITC data analyzed with a model of n identical binding sites. This behavior might have high physiological relevance as possible rupture of cell membranes is unwanted. Circular dichroism experiments yielded insight into structural transitions as part of the driving force of lipid binding. Associated with lipid binding is a transition of the peptide from β–sheet and random coiled to α-helical secondary structure. Tryptophan fluorescence measurements complemented the studies indicating binding to lipids as well. Thermodynamic calculation proved the structural transition of β–sheet and random coiled to α-helix as well as hydrophobic interactions as driving forces of the reaction. Further, we studied binding of the peptide 4F to cholesterol by means of ITC. Our results suggested affinity of 4F towards cholesterol but with lower affinity compared to POPC. This might explain the formation of HDL like particles, mainly consisting of phosphocholine lipids. These particles, in turn, could bind to cholesterol with high affinity. ABCA1 ABCA1 is an ATP binding cassette transporter that flops excess lipids of a cell to the outer membrane leaflet, where it can be picked up by Apo A-1 or HDL particles. Research in the field of ABCA1 is mainly focused on studies in cell culture and in animal models and is therefore rather indirect. Cholesterol efflux by ABCA1 was assumed to be controlled by the copy number of the transporter. The possibility of a direct modulation of the transporter activity by allocrites like in P-glycoprotein (Pgp) as well as the proposed allocrite specificity was rarely investigated in previous studies. Here, we measured the ATPase activity of inside-out vesicles prepared from ABCA1 transfected Human Embryonic Kidney 293 cells by means of a spectroscopic phosphate release assay. Aluminum fluorides were found as strong inhibitor of the nucleotide binding sites (NBD) of ABCA1 in contrast to vanadate. Furthermore, a screening for putative allocrites interacting with the transmembrane domains (TMDs) was performed with numerous compounds. Therewith we found that all compounds with a pegylated chain, a heterocyclic group and a hydrocarbon tail indicated activation of the ABCA1 ATPase

Mesoscopic light transport by very strong collective multiple scattering in nanowire mats

Under the extreme condition of the scattering length being much shorter than the wavelength, light transport in random media is strongly modified by mesoscopic interference, and can even be halted in an effect known as Anderson localization. Anderson localization in three dimensions has recently been realized for acoustic waves and for cold atoms. Mats of disordered, high-refractive-index semiconductor nanowires are one of the strongest three-dimensional scattering materials for light, but localization has not been shown. Here, we use statistical methods originally developed for microwave waveguides to demonstrate that transport of light through nanowire mats is strongly correlated and governed by mesoscopic interference contributions. Our results confirm the contribution of only a few open modes to the transmission

Dynamische Ausführung von Positionstransformationen mittels OpenGL ES 2.0-Shaderprogrammen

Wegen der Forderung nach Isolation und Performanz in eingebetteten Systemen ("embedded Systems") ist ein Konzept notwendig, dass dynamisch bei laufzeitkritischen Grafikanwendungen die Positionstransformation von Vertices mit Hilfe von Vertex Shadern, während der Laufzeit übernimmt. Daher wurde in dieser Arbeit ein Konzept entwickelt, welches die Position einzelner Vertices berechnet, bevor die kompletten Rendering Befehle ausgeführt werden. Dies ist nötig um abschätzen zu können, ob die Deadlines von sicherheitskritischen Anwendungen eingehalten werden. Dazu wird in dieser Arbeit der Vertex Shader während der Laufzeit mit Hilfe von LLVM kompiliert und berechnet, für einen gegebenen Vertex dessen Position

Dynamische Ausführung von Positionstransformationen mittels OpenGL ES 2.0-Shaderprogrammen

Wegen der Forderung nach Isolation und Performanz in eingebetteten Systemen ("embedded Systems") ist ein Konzept notwendig, dass dynamisch bei laufzeitkritischen Grafikanwendungen die Positionstransformation von Vertices mit Hilfe von Vertex Shadern, während der Laufzeit übernimmt. Daher wurde in dieser Arbeit ein Konzept entwickelt, welches die Position einzelner Vertices berechnet, bevor die kompletten Rendering Befehle ausgeführt werden. Dies ist nötig um abschätzen zu können, ob die Deadlines von sicherheitskritischen Anwendungen eingehalten werden. Dazu wird in dieser Arbeit der Vertex Shader während der Laufzeit mit Hilfe von LLVM kompiliert und berechnet, für einen gegebenen Vertex dessen Position

Early phylogenetic estimate of the effective reproduction number of SARS-CoV-2

To reconstruct the evolutionary dynamics of the 2019 novel-coronavirus recently causing an outbreak in Wuhan, China, 52 SARS-CoV-2 genomes available on 4 February 2020 at Global Initiative on Sharing All Influenza Data were analyzed. The two models used to estimate the reproduction number (coalescent-based exponential growth and a birth-death skyline method) indicated an estimated mean evolutionary rate of 7.8 7 10 124 subs/site/year (range, 1.1 7 10 124-15 7 10 124) and a mean tMRCA of the tree root of 73 days. The estimated R value was 2.6 (range, 2.1-5.1), and increased from 0.8 to 2.4 in December 2019. The estimated mean doubling time of the epidemic was between 3.6 and 4.1 days. This study proves the usefulness of phylogeny in supporting the surveillance of emerging new infections even as the epidemic is growing

Spatial and temporal phylogeny of border disease virus in pyrenean chamois (Rupicapra p. Pyrenaica)

Border disease virus (BDV) affects a wide range of ruminants worldwide, mainly domestic sheep and goat. Since 2001 several outbreaks of disease associated to BDV infection have been described in Pyrenean chamois (Rupicapra pyrenaica pyrenaica) in Spain, France and Andorra. In order to reconstruct the most probable places of origin and pathways of dispersion of BDV among Pyrenean chamois, a phylogenetic analysis of 95 BDV 5'untranslated sequences has been performed on chamois and domestic ungulates, including novel sequences and retrieved from public databases, using a Bayesian Markov Chain Monte Carlo method. Discrete and continuous space phylogeography have been applied on chamois sequences dataset, using centroid positions and latitude and longitude coordinates of the animals, respectively. The estimated mean evolutionary rate of BDV sequences was 2.9x10(-3) subs/site/year (95% HPD: 1.5-4.6x10(-3)). All the Pyrenean chamois isolates clustered in a unique highly significant clade, that originated from BDV-4a ovine clade. The introduction from sheep (dated back to the early 90s) generated a founder effect on the chamois population and the most probable place of origin of Pyrenean chamois BDV was estimated at coordinates 42.42 N and 1.9 E. The pathways of virus dispersion showed two main routes: the first started on the early 90s of the past century with a westward direction and the second arise in Central Pyrenees. The virus spread westward for more than 125 km and southward for about 50km and the estimated epidemic diffusion rate was about 13.1 km/year (95% HPD 5.2-21.4 km/year). The strong spatial structure, with strains from a single locality segregating together in homogeneous groups, and the significant pathways of viral dispersion among the areas, allowed to reconstruct both events of infection in a single area and of migrations, occurring between neighboring areas

Ventricular arrhythmias initiated by programmed stimulation in four groups of patients with healed myocardial infarction

Programmed electrical stimulation of the heart was prospectively used in 160 patients with healed myocardial infarction to study the incidence and characteristics of ventricular arrhythmias induced. Thirty-five patients had neither documented nor suspected ventricular arrhythmias (Group A); 37 patients had documented nonsus-tained ventricular tachycardia (Group B); 31 patients had been resuscitated from ventricular fibrillation (Group C); and 57 patients had documented sustained mono-morphic ventricular tachycardia (Group D). No electrophysiologic differences were found between patients in Group A and Group B, but patients in both groups differed significantly from patients in Group C and Group D. In the last two groups, sustained monomorphic ventricular tachycardia was more frequently induced, the cycle length of the induced ventricular tachycardia was slower and a lesser number of premature stimuli was required for induction. No differences were found in the incidence, rate or mode of induction of nonsustained monomorphic ventricular tachycardia, but nonsustained polymorphic ventricular tachycardia and ventricular fibrillation were more frequently induced in Groups A and B.It is concluded that the substrate for sustained ventricular arrhythmia is present in at least 42% of patients after myocardial infarction. The electrophysiologic characteristics of the substrate for ventricular tachycardia seem to be the major determinant of the clinical occurrence of sustained ventricular arrhythmia. Changes in the electrophysiologic properties of the substrate of ventricular tachycardia, either spontaneously with time or induced by ischemia or antiarrhythmic drugs, can contribute to the clinical occurrence of sustained ventricular arrhythmias in patients with an old myocardial infarction

Chikungunya virus, epidemiology, clinics and phylogenesis: A review.

Abstract Chikungunya virus is a mosquito-transmitted alphavirus that causes chikungunya fever, a febrile illness associated with severe arthralgia and rash. Chikungunya virus is transmitted by culicine mosquitoes; Chikungunya virus replicates in the skin, disseminates to liver, muscle, joints, lymphoid tissue and brain, presumably through the blood. Phylogenetic studies showed that the Indian Ocean and the Indian subcontinent epidemics were caused by two different introductions of distinct strains of East/Central/South African genotype of CHIKV. The paraphyletic grouping of African CHIK viruses supports the historical evidence that the virus was introduced into Asia from Africa. Phylogenetic analysis divided Chikungunya virus isolates into three distinct genotypes based on geographical origins: the first, the West Africa genotype, consisted of isolates from Senegal and Nigeria; the second contained strains from East/Central/South African genotype, while the third contained solely Asian. The most recent common ancestor for the recent epidemic, which ravaged Indian Ocean islands and Indian subcontinent in 2004 – 2007, was found to date in 2002. Asian lineage dated about 1952 and exhibits similar spread patterns of the recent Indian Ocean outbreak lineage, with successive epidemics detected along an eastward path. Asian group splitted into two clades: an Indian lineage and a south east lineage. Outbreaks of Chikungunya virus fever in Asia have not been associated necessarily with outbreaks in Africa. Phylogenetic tools can reconstruct geographic spread of Chikungunya virus during the epidemics wave. The good management of patients with acute Chikungunya virus infection is essential for public health in susceptible areas with current Aedes spp activity

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Die corporativen Organisationen im deutschen Studentenleben: Rectorats-Rede am 28. Februar 1876 in der Aula academica

Hauptsächlich Rostocker Verhältnisse, S. 25 ff. Aktenstücke betr. die alten Rostocker Landsmannschaften von 1623-179