The fundamental building blocks of chromatin are the nucleosomes. Each such unit is composed of about 200 bp of DNA, the well-conserved core histones (H2A, H2B, H3 and H4) and a linker histone (H1). The DNA is wound around two dimers of H2A–H2B and a tetramer comprising two molecules each of H3 and H4, and there is approximately one linker histone molecule positioned on the exterior of the DNA–protein octamer complex. The nucleosome directs the various structural transitions in chromatin that are needed for proper transcriptional regulation during differentiation and development of the organism in question. The gene activity can be regulated by different histone variants, DNA–protein interactions, and protein–protein interactions, all of which are influenced by the enormous amounts of post-translational modifications that occur in the histone tails. The research underlying this thesis focused on different aspects of post-translational modifications during aging, differentiation, and progression of the cell cycle, and also on expression of linker histone variants and linker histone-chromatin interactions in a variety of cells and tissues. The present results are the first to show that H4 can be trimethylated at lysine 20 in mammalian cells. The trimethylated H4K20 was found in rat kidney and liver at levels that rose with increasing age of the nimals, and it was also detected in trace amounts in human cell lines. Furthermore, in differentiating MEL cells, trimethylated H4K20 was localized to heterochromatin, and levels of trimethylated H4K20 increased during the course of cell differentiation and were correlated with the increasing compaction of the chromatin. The chromatin of terminally differentiated chicken and frog erythrocytes is highly condensed, and the linker histone variants it contains vary between the two species. Cytofluorometric analyses revealed that the linker histones in the chicken erythrocytes exhibited higher affinity for chromatin than did those in the frog erythrocytes. Characterization of the H1° in frog erythrocytes proved it to be the H1°-2 subvariant. Other experiments demonstrated that normal human B lymphocytes expressed the linker histone variants H1.2, H1.3, H1.4, and H1.5, and that B cells from patients with B-CLL expressed the same variants although in different amounts. The most striking dissimilarity was that amounts of H1.3 in the cells were decreased or undetectable in some samples. Sequencing did not discern any defects in the H1.3 gene, and thus the absence of H1.3 is probably regulated at the post-translational level. It was also observed that the levels of linker histone phosphorylation in EBV-transformed B lymphocytes were already increased in the G1 phase of the cell cycle, which is earlier than previously thought. This increase in phosphorylation is probably responsible for the lower affinity of linker histones for chromatin in EBV-transformed cells in the G1 phase of the cell cycle.

The production of enantiopure compounds can be achieved by using dehydrogenases as biocatalysts catalyzing reduction reactions of prochiral compounds such as ketones, aldehydes and nitriles. These dehydrogenases are cofactor dependent enzyme where cofactor is Nicotinamide dinucleotite having some restrictions that limits usage of dehydrogenases in organic synthesis including instability of cofactor in water and high cost. Therefore suitable regeneration method is needed and developed which are enzymatic and electrochemical. We will use an electrochemical approach for the regeneration of reduced co-factors which has been shown in principal with mediators like pentamethylcyclopentadienyl rhodium bipyridine complexes or ferrocenes. This project is European Union project, whose name is Development of Electrochemical Reactors Using Dehydrogenases for Enantiopure Synthon Preparations. All active compounds / mediator, cofactor and enzyme, will be immobilized on the electrode surface of the constructed reactor surface. Therefore only educts and v products will exist in the reactor medium. A gas diffusion electrode will be employed as a counter electrode / which delivers clear protons to the system. Mediator will carry electrons to the cofactor for cofactor regeneration. Then enzyme will use the cofactor and convert substrates to the product in high stereoselectivity. Our part in this project is the synthesis of mediator and suitable linkers for enzyme, cofactor and mediator immobilization. In the first part of the study, Linkers which contain thiol group and disulfide linkage were synthesized because working electrode made of by gold nano particles and immobilization carried out by the help of these groups on gold nano surface. In the second part of the study, mediators were synthesized which are pentamethylcyclopentadienyl rhodium bipyridine complexes and ferrocene derivatives. Synthesized mediators were reacted with linkers by using Click Chemistry and by imine formation in order to convert mediator to the thiol functionalized form.

Phospholipid vesicles are biocompatible, and have potential for intracellular applications, but minimal membrane integrity limits their use in membrane-rich environments. Stabilized membranes overcome this limitation while maintaining biocompatible surface structures. Additionally, the modularity of phospholipid bilayer makes them ideal components when designing biologically inspired sensors. Membrane composition can be tailored to specific applications, transmembrane proteins can provide added functionalities, and the isolated interior can prevent cytotoxic and interfering detection chemistries from altering the cellular environment. This work has focused on expanding the capabilities of stabilized phospholipid membranes, and determining which formulations hold promise in developing stabilized phospholipid vesicle nanosensors.Current membrane stabilization methods suffer from either incomplete stabilization, or irreversible stabilization limiting the applications of vesicle nanosensors. Therefore, a facile method to prepare robust phospholipid vesicles using commonly available phospholipids stabilized via the formation of an interpenetrating, acid-labile, cross-linked polymer network that imparts controlled polymer destabilization and subsequent vesicle degradation was developed. Upon exposure to acidic conditions, the highly cross-linked polymer network was converted to linear polymers, substantially reducing vesicle stability upon exposure to chemical and physical insults. The resultant transiently stabilized vesicles have potential for enhanced drug delivery and chemical sensing applications requiring minimal membrane defects, and allow for improved physiological clearance.Some vesicle nanosensor schemes may require the passive diffusion of low molecular weight species across the membrane in addition to controllable degradation. Therefore, the acid-degradable, polymer-stabilized, phospholipid vesicle production method was extended to bis-SorbPC membranes by simultaneously polymerizing the vesicle with an acetal-containing cross-linker. The vesicles display prolonged stability under physiological conditions, and significant additional stability compared to vesicles composed of naturally occurring phospholipids. The vesicles demonstrated potential utility for sensing and therapeutic applications.Phospholipid vesicles can also serve as labels to observe movement in macromolecular biological assemblies, but a dearth of caged fluorescent labels limits design and function. Therefore, the first caged fluorescent thiol was synthesized, shown to label amines rapidly, and demonstrated the required photolytic properties. The caged fluorescent thiol has potential as a label in observing the movement of macromolecular biological assemblies and as a fluorescent probe for observing endosomal trafficking and release.

Die Vergrößerung des linken Vorhofes (LA) hat bei Katzen mit Herzerkrankungen eine prognostische Bedeutung und wird mit einem erhöhten Risiko der Entwicklung einer Links-herzinsuffizienz, von supraventrikulären Arrhythmien, von systemischer Thrombembolie und von plötzlichem Herztod in Verbindung gebracht. Bei Katzen mit idiopathischer (HCM) oder sekundärer linksventrikulärer (LV) Hypertrophie kommt es infolge einer LV diastolischen Dysfunktion zu Veränderungen von Größe und Funktion des LA. Ziel der prospektiven Studie war es, die Größe und Funktion des LA in einer Population gesunder Katzen und Katzen mit Kardiomyopathie elektrokardiographisch, radiologisch sowie echokardiographisch zu charakterisieren und die Tiergruppen sowie die diagnostischen Methoden miteinander zu vergleichen. Zur Untersuchung des LA wurden 59 Katzen berücksichtigt. Die Kontrollgruppe bestand aus 26 gesunden Katzen und die Gruppe der kranken Tiere aus 33 Katzen mit HCM oder einer sekundären LV-Hypertrophie. Davon waren 18 Katzen asymptomatisch und 15 hatten eine Linksherzinsuffizienz oder einen aortalen Thrombembolismus. Im EKG wurde die Dauer der P-Welle als diagnostisches Kriterium der LA-Größe untersucht. Bei der radiologischen Untersuchung wurde die Größe des LA in zwei orthogonalen Projektionsebenen subjektiv beurteilt. Außerdem wurde eine neue quantitative Messung des LA im latero-lateralen Strahlengang etabliert. Diese vergleicht die Größe des LA mit der Länge der Thorakalwirbel und wurde als vertebrale Vorhofgröße (LA-VHS) bezeichnet. Mit der transthorakalen zweidimensionalen (2D)- und M-Mode Echokardiographie konnte die Größe des LA durch unterschiedliche Messungen bestimmt werden. Im rechts-parasternalen Vierkammerblick wurden der maximale antero-posteriore Durchmesser des LA (LADs) gemessen und als echokardiographische Bezugsvariable („Gold Standard“) zur Charakterisierung der LA-Größe genutzt. Aus derselben Anschallung wurden die maximale apico-basale Länge des LA und die maximale Vorhoffläche ermittelt. In der rechts-parasternalen Darstellung des LV-Ausflusstraktes wurde unter Anwendung des M-Modes der LA dargestellt und das LA/Ao-Verhältnis berechnet. In der rechts-parasternalen kurzen Achse wurde der maximale Durchmesser des LA (LAmax) bestimmt und sowohl die so genannte „schwedische Methode“ als auch der M-Mode zur Bestimmung der Vorhofgröße und Berechnung der LA/Ao-Verhältnisse angewandt. Der LA wurde als vergrößert definiert, sobald LADs größer 1,60 cm war. Die Ergebnisse der weiteren echokardiographischen Messmethoden zur Charak-terisierung der LA-Größe wurden mit LADs verglichen und folgende Grenzwerte zur Diagnose einer Vergrößerung des LA für die einzelnen unterschiedlichen Messmethoden ermittelt: aus der rechts-parasternalen langen Achse die maximale LA-Länge 1,97 cm, die maximale LA-Fläche 2,80 cm2 und LA/Ao (M-Mode) 1,55, aus der rechts-parasternalen kurzen Achse LA/Ao (M-Mode) 1,54, LA/Ao („schwedische Methode“) 1,44 und LAmax 1,60 cm. Trotz ähnlicher Grenzwerte der LA/Ao-Verhältnisse konnte gezeigt werden, dass die Messwerte der unterschiedlichen Methoden untereinander abweichen und daher diagnostisch nicht austauschbar sind. Die Ergebnisse der elektrokardiographischen und radiologischen Messungen der LA-Größe wurden hinsichtlich ihrer diagnostischen Wertigkeit mit den echokardiographischen Unter-suchungen zur Diagnose einer LA-Vergrößerung verglichen. Das EKG (P-Welle) war wenig sensitiv aber spezifisch bei der Diagnosestellung einer LA-Vergrößerung. Die subjektive radiologische Beurteilung der LA-Größe hatte eine deutlich höhere diagnostische Treffsicherheit, wobei jedoch die Bestimmung der LA-VHS sowohl die größte Sensitivität als auch Spezifität zeigte. Zur Einschätzung der globalen Vorhoffunktion wurden die echokardiographischen Indices LA-Verkürzungsfraktion und LA-Flächenverkürzung herangezogen. Beide Variablen waren in der Gruppe der symptomatischen Katzen deutlich vermindert, verglichen mit der Kontrollgruppe und der Gruppe der asymptomatischen Katzen mit LV-Hypertrophie. Die Reservoirfunktion des LA wurde anhand der S-Welle und des S/D-Verhältnisses des Pulmonalvenenflusses beurteilt. In der Gruppe der symptomatischen Katzen mit LV-Hypertrophie lag das S/D-Verhältnis unter 1,0, was für eine gestörte Reservoirfunktion des LA sprach. Die Weiterleitungsfunktion des LA, charakterisiert durch die D-Welle des Pulmonalvenenflusses war in der Gruppe der symptomatischen Katzen im Gegensatz zu den beiden anderen Gruppen signifikant vermindert. Die Vorhofkontraktion („Booster-Funktion“) wurde anhand der transmitralen A-Welle und der AR-Welle des Pulmonalvenenflusses beurteilt. Hinsichtlich der AR-Welle lag kein Unterschied der Maximalgeschwindigkeit zwischen den drei Tiergruppen vor, jedoch war die Dauer der AR-Welle in der Gruppe der symptomatischen Katzen verlängert. Dieses wurde als Hinweis auf eine erhöhte Nachlast des LA (verminderte LV Dehnbarkeit) und/oder gestörte systolische Funktion des LA gewertet. Die Untersuchung der Blutflussgeschwindigkeit im linken Herzohr zeigte eine deutliche Abnahme in der Gruppe der symptomatischen Katzen und galt als hinweisend für eine gestörte Pumpfunktion und Blutstase. Es wurde ein Zusammenhang zwischen verminderter Blutflussgeschwindigkeit im linken Herzohr (< 0,23 m/s) und dem Risiko eines aortalen Thrombembolismus gefunden. Zusammenfassend kann festgestellt werden, dass eine LV-Hypertrophie bei Katzen zu einer Vergrößerung des LA, verminderter Funktion des LA und Blutstase in LA und im linken Herzohr führt. Somit bestätigt sich die Vermutung, dass der LA sowohl einen diagnostischen als auch prognostischen Wert bei der Untersuchung von Katzen mit HCM oder sekundärer LV-Hypertrophie hat. Die vorliegende Studie liefert einen Beitrag zur echokardiographischen Standarisierung der LA-Größe und LA-Funktion bei der Katze. Weitere Untersuchungen, insbesondere die invasive Validierung echokardiographischer Indices der Größe und Funktion des LA sind notwendig.

Techn. Hochsch., Diss., 2003--Aachen.

A literature study was performed on a new type of cancer medicine: antibody drug conjugates, or ADCs. These consist of a monoclonal antibody, chemically linked to a cytotoxic agent. What makes them unique is their selective toxicity against cancer cells. The first approval of such a pharmaceutical was in the year 2000, with three or four available in different regions of the world today. In the range of 50 registered drugs in clinical development were found, by major and minor corporations. These have been presented in a table in the appendix according to their properties such as type of linker, cytotoxin, development status etc. Furthermore, a detailed study has been done of the chemistry of the linker conjugation as well as an attempt at studying the ADC market. Finally, the mentioned strengths of the drug were compared to its weaknesses, mainly instability and otherwise poor pharmacokinetics. The main conclusion is that these drugs are expected to play a major role in oncology in the future.

Plasmon coupling is known to cause distance dependent red-shifts of the characteristic plasmon resonance and localize strong electric fields to the gap between individual nanoparticles. These effects form the basis of nanoscale plasmonic sensors designed by creating specic structures of coupled nanoparticles. The simplest of these structures, a nanoparticle dimer, can easily be assembled through molecular self-assembly, resulting in a structure called a plasmon ruler. These plasmon rulers are crucial tools for the measurement of nanoscale distances, but the impact of the molecular linker on the plasmonic response of the coupled system remains insufficiently understood. In this dissertation, plasmons rulers composed of 40 nm gold nanoparticles are utilized to systematically investigate the potential effects of one molecular linker, DNA, on the strength of the plasmon coupling at a variety of interparticle separations. The strength of the plasmon coupling is determined based on the shifting of the plasmon resonance, which, at separations below 2.7 nm, is significantly blue-shifted when compared to expected values from electromagnetic simulations and experiments without DNA linkers. This deviation indicates a reduced charge accumulation on the nanoparticles in the gap region and is ascribed to DNA-mediated charge transfer. Enhancements to the charge transfer capabilities of the DNA were also investigated, through the deposition of interstitial palladium nanocrystals on the DNA linkers. The deposition of these nanocrystals results in a variety of structural changes to the plasmon rulers, associated with blue- and red-shifts of the plasmon resonance relative to electromagnetic simulations without gap material and experimental spectra of structures without molecular or metallic linkers. The relative blue-shift of the resonance results from a variety of scenarios, including short interparticle separations bridged by DNA or palladium nanocrystals, the build-up of palladium nanocrystals within the gap, or the incorporation of discrete palladium nanoparticles in the DNA linkers. The underlying mechanisms of the observed spectral shifts are analyzed. The red-shifted resonances resulted from a significant build-up of palladium nanocrystals in the gap, effectively linking the gold nanoparticles and forming a hybrid nanorod-like structure.

The use of autografts is the gold standard for peripheral nerve regeneration (PNR) while biomedical engineering made some contributions to improve PNR. A next generation of nerve guidance conduits (NGC) is required to transmit topographical and biochemical signals towards severed nerves. In this thesis, the gelatin hydrolyzate Collagel® (COL) and anhydride-containing cross-linkers (oPNMA, oPDMA) were used to fabricate crosslinked hydrogels (cGEL) for PNR. At first, established cGEL formulations were adjusted towards an injection-molding tool with static mixer. Therefore, the gelation kinetic was modified by variation of the gelation base. Hence, high reactive oPNMA was available for fabrication of robust cGEL based NGC. Secondly, novel cGEL and molding technique were adapted towards the fabrication of cGEL-based filler for polymer-derived braided NGC. Shear-thinning filler was developed that allowed direct application inside the conduit lumen with minimal mechanical stiffness but sufficient scaffolding properties. Besides pristine filler, chemically modified filler was designed with a small mimetic of the nerve growth factor, LM11A-31, that was grafted to oPNMA. In a rat sciatic nerve model, the performance of this derivatized filler was comparable to the control and underlined the potential of chemical cues in PNR. A number of small diamines were further integrated into oPNMA and oPDMA to modify cGEL bulk. In addition to chemical feasibility, the cytocompatibility and cellular response were tested on L929 mouse fibroblasts and human adipose-derived stem cells. The functionalization showed an impact on the cell behavior with differences in cell proliferation, migration and spreading. Finally, modified oPNMA-derived hydrogels were tested on neonatale Schwann cells. The cell viability and extension was maintained in all hydrogels while the impact of LM11A-31 was not as pronounced. This thesis emphasizes the potential of cGEL hydrogels in nerve implants as fillers or conduits and, thus, is a promising building block for a new generation of NGC.

No description available.

The focus of the project was to alter and optimize the water repellant textile coating formulations to reach enhanced durability. For this purpose, the project was approached with three methods. Firstly, bio-based components were implemented in the mother emulsion to act as surfactant and crosslinking agent and to provide hydrophobic properties. Secondly different binders were added to crosslink and increase the coating resistance towards washes. Lastly additives at nano-scale were added to increase surface roughness in order to obtain higher hydrophobicity and improved of crosslinking capacity due to the presence of more functional groups.  The stability of all emulsions was controlled using different techniques such as optical microscopy to determine particle size, distribution and any observable instability (flocculation etc.), normal aging at room temperature and accelerated aging using higher temperature. All coatings were applied using a laboratory padder on standard PA and PES pieces of textiles and hydrophobic performance was evaluated through ISO 4920 spray test. By standard washing and repeating spray test, durability could be assessed. Further structure and property studies have been run using other tests such as: contact angle measurement, breathability of the coating and SEM observations. Based on the obtained results the incorporation of low HLB, bio-based surfactants in low amount (~0,25%) resulted in an increase in the hydrophobic performance of the tested textiles. However, a decrease in shelf life could be observed with these surfactants at room temperature. Sonication was successfully used to increase both stability and shelf life significantly. Some binders and nanoparticles proved to be successful in increasing the coating quality and thus the durability. Overall many of the developed formulations could enhance performance on PA compared to the already present commercial product. On PES textile, however, the developed strategies yielded hydrophobic effect close to the commercial product.