Gegenstand dieser Diplomarbeit ist die Untersuchung der Dynamik in dünnen Polymerfilmen anhand von einzelnen Molekülen. Zu diesem Zweck wurden dünne Filme (kleiner 100nm) hergestellt und mittels Einzelmoleküldetektion und zeitaufgelöster Einzelphotonenzählung analysiert, was eine orts- und zeitaufgelöste Untersuchung einzelner Farbstoffmoleküle ermöglicht. Ziel war es, festzustellen ob, und auf welchem Weg, die Dynamik der Umgebung in Fluktuationen der Fluoreszenzlebensdauer einzelner Moleküle sichtbar wird. Neben der Evaluierung der Untersuchungsmethoden wurden in dieser Arbeit zwei Arten von Sensormolekülen - DiD und Malachit Grün - näher untersucht. / The subject of this diploma thesis is the analysis of dynamics in thin polymer films using single molecules. Thin polymer films (less than 100nm) were produced and analysed by Single Molecule Detection (SMD) and Time Correlated Single Photon Counting (TCSPC). This allows a spatial and time resolved investigation of the single dye molecule. The aim was to ascertain if, and in which way, the dynamics of the environment are reflected by fluctuations of the fluorescence lifetime of the single molecule. In addition to evaluating the investigation methods two kinds of molecules - DiD and Malachite Green - were also analysed.

Die vorliegende Arbeit hat die Entwicklung und Validierung eines Analysealgorithmus zur objektiven Auswertung der Materialverteilung entlang der Zarge an gezogenen Karton-formziehteilen zum Ziel. Die dafür verwendeten Messdaten werden durch Oberflächenab-tastung mit konfokaler Sensorik erzeugt. Unter Anwendung des Analysealgorithmus wer-den die Einflüsse der Prozessparameter auf die Materialverteilung ermittelt. Abschließend wird aus den gewonnenen Daten ein Ansatz zur Berechnung der Werkzeuggeometrie ent-wickelt. Zur Bestimmung der Materialverteilung wird die Zarge beidseitig abgetastet. Aus der Diffe-renz der Radien von Außen- und Innenkontur wird der Dickenverlauf entlang der Zarge bestimmt, die die Materialverteilung beschreibt. Die Messung mit konfokalen Sensoren wird mit mikroskopischen Aufnahmen von Mikrotomschnitten und röntgentomographi-schen Aufnahmen geprüft. Der Mittelwert und die Standardabweichung des Dickenverlaufs charakterisieren die Mate-rialverteilung. Der Analysealgorithmus ermittelt aus den durch Sensoren aufgenommenen Daten die charakteristischen Werte. In der Auswertung zeigt sich, dass eine Höhere Zieh-stempeltemperatur als Ziehbüchsentemperatur sowie eine hohe Temperatursumme die mittlere Dicke verringern. Eine hohe Faltenhalterkraft und eine hohe Stempeltemperatur bewirken eine gleichmäßigere Materialverteilung. Der ermittelte Zusammenhang von der Beeinflussung des Mittelwertes der Dicke mit den Temperaturunterschieden von Stempel und Büchse wird durch ein Modell der thermischen Ausdehnung von Stempel und Büchse belegt. / The present thesis comprises the development and validation of an algorithm for the objec-tive evaluation of data on material distribution alongside the architrave of drawn cardboard shells. The necessary data are obtained through detection of the surface with confocal sensors. The influences of the process parameters on material distribution are investigated with algorithm. Resulting from this data, the basis for the calculation of the tools geometry is developed. The determination of material distribution is accomplished by double-sided optical scanning of the architraves inner and outer surface. The trend of cardboard thickness alongside the architrave is calculated by the subtracion of the inside radius from the outside radius of the cardboard shell. The cardboard thickness describes material distribution. The measures with confocal sensors are approved of examined microtome cuts under microscope and x-ray analyses. Material distribution is characterised by average of the thickness and standard deviation of thickness. The analysis algorithm calculates with sensor data the characteristic values. A higher temprature of the drawing die as the temprature of the drawing box and the sum of temperatures lead to a decrease of the average thickness. The combination of a high die temperature and a high force applied by the blankholder result in a more even material distribution. The identified correlation of average thickness and temperature gradients between the drawing die and the box is discribed by thermal expansion of die and box.

The Gram-negative bacterium Pseudomonas aeruginosa is a life-threatening nosocomial pathogen due to its generally low susceptibility toward antibiotics. Furthermore, many strains have acquired resistance mechanisms requiring new antimicrobials with novel mechanisms to enhance treatment options. Proline-rich antimicrobial peptides, such as the apidaecin analog Api137, are highly efficient against various Enterobacteriaceae infections in mice, but less active against P. aeruginosa in vitro. Here, we extended our recent work by optimizing lead peptides Api755 (gu-OIORPVYOPRPRPPHPRL-OH; gu = N,N,N′,N′-tetramethylguanidino, O = L-ornithine) and Api760 (gu-OWORPVYOPRPRPPHPRL-OH) by incorporation of Ile-Orn- and Trp-Orn-motifs, respectively. Api795 (gu-O(IO)2RPVYOPRPRPPHPRL-OH) and Api794 (gu-O(WO)3RPVYOPRPRPPHPRL-OH) were highly active against P. aeruginosa with minimal inhibitory concentrations of 8–16 and 8–32 μg/mL against Escherichia coli and Klebsiella pneumoniae. Assessed using a quartz crystal microbalance, these peptides inserted into a membrane layer and the surface activity increased gradually from Api137, over Api795, to Api794. This mode of action was confirmed by transmission electron microscopy indicating some membrane damage only at the high peptide concentrations. Api794 and Api795 were highly stable against serum proteases (half-life times >5 h) and non-hemolytic to human erythrocytes at peptide concentrations of 0.6 g/L. At this concentration, Api795 reduced the cell viability of HeLa cells only slightly, whereas the IC50 of Api794 was 0.23 ± 0.09 g/L. Confocal fluorescence microscopy revealed no colocalization of 5(6)-carboxyfluorescein-labeled Api794 or Api795 with the mitochondria, excluding interactions with the mitochondrial membrane. Interestingly, Api795 was localized in endosomes, whereas Api794 was present in endosomes and the cytosol. This was verified using flow cytometry showing a 50% higher uptake of Api794 in HeLa cells compared with Api795. The uptake was reduced for both peptides by 50 and 80%, respectively, after inhibiting endocytotic uptake with dynasore. In summary, Api794 and Api795 were highly active against P. aeruginosa in vitro. Both peptides passed across the bacterial membrane efficiently, most likely then disturbing the ribosome assembly, and resulting in further intracellular damage. Api795 with its IOIO-motif, which was particularly active and only slightly toxic in vitro, appears to represent a promising third generation lead compound for the development of novel antibiotics against P. aeruginosa.

In dieser Doktorarbeit wird die Hypothese geprüft, ob Osteozyten einen direkten Einfluss auf die Knocheneigenschaften in ihrer unmittelbaren Umgebung haben. Der zentrale Experimentieransatz ist dabei die Korrelation der Organisation des Osteozytennetzwerks mit den Mineraleigenschaften des Knochens auf der Submikrometerebene. Es wird gezeigt, dass bereits die anfängliche Ausrichtung der Osteoblasten entscheidend für die Synthese von hoch ausgerichtetem Knochenmaterial ist. Die dabei entstehenden Osteozytennetzwerke sind so organisiert, dass die Osteozyten und ihre Zellfortsätze jeweils einen möglichst kleinen Abstand zum Knochenmineral haben. Deshalb wird vermutet, dass genau diese Netzwerkorganisation mitentscheidend ist, wie gut die Zellen das Mineral in ih-rer Umgebung beeinflussen können. Messungen der Knochenmineraleigenschaften auf Submikrometerebene mit Röntgenkleinwinkelstreuung bestätigen diese Vermutung. Dabei wird deutlich, dass Knochenmaterial in der Nähe der Osteozyten durch andere Mineraleigenschaften geprägt ist. Um zu klären, wie Osteozyten Mineral in ihrer direkten Umgebung verändern können, werden Mechanismen der passiven Mineralherauslösung aus der mineralisierten Oberfläche des Osteozytennetzwerks untersucht. Es wird gezeigt, dass kalziumarme ionische Lösungen unter physiologischen Bedingungen große Mengen von Kalzium-Ionen aus dem Knochen lösen und diese dann durch die Osteozytennetzwerkstrukturen diffundieren können. Zum Abschluss wurde medullärer Knochen von Hühnern als ein Modellsystem für rasanten Knochenumbau untersucht. Dieser spezielle Knochentyp dient den Hennen als labiles Kalziumreservoir und ermöglicht dadurch die tägliche Eierschalenproduktion. Experimente am medullären Knochen-material zeigen insbesondere die Bedeutung von weniger stabilen Mineralstrukturen die benötigt werden um den Knochen an den schnellen, sich wiederholenden Knochenauf- sowie Abbau optimal anzupassen. / This thesis aims to test the hypothesis whether osteocytes have a direct influence on bone material properties in their vicinity. In this regard, the concomitant ana-lysis of osteocyte network organization and bone ultrastructural properties on the submicron level is the central approach to answer this question. In this work, it is shown that already initial cell-cell alignment during the process of bone formation is crucial for the synthesis of highly organized bone. Furthermore it is proposed that the occurrence of highly ordered osteocyte networks visualized with confocal laser scanning microscopy (CLSM) has a strong impact on the ability of osteocytes to directly influence bone material properties. These highly organized networks are another consequence of initial cell-cell alignment and are found to be arranged such as to feature short mineral cell distances. Examination of sub-micron mineral properties with scanning small angle x-ray scattering (sSAXS) shows that bone material in the direct vicinity of osteocytes and their cell proc-esses shows different mineral properties compared to bone further away in the depth of the tissue. Moreover, mechanisms of passive mineral extraction from the mineralized surface of the osteocyte network, due to the treatment with calcium poor ionic solutions, are investigated. It is shown that this chemical process occurring under physiological conditions leads not only to the dissolution of considerable amounts of calcium, but also to efficient diffusion of these ions through the osteocyte network structures. Finally, medullary bone which is intended as a labile calcium source for daily egg shell formation in hens is used as a model system for rapid bone turnover rates. This bone type in particular indicates the importance of uniquely adapted, less stable mineral structures to fit the requirements for rapid bone resorption as well as reformation.

Ternary lipid mixtures composed of cholesterol, saturated (frequently with sphingosine backbone), and unsaturated phospholipids show stable phase separation and are often used as model systems of lipid rafts. Yet, their ability to reproduce raft properties and function is still debated. We investigated the properties and functional aspects of three lipid raft model systems of varying degrees of biological relevance – PSM/POPC/Chol, DPPC/POPC/Chol, and DPPC/DOPC/Chol – using 2H solidstate nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy, and atomic force microscopy. While some minor differences were observed, the general behavior and properties of all three model mixtures were similar to previously investigated influenza envelope lipid membranes, which closely mimic the lipid composition of biological membranes. For the investigation of the functional aspects, we employed the human N-Ras protein, which is posttranslationally modified by two lipid modifications that anchor the protein to the membrane. It was previously shown that N-Ras preferentially resides in liquid-disordered domains and exhibits a time-dependent accumulation in the domain boundaries of influenza envelope lipid membranes. For all three model mixtures, we observed the same membrane partitioning behavior for N-Ras. Therefore, we conclude that even relatively simple models of raft membranes are able to reproduce many of their specific properties and functions.

No description available.

Sphaerodoridae is a group of benthic marine worms (Annelida) characterized by the presence of spherical tubercles covering their whole surface. They are commonly considered as belonging to Phyllodocida although sistergroup relationships are still far from being understood. Primary homology assessments of their morphological features are lacking, hindering the appraisal of evolutionary relationships between taxa. Therefore, our detailed morphological investigation focuses on different Sphaerodoridae as well as on other members of Phyllodocida using an integrative approach combining scanning electron microscopy (SEM) as well as immunohistochemistry with standard neuronal (anti-5-HT) and muscular (phalloidin-rhodamine) markers and subsequent CLSM analysis of whole mounts and sections. Furthermore, we provide histological (HES) and light microscopical data to shed light on the structures and hypothetical function of sphaerodorid key morphological features. We provide fundamental details into the sphaerodorid morphology supporting a Phyllodocida ancestry of these enigmatic worms. However, the muscular arrangement and the presence of an axial muscular pharynx is similar to conditions observed in other members of the Errantia too. Furthermore, nervous system and muscle staining as well as SEM and histological observations of different types of tubercles indicate a homology of the so called microtubercles, present in the long-bodied sphaerodorids, to the dorsal cirri of other Errantia. The macrotubercles seem to represent a sphaerodorid autapomorphy based on our investigations. Therefore, our results allow comparisons concerning morphological patterns between Sphaerodoridae and other Phyllodocida and constitute a starting point for further comparative investigations to reveal the evolution of the remarkable Sphaerodoridae.

Ternary lipid mixtures composed of cholesterol, saturated (frequently with sphingosine backbone), and unsaturated phospholipids show stable phase separation and are often used as model systems of lipid rafts. Yet, their ability to reproduce raft properties and function is still debated. We investigated the properties and functional aspects of three lipid raft model systems of varying degrees of biological relevance – PSM/POPC/Chol, DPPC/POPC/Chol, and DPPC/DOPC/Chol – using 2H solidstate nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy, and atomic force microscopy. While some minor differences were observed, the general behavior and properties of all three model mixtures were similar to previously investigated influenza envelope lipid membranes, which closely mimic the lipid composition of biological membranes. For the investigation of the functional aspects, we employed the human N-Ras protein, which is posttranslationally modified by two lipid modifications that anchor the protein to the membrane. It was previously shown that N-Ras preferentially resides in liquid-disordered domains and exhibits a time-dependent accumulation in the domain boundaries of influenza envelope lipid membranes. For all three model mixtures, we observed the same membrane partitioning behavior for N-Ras. Therefore, we conclude that even relatively simple models of raft membranes are able to reproduce many of their specific properties and functions.

The Gram-negative bacterium Pseudomonas aeruginosa is a life-threatening nosocomial pathogen due to its generally low susceptibility toward antibiotics. Furthermore, many strains have acquired resistance mechanisms requiring new antimicrobials with novel mechanisms to enhance treatment options. Proline-rich antimicrobial peptides, such as the apidaecin analog Api137, are highly efficient against various Enterobacteriaceae infections in mice, but less active against P. aeruginosa in vitro. Here, we extended our recent work by optimizing lead peptides Api755 (gu-OIORPVYOPRPRPPHPRL-OH; gu = N,N,N′,N′-tetramethylguanidino, O = L-ornithine) and Api760 (gu-OWORPVYOPRPRPPHPRL-OH) by incorporation of Ile-Orn- and Trp-Orn-motifs, respectively. Api795 (gu-O(IO)2RPVYOPRPRPPHPRL-OH) and Api794 (gu-O(WO)3RPVYOPRPRPPHPRL-OH) were highly active against P. aeruginosa with minimal inhibitory concentrations of 8–16 and 8–32 μg/mL against Escherichia coli and Klebsiella pneumoniae. Assessed using a quartz crystal microbalance, these peptides inserted into a membrane layer and the surface activity increased gradually from Api137, over Api795, to Api794. This mode of action was confirmed by transmission electron microscopy indicating some membrane damage only at the high peptide concentrations. Api794 and Api795 were highly stable against serum proteases (half-life times >5 h) and non-hemolytic to human erythrocytes at peptide concentrations of 0.6 g/L. At this concentration, Api795 reduced the cell viability of HeLa cells only slightly, whereas the IC50 of Api794 was 0.23 ± 0.09 g/L. Confocal fluorescence microscopy revealed no colocalization of 5(6)-carboxyfluorescein-labeled Api794 or Api795 with the mitochondria, excluding interactions with the mitochondrial membrane. Interestingly, Api795 was localized in endosomes, whereas Api794 was present in endosomes and the cytosol. This was verified using flow cytometry showing a 50% higher uptake of Api794 in HeLa cells compared with Api795. The uptake was reduced for both peptides by 50 and 80%, respectively, after inhibiting endocytotic uptake with dynasore. In summary, Api794 and Api795 were highly active against P. aeruginosa in vitro. Both peptides passed across the bacterial membrane efficiently, most likely then disturbing the ribosome assembly, and resulting in further intracellular damage. Api795 with its IOIO-motif, which was particularly active and only slightly toxic in vitro, appears to represent a promising third generation lead compound for the development of novel antibiotics against P. aeruginosa.

In dieser Arbeit wird der Aufbau eines Raumtemperatur-Nahfeldmikroskopes und die Implementation eines neuen nichtoptischen Abstandsregelmechanismus beschrieben und die Ergebnisse verschiedener Abbildungsarten dargestellt. Eine theoretische Modellierung des Scherkraft-Detektionsmechanismus erlaubt ein gezieltes Design fuer eine Nachnutzung. Der Aufbau eines Tieftemperatur-Konfokalmikroskopes, das zum Tieftemperatur-Nahfeldmikroskop erweiterbar ist, wird dargelegt. Die Abhaengigkeit des Photobleichens einzelner Terrylen-Molekuele von der Temperatur wird untersucht. Beobachtungen der spektralen Diffusion von Terrylen-Molekuelen werden dargelegt. Den Hauptteil dieser Arbeit bilden Untersuchungen am N-V Farbzentrum im Diamant. Einzelne Defektzentren wurden bei tiefen Temperaturen abgebildet und mittels Fluoreszenz-Anregungsspektroskopie untersucht, die Fluoreszenz-Emissionsdynamik wurde mit Autokorrelationsmessungen studiert. Das Temperaturverhalten der Fluoreszenzintensitaet, die Autokorrelation der Fluoreszenzintensitaet und die verzoegerte Fluoreszenz beweisen die Existenz eines metastabilen Zustandes. Durch Einstrahlen einer zweiten Wellenlaenge verkuerzt sich die Verweilzeit im metastabilen Zustand und die mittlere Fluoreszenzrate wird erhoeht (Deshelving). Die Tieftemperaturspektren widersprechen in mehreren Punkten den aus der Literatur bekannten Werten, die an hoeherkonzentrierten Proben gemessen wurden. Diese Unterschiede werden diskutiert und sind mit einer in hoeherkonzentrierten Proben verstaerkten Verspannung zu erklaeren. Ein Vergleich mit Diamant-Nanokristalliten, in denen eine erhoehte Verspannung auftritt, bestaetigt den Zusammenhang zwischen schmalen Uebergaengen und wirkender Verspannung durch das Auftreten von schmalen Emissionslinien.