NK cells are innate lymphocytes which constitute the first line of organism's defence against infections through their receptor system. These cells represent an important part of antiviral and antitumor immunity, they also play a role in transplant immunity, autoimmunity and reproduction. This diploma thesis inquires into the structure of the transmembrane receptor NKR-P1B of mouse NK cells and the interaction with its ligand Clr-b. The aim was to prepare the expression vector coding the ligand-binding and whole extracellular region of the receptor NKR-P1B and to optimize its production and refolding in vitro. Purified protein samples were analyzed by size-exclusion chromatography, electrophoresis and mass spectrometry. Interaction between NKR-P1B and Clr-b proteins was tested using biophysical (size-exclusion chromatography and surface plasmon resonance) and biological methods (labelling of cellular sample with NKR-P1B proteins marked with fluorescent dye). In vitro binding experiments have not confirmed mutual interaction between NKR-P1B and Clr-b despite the prepared proteins binding to the bone marrow cells.

Im Rahmen dieser Arbeit ist es durch die kombinierte Anwendung von P-31 Flüssigkeits (HR)- NMR-Spektroskopie sowie dynamischer Lichtstreuung (DLS) gelungen, die supramolekulare Struktur von mizellarem Casein aus ultrahocherhitzter (UHT) Milch unter dem Einfluss einer enzymatischen Vernetzung mittels mikrobieller Transglutaminase (mTG) zu charakterisieren. Die P-31 HR NMR-Spektroskopie erweist sich dabei als hervorragende Methode, um sowohl den Einbau von Casein aus dem Milchserum in die mizellaren Aggregate durch die enzymatische Reaktion als auch die bevorzugte mTG Vernetzung des beta-Caseins nachzuweisen. Durch die Kombination von P-31 HR NMR-Spektroskopie und Messungen der dynamischen Lichtstreuung war es weiterhin möglich, das Vorliegen vernetzter Caseinaggregate in Dispersionen mTG-behandelter Caseine zu belegen und besonders den Anteil an nicht vernetztem Casein „sichtbar“ zu machen, der durch EDTA-Zugabe aus den mTG-vernetzten Caseinnetzwerken freigesetzt wird. Es zeigt sich, dass die Caseinnetzwerke nach der EDTA-Behandlung eine geringere Proteindichte als mizellares Casein aufweisen, da sie nur ca. 20 % des Serinphosphats des mizellaren Caseins enthalten. P-31 Festkörper-NMR-spektroskopische Messungen legen außerdem nahe, dass die Beweglichkeit des phosphorylierten Ser149-Restes des kappa-Caseins in der äußeren Schicht der mizellaren Caseinaggregate durch die mTG-Behandlung nicht wesentlich verändert wird. Um die erhaltenen Caseinnetzwerke im Hinblick auf ihr Anwendungspotential zu untersuchen, wurden sie als Proteinkomponente bei der biomimetischen Calciumphosphatfällung sowie als Trägerstrukturen für bioaktives Lysozym verwendet. Durch den Einsatz von Caseinnetzwerken als Fällungsmedium während der Präzipitation von Calciumphosphat (CaP) ist es gelungen, eine hydratisierte, apatitische Phase zu stabilisieren, die sowohl ungeordnete als auch kristalline Bereiche enthält und damit strukturelle Ähnlichkeit zu biologisch und besonders biomimetisch gebildetem Apatit besitzt. Die in den Präzipitaten ebenfalls vorhandenen Phosphoratome in einer relativ ungeordneten OCP (Octacalciumphosphat)-ähnlichen Umgebung stehen höchstwahrscheinlich mit der apatitischen Phase in räumlich engem Kontakt und sind damit entweder Bestandteil dieser Phase oder befinden sich in einer getrennten Phase, die jedoch mit der apatitischen Phase in Form eines Nanokomposits mit sehr kleinen, eng benachbarten Kristalliten vorliegt. Bei der Fällung des Caseinnetzwerk/CaP-Präzipitats wird ebenfalls eine Dicalciumphosphat-Dihydrat (DCPD)-Phase gebildet. Diese ist separiert von den anderen CaP-Phasen und tritt in wesentlich geringerem Maße auf als in einem reinen CaP-Präzipitat, das ohne Proteinkomponente gefällt wurde. Damit konnte gezeigt werden, dass unter Bedingungen, bei denen ohne Proteinkomponente größtenteils DCPD entsteht, die Caseinnetzwerke eine apatitische Phase stabilisieren, die strukturelle Ähnlichkeit zu biologisch und biomimetisch gebildetem Apatit aufweist. Die qualitativ gleichen Ergebnisse konnten für vergleichsweise untersuchtes unvernetztes Casein gefunden werden. Die Caseinnetzwerke zeigen jedoch in Bezug auf die apatitische Phase einen stärkeren Stabilisierungseffekt als unvernetztes Casein. Es ist denkbar, dass dies unter anderem darauf zurückzuführen ist, dass die Phosphatzentren in den Caseinnetzwerken im Gegensatz zu Casein frei von CaP-Brücken sind, da diese durch die EDTA-Behandlung entfernt wurden. Da die Caseinnetzwerke zudem eine geringere Proteindichte und damit eine höhere „Porosität“ als die mizellaren Caseinaggregate aufweisen, kann sich die apatitische Phase möglicherweise auch innerhalb der Netzwerke bilden, während dies für die mizellaren Caseinaggregate wahrscheinlich nur begrenzt möglich ist. In der vorliegenden Arbeit konnte ebenfalls gezeigt werden, dass sich Caseinnetzwerke grundsätzlich als Transportsysteme für Lysozym eignen, da sie eine hohe Stabilität aufweisen und erfolgreich mit Lysozym beladen werden können. Während die Assoziation von Lysozym mit mizellaren Caseinaggregaten, die aus UHT-Milch gewonnenen wurden, zu einem fast vollständigen Verlust der Lysozymaktivität führt, bleibt die Aktivität des Enzyms bei der Bindung an Caseinnetzwerke erhalten. Das Anwendungspotential der Caseinnetzwerk/Lysozym-Assoziate wurde im Rahmen von zahnmedizinischen Versuchen in vitro und in situ untersucht. Es konnte nachgewiesen werden, dass die Caseinnetzwerk/Lysozym-Assoziate in vitro eine dauerhafte Immobilisierung des Lysozyms in der in situ gebildeten Pellikel bewirken. Eine deutliche Anreicherung des Enzyms in situ wird mithilfe der Caseinnetzwerke allerdings nicht beobachtet. Dies könnte darin begründet sein, dass die im Vergleich zu den in vitro vorgefundenen Verhältnissen deutlich komplexeren Bedingungen in situ zu einem selektiveren Anreicherungsprozess von Enzymen in der Pellikel führen.

Užití biologických materiálů k náhradě tkání v plastické chirurgii ! Abstrakt v angličtině Background: Biological meshes are biomaterials consisted of extracellular matrix and used in surgery particularly for hernia treatment or thoracic wall reconstruction. They are capable of vascularization, that decreases risk of infection, expecially when used in contaminated fields. This study compared the strength of incorporation and biocompatibility of two porcine-derived grafts (cross-linked and non-cross-linked) in a rat hernia model. In addition, we hypothesized that combination of extracellular matrices with autologous mesenchymal stem cells used for hernia repair would result in increased vascularization and increased strength of incorporation. Methods: Standardized 2 x 4 cm fascial defect was created in 42 Wistar rats and repaired with a cross-linked or a non-cross-linked graft either enriched or non-enriched with stem cells. The rats were sacrificed 3, 6 and 12 months later. The strength of incorporation, vascularization, cellular invasion, foreign body reaction and capsule formation were evaluated. Results: Comparison of stem cell enriched and non-enriched groups showed no significant differences in the capsule thickness, foreign body reaction, cellularization or vascularization. In the non-cross-linked...

No description available.

Purpose In ophthalmology, data from both eyes of a person are frequently included in the statistical evaluation. This violates the requirement of data independence for classical statistical tests (e.g. t-Test or analysis of variance (ANOVA)) because it is correlated data. Linear mixed models (LMM) were used as a possibility to include the data of both eyes in the statistical evaluation. Methods The LMM is available for a variety of statistical software such as SPSS or R. The application was applied to a retrospective longitudinal analysis of an accelerated corneal cross-linking (ACXL (9*10)) treatment in progressive keratoconus (KC) with a follow-up period of 36 months. Forty eyes of 20 patients were included, whereas sequential bilateral CXL treatment was performed within 12 months. LMM and ANOVA for repeated measurements were used for statistical evaluation of topographical and tomographical data measured by Pentacam (Oculus, Wetzlar, Germany). Results Both eyes were classified into a worse and better eye concerning corneal topography. Visual acuity, keratometric values and minimal corneal thickness were statistically significant between them at baseline (p < 0.05). A significant correlation between worse and better eye was shown (p < 0.05). Therefore, analyzing the data at each follow-up visit using ANOVA partially led to an overestimation of the statistical effect that could be avoided by using LMM. After 36 months, ACXL has significantly improved BCVA and flattened the cornea. Conclusion The evaluation of data of both eyes without considering their correlation using classical statistical tests leads to an overestimation of the statistical effect, which can be avoided by using the LMM.

No description available.

Proteins and their intricate network of interactions are fundamental to many molecular processes that govern life. Mass spectrometry-based structural proteomics represents a powerful set of techniques for characterizing protein structures and interactions. The last decade has witnessed a large-scale adoption in the application of these techniques toward solving a variety of biological questions. Addressing these questions has often been coincident with the further development of these techniques. Insight into the structures of individual proteins and their interactions with other proteins in a proteome-wide context has been made possible by recent developments in the relatively new field of chemical crosslinking combined with mass spectrometry. In these experiments crosslinking reagents are used to capture protein-protein interactions by forming covalent linkages between proximal amino acid residues. The crosslinked proteins are then enzymatically digested into peptides, and the covalently-coupled crosslinked peptides are identified by mass spectrometry. These identified crosslinked peptides thus provide evidence of interacting regions within or between proteins. In this dissertation the development of tools and methods that facilitate this powerful technique are described. The primary arc of this work follows the development and application of mass spectrometry-based approaches for the identification of protein crosslinks ranging from those which exist endogenously to those which are introduced synthetically. Firstly, the development of a novel strategy for comprehensive determination of naturally occurring protein crosslinks in the form of disulfide bonds is described. Secondly, the application of crosslinking reagents to create synthetic crosslinks in proteins coupled with molecular dynamics simulations is explored in order to structurally characterize the intrinsically disordered tau protein. Thirdly, improvements to a crosslinking-mass spectrometry method for defining a protein-protein interactome in a complex sample is developed. Altogether, these described approaches represent a toolset to allow researchers to access information about protein structure and interactions. / Graduate

Soft matter, here, liquids and polymers, have adaptability to a surrounding geometry. They intrinsically have advantageous characteristics from a mechanical perspective, such as flowing and wetting on surrounding surfaces, giving compliant, conformal and deformable behavior. From the behavior of soft matter for heterogeneous surfaces, compliant structures can be engineered as embedded liquid microstructures or patterned liquid microsystems for emerging compliant microsystems. Recently, skin electronics and soft robotics have been initiated as potential applications that can provide soft interfaces and interactions for a human-machine interface. To meet the design parameters, developing soft material engineering aimed at tuning material properties and smart processing techniques proper to them are to be highly encouraged. As promising candidates, Ga-based liquid alloys and silicone-based elastomers have been widely applied to proof-of-concept compliant structures. In this thesis, the liquid alloy was employed as a soft and stretchable electrical and thermal conductor (resistor), interconnect and filler in an elastomer structure. Printing-based liquid alloy patterning techniques have been developed with a batch-type, parallel processing scheme. As a simple solution, tape transfer masking was combined with a liquid alloy spraying technique, which provides robust processability. Silicone elastomers could be tunable for multi-functional building blocks by liquid or liquid-like soft solid inclusions. The liquid alloy and a polymer additive were introduced to the silicone elastomer by a simple mixing process. Heterogeneous material microstructures in elastomer networks successfully changed mechanical, thermal and surface properties. To realize a compliant microsystem, these ideas have in practice been useful in designing and fabricating soft and stretchable systems. Many different designs of the microsystems have been fabricated with the developed techniques and materials, and successfully evaluated under dynamic conditions. The compliant microsystems work as basic components to build up a whole system with soft materials and a processing technology for our emerging society.

Mit Lösungen von 9,10-Diphenylanthracen und N(C2H5)4ClO4 oder N(C4H9)4ClO4 als Leitsalz im Lösungsmittel Acetonitril wurden Elektrochemilumineszenz (ECL)-Experimente durchgeführt. Dazu wurden die Elektroden mit Folgen von jeweils drei in bestimmten zeitlichen Abständen aufeinander folgenden Potentialsprüngen polarisiert. Es wird gezeigt, dass bei entsprechender Wahl der Potentiale und der Haltezeiten anodische und kathodische ECL-Emissionen gleicher Intensität erzeugt werden können. Sodann wurde ECL in den Derivaten von Poly(p-phenylen-vinylen), MEH-PPV und DB-PPV erzeugt. Diese leitfähigen Polymere wurden als dünne Schichten auf Platin-Elektroden aufgebracht und wie bei ECL aus der Lösungsphase in Acetonitril-Elektrolyten mit Tetralkylammonium-Leitsalzen Potentialsprüngen unterworfen. Bei geeigneter Einstellung der Potentialsprünge und Haltezeiten konnten anodische und kathodische ECL gleicher Intensität erhalten werden. Dies ist das erste Mal, dass symmetrische ECL mit polymerbeschichteten Elektroden erhalten wurde. Die Kinetik der ECL weicht deutlich von der aus der Lösungsphase ab. Der ECL-Prozess verläuft langsamer als in der Lösungsphase, und der Leitelektrolyt hat einen signifikanten Einfluss auf das elektrochemische Verhalten der Polymerschicht. Die Ursachen dafür wurden über Modellrechnungen analysiert, mit denen die Ladungstransportprozesse in der Polymerschicht simuliert wurden. In derartigen Simulationsrechnungen konnten die Geschwindigkeitskonstanten der ECL-Reaktion sowohl im Polymer als auch in der Lösung bestimmt werden. Um die Stabilität der Polymerschichten zu erhöhen, wurde versucht, die Polymerketten mit Synchrotronstrahlung zu vernetzen. Diese Experimente brachten nicht das erwartete Ergebnis. Die Ursachen dafür werden auf der Grundlage von Ex-Situ-Raman-spektroskopischen Untersuchungen diskutiert. / Electrochemiluminescence (ECL) has been generated in solution phase using 9,10-diphenylanthracene (DPA) with TEAClO4 (or TBAClO4) in acetonitrile solvent. Triple potential step was used for the generation of ECL. It was found that anodic and cathodic ECL of equal intensities can be generated by proper choice of potential step magnitude, width and the waiting period (tw) between successive triple potential steps. ECL was then generated in conducting polymers poly(2-ethylhexyloxy-5-methoxy-1,4-phenylenevinylene) (MEH-PPV) and poly(2,3-dibutoxy-1,4-phenylenevinylene) (DB-PPV) by coating them on Pt electrodes and subjecting to potential steps in tetraalkylammonium salt solutions with acetonitrile. Similar to the case of solution phase ECL, symmetrical anodic and cathodic ECL could be observed by the appropriate choice of the potential step parameters. But the kinetics of the ECL was found to be different from that of the solution phase ECL. The time scale of the ECL process was found to be longer than that in the solution phase ECL. The nature of supporting electrolyte had a remarkable impact on the electrochemistry of conducting polymers. The reasons were analyzed by theoretical calculations evoking the concept of charge transport characteristics of conducting polymers. The rate constants of the ECL process were calculated by separate simulation procedure in the solution phase as well as in the polymer phase ECL. To enhance the stability of conducting polymers, synchrotron radiation induced cross-linking was performed. The effects were different from expected which were analyzed and rationalized by ex-situ Raman spectroscopic studies.

[Formulae and special characters can only be approximated here. Please see the pdf version of the Abstract for an accurate reproduction.] Several inert platinum complexes were synthesised: [(en)Pt([special character]-dpzm)2Pt(en)]4+, [{Pt(dien)}2[special character]-dpzm]4+, [{Pt(dien)}2[special character]-H2N-(CH2)6-NH2]4+, cis-[(NH3)2Pt([special character]--dpzm)2Pt(NH3)2]4+, trans-[Pt(NH3)2([special character]-dpzm)2]2+. Three active complexes, all with chloro ligands, were also synthesised: trans-[{Pt(NH3)Cl2}2[special character]-dpzm)], trans-[{Pt(NH3)2Cl}2[special character]-dpzm]2+ (di-Pt) and trans-[trans-{Pt(NH3)2Cl}2{trans-[Pt(NH3)2([special character]-dpzm)2]}]4+ (tri-Pt). 1H NMR established that multi-nuclear platinum complexes will preferentially associate in the DNA minor groove with a preference for A/T sequences, and with a binding constant [special character]-105 M-1, regardless of the charge, linking ligand, length or shape. Using [(en)Pt([special character]-dpzm)2Pt(en)]4+ and the oligonucleotide d(GC)5 it was determined that the metal complex binds G/C rich sequences also in the minor groove, but with a much reduced binding constant, 103 M-1. CD studies showed [(en)Pt([special character]-dpzm)2Pt(en)]4+ was able to induce a DNA conformation change from B-type to what appeared to be a partial Z-type. Transcription assays showed that even though the metal complex does not bind DNA covalently, it is still able to inhibit DNA transcription at particular sites. The complexes di-Pt, tri-Pt, [{Pt(dien)}2[special character]-dpzm]4+ and trans-[Pt(NH3)2([special character]-dpzm)2]2+ were tested for anti-cancer activity in the L1210 murine leukaemia cell line, and gave values of 3.8, 2.5, [special character]200 and 64 [special character]M respectively. In the cisplatin resistant line (L1210/DDP), trans-[Pt(NH3)2([special character]-dpzm)2]2+ showed an increase in activity with a drop to 32 [special character]M, while both di-Pt and tri-Pt showed decreases in activity to values of 8.8 and 3.6 [special character]M. In the human ovarian carcinoma 2008 cell line and its cisplatin resistant derivative C13[special character]5, both complexes showed good activity with values of 2.5 and 20.9 [special character]M respectively, but again both showed decreases in activity in the resistant line with values of 17.8 and 37.7 [special character]M respectively. To help explain the difference between activity of these complexes and the complexes BBR3464 and BBR3005, cell uptake and DNA interstrand cross-linking experiments were performed. The cell uptake studies showed that both di-Pt and tri-Pt are taken up by cells at very high levels, when administered at 100 [special character]M, thus indicating that the difference is unlikely to be due to large differences in cell uptake. The DNA interstrand cross-linking studies showed both complexes readily form interstrand adducts (50% interstrand cross-linking at 12 nM and 22 nM respectively, c.f cisplatin 3 [special character]M). These results suggest that the rigid nature of the dpzm linker may be affecting the DNA adducts formed, with more interstrand links being formed than BBR3464. Possibly, it is this that causes the large differences in cytotoxicity. The DNA binding of di-Pt and tri-Pt was examined with the nucleosides adenosine and guanosine and the dinucleotide d(GpG). Both complexes bound at the N7 of guanosine, but 2-fold slower than cisplatin. In addition, di-Pt bound at the N7 and either the N1 or N3 of adenosine, 7-fold slower than guanosine. Di-Pt forms a large variety of cross-links between two d(GpG) molecules, however it could not be established whether the 1,2-intrastrand adduct could be formed. Di-Pt, however, forms a 1,2-GG interstrand adduct with the oligonucleotide d(ATGCAT)2 resulting in a conformation change away from B-type DNA. The sugar pucker of the G3 nucleoside changes from 2[special character]-endo towards 3[special character]-endo, and the position of the nucleotide relative to the sugar changes from anti to syn. The ability of multi-nuclear platinum complexes to form covalent adducts in the DNA minor groove remains unclear. It appears that di-Pt can form up to 33% minor groove adducts with the oligonucleotide d(AT)5, but when added to the oligonucleotide d(GCCAAATTTCCG)2 no definite minor groove adducts are seen and the major adduct appears to be a 1,2-interstrand cross-link between the two A6's or between the G1 and G11. Finally, a study of the encapsulation of platinum complexes within cucurbit[7]uril (Q7) as a means of reducing drug toxicity was made. For complex A and di-Pt, encapsulation of the linker ligand occurred. The effect of Q7 on the rate of hydrolysis of di-Pt was at least a 3-fold reduction as compared to free di-Pt with guanosine. Studies with [{Pt(dien)}2[special character]-dpzm]4+/Q7 and the oligonucleotide d(CGCGAATTCGCG)2 showed that the metal complex could dissociate from the Q7 and associate with the oligonucleotide, where an equilibrium is achieved with 15 % of the metal complex bound to the oligonucleotide and 75 % encapsulated in Q7. Tests in the L1210 and L1210/DDP cancer cell lines showed that di-Pt/Q7 has almost the same activity compared to free di-Pt.