Untersuchung der DNA-Reparatur in humanen Lymphozyten

Oxidative DNA-Schäden, wie 7,8-Dihydro-8-oxoguanin (8-oxoG), werden kontinuierlich in allen Zellen durch endogene und exogene Noxen gebildet. Ohne eine effektive Reparatur können DNA-Schäden nach erfolgter Replikation als Mutationen fixiert werden und somit die Kanzerogenese initiieren.\r\nUntersuchungsgegenstand dieser Arbeit war die Reparatur, vorrangig von oxidativen DNA-Schäden, in humanen Lymphozyten. Dabei sollte ebenfalls überprüft werden, inwiefern eine Aktivierung dieser Immunzellen, die u.a. zu einer Initiierung der Proliferation führt, modulierend auf die DNA-Reparatur wirkt. Für diese Untersuchungen wurden primäre Lymphozyten aus Buffy Coats isoliert. Eine Aktivierung von T Lymphozyten, welche physiologisch Antigen-vermittelt über den T-Zell-Rezeptor verläuft, wurde durch eine ex vivo Stimulation mit Phytohämagglutinin (PHA) nachgeahmt. Die Induktion oxidativer DNA-Basenmodifikationen erfolgte mit Hilfe des Photosensibilisators Acridinorange in Kombination mit sichtbarem Licht. Das Schadensausmaß sowie die Reparatur wurden mittels der Alkalischen Elution unter Nutzung der Reparaturendonuklease Fpg bestimmt.\r\nDie Ergebnisse zeigten, dass global keine Reparatur induzierter oxidativer DNA-Schäden in primären Lymphozyten stattfindet. Eine Aktivierung der Lymphozyten mittels PHA führte hingegen zu einer deutlichen Reduktion der induzierten DNA-Schäden innerhalb einer 24-stündigen Reparaturzeit. Diese verbesserte Reparatur konnte auf eine Steigerung der Transkription und somit eine erhöhte Proteinmenge von OGG1, welches die Reparatur von 8-oxoG DNA-Glykosylase initiiert, zurückgeführt werden. Weiterführende mechanistische Untersuchungen deuten darauf hin, dass der transkriptionellen Regulation von OGG1 eine Aktivierung der JNK-Signalkaskade zugrunde liegt. Als ein verantwortlicher Transkriptionsfaktor konnte NF-YA identifiziert werden. Dessen erhöhte Bindung am OGG1-Promotor in Folge einer PHA-Stimulation konnte durch eine JNK-Hemmung reduziert werden.\r\nDie Ergebnisse dieser Arbeit zeigen, dass eine Aktivierung von Lymphozyten, welche die Proliferation initiiert und dadurch mit dem Risiko für die Entstehung von Mutationen und malignen Entartungen verknüpft ist, gleichzeitig eine transkriptionelle Hochregulation von OGG1 bewirkt, die die Reparatur oxidativer DNA-Schäden sicherstellt. Die Fähigkeit zur Steigerung der DNA-Reparatur unter den gezeigten Bedingungen bietet den proliferierenden Zellen einen Schutzmechanismus zur Erhaltung ihrer genomischen Stabilität.\r\nOxidative DNA modifications such as 7,8-dihydro-8-oxoguanine (8-oxoG) are generated continuously in all cells by different agents of endogenous and exogenous origin. Without an effective repair, DNA damage can cause mutations and thus initiate carcinogenesis. Aim of this study was to investigate the repair mechanisms of - in particular oxidatively generated - DNA modifications in humane lymphocytes. Moreover, the influence of an activation of these immune cells, which stimulates proliferation, on DNA repair was to be investigated.\r\nFor these studies primary lymphocytes were isolated from buffy coats. To mimic the activation of T-lymphocytes, which is mediated physiologically via antigens through T-cell receptors, an ex vivo stimulation by phytohämagglutinin (PHA) was utilized. Oxidative DNA modifications were induced by the photosensitizer acridine orange in combination with visible light. The extent of damage and repair rates were determined by the alkaline elution technique in combination with the repair endonuclease Fpg, which recognizes oxidized purines in DNA.\r\nResults showed that there is no repair of induced oxidative DNA base modifications in primary lymphocytes on a global level. However, an activation of lymphocytes by PHA leads to a significant reduction of the number of oxidatively generated purine modifications detected after 24 hours of repair time. The observed improvement of repair was shown to result from an increased transcription and therefore an increased amount of OGG1 protein, which is the repair glycosylase initiating the repair of 8-oxoG. Further studies suggested that the transcriptional regulation of OGG1 under conditions of PHA stimulation results from an activation of the JNK signaling cascade as the underlying mechanism. In this context, the involvement of the transcription factor NF-YA could be verified: an increased binding of NF-YA to the OGG1 promoter as a result of PHA stimulation could be reduced by JNK inhibition.\r\n\r\nThe results of this study show that the activation of lymphocytes, which initiates proliferation and therefore is linked to a higher risk for the development of mutations, results in a transcriptional up-regulation of OGG1 and thereby a more efficient repair of oxidative DNA damage. The ability to accelerate DNA repair under certain conditions provides proliferating cells with a protective mechanism to maintain their genomic stability.\r\n148 S

Self-organized lattice of ordered quantum dot molecules

Ordered groups of InAs quantum dots (QDs), lateral QD molecules, are created by self-organized anisotropic strain engineering of a (In,Ga)As/GaAs superlattice (SL) template on GaAs (311)B in molecular-beam epitaxy. During stacking, the SL template self-organizes into a two-dimensionally ordered strain modulated network on a mesoscopic length scale. InAs QDs preferentially grow on top of the nodes of the network due to local strain recognition. The QDs form a lattice of separated groups of closely spaced ordered QDs whose number can be controlled by the GaAs separation layer thickness on top of the SL template. The QD groups exhibit excellent optical properties up to room temperature

Single InGaAs Quantum Dot Coupling to the Plasmon Resonance of a Metal Nanocrystal

We report the observation of coupling of single InGaAs quantum dots with the surface plasmon resonance of a metal nanocrystal, which leads to clear enhancement of the photoluminescence in the spectral region of the surface plasmon resonance of the metal structures. Sharp emission lines, typical for single quantum dot emission, are observed, whereas for reference samples, only weak continuous background emission is visible. The composite metal–semiconductor structure is prepared by molecular beam epitaxy utilizing the principle of strain-driven adatom migration for the positioning of the metal nanocrystals with respect to the quantum dots without use of any additional processing steps

Guided self-assembly of lateral InAs/GaAs quantum-dot molecules for single molecule spectroscopy

We report on the growth and characterization of lateral InAs/GaAs (001) quantum-dot molecules (QDMs) suitable for single QDM optical spectroscopy. The QDMs, forming by depositing InAs on GaAs surfaces with self-assembled nanoholes, are aligned along the [] direction. The relative number of isolated single quantum dots (QDs) is substantially reduced by performing the growth on GaAs surfaces containing stepped mounds. Surface morphology and X-ray measurements suggest that the strain produced by InGaAs-filled nanoholes superimposed to the strain relaxation at the step edges are responsible for the improved QDM properties. QDMs are Ga-richer compared to single QDs, consistent with strain- enhanced intermixing. The high optical quality of single QDMs is probed by micro-photoluminescence spectroscopy in samples with QDM densities lower than 108 cm−2