A new double laser pulse pumping scheme for transient collisionally excited plasma soft X-ray lasers

Within this thesis a new double laser pulse pumping scheme for plasma-based, transient collisionally excited soft x-ray lasers (SXRL) was developed, characterized and utilized for applications. SXRL operations from ~50 up to ~200 electron volt were demonstrated applying this concept. As a central technical tool, a special Mach-Zehnder interferometer in the chirped pulse amplification (CPA) laser front-end was developed for the generation of fully controllable double-pulses to optimally pump SXRLs.rnThis Mach-Zehnder device is fully controllable and enables the creation of two CPA pulses of different pulse duration and variable energy balance with an adjustable time delay. Besides the SXRL pumping, the double-pulse configuration was applied to determine the B-integral in the CPA laser system by amplifying short pulse replica in the system, followed by an analysis in the time domain. The measurement of B-integral values in the 0.1 to 1.5 radian range, only limited by the reachable laser parameters, proved to be a promising tool to characterize nonlinear effects in the CPA laser systems.rnContributing to the issue of SXRL pumping, the double-pulse was configured to optimally produce the gain medium of the SXRL amplification. The focusing geometry of the two collinear pulses under the same grazing incidence angle on the target, significantly improved the generation of the active plasma medium. On one hand the effect was induced by the intrinsically guaranteed exact overlap of the two pulses on the target, and on the other hand by the grazing incidence pre-pulse plasma generation, which allows for a SXRL operation at higher electron densities, enabling higher gain in longer wavelength SXRLs and higher efficiency at shorter wavelength SXRLs. The observation of gain enhancement was confirmed by plasma hydrodynamic simulations.rnThe first introduction of double short-pulse single-beam grazing incidence pumping for SXRL pumping below 20 nanometer at the laser facility PHELIX in Darmstadt (Germany), resulted in a reliable operation of a nickel-like palladium SXRL at 14.7 nanometer with a pump energy threshold strongly reduced to less than 500 millijoule. With the adaptation of the concept, namely double-pulse single-beam grazing incidence pumping (DGRIP) and the transfer of this technology to the laser facility LASERIX in Palaiseau (France), improved efficiency and stability of table-top high-repetition soft x-ray lasers in the wavelength region below 20 nanometer was demonstrated. With a total pump laser energy below 1 joule the target, 2 mircojoule of nickel-like molybdenum soft x-ray laser emission at 18.9 nanometer was obtained at 10 hertz repetition rate, proving the attractiveness for high average power operation. An easy and rapid alignment procedure fulfilled the requirements for a sophisticated installation, and the highly stable output satisfied the need for a reliable strong SXRL source. The qualities of the DGRIP scheme were confirmed in an irradiation operation on user samples with over 50.000 shots corresponding to a deposited energy of ~ 50 millijoule.rnThe generation of double-pulses with high energies up to ~120 joule enabled the transfer to shorter wavelength SXRL operation at the laser facility PHELIX. The application of DGRIP proved to be a simple and efficient method for the generation of soft x-ray lasers below 10 nanometer. Nickel-like samarium soft x-ray lasing at 7.3 nanometer was achieved at a low total pump energy threshold of 36 joule, which confirmed the suitability of the applied pumping scheme. A reliable and stable SXRL operation was demonstrated, due to the single-beam pumping geometry despite the large optical apertures. The soft x-ray lasing of nickel-like samarium was an important milestone for the feasibility of applying the pumping scheme also for higher pumping pulse energies, which are necessary to obtain soft x-ray laser wavelengths in the water window. The reduction of the total pump energy below 40 joule for 7.3 nanometer short wavelength lasing now fulfilled the requirement for the installation at the high-repetition rate operation laser facility LASERIX.rn

Gene expression analysis in ‘Candidatus Phytoplasma mali’-resistant and -susceptible Malus genotypes

Apple proliferation (AP) disease is the most important graft-transmissible and vector-borne disease of apple in Europe. ‘Candidatus Phytoplasma mali’ (Ca. P. mali) is the causal agent of AP. Apple (Malus x domestica) and other Malus species are the only known woody hosts. In European apple orchards, the cultivars are mainly grafted on one rootstock, M. x domestica cv. M9. M9 like all other M. x domestica cultivars is susceptible to ‘Ca. P. mali’. Resistance to AP was found in the wild genotype Malus sieboldii (MS) and in MS-derived hybrids but they were characterised by poor agronomic value. The breeding of a new rootstock carrying the resistant and the agronomic traits was the major aim of a project of which this work is a part. The objective was to shed light into the unknown resistance mechanism. The plant-phytoplasma interaction was studied by analysing differences between the ‘Ca. P. mali’-resistant and -susceptible genotypes related to constitutively expressed genes or to induced genes during infection. The cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) technique was employed in both approaches. Differences related to constitutively expressed genes were identified between two ‘Ca. P. mali’-resistant hybrid genotypes (4551 and H0909) and the ‘Ca. P. mali’-susceptible M9. 232 cDNA-AFLP bands present in the two resistant genotypes but absent in the susceptible one were isolated but several different products associated to each band were found. Therefore, two different macroarray hybridisation experiments were performed with the cDNA-AFLP fragments yielding 40 sequences encoding for genes of unknown function or a wide array of functions including plant defence. In the second approach, individuation and analysis of the induced genes was carried out exploiting an in vitro system in which healthy and ‘Ca. P. mali’-infected micropropagated plants were maintained under controlled conditions. Infection trials using in vitro grafting of ‘Ca. P. mali’ showed that the resistance phenotype could be reproduced in this system. In addition, ex vitro plants were generated as an independent control of the genes differentially expressed in the in vitro plants. The cDNA-AFLP analysis in in vitro plants yielded 63 bands characterised by over-expression in the infected state of both the H0909 and MS genotypes. The major part (37 %) of the associated sequences showed homology with products of unknown function. The other genes were involved in plant defence, energy transport/oxidative stress response, protein metabolism and cellular growth. Real-time qPCR analysis was employed to validate the differential expression of the genes individuated in the cDNA-AFLP analysis. Since no internal controls were available for the study of the gene expression in Malus, an analysis on housekeeping genes was performed. The most stably expressed genes were the elongation factor-1 α (EF1) and the eukaryotic translation initiation factor 4-A (eIF4A). Twelve out of 20 genes investigated through qPCR were significantly differentially expressed in at least one genotype either in in vitro plants or in ex vitro plants. Overall, about 20% of the genes confirmed their cDNA-AFLP expression pattern in M. sieboldii or H0909. On the contrary, 30 % of the genes showed down-regulation or were not differentially expressed. For the remaining 50 % of the genes a contrasting behaviour was observed. The qPCR data could be interpreted as follows: the phytoplasma infection unbalance photosynthetic activity and photorespiration down-regulating genes involved in photosynthesis and in the electron transfer chain. As result, and in contrast to M. x domestica genotypes, an up-regulation of genes of the general response against pathogens was found in MS. These genes involved the pathway of H2O2 and the production of secondary metabolites leading to the hypothesis that a response based on the accumulation of H2O2 in MS would be at the base of its resistance. This resembles a phenomenon known as “recovery” where the spontaneous remission of the symptoms is observed in old susceptible plants but occurring in a stochastic way while the resistance in MS is an inducible but stable feature. As additional product of this work three cDNA-AFLP-derived markers were developed which showed independent distribution among the seedlings of two breeding progenies and were associated to a genomic region characteristic of MS. These markers will contribute to the development of molecular markers for the resistance as well as to map the resistance on the Malus genome.

Direct electron transfer to cytochrome c oxidase investigated by electrochemistry and time-resolved surface-enhanced infrared absorption spectroscopy

Cytochrom c Oxidase (CcO), der Komplex IV der Atmungskette, ist eine der Häm-Kupfer enthaltenden Oxidasen und hat eine wichtige Funktion im Zellmetabolismus. Das Enzym enthält vier prosthetische Gruppen und befindet sich in der inneren Membran von Mitochondrien und in der Zellmembran einiger aerober Bakterien. Die CcO katalysiert den Elektronentransfer (ET) von Cytochrom c zu O2, wobei die eigentliche Reaktion am binuklearen Zentrum (CuB-Häm a3) erfolgt. Bei der Reduktion von O2 zu zwei H2O werden vier Protonen verbraucht. Zudem werden vier Protonen über die Membran transportiert, wodurch eine elektrochemische Potentialdifferenz dieser Ionen zwischen Matrix und Intermembranphase entsteht. Trotz ihrer Wichtigkeit sind Membranproteine wie die CcO noch wenig untersucht, weshalb auch der Mechanismus der Atmungskette noch nicht vollständig aufgeklärt ist. Das Ziel dieser Arbeit ist, einen Beitrag zum Verständnis der Funktion der CcO zu leisten. Hierzu wurde die CcO aus Rhodobacter sphaeroides über einen His-Anker, der am C-Terminus der Untereinheit II angebracht wurde, an eine funktionalisierte Metallelektrode in definierter Orientierung gebunden. Der erste Elektronenakzeptor, das CuA, liegt dabei am nächsten zur Metalloberfläche. Dann wurde eine Doppelschicht aus Lipiden insitu zwischen die gebundenen Proteine eingefügt, was zur sog. proteingebundenen Lipid-Doppelschicht Membran (ptBLM) führt. Dabei musste die optimale Oberflächenkonzentration der gebundenen Proteine herausgefunden werden. Elektrochemische Impedanzspektroskopie(EIS), Oberflächenplasmonenresonanzspektroskopie (SPR) und zyklische Voltammetrie (CV) wurden angewandt um die Aktivität der CcO als Funktion der Packungsdichte zu charakterisieren. Der Hauptteil der Arbeit betrifft die Untersuchung des direkten ET zur CcO unter anaeroben Bedingungen. Die Kombination aus zeitaufgelöster oberflächenverstärkter Infrarot-Absorptionsspektroskopie (tr-SEIRAS) und Elektrochemie hat sich dafür als besonders geeignet erwiesen. In einer ersten Studie wurde der ET mit Hilfe von fast scan CV untersucht, wobei CVs von nicht-aktivierter sowie aktivierter CcO mit verschiedenen Vorschubgeschwindigkeiten gemessen wurden. Die aktivierte Form wurde nach dem katalytischen Umsatz des Proteins in Anwesenheit von O2 erhalten. Ein vier-ET-modell wurde entwickelt um die CVs zu analysieren. Die Methode erlaubt zwischen dem Mechanismus des sequentiellen und des unabhängigen ET zu den vier Zentren CuA, Häm a, Häm a3 und CuB zu unterscheiden. Zudem lassen sich die Standardredoxpotentiale und die kinetischen Koeffizienten des ET bestimmen. In einer zweiten Studie wurde tr-SEIRAS im step scan Modus angewandt. Dafür wurden Rechteckpulse an die CcO angelegt und SEIRAS im ART-Modus verwendet um Spektren bei definierten Zeitscheiben aufzunehmen. Aus diesen Spektren wurden einzelne Banden isoliert, die Veränderungen von Vibrationsmoden der Aminosäuren und Peptidgruppen in Abhängigkeit des Redoxzustands der Zentren zeigen. Aufgrund von Zuordnungen aus der Literatur, die durch potentiometrische Titration der CcO ermittelt wurden, konnten die Banden versuchsweise den Redoxzentren zugeordnet werden. Die Bandenflächen gegen die Zeit aufgetragen geben dann die Redox-Kinetik der Zentren wieder und wurden wiederum mit dem vier-ET-Modell ausgewertet. Die Ergebnisse beider Studien erlauben die Schlussfolgerung, dass der ET zur CcO in einer ptBLM mit größter Wahrscheinlichkeit dem sequentiellen Mechanismus folgt, was dem natürlichen ET von Cytochrom c zur CcO entspricht.