Application of CRISPR-Cas9 genome editing systems for improving oilseed rape (Brassica napus) disease resistance against Verticillium longisporum

Modern agriculture requires innovative techniques to advance established strategies in breeding. Traditional approaches have their limitations and are primarily based on the availability of a broad genetic spectrum and the selection of suitable genotypes to improve the desired characteristics. One of these characteristics is the improvement of resistance to biotic stresses or pathogens. In order to achieve this goal in crops with a narrow genetic base such as Brassica napus new strategies are required. An example for this is the disruption of plant factors that are required by the pathogen for a successful colonization of the host (susceptibility factors). Verticillium wilt is caused by the soil-borne and hemi-biotrophic fungus Verticillium longisporum, which is one of the most common fungal diseases in rapeseed cultivation. The disease leads to premature ripening and can cause considerable yield losses. The methods for disease management are very limited. Countermeasures are mainly based on soil hygiene or prevention strategies to reduce the number of spores in the soil. The genetic resources for resistance breeding are limited and no real resistance has been discovered so far. Susceptibility factors in combination with genome modification based on CRISPR/Cas9 can be used as a source of recessive resistance. CRISPR/Cas9 is the most promising system for targeted mutagenesis with the advantage of easy assembly and use as well as multiplexing. The knockout of target genes is possible by the induction of small InDels caused by non-homologous end joining (NHEJ) after a double-strand break in the target site. Another approach possible through this technology is the introduction of a repair template for homology directed repair (HDR), which allows the nucleotide sequence to be altered without disrupting the gene function. In this study we adapt several vector systems containing different nucleases and promoters for application in Brassica napus. After successful establishment of our expression cassettes, the focus relied on the knockout of the candidate genes BnCRT1a and BnHVA22c, which are involved in the V. longisporum -B. napus interaction. Loss-of-function genotypes for the genes BnCRT1a and BnHVA22c were generated and infected with V. longisporum. In addition to this NHEJ approach, miRNA binding sites of fungal miRNAs in the host genome are modified using the HDR approach without affecting the function for the genes BnAGO1 and BnTAO1. For this purpose, the viral genome of the Bean Yellow Dwarf Virus was adapted for use in B. napus hairy roots. In summary, all loss-of-function genotypes showed a greatly reduced expression of symptoms and growth inhibition. These results support our working hypothesis that CRT1a and HVA22c may induce resistance to V. longisporum in their mutated state. Following experiments focused on the function of these genes in plants. Unfortunately, no significant differences in the expression profile of marker genes for molecular and physiological processes between mutants and wild type were found. Only the ethylene marker gene ETR2 showed increased expression in knockout genotypes in the uninfected state, which is consistent with expression data from A. thaliana. The analysis of co-regulated genes, which play a possible role in protein folding / ER stress (for CRT1a) or vesicle transport / callose closure of plasmodesmata (for HVA22c), showed no difference between mutants and the wild type as well. The vector systems for the HDR-based approach were successfully implemented and viral replicons of the Bean Yellow Dwarf Virus could be detected within the cell. The miRNA binding sites in the genes BnAGO1 and BnTAO1, which are used by V. longisporum, were the target of our HDR approach. No positive HDR events could be identified that would be indicated by a sequence exchange. Overall, this work showed that the use of CRISPR/Cas in combination with susceptibility factors is a valuable strategy for generating recessive resistance to pathogens. In the future, this tool can be integrated into existing breeding methods, especially for crops with limited genetic resources

Effects of latent membrane protein 2A expression and constitutive nuclear factor-κB activity on B cell development in mice

Deckblatt-Impressum persönlicher Dank Inhaltsverzeichnis Abkürzungsverzeichnis Einleitung Literatur Material und Methoden Ergebnisse Diskussion Zusammenfassung Summary Literaturverzeichnis Anhang Danksagung Lebenslauf SelbständigkeitserklärungHodgkin Lymphome gehören zu den häufigsten malignen Erkrankungen des Immunsystems. Es konnte gezeigt werden, dass Hodgkin/Reed Sternberg (H/RS) Zellen, die malignen Zellen des Hodgkin Lymphoms, von B-Zellen abstammen. Die molekulare Pathogenese der H/RS Zellen ist bis heute ungeklärt. Wichtige Faktoren, die in der Pathogenese dieser Zellen eine Rolle spielen, sind die permanente Aktivität des Transkriptionsfaktors Nuclear Factor (NF)-kB und das in mehr als 50 % der H/RS Zellen exprimierte Latente Membranprotein 2A (LMP2A) des Epstein-Barr Virus. Ziel dieser Arbeit war es, erstmals ein Mausmodell zu entwickeln, in dem NF-kB permanent aktiv ist und LMP2A-exprimiert wird. Die Auswirkungen der beiden Faktoren auf die B-Zellen der Mäuse sollten untersucht werden. Zur Etablierung des Mausmodells wurden modifizierte fötale hämatopoietische Stammzellen in B-Zell-defiziente Rezipienten transplantiert. Auf diese Weise konnte man Untersuchungen an Mäusen mit definiert veränderten B-Zellen durchführen. Die Spender-Stammzellen stammten aus Föten mit einem knockout des NF-kB-Inhibitors IkB(alpha). Sie wurden vor der Transplantation mit einem LMP2A-kodierenden Retrovirus infiziert. Als Kontrolle dienten mit dem LMP2A-Retrovirus infizierte Stammzellen aus Wildtyp-IkB(alpha)-Föten. Die permanente Aktivität von NF-kB und die Expression von LMP2A konnten bereits fünf Wochen nach der Transplantation in den hämatopoietischen Zellen der Rezipienten nachgewiesen werden. So ermöglichte das Modell erstmals in vivo die Expression von LMP2A und die permanenten Aktivität von NF-kB in Kombination zu untersuchen. Durchflusszytometrische Analysen belegten einen verringerten Anteil an B-Zellen in Zellfraktionen mit LMP2A Expression. Hervorzuheben ist, dass der Anteil an B-Zellen, die B-Zell-Rezeptoren in hoher Dichte exprimierten (IgM high), innerhalb dieser Zellfraktion niedriger war als in Fraktionen ohne LMP2A Expression. Als Ursache hierfür kommt die - im Rahmen dieser Arbeit nachgewiesene - durch LMP2A und permanente NF-kB Aktivität verminderte Expression der B-zellspezifischen Transkriptionsfaktoren EBF und Pax5 in Betracht. Deren Verlust führt in knockout Mausmodellen zur Unterbrechung der B-Zell-Entwicklung im pro-B-Zell-Stadium. Proliferationsversuche bewiesen eine Hemmung der Proliferation der B-Zellen durch LMP2A. Diese Hemmung und die mittels RT-PCR Analysen gezeigte Herunterregulierung von B-Zell-Differenzierungsfaktoren (EBF und Pax5) spiegeln wider, dass die B-Zellen in ihrer normalen Differenzierung und Proliferation gestört sind. Die permanente Aktivität von NF-kB forcierte die Proliferation der B-Zellen nach Stimulation mit B-zellspezifischen Stimulantien. Diese Zellen profitierten also von Überlebensvorteilen, die sie gegenüber Zellen mit physiologischer NF-kB Aktivität hatten. Sowohl NF-kB als auch LMP2A steigerten die Transkription des Rezeptors und Transkriptionsfaktors Notch1. Es ist bereits belegt, dass dieser Faktor in H/RS Zellen aktiv ist und proliferative und anti-apoptotische Effekte aufweist. NF-kB und LMP2A könnten also den Notch1 Signalweg nutzen, um das Überleben der B-Zellen zu sichern. Im Rahmen dieses Mausmodells konnte gezeigt werden, dass die permanente Aktivität des Transkriptionsfaktors NF-kB und die Expression des Epstein-Barr Virus Proteins LMP2A nicht ausreichend sind für die maligne Transformation von B-Zellen in Milz und Knochenmark. Die Untersuchungen konnten allerdings demonstrieren, dass beide Faktoren auf B-Zellen und deren Transformation Auswirkungen haben. NF-kB verstärkt dabei das Tumorpotential der B-Zellen durch forcierte Proliferation. Beide Faktoren verringern die Transkription der B-Zell-Differenzierungsfaktoren EBF und Pax5.Hodgkin lymphoma are among the most frequent malignant lymphoid disorders. The Hodgkin/Reed Sternberg (H/RS) cells, the malignant cells of the lymphoma, are B cell derived. The molecular pathogenesis of these cells is unknown to date. Common features of more than 50 % of H/RS cells are the constitutive activity of the transcription factor Nuclear Factor (NF) -kB und the expression of the Epstein-Barr virus latent membrane protein 2A (LMP2A). Aim of this study was to establish a mouse model with constitutively active NF-kB und LMP2A expression. The effects of these factors on B-cells in mice were investigated.To establish the model modified fetal haematopoietic stem cells were transplanted into B cell deficient recipients. The donor haematopoietic stem cells were extracted from foetuses with a knockout of the NF-kB-inhibitor IkB(alpha). Before transplantation they were transfected with a retrovirus encoding for LMP2A. Haematopoietic stem cells that were infected with the LMP2A retrovirus from wildtype IkB(alpha) foetuses served as controls. Mice with defined genetic changes in their B cell population were analysed. Five weeks after transplantation the recipients showed constitutive NF-kB activity und LMP2A expression in haematopoietic cells. This model enabled us to analyse effects of permanent NF-kB activity und expression of LMP2A in combination in vivo. Flowcytometric analysis showed lower fractions of B cells when LMP2A was expressed. Additionally the fraction of high IgM expressing B cells decreased. Transcriptional downregulations of B cell specific differentiation factors EBF und Pax5 could be the reasons for this. In EBF und Pax5 knockout mice B cell development is blocked in the pro-B cell stage. Additionally, proliferation assays demonstrated decreased proliferative responses of LMP2A expressing B-cells to B cell specific stimuli. The inhibition of proliferation und the down regulation of the transcription of B-cell specific transcription factors (EBF und Pax5, shown in RT-PCR analysis) revealed deregulated differentiation und proliferation of the B-cells. Permanent NF-kB activity increased B-cell proliferation after stimulation. That means it lead to advantages in survival compared to cells with physiological NF-kB activity. NF-kB as well as LMP2A increased transcription of the receptor und transcription factor Notch1. It was already shown that Notch1 is active in H/RS cells und provides proliferative und antiapoptotic effects. NF-kB und LMP2A might use the Notch1 signalling pathway to protect B-cells from cell death. This mouse model enabled us to show that permanent NF-kB activity und LMP2A expression are not sufficient to turn B-cells malignant like H/RS cells. Nevertheless, analysis demonstrated strong effects on B-cells und their transformation. NF-kB increases the tumor potentials of B-cells by increasing proliferation. Both factors decrease transcription of the transcription factors EBF und Pax5

Investigation of the Electrohydraulic Forming Process with respect to the Design of Sharp Edged Contours

The overcoming of design constraints with respect to forming of sharply contoured sheet metal workpieces made of high strength steel or other materials which are difficult to form is an important aspect in sheet metal part production. One interesting solution to extend existing forming limits can be the use of electrohydraulic forming as single forming operation or in combination with quasi-static hydroforming. Apart from promising results regarding the feasible part geometries this process allows a quite efficient production due to its potential to reduce equipment expenses. Current research work at the Chair of Forming and Machining Technology (LUF) at Paderborn University deals with a comparison of investigations on both processes, quasistatic and high speed hydroforming. Recent results show an adequate comparison of achievable edge radii using an oblong die geometry and sheet metal made of thin stainless steel. It can be seen that when using electrohydraulic forming an increase of discharge energy leads to smaller radii than achievable by quasi-static hydroforming. An additional potential can be seen in the process characteristic itself because the very short pressure pulse allows a significant reduction of locking forces using only the inertia of the tooling mass

Annäherung an Blumenbergs Philosophieverständnis

Abstract If one compares Hans Blumenberg with the dominant contemporary German-speaking characters of philosophy and heads of their own schools, Husserl, Heidegger and Adorno, then one sees that Blumenberg’s understanding of philosophy proves tobe emphatically unemphatic, withdrawn, and deeply stacked. He exchanges the big bills of those philosophies for small coins: Philosophy is attention first, thoughtfulness second, consolation third, and memory fourth. – An introduction is evidence of the reorientation that Blumenberg undertook in the 1950s with regard to his Catholic-theological and Heideggerian philosophical beginnings. This led him to redefine the modern age from one of crisis to one of overcoming the crisis

An aeroacoustic investigation into the effect of self-oscillating trailing edge flaplets

The aeroacoustics of a NACA 0012 aerofoil with an array of self-oscillating flexible flaplets attached on the trailing edge has been investigated at low to moderate chord based Reynolds number (50,000 -- 350,000) and at geometric angles of attack from $\alpha_g = 0^\circ$ -- $20^\circ$. When the aerofoil is untripped, tonal peaks are observed on the baseline aerofoil. When the passive flaplets are attached to the pressure side of the aerofoil, the tonal peak is removed. If the flaplets are then placed on the suction side, the tonal peak is reduced, but not removed. It is therefore hypothesised that the flaplets on the pressure side modifies the laminar separation bubble situated on the pressure side of the aerofoil, a key mechanism for tonal noise. Throughout all cases, both tripped and untripped, a low frequency (0.1 kHz -- 0.6 kHz) noise reduction and a slight increase at higher frequencies (>2 kHz) is seen. This gives an average overall sound pressure level (OSPL) reduction of 1.5 -- 2 dB for the flaplets affixed to the pressure side. The cases where the tonal noise component is removed an OSPL reduction of up to 20 dB can be seen

The structure of the QED-Vacuum and Electron-Positron Pair Production in Super-Intense, pulsed Laser Fields

We discuss electron-positron pair-production by super-intense, short laser pulses off the physical vacuum state locally deformed by (stripped) nuclei with large nuclear charges. Consequences of non-perturbative vacuum polarisation resulting from such a deformation are shortly broached. Production probabilities per pulse are calculated.Comment: 10 pages, 1 figure, submitted to Journal of Physics

„Ich bin durch den Dienst Teil meiner Gemeinde“: Ergebnisse einer explorativen Studie zu Selbstverständnis, Motivation, und Religiosität von Ministrant*innen heute

Über 350.000 Kinder und Jugendliche sind in den deutschen Diözesen als Ministrant*innen tätig. Sie „dienen“ regelmäßig in der Messfeier und sind auch sonst eine wichtige Gruppe kirchlicher Jugendpastoral. Die Zahl der wissenschaftlichen Studien über diese mit der Gemeinde verbundenen Kinder und Jugendlichen ist überschaubar. Eine studentische Forschungsinitiative aus Münster hat sich zum Ziel gesetzt, mehr Erkenntnisse über diese wichtige Gruppe junger Menschen zu gewinnen, um einen Beitrag zu einer religionspädagogisch reflektierten Bildungsarbeit für Ministrant*innen zu leisten. Die Ergebnisse einer ersten explorativen Studie sind ermutigend. Over 350,000 children and youngsters are involved in German dioceses as ministrants. They regularly “serve” in Mass and are an important group of church youth pastoral care. There are very few scientific studies on this large number of children and adolescents. A student research initiative from Münster focused on this important group of young people in order to gain knowledge and to reflect on educational work of ministrants. The results of an initial exploratory study are encouraging