Summaries in German and English. / Gegenstand der vorliegenden Forschungsarbeit ist die Dokumantarische Evaluation der missional ausgerichteten Gemeindeberatung des Gemeindeberatungsinstituts Trafo. Dabei soll die transformatorische Wirkung der Kombination von Gemeindeberatung und dem aus der Gemeinwesenarbeit adaptierten und für Gemeindearbeit modifizierten Transformationszyklus auf der Basis von dokumentarischer Sozialforschung und dem Empirisch Theologischen Praxiszyklus intradisziplinär erforscht werden. Die Dokumentarische, qualitative Forschungsmethode generiert ihre Ergebnisse nicht in den Aussagen des Common Sense, sondern durch das atheoretische konjunktive Wissen der Beteiligten. Zugang zu diesem impliziten Wissen verschafft sich die Forschung durch Rekonstruktion kollektiver Handlungslogiken, die auf der Ebene des „modus operandi“ angesiedelt sind. Diese Herangehensweise ermöglicht es der Forschung unbewusste Alltagserfahrungen, wie in diesem Fall die Teilnahme an einem Trafo-Gemeindeberatungsprozess und damit einhergehenden transformatorischen Veränderungen prozesshaft zu reproduzieren und die unbewussten Handlungsmuster für die Auswertung fruchtbar werden zu lassen. Die aus dieser Vorgehensweise gewonnenen Ergebnisse speisen die Erkenntnisfunktionen von Evaluationen, die in Erkenntnis-, Kontroll-, Dialog- und Lernfunktion (Stockmann 2007:25) zu unterteilen sind, und die vor allem zur Verbesserung der Beratungsqualität dient und damit gleichzeitig eine Auswirkung auf die Gemeindetransformation an sich hat. / Subject-matter of the following MTh dissertation is a documentary evaluation of missional church-counsel in the setting of Trafo-Church-Counsel Institute. Aim of this research is to evaluate the impact of transformation within the Trafo-Church-Counsel Program which is conducted of a combination of church-counsel and a transformation cycle of community organizing adapted for churches. Based on documentary social science and the empirical-theological praxis cycle this study will be performed in an intradisciplinary manner. Documentary research doesn’t generate its results based on common sense but on collective tacid knowledge of the participants. Further access will be gained through reconstructing collective implicit logic of action. This specific methodical approach provides insights on missional transformation in general and in the specific case of Trafo-Church-Counsel Institute. The results of this study will provide on one hand answers for the quest of evaluations which include learn functions as: awareness, control, increased insights for dialogue with stakeholders and learn function for quality improvement (Stockmann 2007:25) and will enhance on the other hand church counsel and church transformation. / Christian Spirituality, Church History and Missiology / M. Th. (Missiology)

Knowledge on the human genome and specific sequences associated with human diseases is continuously growing. The ability to connect human genetics to cellular mechanisms and physiology raises the need for medicine to get to gene specific therapeutics. In order to achieve gene-specific modification, tools are required to enable sequence-specific DNA cleavage. Not long ago, the RNA-guided endonuclease Cas9 was shown to effectively facilitate gene editing in humans. Cas9 endonuclease, which is naturally part of an adaptable bacterial immune system, can be easily adjusted to recognize and cleave specific DNA sequences in a 20 nt RNA-DNA complementary manner. The easy adjustability and high efficiency of Cas9 gave rise to hopes that this genome engineering tool could pave the way to ‘gene surgery’ in humans. However, to achieve DNA cleavage, the endonuclease and its guiding RNA need to be sufficiently accessible in the nucleus of target cells. Viruses, which evolution has made well adapted to transfer their own genetic information into cells can be exploited for transfer of foreign genetic material. Replication deficient retroviruses therefore represent interesting vehicles for gene delivery. Retroviruses preferentially incorporate their own genetic information in the form of RNA into viral particles. Typically, viral RNA of retroviruses is reverse transcribed into DNA during viral infection and integrated into host cell chromosomes. In this respect, integration-competent or integration-deficient lentiviral (HIV-derived) vectors (ICLV/IDLV) were reported to be efficient ‘gene shuttles’ for Cas9 delivery. In contrast, up to now Foamy viruses (FV), which represent a distinct subfamily in the family of retroviruses have not previously been tested for their efficiency to transduce CRISPR/Cas9 components. FV show several unique characteristics some of which make them interesting candidates for gene therapy, such as high transduction efficiency on a wide variety of human cell lines or a special capability to efficiently transfer and provide non-viral RNA in target cells. In this thesis the unique characteristic of FVs, which allow for the efficient transduction of non-viral RNAs, was exploited for transient FV mediated (TraFo) Cas9 expression. It is shown in this thesis that gene knock-out (KO) achieved with TraFo Cas9 particles appears to have several advantages over ICLV or IDLV mediated Cas9 transduction. In this work, it could be demonstrated that a single application of TraFo Cas9 supernatant results in high efficiency of GFP KO in osteosarcoma cells (U2OS). The efficiency of gene KO with TraFo Cas9 particles exceeded gene KO frequencies achieved with similar volumes of ICLV or IDLV supernatant for Cas9 transduction. In addition, transient Cas9 delivery achieved with TraFo particle supernatant resulted in remarkably reduced Cas9 off-target cleavage compared to corresponding infections with ICLV or IDLV particles. The results show, that TraFo Cas9 represents an interesting addition to the currently utilized methods for transient Cas9 delivery. One particular feature of TraFo particle transduction is especially noteworthy – TraFo mediated transduction does not depend on any particular adjustment on the encapsidated non-viral RNA sequence (such RNA only needs to be present in sufficient amounts during virus assembly) nor does it depend on any modification of viral proteins. The easy adaptability of TraFo mediated non-viral RNA transfer is an especially remarkable feature, since science continues to both developing new variants of Cas9 and continues to find new and interesting members of the pool of CRISPR enzymes. In this regard TraFo particles represent interesting vehicles to transiently provide mRNA transcripts of such new protein candidates in cells. The ability of TraFo particles to provide the RNA sequence needed to guide Cas9 (termed sgRNA) to its target DNA sequence in cells was additionally investigated. It was assumed that typically engaged RNA polymerase (RNAP) III transcription of sgRNAs hampers transduction with TraFo particles, since RNAP III-derived transcripts are not actively exported into the cytoplasm and show low stability. An additional CRISPR enzyme Csy4 was used, which is able to specifically cleave RNA. This enabled TraFo mediated transfer of RNAP II transcripts (with active nuclear export and higher stability than RNAP III transcripts) with embedded sgRNA sequences. It was demonstrated that a simultaneous infection of cells with TraFo particles providing bicistronic transcripts of Cas9 and Csy4 on the one side and RNAP II-derived transcripts with embedded sgRNA sequences on the other, enabled reasonable GFP gene inactivation in U2OS cells. Gene KO with RNAP II transcripts as a result significantly exceeds TraFo transduction of RNAP III-derived sgRNA. Interestingly, with regard to gene KO, it was found that de novo transcription of sgRNAs from viral DNA (by integration-competent or integration-deficient retroviral vector [ICRV/IDRV] transduction) when combined with TraFo Cas9 transduction was superior to a TraFo transduction of sgRNA transcripts. IDRV mediated transduction was optimized in order to minimize the risk of unfavorable genome modification of cells by viral DNA integration. By adding the coding sequence of a fluorescent marker to the viral vector, it was demonstrated that a smaller number of viral particles helps to significantly lower the frequency of viral DNA integration. In addition, the expression of a fluorescent marker opened up the opportunity to further reduce the cell fraction with continuous marker gene expression by flow cytometric cell sorting. The IDRV/ICRV sgRNA and TraFo Cas9 delivery system was then challenged for use on immortalized and primary T cells. Primary T cells represent interesting targets for genetic engineering since modified T cells can be utilized as ‘living drugs’ (by expression of chimeric antigen receptors – CARs) against cancer cells. Efficient gene inactivation was observed on the immortalized T cell line – Jurkat. Transduction of primary T cells pointed to certain restrictions of the split two-virus delivery system for sgRNA and Cas9 transduction. However, despite certain limitations, it was possible to demonstrate that this FV-derived Cas9 delivery system is also feasible on primary tissue, and further optimization could make it an interesting alternative delivery method for CAR therapy. The ability of IDRV vector genomes to provide repair template donor DNA to induce homologous recombination (HR) was additionally investigated. DNA double-strand breaks in eukaryotic cells are typically repaired by the error prone non-homologous end joining pathway (often leading to frame-shift mutations by small insertions or deletions) or HR. Delivery of a homologous DNA sequence during DNA cleavage enables site-specific integration of exogenous DNA sequences. The work of this thesis showed that IDRV vector genomes providing repair template donor DNA allow for HR in a homology length dependent manner. Besides the length of homology, it was also observed, that the length of sequence which should be integrated (KI) remarkably influences the frequency of HR. HR is therefore engaged significantly more frequently if single nucleotides, rather than a whole gene, are provided as sequences within a repair template. In addition, viral vectors were augmented with additional fluorescent marker sequences. It could subsequently be demonstrated that the majority of cells showed accurate sequence-specific DNA integration. Furthermore, several indications were found, which lead to the assumption that the ratio of KI to homologous sequence markedly influences the accuracy of HR. Using the previously obtained knowledge it was further possible to tag an essential human protein by FV vector mediated transient Cas9 and repair template transduction. It was found that the large packaging capacity of FV vectors can be exploited to enable selection and flow cytometric sorting of cells with correct site-specific DNA integration. In summary, the results of this thesis demonstrate for the first time that FV mediated non-viral mRNA Cas9 transduction in combination with retroviral delivery of sgRNA (and repair template sequence) are a promising basis for several different interesting applications with relevance for not only basic research, but also for gene therapy.:1. Introduction 1 1.1 Gene therapy 1 1.2 Viral vectors for gene therapy 2 1.3 History of retroviral research 2 1.4 Taxonomy of Retroviruses 3 1.5 Foamy Viruses 4 1.5.1 Morphology of Foamy Virus 6 1.5.2 Foamy Virus replication 7 1.5.3 Foamy virus proteins, as part of a viral vector system 10 1.6 Genetic engineering 14 1.6.1 ‘DNA scissors’ – Zinc-finger and Transcription-activator like effector nucleases 15 1.6.2 History of CRISPR/Cas9 as a tool for genetic engineering 16 1.7 CRISPR/Cas immunity in prokaryotes 18 1.8 CRISPR/Cas9 functioning 21 1.9 Double-strand break repair in eukaryotic cells 21 1.9.1 Classical NHEJ 23 1.9.2 Homologous recombination 24 1.9.3 DSB repair in vertebrates 26 1.10 DSBs in context of CRISPR/Cas9 cleavage 27 1.11 Thesis Aim: CRISPR/Cas9 transduction with FV particles 28 2. Materials and Methods 30 2.1 Materials 30 2.1.1 Chemicals 30 2.1.2 Buffers and Solutions 30 2.1.3 Bacterial Growth Media 33 2.1.4 Cell Culture Media 34 2.1.5 Antibodies 34 2.1.6 Enzymes 35 2.1.7 Commercial Kits and additional reagents 36 2.1.8 Size Markers 36 2.1.9 Antibiotics 36 2.1.10 Bacterial strains 37 2.1.11 Cell lines 37 2.1.12 Devices and Software 37 2.1.13 Oligonucleotides 38 2.1.14 Plasmids 46 2.1.15 sgRNA sequences 56 2.1.16 Consumable material 57 2.2 Molecular Biology Methods 58 2.2.1 Restriction of DNA 58 2.2.2 Polymerase chain reaction 59 2.2.3 Gibson assembly 60 2.2.4 Agarose gel electrophoresis 60 2.2.5 Ligation 61 2.2.6 Cultivation of bacteria 62 2.2.7 Transformation 62 2.2.8 Plasmid Preparation 63 2.2.9 Sequencing 65 2.3 Cell culture methods 66 2.3.1 Passaging of cells 66 2.3.2 Cell counting 66 2.3.3 Freezing and thawing of cells 66 2.3.4 Seeding and fixation of cells for microscopy 67 2.4 Virological Methods 67 2.4.1 Polyethyleneimine transfection 67 2.4.2 Integration-competent, integration-deficient and ‘transient’ retroviral vectors 68 2.4.3 Infection of adherent cells 70 2.4.4 Infection of suspension cells 71 2.4.5 Flow cytometry 72 2.4.6 Multiplicity of infection (MOI) 72 2.4.7 Particle preparation 73 2.5 Nucleic acid composition in viral particles and culture cells 73 2.5.1 Isolation of total RNA from viral particles 73 2.5.2 RNA isolation from culture cells 73 2.5.3 Reverse transcription of viral or cellular RNA 73 2.5.4 DNA isolation from culture cells 74 2.5.5 Quantitative PCR (qPCR) analysis 74 2.5.6 T7 endonuclease assay 75 2.6 Protein biochemistry methods 76 2.6.1 Cell lysates 76 2.6.2 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 76 2.6.3 Semi-dry Western Blot 77 2.6.4 Immunodetection 78 2.6.5 Stripping of Western blot membranes 78 2.6.6 Immunostaining of cells for FACS analysis 78 2.7 Microscopy methods 79 2.7.1 Fluorescence microscopy 79 2.7.2 Confocal Laser scanning Microscopy (CLSM) 79 2.7.3 Live-cell imaging 79 3. Results 80 3.1 Transient foamy virus transduction of non-viral mRNA transcripts 80 3.2 Transient foamy virus transduction of Cas9-encoding mRNA transcripts 81 3.3 Cas9-encoding nucleic acids and their ‘effects’ in cells after retroviral transduction 84 3.4 Off-target analysis after TraFo Cas9 delivery 87 3.5 Transient fomy virus transduction of Cas9 and sgRNAs 89 3.6 Retroviral vectors providing sgRNAs and a fluorescent protein 92 3.6.1 Gene knock-out with retroviral vectors under saturated conditions 92 3.6.2 MOI adjusted ID sgRNA vector supernatants for comparison of residual vector integration 94 3.6.3 Gene knock-out in murine embryonic fibroblasts 95 3.7 Influence of Cas9 expression on IDRV vector genome integration 96 3.8 TraFo Cas9 mediated T cell receptor knock-out in immortalized and primary human T cells 97 3.9 Homology-directed repair after FV CRISPR/Cas9 mediated double-strand breaks 99 3.9.1 Length of homologous donor DNA and its influence on HDR 100 3.9.2 Effect of freezing viral supernatants on the frequency of HDR 102 3.9.3 Effect of donor DNA mismatches on the frequency of HDR 104 3.10 Investigation on donor DNA integration with additional fluorescent markers 105 3.11 Lentiviral and foamyviral transduction of HDR donor DNA 107 3.12 HDR mediated single nucleotide substitutions after TraFo CRISPR/Cas9 delivery 109 3.13 Tagging of an endogenous protein after TraFo CRISPR/Cas9 delivery 111 3.13.1 Specific CRISPR/Cas9 mediated cleavage of endogenous hPLK1 gene 111 3.13.2 Homology-directed repair of the hPLK1 gene for endogenous gene tagging 113 3.13.3 Confocal fluorescence microscopy analysis of GFP-Plk1 HeLa cell populations 118 4. Discussion 120 4.1 Genetic engineering – potential and risks 120 Chapter I Transient FV vectors – mRNA delivery vehicles for CRISPR/Cas9 mediated gene editing 122 4.2 Non-viral Cas9-encoding mRNA transfer in foamy virus particles 122 4.2.1 Fate of Cas9-encoding nucleic acids in cells after TraFo Cas9 transduction 124 4.2.2 Potential adjustments to further improve TraFo Cas9 transduction 125 4.2.3 Lentiviral in contrast to TraFo transduction of Cas9-encoding nucleic acids 126 4.3 Efficiency of Cas9-mediated gene knock-out with different retroviral vectors 127 4.4 Type of retroviral Cas transduction and its influence on the specificity of Cas9 cleavage 127 4.5 Alternative approaches to deliver Cas9-encoding mRNA in human cells 129 4.6 Transient sgRNA transduction with TraFo particles 131 Chapter II Delivery of foreign DNA with FV-derived vectors – enabling gene knock-out and homology-directed repair 133 4.7 Gene inactivation by TraFo Cas9 transduction and sgRNA expression from retroviral vector genomes 133 4.7.1 Gene editing in immortalized and primary T cells 135 4.8 Homology-directed repair with IDRV genomes 137 4.8.1 The influence of the length of sequence homology on HR 138 4.8.2 The influence of freezing viral supernatants on HR 139 4.8.3 Widening the applicability of TraFo vector particles for improved HR 140 4.8.4 The influence of mismatching nucleotides on HR 140 4.8.5 Visualization of inaccurate HR or additional dsDNA integration 141 4.8.6 The influence of the ratio of knock-in and homologous sequence on the accuracy of HR 142 4.8.7 Alternatives to double-stranded donor DNA 143 4.9 Endogenous gene tagging with IDPV donor DNA transduction 145 4.9.1 Alternative approaches for endogenous protein tagging 146 5. Conclusion 148 6. Summary 150 6.1 Summary 150 6.2 Zusammenfassung 153 7. Supplementary 157 8. References 159 9. Appendices 182 9.1 Indices 182 9.1.1 Abbreviations 182 9.1.2 Index of Figures 185 9.1.3 Index of Tables 187 9.2 Curriculum Vitae 188 9.3 Publication Record 189 9.4 Congress Contributions 189 9.5 Patent Applications 189 10. Statement of Authorship 190

The dissertation thesis deals with the analysis of prospective environmentally compatible electrical insulating fluids for electrical engineering in relation to their chemical structure. The thesis starts with the overview of the current state of the art and of the latest trends in the use of synthetic and biodegradable natural oils. In the experimental part were studied these oils: mineral oils, rapeseed oil, sunflower oils, soybean oil, methyl oleate, peanut oil, MCT oil, castor oil and other. Dielectric properties were measured using LRC meter Agilent 4980A including dielectric liquid test fixture Agilent 16452A and also by the Novocontrol Alpha-A analyzer. Electrical properties are presented in the frequency range 10 mHz – 1 MHz range in the temperature interval 253 K to 363 K. The work goes on with the study of the suitability of individual oils for lower temperature, including the impact of the chemical structure and formulation on electrical properties.

This Master’s thesis is dealing with theoretical analysis of vegetable, mineral oils and synthetic fluids, and with measuring of density and viscosity of selected samples of electric insulating fluids. The main part of the thesis is focused on vegetable oils and their elements called fatty acids. Those are more detail described in separate capitols. In those capitols is described their utilization in engineering practice with focus on energetics, where vegetable oils are used in larger scale. In experimental part of the work is measurement of density and dynamic viscosity. Viscosity was measured on two different machines, first the Hoppler viscometer and on vibrating viscometer. Results of these measurement are evaluated in tables and graphs.