Search

The overarching objective of this thesis was the development of an improved expression cell line for recombinant proteins, in which a transgene of interest can be inserted into a highly active gene locus using recombinase mediated cassette exchange. Random integration of transgenic DNA is a common route to achieve transgene expression. However, randomly integrated transgenes are susceptible to gene silencing over time, and do not show stable expression for extended periods in culture. Furthermore, every new cell clone generated requires regulatory approval. The improvement of expression strategies may significantly reduce the time required to bring novel recombinant protein products to the market. In order to identify a suitable expression locus for the integration of transgene expression cassettes, total protein samples were derived from the production cell lines HEK293 and CHO. Highly expressed proteins were isolated via 2D PAGE, and identified via peptide mass fingerprinting. Their promoter regions were then validated to express a recombinant transgene in HEK293 cells. The long term stability of these promoter regions was also assessed. Direct gene targeting of the highly active gene loci may or may not be possible in typical producer cell lines. Targeting of a murine homologue to these highly expressed CHO/HEK293 loci may be more efficient in a murine stem cell line. The transfer of a modified allele from HM1 murine embryonic stem cells, into a somatic cell line (HC11) was demonstrated in this thesis. These validated methods were then explored for the generation of viable HM1-HEK293 and HM1-CHO fusion hybrids. For these experiments, a fluorescence based fusion assay was generated, validated and used for in-situ monitoring of the cell fusion process. The random integration of transgenic DNA into mammalian genomes typically results in a highly unpredictable integration architecture. RMCE at such loci would be inefficient. However, a highly efficient RMCE reaction at (rare) single copy transgene integrations, may be possible under the correct conditions. RMCE at randomly integrated loci could therefore be more beneficial (for transgene expression) than random integration alone. This thesis explores this concept with the use of a randomly integrated RMCE platform, and subsequent selection of cell lines post RMCE attempts at these loci CRISPR/Cas9 technology was also applied to a highly expressed locus in HEK293 cells. A framework for successful direction of double strand breaks to a defined locus is demonstrated in this work. The methods used to achieve this can therefore be built upon for the homologous recombination of a transgenic cassette, into a highly expressed locus in HEK293 cells. Monoclonal antibodies have dominated the biologics market for over two decades, and mammalian expression systems are well suited to their production. The work in this thesis attempts to raise and verify antibody molecules against a potential tumour marker using hybridoma and phage display technologies.

This thesis examines the contribution made by twelve important and influential individuals to the development of a policy of physical and economic reconstruction, political renewal and personal reconciliation in the British Zone of occupied Germany in the first three years after the end of the Second World War. The selected individuals all possessed power, authority and influence, at different levels of the hierarchy, and collectively represent the view of the ‘governing elite’ of the occupation, including some of its internal differences. They have been categorised in three groups of four: those at the top of the hierarchy, the three Military Governors and one of their senior generals; four senior civilian diplomats and administrators responsible for promoting democracy in Germany; and four young officers with no adult experience but war, who held responsible and influential positions despite their youth. A biographical approach is a novel methodology for studying the British occupation of Germany. It highlights the diversity of aims and personal backgrounds and in so doing can explain some of the apparent contradictions in occupation policy. Personal influences were especially important in a period of transition from war to peace, when official policy guidelines appeared unclear or inappropriate and organisational structures created for the occupation were short-lived and changed rapidly. A wide range of sources has been used including memoirs and autobiographies, official documents, personal papers and oral history interviews. Although sources were created at different times for different purposes, most accounts were found to be remarkably consistent, both internally and with each other. Subjective accounts have been placed in their historical context in order to understand individuals’ perceptions, motivations and personal interests, together with the limitations and constraints on their scope for action.

(1) Respiratory particles of Azotobacter vinelandii grown on nitrogen-free medium contained ubiquinone (Q-8), flavoprotein, cytochromes c4 + c5, b1, a1, a2 and o. Growth with urea as nitrogen source resulted in a loss of cytochromes a2 and o. (2) The aerobic steady-state reductions of cytochromes c4 + c5, b1 and a2 were 39-41%, 18-25% and 0% for the succinate, L-malate or NADH oxidases and 80-100% in the anaerobic state. The anaerobic reduction of cytochrome b1 was 56-69% in the "urea" particles. (3) The concentrations of the electron carriers were unaffected by aeration levels on nitrogen-free medium. With transfer from urea to nitrogen-free medium cytochrome a2 was linearly synthesised with no lag in growth. (4) The redox state and kinetics of ubiquinone were examined by solvent extraction and direct spectrophotometry, with excellent correlation. Ubiquinone was 35-44% reduced in the aerobic steady-state and 72-77% reduced in the anaerobic state, but the ubiquinone reductase rates were only 40-60% of the oxidase rates. The anaerobic reduction was only 65-69% in the "urea" particles. (5) 96% acetone extracts ubiquinone and cytochromes a1 and a2. Low oxidase activities remained in the extracted particles and there was no stimulation with ubiquinone. Assay of extracted and unextracted particles with artificial electron acceptors showed specific stimulation with added ubiquinone. (6) Succinate, L-malate or NADH oxidases were 70-80% inhibited by 60 microg. piericidin A/mg. protein and activity was restored by ubiquinone (Q-3). Cytochromes c4 + c5 and b1 were more reduced in the aerobic steady-states by partial piericidin A inhibition. (7) Electron micrographs showed that the respiratory particles were flat membrane fragments of 35-75 mmicro and studded with spherical knobs of 30-48°A diameter. Some differentiation between the "nitrogen-free" and "urea" particles was noticed. (8) The results were discussed in the light of a branched electron transport system.