The objective of this study was to optimize tissue culture, transformation and regeneration parameters of mature embryo based culture of Triticum durum cv. Mirzabey 2000 and Triticum aestivum cv. Y&uuml / regir 89. The effects of auxin type of hormone at different concentrations and dark incubation periods on regeneration capacity were evaluated. Two different hormone types 2,4- dichlorophenoxyacetic acid and picloram were used at three different concentrations 2, 4 and 8 mg/l. Mature embryo derived calli were incubated in 6 different induction media at dark for 4 and 6 weeks for initiation of primary callus induction. After dark incubation periods, average callus fresh weight and primary callus induction rate were determined. The primary callus induction rates for 4 weeks and 6 weeks old dark adapted Mirzabey calli incubated was found to be 91 % and 93.25 % respectively. Y&uuml / regir primary callus induction rate was 92.5 % for 6 weeks old calli in 6W2D medium and 86.75 % for 4 weeks old calli in 4W8P medium. The primary calli were transferred to embryogenic callus induction medium. The embryogenic callus formation was 94.88 in 6W2D medium for Mirzabey cultivar. The necrosis was observed at high concentration of 2,4-D for both of cultivars. After embryogenic callus induction, embryogenic calli were transferred into hormone free regeneration medium. The maximum regeneration rate (62.31 %) and culture efficiency (44.13 %) were observed in 4W2D medium for Mirzabey. However, the low regeneration rate was observed for Y&uuml / regir (5 %) in 6W2D medium. The transformation studies were performed by using Obitek Biolab Gene Transfer System. The old and the modified loading units were used for optimization of bombardment pressure and distance for mature embryo based calli transformation. After bombardment of pAHC25 coated gold particles, histochemical GUS assay was performed and blue spots were counted. The transformation efficiency increased to 0.65 fold for 30 bar bombardment pressure and 5.5 fold for 35 bar bombardment by the modified loading unit. The modified loading unit could be used for further transformation studies.

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Schistosomen verursachen die Tropenparasitose Schistosomiasis mit 250-300 Millionen infizierten Menschen weltweit. Zur Analyse von Genfunktionen, durch die wirksame Medikamente entwickelt werden könnten, wird ein System zur Herstellung transgener Schistosomen benötigt. In dieser Arbeit wurde daher versucht, ein System zur Transfektion von Schistosoma mansoni von der transienten Expression von Reportergenen ausgehend zu entwickeln. Die Funktionalität der hergestellten Plasmide zur transienten Transfektion wurde durch die Expression der Reporter Enhanced Green Fluorescent Protein (EGFP) und beta-Galatosidase unter der Kontrolle der schistosomalen Promotoren für das Calreticulin, die 28 kDa-Glutathion-S-Transferase und das 70 kDa-Hitzeschockprotein in cos7-Zellen nachgewiesen. Für den Gentransfer in S. mansoni wurden Mikroinjektion, Elektroporation und Mikroprojektilbeschuss eingesetzt. Der Mikroprojektilbeschuss erwies sich als geeignete Methode, Plasmid-DNA zur transienten Expression von EGFP und beta-Galaktosidase in adulte und larvale Stadien zu transferieren. Da die beobachtete Reportergenexpression schwach war, wurde durch den Einsatz von Discosoma Red Fluorescent Protein (DsRed) als Reportergen versucht, das Transfektionsssytem zu optimieren. Außerdem wurden die potentiellen Promotoren für das Cathepsin D und für ein Aktin durch inverse PCR aus genomischer DNA isoliert. Die Funktionalität dieser Promotoren wurde in cos7- bzw. HeLa-Zellen nachgewiesen, eine verbesserte Expression in S. mansoni dagegen wurde mit diesen zwei Promotoren und unter Einsatz von DsRed nicht erreicht. Da S. mansoni sich in vitro nicht kultivieren lässt, muss für die Etablierung eines Transfektionssystems ein in seinen Keimzellen transgenes Stadium in den Lebenszyklus eingeschleust werden. Durch Mikroprojektilbeschuss transfizierte Mirazidien (freilebende Stadien des Parasiten) infizierten Zwischenwirtsschnecken und entwickelten sich zu transgenen Sporozysten, was durch die Transkription von EGFP nachgewiesen wurde. Für eine stabile Integration in das Genom werden unter anderem mobile genetische Elemente eingesetzt. Boudicca ist ein endogenes long terminal repeat (LTR) Retroelement. In dieser Arbeit wurde gezeigt, dass es in adulten Würmern, Sporozysten und Zerkarien transkribiert wird. Das LTR kann in vitro als Promotor zur Expression von EGFP eingesetzt werden. Außerdem wurde eine transkribierte Kopie kloniert und analysiert. Die in dieser Arbeit gewonnen Ergebnisse weisen darauf hin, dass Boudicca als Vektor zur stabilen Transfektion von S. mansoni eingesetzt werden kann. / Schistosomiasis caused by schistosomes affects about 250-300 million people world wide. The establishment of a transgenesis system for schistosomes is a pre-requisite to the identification of new drug targets and to the analysis of gene regulation and will add to our knowledge of parasite physiology and development. In this study, plasmids expressing Enhanced Green Fluorescent Protein (EGFP) and beta-galactosidase driven by the schistosomal promoters of the calreticulin, the 28kDa-glutathione-S-transferase and the 70kDa-heatshock protein were cloned and successfully tested in cos7-cells. Microinjection, electroporation and particle bombardment were used to introduce plasmid DNA into Schistosoma mansoni. Adult and larval stages of S. mansoni subjected to particle bombardment transiently expressed the reporter genes, although the observed transfection rates were very low. To optimize the transfection system Discosoma Red Fluorescent Protein (DsRed) was used as a reporter gene. Furthermore, the schistosomal promoters of the aspartic protease cathepsin D and a cytoplasmatic actin gene were isolated from genomic DNA using inverse PCR. The isolated promoters were able to drive EGFP and DsRed expression in cos7- or HeLa-cells, but improved expression could not be observed in S. mansoni. Due to the complexity of the life cycle S. mansoni cannot be cultured in vitro. In order to develop a transgenesis system for schistosomes, the life cycle must consequently be completed by genetically modified parasites. In this study it was shown that the free-living and mobile miracidium that infects the intermediate host snail could be transformed by particle bombardment and reintroduced in the life cycle using the natural path of infection. Development into transgenic sporocysts was shown through the isolation of EGFP transcripts from intermediate host snails. Mobile genetic elements are used as molecular tools to achieve stable integration of a foreign gene into a genome. Boudicca is an endogeneous long-terminal repeat (LTR) transposon in the schistosomal genome. In this study it was shown that it is transcribed in adult worms, sporocysts and cercariae. The LTR drives EGFP expression in cell cultures. Furthermore, a Boudicca transcript was cloned and analyzed by sequencing. The results suggest that Boudicca can be used as a molecular tool for stable integration of transgenes into the schistosomal genome.

The nuclear pore complex (NPC) is perhaps the largest protein complex in the eukaryotic cell, and controls the movement of molecules across the nuclear envelope. The NPC is composed of up to 30 proteins termed nucleoporins (Nups), each grouped in different sub-complexes. The transmembrane ring sub-complex is composed of Nups responsible for anchoring the NPC to the nuclear envelope. Bioinformatic analysis has traced all major sub-complexes of the NPC back to the last eukaryotic common ancestor, meaning that the nuclear pore structure and function is conserved amongst all eukaryotes. In this study Arabidopsis T-DNA knockout lines for these genes were investigated to characterise gene function. Differences in plant growth and development were observed for the ndc1 knockout line compared to wild-type but gp210 plants showed no phenotypic differences. The double knockout line gp210 ndc1 was generated through crosses to observe plant response to the knockout of two anchoring-Nup genes. No synergistic affect from this double knockout was observed, suggesting that more, as yet unidentified Nups function the transmembrane ring in plants. The sensitivity to nuclear export inhibitor leptomycin B (LMB) was tested also for knockout lines, although growth sensitivity to the drug was not observed. Nucleocytoplasmic transport of knockout lines was measured in cells transformed by particle bombardment. To express fluorescent protein constructs actively transported through the NPC, localisation of protein determined the nucleocytoplasmic transport of the cell. The ndc1single knockout and the double knockout gp210 ndc1 exhibited decreased nuclear export. Further experiments in determining NDC1 localisation and identification of other Nups in the transmembrane ring sub-complex would bring a more comprehensive understanding to the plant NPC.