Generation and phenotypic analyses of stzA gene deletion strains of Aspergillus nidulans implies that stzA is allelic to sltA, with the encoded transcription factor regulating tolerance to cations, DNA-damaging agents and high arginine concentrations. The similar severe sensitivity of a sltA1 mutant (GO281) and stzA deletion mutants to these stresses indicated that the premature termination codon in sltA1 represents a total loss-of-function mutation. It was also verified that StzA has no regulatory role in the utilisation of carbon sources. Findings were supported by phenotypic analyses of recombinant progeny resulting from sexual crosses between sltA1 and sltA+ strains. Bioinformatic analysis of genes involved in the osmotic stress response revealed that their promoters were significantly enriched for StzA binding site motifs compared to those of the control group, indicating that StzA may regulate many of these genes that comprise the High Osmolarity Glycerol (HOG) pathway. Although this pathway is activated by fludioxonil, stzA deletants and stzA+ strains showed similar sensitivities to this fungicide. Phenotypic analyses indicate that StzA does not regulate tolerance to sources of oxidative stress, non-ionic osmotic stress or components of the Cell Wall Integrity (CWI) pathway. A. nidulans StzA appears to have no role in cellulase or xylanase expression as revealed by the results of a dinitrosalicylic acid (DNS) assay. Trichoderma reesei ace1 deletant and wild-type strains showed similar sensitivities to cations, DNA-damaging agents, arginine, neomycin, acidic and alkaline pH. These results confirm that A. nidulans StzA and T. reesei Ace1 regulate the distinct phenotypes of abiotic stress tolerance and cellulase and xylanase expression, respectively, despite these two proteins sharing 58% overall amino acid similarity. All twenty-nine StzA orthologues identified are restricted to filamentous ascomycetes of the Pezizomycotina subphylum and may therefore represent specific and novel antifungal drug targets. The C-termini of StzA proteins are highly variable in both length and sequence, with no apparent conservations in amino acids or predicted secondary structure. This region is considered most likely to influence the divergent functions of StzA proteins. Conservations of individual residues in the N-termini correspond to conserved secondary structure (alpha helices) among StzA proteins, implying shared functions for StzA proteins in this region. Regulators of two major nitrogen metabolic pathways (CpcA and AreA) may regulate stzA expression. Statistically significant putative CpcA binding sites were positionally conserved in 26 out of 29 stzA orthologue promoters, indicating an interaction between stzA and CpcA, a transcription factor that mediates the cross pathway control of amino acid biosynthesis. REALALE sequences, likely to be of retrotransposon origin, containing putative overlapping binding sites for StzA and AreA, were found in eleven stzA promoters of the Eurotiomycetes class, indicating an interaction between stzA and the global nitrogen metabolite repressor AreA. Intriguingly, REALALE-containing promoters identified across the genome of A. nidulans were significantly enriched for StzA binding site motifs when compared to a control group of genes. Hence, REALALE may have regulatory significance that extends to other A. nidulans genes.

No description available.

Bis zum heutigen Zeitpunkt ist wenig über die physiologischen Effekte von Ethanol auf Cyanobakterien bekannt. Dies ist nicht überraschend, da es unwahrscheinlich ist, dass Cyanobakterien in ihrer natürlichen Umwelt auf Wachstums inhibierende Konzentrationen stoßen, und deswegen war die Stressantwort auf Ethanol nur von geringerem Interesse für die Forschungsgemeinschaft. Nichts desto weniger sind durch neue Entwicklungen im Biofuel- Sektor, insbesondere im Kontext der Produktion von Ethanol mit Hilfe von genetisch manipulierten Cyanobakterien, Kenntnisse über die zelluläre Toleranz und Zellantwort gegenüber dem gewünschten Produkt von grundlegender Bedeutung. Microarray-Experimente, die einen Einblick in die zelluläre Antwort durch Änderung der Genexpression auf Ethanolproduktion bringen sollten, zeigten, dass Gene des Phycocyanin-Operons als die am signifikantesten und stärksten betroffenen funktionalen genetischen Elemente. Weitere Microarray-Experimente mit verschiedenen Konzentrationen von extern zugefügtem Ethanol zeigten eine zeitverzögerte (24h) Hochregulation von PS II-Genen und dem Transkript cpcG2. Diese Arbeit beschreibt weiterhin die Ergebnisse eines Experiments zur "Evolution im Labor", das die intrinsische Kapazität von Synechocystis sp. PCC 6803 zur Erweiterung der Toleranz gegenüber Ethanol aufzeigen sollte. Die erhöhte Ethanoltoleranz führte zu einer Optimierung der endogenen Ethanolproduktion. Derartige Versuche zur Stammoptimierung durch "Evolution im Labor" sollten daher geeignete Mittel sein, um bestimmte Eigenschaften von Organismen für biotechnologische Ziele zu verbessern. In der Gesamtheit geben die Ergebnisse dieser Arbeit Einblicke in die Antwort der Synechocystis-Zellen auf Ethanol auf den Ebenen des Stoffwechsels und der Genexpression und stellen eine wertvolle Datensammlung für zukünftige Versuche mit dem Ziel dar, die Ethanolproduktionsrate in Cyanobakterien durch genetic engineering zu erhöhen. / Until recently, little has been known about the effects of ethanol on the physiology of cyanobacteria. This is not surprising as it is unlikely that cyanobacteria encounter growth inhibiting concentrations of ethanol in their natural environment, and thus the ethanol stress response used to be of limited interest to the scientific community. Nevertheless, for recent biotechnological approaches in the field of biofuel production, and in particular for the attempts to produce ethanol with the help of genetically modified microalgae and cyanobacteria, knowledge of cellular tolerance and response to the desired product is pivotal. Microarray analysis demonstrating that a specific part of the phycocyanin operon is the most significantly and strongly affected functional genetic subsystem under ethanol producing conditions. Additional microarray experiments with different concentrations of external ethanol showed a time-delayed (24h) characterized by a prominent up-regulation of PS II genes with phycocyanin linker proteins playing a major role in the transcriptional response. Another aspect of this work was an artificial evolution experiment, which was performed to delineate the intrinsic capacity of Synechocystis sp. PCC6803 to tolerate ethanol. In addition, the evolved strain proved to be a superior background for endogenous ethanol production showing that artificial evolution experiments are a suitable method to improve certain features of organisms for biotechnological purposes. Overall, the results of this work give new insight into physiological and gene regulatory responses of Synechocystis sp. PCC6803 exposed to ethanol and will be a very valuable dataset for future attempts to improve cyanobacterial ethanol production by the means of genetic engineering.

No description available.

<p>CYP2C9 is one of our major drug metabolising enzymes and belongs to the cytochrome P450 (CYP) super family. The aim of this thesis was to gain an understanding of the quantitative structure–activity relationships (QSAR) of CYP2C9 substrates and inhibitors. This information will be useful in predicting drug metabolism and the potential for drug–drug interactions. To achieve this, a well characterised data set of structurally diverse, competitive CYP2C9 inhibitors was identified in our laboratory. Several computational methodologies, many based on GRID molecular interaction fields, were applied or developed in order to handle issues such as compound alignment and bioactive conformer selection. First, a traditional 3D QSAR was carried out in GOLPE, generating a predictive model. In this model the selection of a bioactive conformer and alignment was based on docking in a homology model of CYP2C9. Secondly, we introduced the concept of alignment independent descriptors from ALMOND. These descriptors were used to generate quantitatively and qualitatively predictive models. We subsequently derived conformation independent descriptors from molecular interaction fields calculated in FlexGRID. This enabled the derivation of 3D QSAR models without taking into account the selection of an alignment or a bioactive conformer. A subsequent programming effort enabled the conversion of this model back to 3D aligned pharmacophores. Similar alignment independent descriptors were also used in the development of the software MetaSite® that predicts the site of metabolism for CYP2C9 ligands. Finally, as crystal information on this isoform emerged, the performance of molecular dynamics simulations and homology models and the flexibility of the protein were evaluated using statistical analyses.</p><p>These modelling efforts have resulted in detailed knowledge of the structural characteristics in ligand interactions with the cytochrome P450 2C9 isoform.</p>

CYP2C9 is one of our major drug metabolising enzymes and belongs to the cytochrome P450 (CYP) super family. The aim of this thesis was to gain an understanding of the quantitative structure–activity relationships (QSAR) of CYP2C9 substrates and inhibitors. This information will be useful in predicting drug metabolism and the potential for drug–drug interactions. To achieve this, a well characterised data set of structurally diverse, competitive CYP2C9 inhibitors was identified in our laboratory. Several computational methodologies, many based on GRID molecular interaction fields, were applied or developed in order to handle issues such as compound alignment and bioactive conformer selection. First, a traditional 3D QSAR was carried out in GOLPE, generating a predictive model. In this model the selection of a bioactive conformer and alignment was based on docking in a homology model of CYP2C9. Secondly, we introduced the concept of alignment independent descriptors from ALMOND. These descriptors were used to generate quantitatively and qualitatively predictive models. We subsequently derived conformation independent descriptors from molecular interaction fields calculated in FlexGRID. This enabled the derivation of 3D QSAR models without taking into account the selection of an alignment or a bioactive conformer. A subsequent programming effort enabled the conversion of this model back to 3D aligned pharmacophores. Similar alignment independent descriptors were also used in the development of the software MetaSite® that predicts the site of metabolism for CYP2C9 ligands. Finally, as crystal information on this isoform emerged, the performance of molecular dynamics simulations and homology models and the flexibility of the protein were evaluated using statistical analyses. These modelling efforts have resulted in detailed knowledge of the structural characteristics in ligand interactions with the cytochrome P450 2C9 isoform.