La régulation redox est un mécanisme ancien présent chez les organismes biologiques et impliquée dans diverses voies métaboliques. En particulier chez les organismes photosynthétiques elle est responsable des mécanismes d‘adaptation rapide dans un environnement constamment modifié. Dans les chloroplastes le système ferrédoxine/thiorédoxine est la cascade redox principale qui relie l‘activité de plusieurs enzymes plastidiales à la source lumineuse. Le rôle central dans ce système est joué par la ferrédoxine-thiorédoxine réductase (FTR), une protéine hétérodimérique qui récupère des électrons à partir de la ferrédoxine photoréduite et les transfère pour réduire des thiorédoxines plastidiales. Ces protéines peuvent alors réduire des enzymes cibles, requérant l‘accessibilité de paires de cystéines dans un disulfure dont la réduction résulte en une activation/ inactivation de la cible. Jusqu‘à présent des plantes viables n‘ont pu être obtenues en l‘absence de ce système de régulation. Dans cette thèse des secteurs du système redox ont été explorés chez la plante modèle Physcomitrella patens (une mousse). Par manipulation de gènes l‘influence de l‘enzyme FTR sur la croissance et le développement de la plante a été analysée suivant différents paramètres. De manière à impacter la fonction de la réductase des changements nucléotidiques simples ont été introduits au niveau des codons programmant les cystéines catalytiques et dans un deuxième temps le gène complet a été supprimé. De façon inattendue nous n‘avons observé aucun effet significatif sur la viabilité et le développement des plantes mutantes. De plus, nous avons détecté dans P. patens des thiorédoxines additionnelles absentes chez les plantes à graine qui sont fonctionnelles vis à vis des enzymes cibles mais non-réduites par la FTR. Ceci rend possible un scénario de compensation chez les mutants via un système de réduction FTR-indépendant qui reste à caractériser. Deux des cibles photorégulées, la fructose-1,6-bisphosphatase (FBPase) et la sédoheptulose-1,7-bisphosphatase (SBPase), fonctionnent dans la phase de régénération du cycle de Calvin-Benson cycle et elles possèdent plusieurs caractéristiques de catalyse et de régulation similaires. En combinant des approches biochimiques et structurales, une comparaison fonctionnelle et structurale des deux phosphatases de P. patens a été conduite. De plus l‘analyse phylogénétique a révélé une origine procaryotique indépendante des deux séquences en dépit de leurs similitudes structurales et catalytiques. De plus trois articles de revue résument la plasticité et la représentativité du modèle P. patens pour la recherche forestière, les principes généraux de la régulation redox relativement aux aspects évolutifs et fonctionnels chez les plantes ainsi que l‘ état de l‘art de la régulation redox chez les espèces ligneuses en utilisant principalement le peuplier comme modèle / Redox regulation is an ancient mechanism present in biological organisms and is involved in diverse cellular pathways. In particular in photosynthetic organisms it is responsible for fast adaption mechanisms to a constantly changing environment. In chloroplasts the ferredoxin/thioredoxin system represents the main redox regulatory cascade which links the activity of several plastid enzymes to the energy source, light. A central role in this system is played by the heterodimeric ferredoxin-thioredoxin reductase (FTR), which gains electrons from the photo-reduced ferredoxin and transfers those further on via reduction to plastidal thioredoxins. Those proteins in turn reduce their target enzymes and require therefore the availability of redox sensitive cysteine pairs whose reduction results in an inactivation/activation switch of the targets. So far no viable plants could be obtained in complete absence of this redox regulation system. In this thesis single sections of the system were explored in the model plant Physcomitrella patens. Through gene manipulation the influence of the FTR enzyme on plant growth and development was analysed. In order to impact on the function of the reductase, firstly single nucleotide exchange of the catalytic cysteines was performed and later on the gene was completely deleted. Surprisingly, no significant effect could be observed on the viability and development of mutant lines compared to WT plants. Furthermore we found that P. patens possesses in contrast to seed plants additional thioredoxins which are functional for reduction of FTR target enzymes but are most likely not supplied with electrons by this reductase. Thus a possible rescue scenario independent of FTR could be assumed for P. patens and also by other redox regulation systems present in chloroplasts. Two of the FTR target enzymes, fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase, are functional in the regeneration phase of the Calvin-Benson cycle and share similar characteristics in regulation and catalysis. By combining biochemical and structural approaches, a functional comparison of both phosphatases was conducted using cDNAs from P. patens. A stricter TRX-dependent regulation and catalytic cleavage ability for both substrates, FBP and SBP, could be observed for PpSBPase, whereas PpFBPase is only capable of cleaving FBP. By obtaining the oxidized X-ray structure of both enzymes these observations can be associated with the distinct positions of regulatory sites and the various sizes of the substrate binding pocket. In addition, the phylogenetic analysis revealed an independent prokaryotic origin for both phosphatases. Furthermore we summarized in three review articles the amenability of P. patens as model plant for forest research, the general principles of redox regulation in respect of evolution and functional mechanisms in plants, and the current state of the art in forest redox regulation using poplar as exemplary model

High aerobic glycolytic capacity is correlated with poor prognosis and increased tumour aggressiveness. 6Phosphofructo-1-kinase catalyses the first irreversible step of glycolysis, and is activated by fructose-2,6-bisphosphate, a product of the kinase activity of four bifunctional isoenzymes, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFK-2/FBPase-2:PFKFB1-4). These are potential anti-tumour targets, but their individual and collective role requires further investigation. This thesis had three aims; to validate the PFK-2/FBPase-2 isoenzymes as anti-cancer targets, to investigate the requirement for isoenzyme-specific targeting, and to initiate assay development, enabling future identification of novel inhibitors. A panel of cancer cell lines was examined and PFKFB3 and PFKFB4 were confirmed to be the most strongly induced isoenzymes in hypoxia, regulated by HIF-1α. Basal and hypoxic relative PFKFB3/PFKFB4 expression varied markedly, and three cell lines with varying expression ratios (MCF-7, U87, PC3) were selected for further study. siRNA knockdown of each isoenzyme individually, markedly reduced 2D and 3D cell growth. The effect of PFKFB3 knockdown was consistently more pronounced, particularly in hypoxia. Double PFKFB3/PFKFB4 knockdown was significantly less effective than PFKFB3 knockdown alone. Direct antagonism of PFKFB3 and PFKFB4 on F-2,6-BP concentration was observed, with PFKFB3 exhibiting high kinase activity, as anticipated, and PFKFB4 exhibiting high bisphosphatase activity. The degree of antagonism was dependent on the relative PFKFB3/PFKFB4 expression ratio. Extensive efforts were made to examine the wider metabolic effect of PFKFB3/PFKFB4 on flux towards glycolysis or the pentose phosphate pathway (PPP), including using metabolite, lipid droplet, ¹³C NMR and mass spectrometry assays. No significant change in metabolic flux was detected, the evidence presented therefore suggesting the impact of the antagonistic effects of the isoenzymes on [F-2,6-BP] extends beyond regulation of metabolic flux alone. This study concluded that the most effective therapeutic strategy will be one that involves a PFKFB3-specific inhibitor, preferably hypoxia-targeted. Accordingly, steps were taken to validate and optimise a robust medium-throughput assay system.

Während der Entwicklung von Früchten der Tomate (Sorte Micro-Tom) wurde der Kohlenhydrat-Stoffwechsel untersucht. Es wurde ein Unterschied zwischen dem Metabolismus im Perikarp und dem des Plazenta-Gewebes gefunden. Stärke wurde in der Plazenta langsamer abgebaut als im Perikarp, während lösliche Zucker im Perikarp stärker akkumulierten. Die Aktivitäten der glykolytischen Enzyme tendierten zu einem Maximum 40 Tage nach der Blüte. Weiterhin wurde die Expression einiger plastidärer Transporter untersucht. Sowohl der Triosephosphat-Tranporter (TPT) als auch der Glucose-6-phosphat-Transporter wurden am stärksten in grünen Früchten exprimiert, während der Reife nahm die Expression ab. Der ATP/ADP-Transporter wurde während der Fruchtentwicklung nur schwach exprimiert.Es besteht die Hypothese, daß die Rolle der drei Enzyme plastidäre Fructose-1,6-Bisphosphatase (cp-FBPase), ADP-Glucose Pyrophosphorylase (AGPase) und Glucan Wasser Dikinase (GWD) darin besteht, die Stärke-Akkumulation in der frühen Entwicklung der Tomaten-Frucht zu beeinflussen. Diese Hypothese wurde unter Verwendung der Antisense-Technik für die plastidären FBPase (unter der Kontrolle des B33 Promoters), sowie für die AGPase und die GWD (beide unter der Kontrolle des CaMV 35S-Promoters) in der Tomaten-Kultivar Moneymaker untersucht. Die Repression von plastidärer FBPase oder AGPase in der Frucht der Tomate scheint die Metaboliten-Konzentrationen nicht so stark wie in den Blättern zu beeinflussen. Der Grund hierfür ist wahrscheinlich, daß jede Veränderung durch die Fähigkeit der Frucht, Zucker zu importieren, abgepuffert wird. Auf der anderen Seite hatte die Repression des GWD Proteins in der Frucht der Tomate starke Effekte auf die Metaboliten-Konzentrationen. / Carbohydrate metabolism was studied during the development of fruits of the tomato cultivar Micro-Tom. The metabolism of the pericarp and placental tissues was found to be different. Starch being degraded more slowly in the placenta than in the pericarp, while soluble sugars accumulated to a greater extent in the pericarp. The activities of glycolytic enzymes tended to peak at 40 days after flowering. The expression of some plastidial transporters was also studied. Both the triose phosphate transporter (TPT) and Glucose-6-Phosphate (Glc-6-P) transporter were expressed greatest in green fruits, before declining. The expression of the triose phosphate transporter (TPT) was greater than that of Glc-6-P transporter. The ATP/ADP transporter was expressed to a low level throughout fruit development. The role of three enzymes Chloroplastic Fructose-1,6-bisphosphatase (cp-FBPase), ADP-glucose Pyrophosphorylase (AGPase) and Glucan Water Dikinase (GWD) protein are thought to influence the accumulation of starch in early development in tomato fruit were studied in normal sized tomatoes of the cultivar Moneymaker using antisense technique under the control of the patatin B33 promoter in the case of cp-FBPase, and the CaMV 35S promoter in the case of AGPase and GWD protein. It appears that repression of cp-FBPase and AGPase in tomato fruits does not influence metabolite levels as greatly as it does in leaves, possibly because any alterations are buffered by the ability of the fruit to import sugars. On the other hand, the repression of GWD protein in tomato fruits has a strong effect on metabolite levels.