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A highly branched hyphal morphology was observed in early stage (high nutrient availability) batch liquid culture and high dilution rate chemostat culture of Saccharopolyspora erythraea and Streptomyces coelicolor. In contrast, nutrient exhausted batch cultures and low dilution rate chemostat cultures contained unbranched myceUa. Formation of un-branched mycelia is thought to have evolved to expedite rapid "searching" of the soil environment, in order to locate nutrients. The increased frequency of branching is assumed to provide a structure that can exploit nutrient rich micro-habitats (once located) effectively.

The palmerolides are an emerging class of polyketide natural products isolated from marine organisms inhabiting the Antarctic Sea. Although there has been a substantial effort to prepare the most active family member palmerolide A, no synthetic work has been performed in the area of the other members of the family; palmerolides B and C of different ring structure. These compounds are particularly interesting due to their complex structures with undefined stereochemistry and both display high selectivity towards human melanoma cancer cell lines. A highly convergent strategy to access palmerolide C has been developed, relying on the assembly of three fragments using key bond couplings at C6-C7 (boron-mediated aldol reaction/dehydration) and C14-C15 (Julia-Kocienski olefination), followed by a suitable macrolactonisation step and Buchwald enamide formation. Prior to subunit synthesis, the relative configuration of the C8-C10 stereotriad was resolved via the synthesis of diastereomeric degradation fragments by the application of organocatalytic cross aldol reactions and a suitable 1,3-selective reduction.

Polyketides are a diverse group of biologically active natural products synthesized by the poiyketide synthase (PKS) family of enzymes. Bacterial Type II PKS are discrete multienzyme systems consisting of acyl carrier protein (ACP), ketosynthase (KS), chain length factor (CLF), ketoreductase (KR), aromatase (ARO) and cyclase (CYC) amongst other enzymes required for tailoring of the chain. Understanding the structure and biochemistry of the enzymes of the PKS has the potential to allow the targeted biosynthesis of novel compounds. In order to enable such modification the mechanisms by which the PKS controls chain-length, cyclization and reduction must be fully understood.

Polyketides are a class of secondary metabolites that includes compounds with antibiotic and anticancer properties. In type I polyketide synthesis, the individual activities are arranged into large | polyproteins, while in type II polyketide synthesis enzymes are encoded separately. Streptomyces coelicolor actinorhodin acyl carrier protein synthase (AcpS) IS a promiscuous enzyme involved in type II polyketide synthesis that catalyses the transfer of the phosphopantetheine chain from coenzyme A (CoA) to acyl carrier protein (ACP).

Nitric oxide (NO) is a highly reactive signaling molecule that plays diverse and important bioregulatory roles. In endothelial cells, NO is synthesized by eNOS, a member of the NO synthase (NOS) family, whose activity is tightly regulated at the posttranslational level by acylation, phosphorylation, S-nitrosylation and protein-protein interactions. The biological actions of NO arise as a direct consequence of chemical reactions between NO, or NO-derived species, and protein targets. S-nitrosylation of specific cysteine residues in target proteins is emerging as an important mechanism of NO signaling. Although eNOS is known to undergo a complex pattern of trafficking within endothelial cells, the distinct activities, modes of regulation, and protein targets of NO at eNOS-containing organelles are largely unexplored. We hypothesize that eNOS is differentially regulated at alternative subcellular loci and NO is preferentially transferred to protein acceptors that neighbor NO synthases. To test these hypotheses, we sought to develop a conditional heterodimerization approach that can target eNOS, on-demand, to discrete subcellular loci (via specific targeting sequences e.g. nuclear, mitochondrial, Golgi and cell membrane) and assess the consequences for eNOS activity, regulation and protein S-nitrosylation patterns. Furthermore, we sought to devise a proteomic strategy for unbiased identification of S-nitrosylation sites on proteins that mediate NO bioactivities. Use of these new tools should enable the resolution of controversies regarding the role of diffusion in NO biology, as well as the identification of novel protein targets and mechanism that regulate NO biosynthesis at distinct loci within endothelial cells. In this thesis research, I describe the development and application of "eNOS CID" and "SNOSID" as powerful new tools to address the impact of eNOS compartmentalization on NO signalling in endothelial cells.

In plants, the need to efficiently respond to constant environmental fluctuations has led to the evolution of photoprotective mechanisms to regulate photosynthetic light reactions. One of such mechanism is cyclic electron transport (CET) which generates a pH gradient inducing the nonphotochemical quenching (NPQ) of excess excitation energy, synthesis of ATP and maintenance of balanced redox state of the photosynthetic electron transport (PET) chain. Two major pathways of CET involve the NADH dehydrogenase-like complex (NDH)-dependent pathway, and the Proton Gradient Regulation 5 (PGRS)-dependent pathway. However, the mechanism and function of the PGRS-dependent CET to adapt the photosynthetic process in response to environmental variations and its impact in rice photosynthesis remains unclear. This study aims to determine the role of the photo protective protein, PGRS in the regulation of photoprotection and photosynthesis under constant as well as dynamic conditions and its impact on plant growth and development. Oryza sativa var. Kaybonnet, PGRS RNA interference (RNAi) and overexpression (OE) transgenic rice plants were characterised based on their physiological and morphological traits in a controlled environment and in glasshouse under constant and fluctuating light intensities. This study showed that PGRS-dependent CET was actively occurring in rice leaves grown under non saturating light but its rate increased significantly with overexpression of the PGRS protein in saturating light. Under constant and fluctuating light, elevated NPQ observed in the OE was absent in the RNAi lines indicating that PGRS is essential for cyclic electron flow in rice both during induction and at steady state photosynthesis. CO2 assimilation in both OE and RNAi were limited by maximum rate of carboxylation (Vcmax) although the RNAi was also photoinhibited. Experiments under dynamic condition of low atmospheric moisture indicated that the PGRS-dependent CET plays an important role in photoprotection of PSI and PSI!. Results from this experiment further suggest that PGRS could function efficiently in stress alleviation both in the short- and long term. Growth and biomass accumulation in the glasshouse and controlled environment showed that the PGRS protein is important in rice leaf photosynthesis. However, in a biomass experiment, significant photosynthetic advantage attributed to overexpression of PGRS could not be identified except in photoprotection and regulation of the PET chain. This study indicates that the ATP/NADPH ratio may modulate PGRS dependent CET response in rice, however, its regulation is likely to depend on the availability of PSI electron acceptors such as ferredoxin. PGRS may be important in meeting the demand for ATP in rice. In conclusion, this study has shown that the protein PGRS plays a very important role in enhancing photoprotection and is required for efficient photosynthesis in rice. However, there may be need for balance between enhancing photo protection and crop productivity in future improvement programmes.

Les polykétides sont des composés naturels. Ces composés possèdent des rôles thérapeutiques variés tels que antifongiques, antibiotiques, anticancéreux, immunosuppresseurs ou encore anticholestérolémiques. Par conséquent, la recherche de nouvelles structures possédant des bioactivités diverses se révèlent être intéressante. Une stratégie prometteuse pour créer des nouveaux polykétides est l’ingénierie génétique des enzymes synthétisant ces molécules, les polykétide synthases modulaires (PKS), une approche désignée sous le terme de « biologie synthétique ». Pour ce faire, il faut comprendre de façon détaillée le fonctionnement de ces systèmes multienzymatiques. Plusieurs points restent à éclaircir, dont : i) le contrôle de la stéréochimie du polykétide ; et ii) l’interaction des sous-unités composant la PKS. Lors de ma thèse, j’ai identifié deux kétoréductases (KR) qui, introduites dans un contexte modulaire intrinsèquement non-épimérisant, sont capables d’épimériser le méthyle en Cα de façon efficace. Cependant, la modification de la stéréochimie du polykétide ne dépend pas exclusivement des propriétés intrinsèques de la KR mais aussi du contexte modulaire. J’ai également contribué à la réalisation d’un second projet, pour lequel notre équipe a mis en évidence une nouvelle classe de domaine de docking de PKS de type trans-AT présentant une nouvelle topologie. L’un des DD étudié est une protéine intrinsèquement désordonnée dont le repliement est induit par son partenaire. Nous avons caractérisé l’interface complète entre deux sous-unités de PKS de type trans-AT, révélant une chambre de réaction protégée dans laquelle les chaînes de polykétide peuvent croître / Polyketides are natural products which exhibit a variety of therapeutic activities, including anti-fungal, antibiotic, anticancer, immunosuppressant and anti-cholesterolemic properties. Given their medical and economic importance, there is significant interest in identifying new structures with new biological activities. A promising strategy to create such analogues is to genetically engineer the enzymes responsible for synthesizing these molecules, the modular polyketide synthases (PKSs), an approach referred to as ‘synthetic biology’. However, in order to increase the efficacy of this approach, we must understand in detail how the PKS multienzymes work. A number of issues remain to be clarified, including: i) polyketide stereocontrol, ii) the interaction of the component subunits PKS. During my thesis, I identified two ketoreductase (KR) domains which when introduced into an intrinsically non-epimerizing modular context, were able to efficiently epimerise at the Cα of a model polyketide. I also showed that the modular context in which the KR functions has an influence on the ultimate stereochemical outcome. I also made essential contributions to a second project, in which the group identified a novel family of docking domains (DD) in the trans-AT type of PKS which present a novel topology. One of the two model DDs studied is an intrinsically disordered protein whose folding is induced by its partner. Finally, we were able to visualize a complete intersubunit interface within a trans-AT PKS, revealing a protected reaction center in which polyketide chains can grow.

Certains antagonistes du récepteur AT1 (« angiotensin II type 1 receptors ») utilisés dans le traitement de l'hypertension artérielle ont par la suite également montré une activité au niveau de PPAR[gamma] (« Peroxisome Proliferator Activated Receptor [gamma] »), un récepteur impliqué dans la régulation du métabolisme du glucose. Cela constitue une nouvelle approche thérapeutique vers un traitement concomitant de l'hypertension artérielle et du diabète de type II, qui sont souvent associés. Dans ce contexte, nous nous sommes dirigés vers la conception rationnelle de molécules capables d'interagir à la fois sur les deux récepteurs impliqués dans ces affections. Pour cela, nous avons utilisé le concept de « designed multiple ligand » tel qu'il a été défini par certains chercheurs de l'Industrie pharmaceutique. Nous l'avons appliqué en combinant les pharmacophores propres aux antagonistes du récepteur AT1 et aux agonistes de PPAR[gamma], afin de générer une série diversifiée de composés. Afin de diriger la conception de ces molécules, nous avons par ailleurs réalisé des expérimentations de modélisation moléculaire (« docking ») sur PPAR[gamma]. Cela nous a conduits à développer une nouvelle méthode dans le but de prédire le caractère agoniste des composés étudiés. Enfin les molécules ont été évaluées pour leurs propriétés antagonistes AT1 et agoniste PPAR[gamma]. D'intéressantes relations structures activités ont été dégagées / Some angiotensin II-type 1 receptor (AT1) antagonists, used for blood pressure control, exhibit also an activity on peroxisome proliferator activated receptor [gamma] (PPAR[gamma]), which is involved in the control of glucose metabolism. Such compounds could be promising drugs for the treatment of both hypertension and type II diabetes, which are often concomitant. Therefore, we have rationally designed molecules potentially able to interact with both receptors involved in these diseases. We used the "design multiple ligands" concept, as previously developed by industrial pharmacists, to build up a diversified molecule set via combination of both pharmacophores of AT1 and PPAR[gamma] receptor antagonists and agonists, respectively. Molecular modeling experiments (docking) on PPAR[gamma] were conducted to rationalise the synthesis and allow us to predict in some extent the agonistic activity of the studied compounds