Malaria is a life-threatening vector borne infectious disease caused by protozoan parasites of the genus Plasmodium. More than 100 species of Plasmodium can infect numerous animal species such as reptiles, birds and various mammals. However, human malaria is caused by four Plasmodium species -Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae, and occasionally by the simian malaria parasite, Plasmodium knowlesi. Of these, P. falciparum and P. vivax are the major causative agents and P. falciparum is the most virulent. About 300-500 million malaria infections occur every year leading to over 1-2 million deaths, of which 75% occur in African children of less than 5 years infected with P. falciparum. In spite of major global efforts to eliminate this disease over the past few decades, it continues to persist as a major affliction of human-kind imposing serious health and economic burden, especially to the poor countries. In India, the present scenario is about 2 million malaria positive cases every year, with almost 50% being caused by P. falciparum. Although remarkable attempts have been made over the years to develop vaccines against sexual and asexual stages of malaria parasite, an effective vaccine is still not in sight and remains as a distant goal. Hence, highly potent, less toxic and affordable antimalarial drugs remain as a first line therapy for malaria. Unfortunately, these parasites have been evolving against every known antimalarial drug and many of these drugs have lost their potency due to rapid emergence and spread of drug resistant strains. With development of resistance against frontline antimalarials such as chloroquine and antifolates, artemisinin and its derivatives seem to be the only effective antimalarials. However, recent reports on the possible emergence of artemisinin resistant strains, have led to the implementation of artemisinin-based combination therapies as a strategy to prevent drug resistance. Also, this continuous emergence of drug resistance has necessitated the development of new antimalarial drugs to combat this disease. While, Anopheles mosquitoes transmit parasites that infect humans, monkeys and rodents, Culex and Aedes mosquitoes predominate in the natural transmission to birds, and vectors of reptilian parasites are largely unknown. Of the approximately 400 species of Anopheles throughout the world, about 60 are malaria vectors under natural conditions, and 30 of which are of major importance. Ironically, the strategies implemented for controlling Anopheles, have also been hampered by insecticide resistance and other practical difficulties that exist in the scope of their applicability. In the past few years several milestones have been achieved in parasite genome, transcriptome and proteome studies, which could be exploited for the development of new drugs and drug targets. One such promising target includes the metabolic pathways of the malaria parasite which differ significantly from its human host. This thesis entitled “Unique Features of the Heme-Biosynthetic Pathway in Human Malaria Parasite, Plasmodium falciparum” unravels the unique biochemical features of heme-biosynthetic enzymes of P. falciparum, which have the potential for being drug targets. This pathway was first identified in this laboratory over 15 years ago. In the present study, five of the 7 enzymes of this pathway have been cloned, expressed, properties studied and sites of localization identified. With the knowledge on the first two enzymes coming from earlier studies, it is now possible to depict the unique hybrid pathway for heme biosynthesis in P. falciparum with full experimental validation.

Plus de 300 alcaloïdes d’Amaryllidaceae doués d’activités biologiques ont été isolés à partir des plantes appartenant à cette famille. De nos jours, seule la galanthamine, utilisée pour le traitement palliatif de la maladie d’Alzheimer, est commercialisée. L’accumulation de ces alcaloïdes dans les plantes est limitée. La culture in vitro est une méthode alternative intéressante pour l’obtention plus aisée de ces alcaloïdes à haute valeur ajoutée. Le premier objectif de ce travail vise à développer une méthode de purification efficace, simple et rapide des extraits de plantes préalablement à leur analyse en LCMS et GCMS. Le second objectif est d’étudier l’effet de plusieurs facteurs exogènes, ajoutés au milieu de culture de bulbilles de Leucojum aestivum et de sa variété Gravety Giant en bioréacteurs RITA®, sur les voies de biosynthèse de la galanthamine et de la lycorine. La variation des paramètres exogènes a permis une accumulation accrue en galanthamine et en lycorine (0,814 mg/g et 1,54 mg/g de matière sèche respectivement) dans les bulbilles. Le troisième objectif porte sur l’isolement et l’identification d’endophytes à partir de bulbes in vivo et in vitro de trois espèces d’Amaryllidaceae (L. aestivum, Narcissus pseudonarcissus et Galanthus elwesii). Des bactéries endophytes du genre Bacillus ont été identifiées. Un nouvel alcaloïde a été isolé à partir des cultures bactériennes / Over 300 Amaryllidaceae alkaloids possessing a wide range of biological activities have been isolated from plants belonging to this family. Galanthamine, used for the palliative treatment of Alzheimer’s disease, is the only one commercialized. The biodisponiblity of these alkaloids is low. In vitro culture offers an alternative yet interesting approach for the biotechnological production of these valuable alkaloids. The aim of this work was, first, to develop a fast, efficient and easy purification method of plant extracts prior to their phytochemical analysis both in LCMS and GCMS. Second, the combined effects of bioreactor RITA® culture and feeding with different exogenous factors on the biosynthetic pathway of both galanthamine and lycorine were studied. The experiments were conducted both with Leucojum aestivum and L. aestivum ‘Gravety Giant’ bulblets. The variation of several exogenous parameters resulted in a better accumulation of galanthamine and lycorine (0.814 mg/g and 1.54 mg/g dry weight respectively) in the bulblets. The third aim was to isolate and identify alkaloid producing endophytes from in vivo and in vitro bulbs of three Amaryllidaceae species (L. aestivum, Narcissus pseudonarcissus and Galanthus elwesii). Bacterial endophtes belonging to the Bacillus genus were identified. A new alkaloid was isolated from bacterial liquid cultures

La pourriture bleue causée par Penicillium expansum est l'une des maladies les plus dommageables des fruits pomaceae (pommes et poires). Outre des dégâts directs, cette maladie pose un problème de santé publique car l'agent pathogène produit des mycotoxines nocives pour l'homme et les animaux dont la plus sérieuse est la patuline. La croissance du champignon pathogène et la production de patuline requièrent des conditions physico-chimiques particulières. Les informations existantes à ce propos demeurent cependant modestes et insuffisantes pour envisager de développer des moyens de lutte contre l'apparition du champignon. Par ailleurs, la patuline reste avec l'ochratoxine A, les seules toxines dont la voie de biosynthèse n'a pas encore été complètement établie, tant sur le plan chimique que moléculaire. Cette étude apporte dans un premier temps des données complémentaires sur les facteurs physico-chimiques (température, pH….) qui conditionnent la croissance de P. expansum de même que sa capacité à produire la patuline. La connaissance de ces besoins et de ces conditions conduit en pratique à lutter et contrôler la contamination par la patuline tout le long de la chaine alimentaire. Dans un deuxième temps, cette thèse apporte des améliorations spectaculaires sur le plan fondamental, en termes d'élucidation de la voie de biosynthèse de la patuline. Le cluster des gènes impliqués dans la biosynthèse de cette mycotoxine chez l'espèce la plus préoccupante P. expansum a été entièrement identifié et caractérisé. Pour lever encore plus le voile sur la biosynthèse de cette mycotoxine, la caractérisation du facteur de régulation spécifique de cette voie (patL) a été également établie. Une perturbation de ce gène a provoqué une incapacité de production de patuline et une sévère diminution de l'expression des gènes Pat. De même, grâce à ce mutant déficient, il a été montré que la patuline pourrait agir comme facteur de virulence lors du développement de la moisissure dans les pommes. La caractérisation de la dernière étape de la voie de biosynthèse de la patuline a ensuite été entreprise par mutagenèse dirigée du gène patE du cluster de la patuline, chez la même espèce. Ce dernier code pour une Glucose méthanol Choline (GMC) oxydoréductase responsable de la conversion de l'ascladiol en patuline. L'ascladiol est également une molécule clé de la dégradation de la patuline par diverses espèces bactériennes ou de levures et plus particulièrement lors de la fermentation alcoolique. La non-toxicité de l'ascladiol accumulé chez le mutant ∆patE a été démontrée sur une lignée cellulaire intestinale humaine (Caco-2), suggérant que la patuline perd sa toxicité avec l'ouverture du deuxième cycle. Finalement, un système de détection et de quantification de P. expansum par PCR en temps réel a été développé en ciblant un gène hautement spécifique de la voie de biosynthèse de la patuline, patF. Cette approche préventive nous a ainsi permis d'avoir une estimation rapide de la contamination en patuline dans les pommes à partir de la quantification d'ADN de P. expansum. En conclusion, l'ensemble de ces travaux qui s'inscrivent dans le cadre de la gestion du risque « patuline » dans la filière fruit a permis d'amener des réponses tant sur le plan fondamental que sur le plan appliqué avec le séquençage du cluster, le développement d'un outil de diagnostic et la démonstration que l'ascladiol ne présentait aucune cytotoxicité. / Among diseases affecting apples, blue mold caused by Penicillium expansum is a major concern causing yield and quality losses due to the production of mycotoxins, of which patulin is the most alarming one. This mycotoxin was proven to be harmful for humans and animals. The pathogen growth and the patulin production occur under specific physico-chemical conditions (temperature, pH…). However, the description of these conditions in literature remains largely insufficient for the development of strategies to fight the development of the fungus. Furthermore, patulin remains, along with ochratoxin A, the only toxins for which the biosynthetic pathway is not fully established yet at both chemical and molecular levels. Firstly, this study provides supplementary data on the physico-chemical factors that modulate P. expansum growth and its ability to produce patulin. The acquaintance of these conditions leads, in practice, to the control of the patulin contamination along the food chain. Secondly, significant improvements were brought on the fundamental level, especially by elucidating the patulin biosynthetic pathway. The cluster of genes involved in the biosynthesis of this mycotoxin was fully identified and characterized in the species of greatest concern P. expansum. In order to reveal additional info on the biosynthesis of this mycotoxin, the specific factor of the pathway (patL) was characterized. The disruption of this gene has led to failure in patulin production and an important decrease in Pat genes expression. Furthermore, pathogenesis studies, using this same deficient strain showed that patulin potentially acts as a virulence factor during P. expansum development on apples. The last step of the patulin biosynthetic pathway was later characterized by site-directed mutagenesis of the patE gene in the same species. This gene encodes a Glucose Methanol Choline (GMC) oxidoreductase that is responsible for the conversion of ascladiol to patulin. Ascladiol is not only the last intermediate in the patulin pathway but also the main product of patulin degradation during the alcoholic fermentation of apple juice. The non-toxicity of ascladiol accumulated by the ΔpatE strain was proved against the human Caco-2 cell line. Finally a Real time PCR assay was developed to specifically detect and quantify P. expansum. This was done by targeting a highly specific gene from the patulin gene cluster in P. expansum, patF. This predictive approach allowed the quick estimation of the patulin content via the quantification of the P. expansum DNA in apples. To conclude, this thesis is part of the patulin's risk management study in the fruit sector; it provides significant improvements on both fundamental and practical levels. These advances are mainly characterized by the sequencing of the patulin gene cluster, the development of a molecular diagnostic tool and the demonstration of the non-cytotoxicity of ascladiol.

Natural products are bioactive secondary metabolites produced by living organisms and are prevalently utilized as pharmaceutical drugs. Marine adapted organisms are a promising source of new natural products possessing unique chemical structures and biological activities. By studying the biosynthetic pathways employed by living organisms to produce natural products, insights into new strategies to generate molecules to combat disease and overcome drug resistance may be gained. This thesis study aimed to uncover the biosynthetic pathway employed by a marine actinomycete, Nocardiopsis sp. CMB-M0232, to catalyze the assembly of the nocardioazines. These molecules are a group of 2,5-diketopiperazine natural products that feature structurally unique functional groups. Nocardioazine A, the hypothesized end product of the nocardioazine biosynthetic pathway, exhibits anticancer activity. Bioinformatics analyses revealed three biosynthetic gene clusters from Nocardiopsis encoding proteins with hypothesized roles in nocardioazine A biosynthesis. Two cyclodipeptide synthases (CDPSs), NozA and NcdA, were biochemically characterized in vivo and in vitro to reveal that both are substrate specific enzymes that utilize tryptophan-charged tRNA substrates to catalyze assembly of cyclo(L-Trp-L-Trp), a proposed precursor of nocardioazines. Fidelity is uncommon amongst characterized CDPSs, making NozA and NcdA important CDPS family additions. This study also aimed to characterize NozD and NozE, two cytochrome P450 homologs with predicted roles as diketopiperazine-tailoring enzymes. Heterologous expression of these enzymes in Streptomyces strains was not able to confirm the functions of NozD and NozE but set the stage for future studies to optimize conditions for probing their roles in nocardioazine A biosynthesis. The results gathered from this study, along with future work to better understand the engineering of unique functional groups from Nocardiopsis may provide opportunities to produce new bioactive molecules.

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Tumor-induced immunosuppression can occur by multiple mechanisms, each posing a significant obstacle to immunotherapy. Evidence presented in this dissertation suggests that aberrant cytokine signaling, as a result of altered metabolism of Chronic Lymphocytic Leukemia (CLL) cells, confers a selective advantage for tumor survival and growth. Cells from CLL patients with aggressive disease (as indicated by high-risk cytogenetics) were found to exhibit prolongation in Interferon (IFN)-induced STAT3 phosphorylation, and increased levels of reactive oxygen species (ROS) in these cells reflected these signaling processes. Changes in the relative balance of phospho-STAT3 and phospho-STAT1 levels, in response to combinations of IL-2 + Toll-like receptor (TLR)-7 agonist + phorbol esters, as well as IFN, were associated with the immunosuppressive and immunogenic states of CLL cells. In addition, immunosuppressive leukemic cells were found to express high levels of proteins with O-linked N-acetylglucosamine (O-GlcNAc) modifications, due to increased metabolic activity through the Hexosamine Biosynthetic Pathway (HBP), which caused impaired intracellular signaling responses and affected disease progression. A conclusion of the studies presented here is that the intrinsic immunosuppressive properties of leukemic cells may be overcome by agents such as Resveratrol that target metabolic pathways of these cells.

Tumor-induced immunosuppression can occur by multiple mechanisms, each posing a significant obstacle to immunotherapy. Evidence presented in this dissertation suggests that aberrant cytokine signaling, as a result of altered metabolism of Chronic Lymphocytic Leukemia (CLL) cells, confers a selective advantage for tumor survival and growth. Cells from CLL patients with aggressive disease (as indicated by high-risk cytogenetics) were found to exhibit prolongation in Interferon (IFN)-induced STAT3 phosphorylation, and increased levels of reactive oxygen species (ROS) in these cells reflected these signaling processes. Changes in the relative balance of phospho-STAT3 and phospho-STAT1 levels, in response to combinations of IL-2 + Toll-like receptor (TLR)-7 agonist + phorbol esters, as well as IFN, were associated with the immunosuppressive and immunogenic states of CLL cells. In addition, immunosuppressive leukemic cells were found to express high levels of proteins with O-linked N-acetylglucosamine (O-GlcNAc) modifications, due to increased metabolic activity through the Hexosamine Biosynthetic Pathway (HBP), which caused impaired intracellular signaling responses and affected disease progression. A conclusion of the studies presented here is that the intrinsic immunosuppressive properties of leukemic cells may be overcome by agents such as Resveratrol that target metabolic pathways of these cells.