Les analogues nucléosidiques constituent une famille importante d'agents thérapeutiques dans le traitement de maladies d'origine virale. Parmi ces composés, les nucléosides carbocycliques possèdent des propriétés biologiques intéressantes. Le premier chapitre de cette thèse est consacrée à la famille des neplanocines qui sont des carbonucleosides naturels. Nous avons détaillé l'interaction de ces composés avec leur principale cible, la S-adénosylhomocystéine hydrolase, ainsi que les différentes approches de synthèses de ces carbonucléosides et de leurs énantioméres avant de passer en revue leurs activités biologiques. Nous avons présenté dans le deuxième chapitre la première synthèse énantiosélective de la (éD)-néplanocine B. Le troisième chapitre est quant à lui axé sur la mise au point d'une synthèse de 3 '-halo-5'-norcarbonucléosides phosphonates ainsi qu’à l'évaluation de leurs activités antivirales. / Nucleosides analogues constitute an important family of therapeutic agents in the treatment of viral diseases. Among these compounds, carbocyclic nucleosides have interesting biological properties. The first chapter of this thesis is dedicated to a family of natural carbonucleosides, the neplanocins. We have presented their mode of action against S-adenosylhomocysteine hydrolase, as well as various syntheses of natural neplanocins and their enantiomers before reviewing their biological activities. In the second chapter, we described the first enantioselective synthesis of (¨D)-neplanocine B. The third chapter is devoted to the development of the synthesis of 3 '-halo-5¡¯-norcarbonucleosides phosphonates as well as the evaluation of their antiviral activities.

This thesis documents the subcellular localization and protein-protein interactions of two methyl recycling enzymes. These two enzymes, adenosine kinase (ADK) and S-adenosyl-L-homocysteine hydrolase (SAHH), are essential to sustain the hundreds of S-adenosyl-L-methionine (SAM)-dependent transmethylation reactions in plants. Both ADK and SAHH are involved in the removal of a competitive inhibitor of methyltransferases (MTs), S-adenosyl-L-homocysteine (SAH), that is generated as a by-product of the each transfer of a methyl group from SAM to a substrate. This research focused on understanding how SAH is metabolized in distinct cellular compartments to maintain MT activities required for plant growth and development. Localization studies using green fluorescent protein (GFP) fusions revealed that both ADK and SAHH localize to the cytoplasm and the nucleus, and possibly to the chloroplast, despite the fact that the primary amino acid sequence of neither protein contains detectable targeting signals. This suggested the possibility that these methyl-recycling enzymes may be targeted by specific protein-protein interactions. Moreover, deletion analysis of SAHH1 indicated that the insertion region (IR) of 41 amino acids (Gly150-Lys190), which is present only in plants and parasitic protozoan SAHHs among eukaryotes, is essential for nuclear targeting. This result suggested that the surface-exposed IR loop may serve as a binding domain for interactions with other proteins that may direct SAHH to the nucleus. To investigate protein-protein interactions, several methods were performed including co-immunoprecipitation, bimolecular fluorescence complementation, and pull-down assays. These results not only revealed that ADK and SAHH possibly interact through the IR loop of SAHH in planta, but also suggested that this interaction is either dynamic or indirect, requiring a cofactor/another protein(s) or post-translational modifications. Moreover, possible interactions of both ADK and SAHH with a putative Arabidopsis mRNA cap methyltransferase (CMT), which is localized predominantly in the nucleus, were also confirmed. These results support the hypothesis that the nuclear targeting of both SAHH and ADK can be mediated by the interaction with CMT. In addition, purification of Strep-tagged SAHH1 expressed in Arabidopsis identified a novel interaction between SAHH and aspartate-semialdehyde dehydrogenase (ASDH), an enzyme that catalyzes the second step of the aspartate-derived amino acid biosynthesis pathway. Analysis of ASDH-GFP fusions revealed that ASDH localizes to the chloroplast and the stromule-like structure that emanates from chloroplasts. Moreover the mutation in three amino acids (Pro164-Asp165-Pro166) located within the IR loop of SAHH disrupted its binding to ASDH which affected the plastid localization of SAHH, suggesting that the interaction between SAHH and ASDH is required for plastid-targeting of SAHH. Taken together, this thesis demonstrated that the localization of ADK and SAHH in or between compartments is possibly mediated by specific protein interactions, and that the surface-exposed IR loop of SAHH is crucial for these interactions.

This thesis documents the subcellular localization and protein-protein interactions of two methyl recycling enzymes. These two enzymes, adenosine kinase (ADK) and S-adenosyl-L-homocysteine hydrolase (SAHH), are essential to sustain the hundreds of S-adenosyl-L-methionine (SAM)-dependent transmethylation reactions in plants. Both ADK and SAHH are involved in the removal of a competitive inhibitor of methyltransferases (MTs), S-adenosyl-L-homocysteine (SAH), that is generated as a by-product of the each transfer of a methyl group from SAM to a substrate. This research focused on understanding how SAH is metabolized in distinct cellular compartments to maintain MT activities required for plant growth and development. Localization studies using green fluorescent protein (GFP) fusions revealed that both ADK and SAHH localize to the cytoplasm and the nucleus, and possibly to the chloroplast, despite the fact that the primary amino acid sequence of neither protein contains detectable targeting signals. This suggested the possibility that these methyl-recycling enzymes may be targeted by specific protein-protein interactions. Moreover, deletion analysis of SAHH1 indicated that the insertion region (IR) of 41 amino acids (Gly150-Lys190), which is present only in plants and parasitic protozoan SAHHs among eukaryotes, is essential for nuclear targeting. This result suggested that the surface-exposed IR loop may serve as a binding domain for interactions with other proteins that may direct SAHH to the nucleus. To investigate protein-protein interactions, several methods were performed including co-immunoprecipitation, bimolecular fluorescence complementation, and pull-down assays. These results not only revealed that ADK and SAHH possibly interact through the IR loop of SAHH in planta, but also suggested that this interaction is either dynamic or indirect, requiring a cofactor/another protein(s) or post-translational modifications. Moreover, possible interactions of both ADK and SAHH with a putative Arabidopsis mRNA cap methyltransferase (CMT), which is localized predominantly in the nucleus, were also confirmed. These results support the hypothesis that the nuclear targeting of both SAHH and ADK can be mediated by the interaction with CMT. In addition, purification of Strep-tagged SAHH1 expressed in Arabidopsis identified a novel interaction between SAHH and aspartate-semialdehyde dehydrogenase (ASDH), an enzyme that catalyzes the second step of the aspartate-derived amino acid biosynthesis pathway. Analysis of ASDH-GFP fusions revealed that ASDH localizes to the chloroplast and the stromule-like structure that emanates from chloroplasts. Moreover the mutation in three amino acids (Pro164-Asp165-Pro166) located within the IR loop of SAHH disrupted its binding to ASDH which affected the plastid localization of SAHH, suggesting that the interaction between SAHH and ASDH is required for plastid-targeting of SAHH. Taken together, this thesis demonstrated that the localization of ADK and SAHH in or between compartments is possibly mediated by specific protein interactions, and that the surface-exposed IR loop of SAHH is crucial for these interactions.

N-methylputrescine oxidase (MPO) is a member of the diamine oxidase (DAO) class of enzymes believed to be responsible for synthesis of the alkaloid nicotine in the roots of Nicotiana tabacum (Mizusaki et al., 1972). A purportedly pure MPO protein from tobacco root culture extracts was used to generate immune antiserum in rabbits (McLauchlan et al., 1993). In an attempt to clone a cDNA encoding MPO, we used this antiserum to screen a tobacco cDNA expression library. Unexpectedly, two previously unreported genes with strong homology to members of a gene family encoding S-adenosylhomocysteine hydrolase (SAHH) in N. sylvestris and a gene encoding SAHH in N. tabacum were cloned instead. SAHH is an enzyme of the S-adenosylmethionine (SAM) recycling pathway, which also includes SAM synthetase (SAMS) and methionine synthase (MS). These results led to the hypothesis of a multi-enzyme complex, or metabolon, of at least one member of the nicotine biosynthesis pathway, i.e., MPO, and at least one member of the SAM recycling pathway, i.e., SAHH, during nicotine biosynthesis. Metabolons are stable noncovalent complexes in cells that ensure sufficient passage of the product of one enzyme reaction to the next enzyme in the pathway via a "channel" without equilibrating with the bulk solution (Ovádi, 1991). My research employed co-immunoprecipitation studies to determine if other SAM recycling enzymes are associated in a complex with MPO and SAHH, as well as Northern and Western blot analyses to determine if the genes encoding SAM recycling pathway enzymes are coordinately regulated during nicotine biosynthesis. Our results indicate that nicotine biosynthesis-inducing conditions result in differential mRNA accumulation patterns of the three enzymes of the SAM recycling pathway, although to different extents. However, protein levels of SAM recycling pathway members do not appear to reflect the differential mRNA accumulation patterns. We have firmly established an association of SAHH and an enzyme with DAO activity, purportedly MPO. If the enzyme is proven to be MPO, then our data would constitute the first documentation of an alkaloid metabolon. Finally, using a degenerate primer PCR approach, we have cloned a 986-bp gene fragment with homology to copper amine oxidases, the class to which MPO belongs. / Master of Science

Alkaloids are a class of plant secondary metabolites produced in about 20% of plant families. Domesticated tobacco, Nicotiana tabacum produces nicotine as the predominant alkaloid. The biosynthesis of nicotine occurs exclusively in the roots of tobacco, yet accumulates in the leaves of tobacco where it is acts as a defense compound to deter insect herbivory. The research detailed in this dissertation addresses two aspects of nicotine physiology in tobacco: 1) an investigation of hypothesized protein-protein interactions among nicotine biosynthetic enzymes and 2) the characterization of a novel nicotine transporter. A hypothesized metabolic channel including the two nicotine biosynthetic enzymes putrescine N-methyltransferase (PMT), N-methylputrescine Oxidase (MPO) and the S-adenosylmethionine (SAM) recycling enzyme S-adenosylhomocysteine hydrolase (SAHH) has been proposed. To further explore this hypothesis, protein-protein interactions among nicotine biosynthetic enzymes PMT, MPO and SAHH were investigated using yeast two-hybrid assays and co-immunoprecipitation experiments. The yeast two-hybrid was conducted as both a directed screen to detect interactions between the hypothesized metabolic channel members and as a library screen to detect interactions between hypothesized metabolic channel members and proteins from a tobacco root cDNA library. Co-immunoprecipitation experiments were conducted using proteins produced in an in vitro transcription/ translation system and using native proteins from a tobacco root extract. The outcome of these experiments provided no further evidence of a nicotine metabolic channel and a discussion of the methods and outcomes of the experiments conducted is presented. The nicotine uptake permease, NUP1, was identified in tobacco roots and was shown to preferentially transport nicotine when expressed in Schizosaccharomyces pombe. This report presents the characterization of tobacco plants and hairy roots with diminished NUP1 transcripts created by using RNAi. The NUP1-RNAi hairy roots and plants showed a decreased level of nicotine and the hairy root cultures displayed an altered distribution of nicotine from the root to the culture medium. Additionally NUP1-GFP was used to determine that NUP1 localized to the plasma membrane of tobacco BY-2 protoplasts. Potential models for the role of NUP1 in nicotine physiology will be discussed. / Ph. D.