Le glutamate est le principal neurotransmetteur excitateur du système nerveux central. Il agit notamment sur huit récepteurs métabotropiques (mGluR) qui sont des récepteurs couplés aux protéines G (RCPG) responsables de la modulation de la transmission synaptique. Les mGluR constituent des cibles de choix pour le traitement de maladies neurologiques, psychiatriques et neurodégénératives comme la schizophrénie, la dépression, ou la maladie de Parkinson. Aucun médicament agissant sur les mGluR n'existe à l'heure actuelle, mais plusieurs molécules sont en phase clinique pour différentes pathologies. L'objectif principal de mon travail de thèse a été d'étudier la dynamique structurale des mGluR, pour lesquels le mécanisme moléculaire d'activation reste mal connu. Ces récepteurs forment des homodimères constitutifs, dont chaque sous-unité possède un grand domaine extracellulaire qui lie le glutamate et un domaine transmembranaire responsable de l'activation des protéines G et où se fixent des modulateurs allostériques synthétiques. Une des étapes clé de l'activation serait la réorientation relative des deux domaines extracellulaires induite par le glutamate au sein du dimère. En développant tout d'abord une stratégie de marquage orthogonale des sous-unités de mGlu par fusion avec des enzymes suicide (SNAP-/CLIP-tag) combinée à une technique de transfert d'énergie par résonance de type Förster en temps résolu (TR-FRET), nous avons montré qu'en système hétérologue, les mGluR peuvent s'associer sous forme d'hétérodimères fonctionnels. De plus, nos expériences ont révélé une spécificité d'association au sein de la famille des mGluR : les sous-unités mGlu du group I, mGlu1 et mGlu5, peuvent former des hétérodimères entre elles, mais pas avec celles du groupe II et III, qui elles peuvent toutes s'associer entre elles. Puis nous avons fait évoluer la technologie précédente pour développer le premier biosenseur conformationnel de l'activation des mGluR. Nous avons ainsi identifié sur cellules vivantes les changements conformationnels nécessaires à l'activation du récepteur, et démontré que la variation de signal de FRET entre les deux sous-unités au sein du dimère correspondant au réarrangement relatif des domaines externes est corrélée avec l'état d'activation du récepteur. Nous avons ainsi confirmé le modèle d'activation des mGluR initialement proposé à partir des premières structures cristallines des domaines extracellulaires isolés. D'autre part, ce senseur permet de discriminer facilement les agonistes partiels des agonistes complets, et permet de mieux comprendre les mécanismes allostériques régulant l'activité au sein des mGluR (notamment le mode d'action des modulateurs allostériques positifs et négatifs qui se lient dans le domaine membranaire). Cette stratégie de senseurs conformationnels a également pu être appliquée à l'étude d'autres récepteurs membranaires (RCPG et récepteurs tyrosines kinases), et au développement de tests de criblage à haut débit. Enfin, nous nous sommes attachés à développer de nouveaux types de molécules ciblant les mGluR, en utilisant des anticorps simple domaine provenant de lamas. Ces ligands qui agissent sur de nouveaux sites d'activation à la surface des mGluR, représentent de nouvelles pistes pour développer de meilleures solutions thérapeutiques. / Glutamate is the main excitatory neurotransmitter in the central nervous system. It notably acts on eight metabotropic glutamate receptors (mGluR), which are G protein coupled receptor responsible for the modulation of synaptic transmission. mGluRs are promising pharmacological targets to treat neurological, psychiatric or neurodegenerative diseases such as depression, schizophrenia or Parkinson's disease. Unfortunately, so far, no drug acting at mGluR is accessible to patients, but several molecules are in clinical trials. The main objective of my thesis has been the study of the structural dynamics of mGluR, for which the molecular mechanism allowing activation are still poorly understood. These receptors are known to form constitutive dimers, with each subunit composed of a large extracellular domain which bind glutamate and a transmembrane domain responsible for G protein activation and where synthetic allosteric modulators bind. A key step in the activation process could be the relative reorientation of the two extracellular domains in the dimer upon glutamate binding. We first developed an orthogonal labeling method of each mGlu subunits by fusion with a suicide enzyme (SNAP-/CLIP-tag) that we combined with time-resolved Förster resonance energy transfer measurements to show that in a heterologous system, mGlu subunits can associate as strict and functional heterodimers. Our experiments also revealed a specific association pattern: mGlu subunits from group I, mGlu1 and mGlu5, can associate with each other, but not with those from group II and III, which can also associate with each other. Then we improved the technology to develop the first conformational sensor to monitor mGluR activation. We were able to monitor in real time in live cells the conformational changes occurring in the mGlu receptor upon activation, and we proved that the variation in FRET signal is correlated with the activation state of the receptor. This allowed us to confirm the activation model proposed based on the crystal structures of the isolated extracellular domains, which consist of a relative movement of the dimer extracellulair domains upon activation. Moreover, this sensor makes it possible to easily discriminate between full and partial agonists, and to better understand the allosteric mechanisms occurring in the mGluR (especially the action mode of positive and negative allosteric modulators binding in the transmembrane domain). This conformational sensor strategy was further applied to study the activation of other receptors (GPCR or tyrosine kinase receptors), and to develop screening assays compatible with high-throughput formats. Finally, we developed innovative ligands acting on mGluRs using single-domain antibodies from llamas. These activating ligands seem to bind to a new site on the surface of the receptor, offering new possibilities to develop better treatment acting at mGluRs.

The first chapter will provide an introduction to the physiology, pathogenesis and biology of mycobacteria. Host-pathogen interactions, different modes of resistance of the bacteria, adaptations for survival under nutrient and oxygen depleted conditions has been discussed. This is followed by a general discussion on gene expression and regulation in the microbe. The physiology of bacteria under stresses from the view of the transcriptional regulation of specific genes has also been discussed. The scope and objective of the present study in M. smegmatis covered in the thesis has been considered at the end. The next chapter discusses the characterization of msdps promoter in vivo with the help of reporter gene assay technologies. With the advent of promoterless E. coli-mycobacterium shuttle vectors, activity assays can be easily performed to characterize unknown upstream putative promoter sequences of genes. Both the 1 kb upstream as well as a 200bp upstream region of msdps gene has been characterized by. Primer extension analysis and subsequent site directed mutagenesis studies reveal +1 transcription start site and the promoter consensus sequence for the msdps gene respectively. Next chapter comprises of the method of constructing heterologous in vitro transcription machinery in mycobacteria. It is followed by characterization of transcription initiation at two dps promoters of M. smegmatis. A novel pull-down assay has been designed which enabled us to identify the sigma factors in the reconstituted RNA polymerases to be associated with the respective dps promoters and to compare the regulation of the two genes at transcription level. Further characterization through single round in vitro transcription at mycobacterial promoters has been attempted. The following two chapters provide some newer insights into the structure-function relationship of the first Dps molecule, MsDps (MsDps1) with respect to its DNA binding activity. The DNA binding activity is associated with the higher oligomeric form only. With the help of time resolved anisotropy and Förster Resonance Energy Transfer (FRET) experiments, we have monitored the nature of Dps dodecamer-DNA complex and mapped the distance between the N and C169 position in the absence and the presence of DNA. A new computational programme, Maximum Entropy Method (MEM) has been applied successfully to analyze data obtained from phase-modulation (Phi-M) lifetime experiments in order to get distribution of lifetime. In the last chapter a new method is adopted to predict amino acids important for stabilizing the interface in a trimeric structure. Subsequently, single and double amino acid mutants of the native MsDps protein has been constructed through site directed mutagenesis and are scored for the ability of the mutants to oligomerize under conditions similar to that of the native protein. This helped us to propose a hypothetical model of the overall mechanism of the protein oligomerization process in solution.

Biological membranes are heterogeneous two-dimensional fluids composed of lipids, sterols and proteins that act as complex gateways and define the cell boundary. The functions of these interfaces are diverse and specific to individual organisms, cell types, and tissues. Membranes must take up nutrients and small molecules, release waste products, bind ligands, transmit signals, convert energy, sense the environment, maintain cell adhesion, control cell migration, and much more while forming a tight barrier around the cell. The molecular mechanisms and structural details responsible for this diverse set of functions of biological membranes are still poorly understood, however. Developing new tools capable of probing and determining the local molecular organization, structure, and dynamics of membranes and their components is critical for furthering our knowledge about these important cellular processes that are often linked to health and diseases. Combinatorial microscopy takes advantage of the rich properties of light (intensity, wavelength, polarization, etc.) to create new forms of imaging that quantify the motions, orientations, and binding kinetics of the sample’s biomolecular constituents. These new optical imaging modalities can also be further combined with other types of microscopy to produce spatially correlated micrographs that provide complementary pieces of information about the sample under investigation that would otherwise remain hidden from the observer if the two imaging techniques were applied independently. The first part of this thesis provides a detailed account of the construction of a specialized hybrid microscopy platform that combines polarized total internal reflection fluorescence microscopy (pTIRFM) with atomic force microscopy (AFM) for the purpose of studying fundamental sterol-lipid and antimicrobial peptide-lipid interactions in model membranes. The second half describes a combined pTIRFM and Förster resonance energy transfer (FRET) imaging method to elucidate the oligomeric state and spatial distribution of carcinoembryonic-antigen-related cell-adhesion molecules (CEACAMs) in the membranes of living cells.

Biological membranes are heterogeneous two-dimensional fluids composed of lipids, sterols and proteins that act as complex gateways and define the cell boundary. The functions of these interfaces are diverse and specific to individual organisms, cell types, and tissues. Membranes must take up nutrients and small molecules, release waste products, bind ligands, transmit signals, convert energy, sense the environment, maintain cell adhesion, control cell migration, and much more while forming a tight barrier around the cell. The molecular mechanisms and structural details responsible for this diverse set of functions of biological membranes are still poorly understood, however. Developing new tools capable of probing and determining the local molecular organization, structure, and dynamics of membranes and their components is critical for furthering our knowledge about these important cellular processes that are often linked to health and diseases. Combinatorial microscopy takes advantage of the rich properties of light (intensity, wavelength, polarization, etc.) to create new forms of imaging that quantify the motions, orientations, and binding kinetics of the sample’s biomolecular constituents. These new optical imaging modalities can also be further combined with other types of microscopy to produce spatially correlated micrographs that provide complementary pieces of information about the sample under investigation that would otherwise remain hidden from the observer if the two imaging techniques were applied independently. The first part of this thesis provides a detailed account of the construction of a specialized hybrid microscopy platform that combines polarized total internal reflection fluorescence microscopy (pTIRFM) with atomic force microscopy (AFM) for the purpose of studying fundamental sterol-lipid and antimicrobial peptide-lipid interactions in model membranes. The second half describes a combined pTIRFM and Förster resonance energy transfer (FRET) imaging method to elucidate the oligomeric state and spatial distribution of carcinoembryonic-antigen-related cell-adhesion molecules (CEACAMs) in the membranes of living cells.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Sous l'effet du stress oxydatif, la production des espèces réactives de l'oxygène (ERO) est accrue. Les ERO peuvent réagir avec différentes molécules biologiques dont les acides gras polyinsaturés libres ou provenant des lipides membranaires engendrant la formation d'hydroperoxydes d'acides gras. Dans le chloroplaste, ces molécules sont sujettes à de nombreuses modifications enzymatiques ou chimiques aboutissant à la formation d'oxylipines. Les oxylipines participent à la signalisation cellulaire (précurseurs de la voie du jasmonate par exemple), à la défense de la plante (activité antimicrobienne par exemple). Certaines de ces molécules très réactives et toxiques doivent être métabolisées en des produits moins réactifs, par des réactions d'oxydoréduction par exemple.La ceQORH (chloroplast envelope Quinone OxydoReductase Homolog) et IEP32 (Inner Envelope Protein 32) d'Arabidopsis thaliana sont des oxydoréductases chloroplastiques impliquées dans la détoxification de la plante. Elles sont transportées au travers de l'enveloppe du chloroplaste sans clivage de leur peptide de transit par une voie d'import alternative à la voie TOC/TIC (Translocon at the Outer envelope membrane of Chloroplasts et Translocon at the Inner envelope membrane of Chloroplasts). Ces particularités ont fait de la ceQORH et d'IEP32 des enzymes intéressantes à étudier.IEP32 a été purifiée et cristallisée. Les structures de la ceQORH sous forme apo, liée au NADPH et liée au NADP+ et à des inhibiteurs ont été déterminées en utilisant la cristallographie aux rayons X. Leur analyse a permis de mettre en évidence que la ceQORH existe sous différents états oligomériques dans les conditions expérimentales utilisées. Ces observations ont été confirmées par des expériences d'ultracentrifugation analytique. La ceQORH est une enzyme monomérique qui lie le NADPH par l'intermédiaire de son domaine de Rossmann . Le site catalytique, large et hydrophobe, permet à la ceQORH d'accommoder et de réduire un grand nombre de substrat dont le motif commun est la présence d'une fonction alcène en α, β d'un groupement carbonyle. Les constantes d'efficacité et d'affinité étant meilleures pour les γ-kétols que pour les quinones, nous proposons que la ceQORH soit renommée « γ-kétol réductase ».Mots clefs : chloroplaste - Medium chain dehydrogenase/reductase - γ-kétol réductase - oxylipines - oligomérisation - inhibition - cristallographie aux rayons X - ultracentrifugation analytique / Under the influence of the oxidative stress, the production of the Reactive Oxygen Species (ROS) is increased. These compounds can react with various biological molecules like free polyunsaturated fatty acids or derived from lipids producting hydroperoxides of fatty acids. In chloroplast, these molecules are subject to numerous enzymatic or chemical modifications resulting in oxylipins. Oxylipins participate in cell signaling (precursors of the way of the jasmonate for example), or in the defense of the plant (antimicrobial activity). However some of them are very reactive and toxic so they are metabolized in less reactive molecules by oxidoreduction reactions.The ceQORH (chloroplast envelope Quinone OxydoReductase Homolog) and IEP32 (Inner Envelope Protein 32) of Arabidopsis thaliana are chloroplast oxidoreductases involved in the plant detoxification. They are transported through the envelope of the chloroplast without cleavage of their transit peptide by an alternative import pathway of TOC/TIC (Translocon at the Outer envelope membrane of Chloroplasts and Translocon at the Inner envelope membrane of Chloroplasts). In order to better understand their roles, we studied their enzymatic properties and structures.IEP32 was purified and crystallized. The structures apo-ceQORH and bound to the NADPH/ NADP + with inhibitors were determined using X-ray crystallography. The analysis allowed us to show that ceQORH exists under different oligomerization states what was confirmed by results of analytical ultracentrifugation. The NADPH binding to the Rossmann fold induces the monomerization. The catalytic site is large and hydrophobic allowing to ceQORH to reduce many α, β-unsaturated carbonyls of various chain lengths. CeQORH was shown to reduce with high efficiency the reactive double bond of γ-ketols that's why we propose to rename ceQORH by “γ- ketol reductase”.Keywords: chloroplast – Medium chain dehydrogenase/reductase – γ-ketol reductase – oxylipins – oligomerization state – inhibition – X-ray crystallography – analytical ultracentrifugation

L'oligomérisation enzymatique de la rutine et esculine a donné lieu à cinq fractions d'oligomères de masse moléculaire moyenne entre 2127,42 et 8331,85 g/mol pour la rutine et 688,12 et 6973 g/mol pour l'esculine. L'analyse de ces fractions par FTIR montre que les fractions d'oligorutines sont obtenues à travers des liaisons C-C, C-O et C=O. Les fractions d'oligoesculines sont obtenues à travers des liaisons C-C. Une meilleure solubilité des oligorutines et des oligoesculines dans l'eau et une plus faible solubilité de ces oligomères dans l'éthanol comparé à leurs monomères a été mis en évidence. Une diminution de l'activité antiradicalaire vis-à-vis de DPPH., ABTS+. et OH. proportionnelle à la masse moléculaire moyenne des fractions d'oligorutines a été observé, contrairement aux fractions d'oligoesculines qui montrent un important pouvoir chélateur de ces mêmes radicaux comparé à leurs monomère. Une augmentation du pouvoir chélateur de fer, inhibiteur de la xanthine oxydase, réducteur du cuivre (CUPRAC), de l'activité antigénotoxique, ainsi que de l'activité stimulatrice de la prolifération des splénocytes, et des lymphocytes (B et T) proportionnelle au degré d'oligomérisation des oligomères étudiées a été noté. L'effet des fractions d'oligorutines et oligoesculines étudiées sur les macrophages en suivant la production de monoxyde d'azote (NO) montre un pouvoir anti-inflammatoire comparé à leurs monomères. L'étude de l'activité lysosomale induite par les fractions d'oligorutine révèle un pouvoir immunostimulateur proportionnelle à la masse moléculaire moyenne des oligorutines, et inversement proportionnelle à celle-ci pour les oligoesculines / Rutin and esculin have been polymerized by laccase. Five fractions with between 2127.42 and 8331.85 g/mol for oligorutins, and between 688.12 and 6973 g/mol for oligoesculins, were obtained. Fourier transformed infrared analysis showed that oligorutins were formed through C-C, C-O and C=O linkages, while oligoesculins were obtained through C-C linkages. Oligorutins and oligoesculins show a higher solubility in water and a lower solubility in ethanol compared to their monomers. The oligomerization of rutin decrease its antiradical capacity, while oligoesculin fractions demonstrated a high antiradical activity compared to monomeric esculin. Oligomer fractions showed a better iron chelating power, xanthine oxidase inhibition, copper reducing power (CUPRAC), antigenotoxic activity, and splenocytes stimulator activity compared to their monomers. Oligorutin and oligoesculin exhibited an important anti-inflammatory capacity through the nitric oxide inhibition. Moreover, oligorutin fractions demonstrated an immunostimulatory effect proportional to their degree of oligomerization, while oligoesculin fractions showed an immunostimulatory effect inversely proportional to their degree of oligomerization

L’objectif de ce travail fut la synthèse de ligands fonctionnels de type N,N'-diphosphanyl-NHC (NHC = carbènes N-hétérocycliques) et l’étude de leur chimie de coordination. La synthèse du nouveau ligand tridentate, stable et rigide, N,N'-diphosphanyl-imidazol-2-ylidene a permis des études expérimentales et théoriques et l’accès à des complexes mono-, di-, tri-, penta-, et hexanucléaires des métaux du groupe 11 (Cu, Ag et Au) originaux et aux propriétés structurales uniques. Les complexes mono- et dinucléaires avec un ou deux atomes de phosphore libres ont permis d’accéder à des complexes hétérotrinucléaires à interactions d10-d10 qui sont luminescents. La transmétallation partielle ou totale des complexes homotrinucléaires de Cu ou d’Ag avec des réactifs contenant du Pd(0) ont conduit à des complexes hétérotrinucléaires à interactions d10-d10. En plus de son comportement pontant, ce ligand peut se agir en chélate dans des complexes du palladium et du chrome. Dans le cas du Cr(III), ils montrent une activité catalytique en oligomérisation de l’éthylène supérieure à celle des complexes du Cr(II) et conduisent principalement à des oligomères. / The purpose of this work was the synthesis of N,N'-diphosphanyl-functionalized NHC ligands andtheir coordination chemistry. The novel stable and rigid tridentate N,N'-diphosphanyl-imidazol-2-ylidene was synthesized and experimental and computational information on its stability weregained. It served as a unique platform for the synthesis of novel mono-, di-, tri-, penta-, hexanuclear complexes with the coinage metals (Cu, Ag and Au), exhibiting rare structural features. The mono- and dinuclear complexes with one or two dangling P-donors provided rational access to heterotrinuclear complexes. All these coinage metal complexes have short metal-metalseparations, indicating the presence of d10-d10 interactions, and display excellent luminescentproperties. Partial or complete transmetallation of the homotrinuclear Cu or Ag complexes withPd(0) precursors led to hetero-trinuclear complexes with d10-d10 interactions. In addition to itsbridging behavior, this ligand also showed its chelating behavior in Pd or Cr(III) complexes. Thelatter displayed superior performance in ethylene oligomerization than the Cr(II) complexes andgave mostly oligomers.

Dans la nature, les plantes sont constamment exposées à des modifications de leur environnement générant un stress oxydant auquel elles doivent s’adapter du fait de leur immobilité. Les végétaux ont donc développé un grand nombre de mécanismes antioxydants permettant de protéger leurs fonctions vitales. L’étude d’une enzyme de type peroxyrédoxine (PRX) localisée dans le chloroplaste montre que son activité antioxydante est importante pour la croissance et la tolérance des végétaux aux contraintes environnementales. De plus, cette PRX pourrait interagir avec d’autres protéines impliquées notamment dans des mécanismes antioxydants et dans le métabolisme du carbone. Parmi elles, une protéine de type fibrilline participant à la protection des structures photosynthétiques, pourrait avec la PRX protéger la photosynthèse, fonction propre aux végétaux et essentielle pour la vie sur terre. / In nature, plants are constantly exposed to environmental changes leading to oxidative stress, to which they must adapt due to their immobility. Plants have developed many antioxidant systems allowing them to maintain their vital functions. The study of a peroxiredoxin, enzyme (PRX) localized in chloroplasts, shows that its direct antioxidant activity is essential for growth and tolerance of plants to environmental constraints. In addition, this PRX could interact with other proteins especially involved in antioxidant mechanisms and carbon metabolism. Among them, fibrillin proteins, which participate in the protection of the photosynthetic structures, could preserve with the PRX the plant photosynthesis, which is essential for the life on earth.

The DNA binding proteins under starvation (Dps) from M. smegmatis are cage-like structures which internalize iron and bind DNA. They provide resistance to the cells from free radical damage, and physically protect the DNA from the harmful effects of reactive oxygen species by DNA compaction. The work compiled in this thesis has been an effort to study oligomerization and dynamics of iron metabolism by these nano-protein compartments. Chapter 1 gives a general introduction on stress, especially oxidative stress, and the ways bacteria fight back the host resistance systems. This has been elaborated from the point of view of the Dps proteins which is the focus of our work. Also, the competition for iron among the host and pathogens, and the modes of iron trafficking of the pathogens from host organisms has been summarized. Finally, the structural aspects of ferritin family proteins to which Dps belongs, has been discussed. Chapter 2 elaborates on the oligomerization pathways of the first M. smegmatis Dps MsDps1, which exists in vitro as two oligomeric forms. The GFP-tagging has been used to locate the Dps1 proteins by live cell imaging and the over-expression of these proteins during nutrient limiting conditions has been studied. The crystal structure of a point mutant F47E in the background of MsDps1, which shows no dodecamerization in vitro, has been solved. The possible ways of dodecamerization of MsDps1 has been concluded by analyzing the intermediates via glutaraldehyde cross-linking and native electrospray mass spectrometry. Chapter 3 documents the gating machinery of iron in MsDps2 protein, the second M. smegmatis Dps protein. Through graph theoretical approaches, a tight histidine-aspartate cluster was identified at the ferritin-like trimeric pore which harbors the channel for the entry and exit of iron. Sitespecific variants of MsDps2 were generated to disrupt this ionic knot, and the mutants were further assayed for ferroxidation, iron uptake and iron release properties. Our studies in MsDps2 show the importance of counter-acting positive and negatively charged residues for efficient assimilation and dispersion of iron. Chapter 4 describes crystallization studies of MsDps2 pore variants, done in an attempt to connect the changes in functional properties described in chapter 3, with structural alterations of the point mutants. We show here that the gating mechanism happens by alterations in side chain configuration at the pore and does not alter the over-all stability of the proteins. Chapter 5 is the final section where we have employed site specific mutations and cocrystallization studies to elucidate the behaviour of MsDps2 proteins upon the addition of iron. By studying the effect of substitutions at conserved sites near ferroxidation center, we attempt to arrive at a pathway which iron atoms take to reach the ferroxidation site. Also, by crystallization of proteins loaded with varying amounts of iron we tried to map the changes in the protein structure in the presence of its ligand. Chapter 6 concludes briefly the work that has been documented in this thesis. Appendix I relates the role of N-terminal tail for DNA binding in MsDp2. Appendix II gives the technical details of a modified protein preparation and oligomerization process for his-tagged MsDps1 protein. Appendix III gives the maps of the plasmids used in this study.