Room-temperature chemisorption of hexacyclic aromatic hydrocarbons on the 2x1, sputtered, oxidized and H-terminated Si(100) surfaces, as well as those upon post treatments of hydrogenation, oxidization and electron irradiation have been investigated by using thermal desorption spectrometry (TDS), Auger electron spectroscopy (AES) and low energy electron diffraction (LEED). This work focuses on the effects of the functional groups (phenyl, methyl, vinyl, heteroatom, and H atom) in the chemisorbed aromatic hydrocarbons (benzene, toluene, xylene isomers, styrene and pyridine) on organic functionalization of the Si(100) surface, particularly on such surface processes as cycloaddition, dative adsorption, hydrogen abstraction, desorption, dissociation, diffusion, and condensation polymerization. Unlike the earlier notion that hydrogen evolution in the hydrocarbon/Si(100) systems is the result of hydrocarbon dissociation (into smaller hydrocarbon fragments and H atoms) on the surface, condensation polymerization of the adsorbed aromatic hydrocarbons is proposed in the present work, in order to explain the higher-temperature hydrogen evolution feature in the toluene/Si(100) system. This hypothesis is supported by our TDS results for other hydrocarbon adsorbates, especially in the pyridine/Si(100) system where electron-induced condensation polymerization has been observed at room temperature. The improved techniques in the TDS experiments developed in the present work have enabled us to observe condensation polymerization and the effect of H on the surface processes (via surface reconstruction) on Si(100) for the first time. New analysis methods have also been developed to determine the adsorption coverage from the AES data, and this work has not only improved the accuracy of the elemental-coverage evaluation, but also provided a means to estimate the rate and the order of chemisorption. By using the density functional theory with the Gaussian 98 program, the adsorption geometries and the corresponding adsorption energies of various adsorption phases have been calculated. These computational results have provided useful insights into the chemisorption structures on the Si(100) surface. The present work also presents the development of three kinetics models for hydrogen evolution in the aforementioned aromatic-hydrocarbon systems on Si(100). Based on a modified collision theory with consideration of diffusion, these theoretical models have proven to be quite successful in simulating the observed TDS profiles and in estimating the kinetic parameters for the analysis of condensation polymerization in 2-dimensional diffusion systems. The present work illustrates that TDS experiments can be used effectively with quantum computation and theoretical kinetics modelling to elucidate the intricate nature of organosilicon surface chemistry.

I formally joined Prof. M. R. N. Murthy’s laboratory at the Molecular Biophysics Unit, Indian institute of Science, on 1st August 2001. During that time, the interest in the laboratory was mainly focused on structural studies on a number of capsid mutants of two plant viruses, sesbania mosaic virus and physalis mottle virus, to gain an insight into the virus structure and its assembly. Besides these two projects, there were a few other collaborative projects running in the lab at that time such as NIa protease from pepper vein banding virus and diaminopropionate ammonia lyase from Escherichia coli with Prof. H. S. Savithri, triosephosphate isomerase from Plasmodium falciparum with Prof. P. Balaram and Prof. H. Balaram and a DNA binding protein (TP2) with Prof. M. R. S. Rao. During my first semester, along with my course work, I was assigned to make an attempt to purify and crystallize recombinant NIa protease and TP2 protein. I started with NIa protease which could be purified using one step Ni-NTA affinity column chromatography. Although the expression and protein yield were reasonably good, protein precipitated with in a couple of hours after purification. Attempts were made to prevent the precipitation of the purified enzyme and towards this end we were successful to some extent. However, during crystallization trials most of the crystallization drops precipitated completely even at low protein oncentration. TP2 protein was purified using three-step chromatographic techniques by one of the project assistant in Prof. M. R. S. Rao’s laboratory. Because of low expression level and three step purification protocol, protein yield was not good enough for complete crystallization screening. Hits obtained from our initial screening could not be confirmed because of low protein yield as well as batch to batch variation. My attempts to crystallize these two proteins remained unsuccessful but in due course I had learnt a great deal about the tips and tricks of expression, purification and mainly crystallization. To overcome the problems faced with these two proteins, we decided to make some changes in the gene construct and try different expression systems. By this time (beginning of 2002), I had finished my first semester and a major part of the course work, so we decided to start a new project focusing on some of the unknown enzymes from a metabolic pathway. Dr. Parthasarathy, who had finished his Ph. D. from the lab, helped me in literature work and in finding targets for structural studies. Finally, we decided to target enzymes involved in the propionate etabolism. The pathways for propionate metabolism in Escherichia coli as well as Salmonella typhimurium were just established and there were no structural information available for most of the enzymes involved in these pathways. Since, propionate metabolic pathways were well described in the case of Salmonella typhimurium, we decided to use this as the model organism. We first started with the enzymes present in the propionate catabolic pathway “2-methylcitrate pathway”, which converts propionate into pyruvate and succinate. 2-methylcitrate pathway resembles the well-studied glyoxylate and TCA cycle. Most of the enzymes involved in 2-methylcitrate pathway were not characterized biochemically as well as structurally. First, we cloned all the four enzymes PrpB, PrpC, PrpD and PrpE present in the prpBCDE operon along with PrpR, a transcription factor, with the help of Dr. P.S. Satheshkumar from Prof. H. S. Savithri’s laboratory. Since these five proteins were cloned with either N- or C-terminal hexa-histidine tag, they could be purified easily using one-step Ni-NTA affinity column chromatography. PrpB, PrpC and PrpD had good expression levels but with PrpE and PrpR, more than 50% of the expressed protein went into insoluble fraction, probably due to the presence of membrane spanning domains in these two enzymes. Around this time, crystallization report for the PrpD from Salmonella was published by Ivan Rayment’s group, so after that we focused only on the remaining four proteins leaving out PrpD. Our initial attempts to crystallize these proteins became successful in case of PrpB, 2-methylisocitrate lyase. We collected a complete diffraction data to a resolution of 2.5 Å which was later on extended to a resolution of 2.1 Å using another crystal. Repeated crystallization trials with PrpC also gave small protein crystals but they were not easy to reproduce and size and diffraction quality always remained a problem. Using one good crystal obtained for PrpC, data to a resolution of 3.5 Å could be collected. Unfortunately, during data collection due to failure of the cryo-system, a complete dataset could not be collected. Further attempts to crystallize this protein made by Nandashree, one of my colleagues in the lab at that time, was also without much success. Attempts to purify and crystallize PrpE and PrpR were made by me as well as one of my colleagues, Anupama. In this case, besides crystallization, low expression and precipitation of the protein after purification were major problems. Our attempt to phase the PrpB data using the closest search model (phosphoenolpyruvate mutase) by molecular replacement technique was unsuccessful,probably because of low sequence identity between them (24%). Further attempts were made to obtain heavy atom derivatives of PrpB crystal. We could obtain a mercury derivative using PCMBS. However, an electron density map based on this single derivative was not nterpretable. Around this time, the structure of 2-methylisocitrate lyase (PrpB) from E. coli was published by Grimm et. al. The structure of Salmonella PrpB could easily be determined using the E. coli PrpB enzyme as the starting model. We also solved the structure of PrpB in complex with pyruvate and Mg2+. Our attempts to crystallize PrpB with other ligands were not successful. Using the structures of PrpB and its complex with pyruvate and Mg2+, we carried out comparative studies with the well-studied structural and functional homologue, isocitrate lyase. These studies provided the plausible rationale for different substrate specificities of these two enzymes. Due to unavailability of PrpB substrate commercially and the extensive biochemical and mutational studies carried out by two different groups made us turn our attention to other enzymes in this metabolic pathway. Since our repeated attempts to obtain good diffraction quality crystals of PrpC, PrpE and PrpR continued to be unsuccessful, we decided to target other enzymes involved in propionate metabolism. We looked into the literature for the metabolic pathways by which propionate is synthesized in the Salmonella typhimurium and finally decided to target enzymes present in the metabolic pathway which converts L-threonine to propionate. Formation of propionate from L-threonine is the most direct route in many organisms. During February 2003, we initiated these studies with the last enzyme of this pathway, propionate kinase (TdcD), and within a couple of months we could obtain a well-diffracting crystal in complex with ADP and with a non-hydrolysable ATP analog, AMPPNP. TdcD structure was solved by molecular replacement using acetate kinase as a search model. Propionate kinase, like acetate kinase, contains a fold with the topology βββαβαβα, identical with that of glycerol kinase, hexokinase, heat shock cognate 70 (Hsc70) and actin, the superfamily of phosphotransferases. Examination of the active site pocket in propionate kinase revealed a plausible structural rationale for the greater specificity of the enzyme towards propionate than acetate. One of the datasets of TdcD obtained in the presence of ATP showed extra continuous density beyond the γ-phosphate. Careful examination of this extra electron density finally allowed us to build diadenosine tetraphosphate (Ap4A) into the active site pocket, which fitted the density very well. Since the data was collected at a synchrotron source to a resolution of 1.98 Å, we could identify the ligand in the active site pocket solely on the basis of difference Fourier map. Later on, co-crystallization trials of TdcD with commercially available Ap4A confirmed its binding to the enzyme. These studies suggested the presence of a novel Ap4A synthetic activity in TdcD, which is further being examined by biochemical experiments using mass-spectrometry as well as thin-layer chromatography experiments. By the end of 2004, we shifted our focus to the first enzyme involved in the anaerobic degradation of L-threonine to propionate, a biodegradative threonine deaminase (TdcB). Sagar Chittori, who had joined the lab as an integrated Ph. D student, helped me in cloning this enzyme. My attempt to crystallize this protein became finally successful and datasets in three different crystal forms were collected. Dataset for TdcB in complex with CMP was collected during a synchrotron trip to SPring8, Japan by my colleague P. Gayathri and Prof. Murthy. TdcB structure was solved by molecular replacement using the N-terminal domain of biosynthetic threonine deaminase as a search model. Structure of TdcB in the native form and in complex with CMP helped us to understand several unanswered questions related to ligand mediated oligomerization and enzyme activation observed in this enzyme. The structural studies carried out on these three enzymes have provided structural as well as functional insights into the catalytic process and revealed many unique features of these metabolic enzymes. All these have been possible mainly due to proper guidance and encouragement from Prof. Murthy and Prof. Savithri. Prof. Murthy’s teaching as well as discussions during the course of investigation has helped me in a great deal to learn and understand crystallography. Collaboration with Prof. Savithri kept me close to biochemistry and molecular biology, the background with which I entered the world of structural biology. The freedom to choose the project and carry forward some of my own ideas has given me enough confidence to enjoy doing research in future.

Des ligands hydrides potentiellement bidentes (possédant un donneur N-hétérocyclique (NHC) associé à un groupement donneur éther ou amine) ainsi que des ligands tritopiques de type pinceur (possédant un groupement (NHC) flanqué de deux types de donneurs azotés différents Nimine et Namine) ont été préparés et utilisés pour la coordination de métaux de transition tels que le Ni, Cr, Cu et Ir. L’influence de la longueur de la chaine alkylée -(CH2)2- ou -(CH2)3- reliant le groupe éther ou amine au groupe hétérocyclique (NHC) a été examinée. Dans le but d’accéder aux complexes des métaux de transition différentes méthodologies ont été adoptées : a) déprotonation préalable du sel d’imidazolium suivie de l’addition des précurseurs métalliques correspondants ; b) transmétallation à partir des complexes (NHC) de l’argent correspondants ; c) réaction d’addition oxydante des sels d’imidazolium ou de leurs sels protonés avec du Ni(0). Une série de complexes du Ni(II) et du Cr(III) a été testée dans la réaction catalytique d’oligomérisation de l’éthylène. / Potentially bidentate hybrid ligands (containing a NHC donor associated with an ether or an amine) and tridentate NCN pincer-type ligands (containing a central NHC donor flancked by two chemically-different nitrogen donors (Nimine and Namine)) have been prepared and used for coordination to transition metals, such as Ni, Cr, Cu, Ir. The influence of the length of the alkyl chain, -(CH2)2- or -(CH2)3- connecting the ether or the amine group to the heterocycle NHC was examined. In order to have access to the transition metal complexes, several methodologies were adopted: a) deprotonation of the corresponding imidazolium salts followed by addition of transition metal precursors; b) transmetalation from NHC silver complexes; c) oxidative-addition reaction of Ni(0) with imidazolium salts or the corresponding protonated salts. A series of Ni(II), Cr(III) complexes were tested in the catalytic ethylene oligomerization reaction.

Os receptores ativadores da proliferação de peroxissomos (PPARs) pertencem à superfamília de receptores nucleares que funcionam como fatores transcricionais. Eles exercem um papel fundamental em processos que envolvem, principalmente, o metabolismo lipídico, em resposta à ativação por ligantes naturais e sintéticos como os ácidos graxos e os fibratos, respectivamente. A crescente descoberta de importantes funções fisiológicas, coordenadas pelos PPARs, e a necessidade de se conhecer como os agonistas, atualmente disponíveis, atuam nesses receptores, têm incitado pesquisas que vislumbram sua melhor exploração nos tratamentos de doenças metabólicas e inflamatórias, minimizando os efeitos adversos de ativações suprafisiológicas. Nesse cenário, o presente trabalho buscou compreender melhor as bases estruturais envolvidas nas funções atribuídas aos PPARs e explicar como as interações com seus ligantes ocorrem. Para isso, foram realizadas a subclonagem do domínio de ligação ao ligante do PPARα, sua expressão e purificação, seguidas de ensaios cristalográficos e biofísicos, além da abordagem de testes funcionais. Uma vez que a formação de oligômeros está relacionada à funcionalidade desses receptores, foram abordados estudos de oligomerização dos PPARs α e γ, compreendendo tanto o processo de homo- quanto o de heterodimerização. Os ensaios de cristalização do hPPARα LBD complexado a ligantes naturais e sintéticos, resultaram em estruturas cristalográficas que permitiram a identificação dos resíduos envolvidos no reconhecimento dos ligantes e a caracterização de sítios de ligação nunca antes descritos. A presença de ligantes nessas regiões afeta a conformação da proteína e, consequentemente, a modulação de sua função e o recrutamento da maquinaria transcricional. Adicionalmente, as estruturas cristalográficas da proteína complexada a ácidos graxos auxiliaram na compreensão de como essa importante classe de ligantes naturais possui efeitos farmacológicos similares aos de ligantes sintéticos. Esses resultados têm imediato impacto na procura racional de agonistas para esses receptores e se inserem em uma perspectiva de promoção do desenvolvimento científico-tecnológico na área de endocrinologia molecular. / The peroxisome proliferation-activated receptors (PPARs) belong to the nuclear receptors superfamily, acting as transcriptional factors. They play a key role in processes involving essentially lipid metabolism in response to activation by natural and synthetic ligands such as fatty acids and fibrates, respectively. The rising discovery of important physiological functions coordinated by PPARs and the necessity to know how the currently available agonists act on these receptors, have encouraged researches envisioning a better receptor exploration in the treatment of metabolic and inflammatory diseases, minimizing the adverse effects of supraphysiological activations. In this scenario, the present study aimed to better understand the structural basis involved in PPARs functions and elucidates how the interactions with their ligands takes place. For this, the ligand-binding domain of PPARα was subjected to subcloning, expression and purification steps, followed by crystallographical and biophysical assays, in addition to functional testing approaches. Since the degree of oligomerization is related to the functionality of these receptors, oligomeric studies of PPARs α and γ oligomerization were also achieved, comprising both homo- and hetero-dimerization. The co-crystallization assays of hPPARα LBD complexed with natural and synthetic ligands resulted in crystallographic structures that allowed the identification of residues involved in ligand recognition and the characterization of novel binding sites. The presence of ligands in these regions affects the conformation of the protein and thereby modulates their function and transcriptional machinery recruitment. Additionally, the crystallographic structures of the protein complexed to fatty acids were valuable for the understanding of how this important class of natural ligands has similar pharmacological effects to those of synthetic ligands. These results have direct impact on rational agonists design to these receptors and are inserted in a perspective of scientifical promotion and technological development in the field of molecular endocrinology.

A eletroquímica direta de enzimas redox depende da distância entre os sítios redox da proteína e a superfície do eletrodo e também da eficiência na imobilização dessas enzimas na superfície eletródica. Dessa forma, a obtenção de enzimas mais hidrofóbicas possibilita a melhora na interação entre elas e a superfície de eletrodos sólidos, como os de carbono. Neste estudo, foi desenvolvida uma rota para a obtenção da glicose oxidase oligomerizada (Ol-GOx) com o objetivo de melhorar a interação entre a enzima e a superfície de fibras de carbono, uma vez que enzimas oligomerizadas contêm suas porções hidrofóbicas expostas.Para tanto, diferentes ácidos de Brønsted foram utilizados, sendo que a enzima obtida a partir da reação com o ácido trifluorometanosulfônico (TFMS) foi a que se manteve ativa cataliticamente. A Ol-GOx se mostrou um biocatalisador promissor devido a sua hidrofobicidade e seu tamanho, os quais permitiram uma imobilização mais eficiente em superfícies de carbono. Após a caracterização estrutural, concluiu-se que a Ol-GOx é formada por um oligômero composto por 10 unidades de GOx nativa com raio hidrodinâmico de aproximadamente 96 nm. Por voltametria cíclica estudou-se a transferência direta de elétrons (TDE) entre o cofator dinucleotídeo de flavina e adenina (FAD) e a superfície das fibras de carbono, sendo observado um aumento de 7 vezes nas correntes faradaicas em relação ao obtido para a GOx nativa. Além disso, as propriedades bioeletrocatalíticas foram melhoradas em 30% quando analisada a oxidação da glicose. Concluiu-se ainda que quanto maior a quantidade de folhas-β presente na estrutura proteica, maior a TDE observada entre a enzima e a superfície das fibras de carbono. / The direct electrochemistry of redox enzymes is dependent on the distance between the active centers of the protein and the electrode surface, and also on the efficiency in the immobilization of these enzymes on the electrodic surface. Thus, the synthesis of more hydrophobic enzymes could lead to better interaction between the redox enzymes and the solid electrode surfaces, such as carbon electrodes. In this study, it was proposed a chemical route to obtain oligomerized glucose oxidase (Ol-GOx), aiming to improve the interaction between the enzyme and the surface of carbon fibers, since oligomerized proteins have their hydrophobic chains exposed. After structural characterization, it was concluded that Ol-GOx is formed by 10 dimeric units of native GOx with a hydrodynamic radius corresponding to approximately 96 nm. By cyclic voltammetry, it was studied the direct electron transfer (DET) between the flavin adenine dinucleotide (FAD) cofactor and the surface of carbon fibers, where it was observed an increase of 7-fold in the faradaic currents in comparison to that observed for native GOx. Besides, bioelectrocatalytic properties are 30% improved, when analyzed the glucose oxidation by cyclic voltammetry. It was also concluded that the greater the β-sheet content in protein structure, the higher the DET observed between the enzyme and the carbon fibers surface.

Dans cette thèse nous avons eu pour but d’étudier la réactivité des complexes à bas degré d’oxydation du fer stabilisés par des ligands phosphines vis-à-vis des substrats insaturés. Cet objectif s’inscrit dans une démarche plus large d’accès à des systèmes catalytiques au fer capables de transformer de manière sélective l’éthylène, par la réaction d’oligomérisation, vers des alpha-oléfines linéaires courtes (butène-1, héxène-1, octène-1), ce qui n’a jamais été rapporté dans la littérature. Pour se faire, le passage par le mécanisme métallacyclique de transformation de l’éthylène est la voie privilégiée. Cependant, les propriétés électroniques et géométriques de ligand requises pour suivre un tel mécanisme, et en particulier son étape clé de couplage oxydant de deux molécules d’éthylène sur le centre métallique, ne sont pas clairement identifiées. Nous rapportons ici dans un premier temps la synthèse d’une bibliothèque de complexes de fer(II) et fer(III) à base de ligands phosphines présentant des propriétés électroniques et géométriques variées. Ces nouveaux complexes sont opportunément testés en tant que catalyseurs, d’une part pour la réaction d’oligomérisation de l’éthylène; mais également en tant qu’espèces réduites in situ en association avec des diènes (isoprène, butadiène) conduisant à leur polymérisation. Afin de nous rapprocher davantage de notre objectif d’accès aux espèces à bas degré d’oxydation du fer, l’emploi d’une voie de réduction en une étape et en conditions douces est rapportée; conduisant à l’isolement et la caractérisation de neuf complexes phosphorés de fer(0) à 18 et à 16 électrons. Bien qu’a priori inactifs vis-à-vis de l’éthylène ou des oléfines plus longues, nous montrons la capacité de ces complexes à promouvoir des réactions de couplage oxydant de substrats insaturés (alcynes) le couplage catalytique de l’éthylène et du butadiène et l’activation de divers composés tels que les silanes ou les halogénures d’alkyles, posant ainsi un premier jalon vers leur optimisation et application en tant que catalyseurs d’autres réactions, et potentiellement dans l’avenir, pour la transformation de l’éthylène. / In this thesis our aim was to study the reactivity of phosphine-based low-valent iron complexes towards unsaturated substrates. This goal is part of a wider approach of access to an iron catalytic system able to transform ethylene in a selective manner, by the reaction of oligomerization, towards short linear alpha-olefins (1-butene, 1-hexene, 1-octene), which is unreported in the literature. To achieve this, going through the metallacyclic mechanism of ethylene transformation is the most likely way. However, the electronic and geometrical features of the ligand required to follow this mechanism, and in particular the key step of the oxidative coupling of two molecules of ethylene to the metal center, are ill-defined. We thus report here in a first part the synthesis of a library of P-based iron(II) and iron(III) complexes bearing varied electronic and geometrical features. These novel complexes are opportunistically screened as catalysts for ethylene oligomerization on one hand, and as in situ reduced species in association with dienes (isoprene, butadiene) leading to the polymerization of the latter substrates, on the other hand. In order to get closer to our goal of access to low-valent iron complexes, the use of a one-pot reduction methodology under mild conditions is reported, leading to the isolation and characterization of nine 18- and 16-electron iron(0) complexes. Even though they do not show reactivity towards ethylene or longer olefins, we demonstrate the ability of these complexes to promote reactions involving the oxidative coupling elementary step of unsaturated substrates (alkynes), the catalytic coupling of ethylene and butadiene and the activation of various compounds such as silanes and organic halides, representing a first milestone towards their optimization and application as catalysts for other reactions, including potentially in the near future, for ethylene transformation

L’intégrase (IN) du VIH-1 est une enzyme clé catalysant l’insertion de l’ADN proviral dans le génome cellulaire au sein d’un complexe nucléoprotéique d’intégration.Les nombreux efforts apportés à l’étude du mécanisme d’intégration ont permis d’accumuler de multiples données sur ce processus complexe. Cependant plusieurs questions essentielles demeurent sans réponses en particulier concernant l’architecture fonctionnelle des complexes d’intégration de VIH-1. En effet même si les complexes actifs pour l’intégration sont relativement bien définis leur chronologie précoce de formation demeure inconnue. De même les phases tardives de leur association au substrat naturel d’intégration que constitue la chromatine restent floues. Enfin le rôle des facteurs du complexe de préintégration (CPI) dans la régulation des ces mécanismes doit être déterminé.Mon travail de thèse s’est reposé sur trois axes s’articulant autour de ces questions:-Comment est régulée la dynamique des phases précoces d’attachement des oligomères d’intégrase sur les extrémités virales ?-Quel est l’impact de la structure de l’ADN cible et de la chromatine sur l’association active des complexes d’intégration ?-Quel(s) rôle(s) joue(nt) les facteurs du CPI dans ces phases ? / HIV-1 integrase (IN) is a key enzyme catalyzing the proviral DNA insertion into the cellular genome within a nucleoprotein integration complex.The numerous efforts made on the mechanism of integration have led to the accumulation of substantial data on this process. However many important questions are still open particularly on the functional architecture of the HIV-1 integration complexes. Indeed even if the active complexes involved in the catalysis of the integration are well described, the chronology of their early formation is still unknown. Moreover, the late association of these complexes with the host chromatin is also obscure. Finally the involvement of the IN cofactors inside the preintegration complex on these steps has to be elucidated.The project of my PhD relied on three axis articulated on these questions:-What is the dynamic of the IN oligomers association on the DNA viral ends?-What is the impact of the target DNA chromatin structure on the functional association of the integration complexes?-Involvement of the PIC factors on these steps?

L’Adrénoleucodystrophie liée à l’X est une maladie neurodégénérative rare due à des mutations dans le gène ABCD1. Ce gène code un demi-transporteur ABC peroxysomal, impliqué dans l’importation d’acides gras à très longue chaîne. Deux autres demi-transporteurs sont localisés dans la membrane peroxysomale : ABCD2 et ABCD3. La surexpression d’ABCD2 permet de compenser la déficience en ABCD1, ouvrant ainsi des perspectives thérapeutiques. Dans cette optique, l’objectif principal de ma thèse était d’étudier la fonction et la structure d’ABCD2, et plus largement des transporteurs ABC peroxysomaux.Les demi-transporteurs doivent au minimum se dimériser pour constituer un transporteur fonctionnel. Leur dimérisation alternative pourrait moduler leur spécificité de substrat. Afin de tester cette hypothèse, nous avons réalisé des constructions plasmidiques codant différents dimères chimériques, dont la fonctionnalité a été vérifiée par transfection transitoire dans deux modèles cellulaires (fibroblastes humains et levures). D’après nos résultats, ABCD1 et ABCD2 seraient fonctionnels quel que soit leur agencement dimérique. De plus, comme d’autres transporteurs ABC, les transporteurs ABC peroxysomaux pourraient s’oligomériser. En utilisant différentes techniques biochimiques (co-immunoprécipitation, sédimentation sur gradient de sucrose et électrophorèse en conditions natives), sur un modèle cellulaire surexprimant ABCD2-EGFP, nous démontrons qu’ABCD2-EGFP interagit avec ABCD1 et ABCD3, et que les transporteurs ABC peroxysomaux sont capables de s’oligomériser. Il reste désormais à déterminer les facteurs qui contrôlent cette oligomérisation et comprendre la valeur fonctionnelle de ces interactions. / X-linked Adrenoleukodystrophy (X-ALD) is a rare neurodegenerative disease caused by deficiency of the peroxisomal half-transporter ABCD1, implicated in very long chain fatty acids import. Two additional half-transporters are located in the peroxisomal membrane: ABCD2 and ABCD3. Over-expression of ABCD2 is known to compensate for ABCD1 deficiency, making ABCD2 a therapeutic target for X-ALD treatment. In this context, the main objective of my thesis was to investigate the function and the structure of ABCD2, and more broadly, of peroxisomal ABC transporters.Half-transporters must at least dimerize to form a functional transporter. Alternative dimerization could modulate substrate specificity. In order to test this hypothesis, we engineered plasmidic constructs encoding chimeric ABCD dimers, whose functionality has been evaluated by transient transfection in two cell models (human fibroblasts and yeasts). Our results show that, ABCD1 and ABCD2 are functional whatever their dimeric organization. Besides, like other ABC transporters, peroxisomal ABC transporters could oligomerize. By using a multi-technical approach (co-immunoprecipitation, velocity sucrose gradient and native polyacrylamide gel electrophoresis experiments) on stably transfected hepatoma cells expressing ABCD2-EGFP, we demonstrate that ABCD2-EGFP interacts with ABCD1 and ABCD3, and that peroxisomal ABC transporters oligomerize. The perspectives will consist in determining which factors control the oligomerization process and understanding the functional value of these interactions.

<p>Dimerization and oligomerization reactions of alkenes are promising catalytic strategies to convert light alkenes, which can be derived from light alkane hydrocarbons (ethane, propane, butane) abundant in shale gas resources, into heavier hydrocarbons used as chemical intermediates and transportation fuels. Nickel cations supported on aluminosilicate zeotypes (zeolites and molecular sieves) selectivity catalyze ethene dimerization over oligomerization given their mechanistic preference for chain termination over chain propagation, relative to other transition metals commonly used for alkene oligomerization and polymerization reactions. Ni-derived sites initiate dimerization catalytic cycles in the absence of external activators or co-catalysts, which are required for most homogeneous Ni complexes and Ni²⁺ cations on metal organic frameworks (MOFs) that operate according to the coordination-insertion mechanism, but are not required for homogeneous Ni complexes that operate according to the metallacycle mechanism. Efforts to probe the mechanistic details of ethene dimerization on Ni-containing zeotypes are further complicated by the presence of residual H⁺ sites that form a mixture of 1-butene and 2-butene isomers in parallel acid-catalyzed pathways, as expected for the coordination-insertion mechanism but not for the metallacycle mechanism. As a result, the mechanistic origins of alkene dimerization on Ni cations have been ascribed to both the coordination-insertion and metallacycle-based cycles. Further, different Ni site structures such as exchanged Ni²⁺, grafted Ni²⁺ and NiOH⁺ cations are proposed as precursors to the dimerization active sites, based on analysis of kinetic data measured in different kinetic regimes and corrupted by site deactivation, leading to unclear and contradictory proposals of the effect of Ni precursor site structures on dimerization catalysis.</p> <p> Dimerization of ethene (453 K) was studied on Ni cations exchanged within Beta zeotypes in the absence of externally supplied activators, by suppressing the catalytic contributions of residual H⁺ sites via selective pre-poisoning with Li⁺ cations and using a zincosilicate support that contains H⁺ sites of weaker acid strength than those on aluminosilicate supports. Isolated Ni²⁺ sites were predominantly present, consistent with a 1:2 Ni²⁺:Li⁺ ion-exchange stoichiometry, CO infrared spectroscopy, diffuse reflectance UV-Visible spectroscopy and <i>ex-situ</i> X-ray absorption spectroscopy. Isobutene serves a kinetic marker for alkene isomerization reactions at H⁺ sites, which allows distinguishing regimes in which 2-butene isomers formed at Ni sites alone, or from Ni sites and H⁺ sites in parallel. 1-butene and 2-butenes formed at Ni sites were not equilibrated and their distribution was invariant with ethene site-time, revealing the primary nature of butene double-bond isomerization at Ni sites as expected from the coordination-insertion mechanism. <i>In-situ</i> X-ray absorption spectroscopy showed that the Ni oxidation state was 2+ during dimerization, also consistent with the coordination-insertion mechanism. Moreover, butene site-time yields measured at dilute ethene pressures (<0.4 kPa) increased with time-on-stream (activation transient) during initial reaction times, and this activation transient was eliminated at higher ethene pressures (≥ 0.4 kPa) and while co-feeding H₂. These observations are consistent with the <i>in-situ</i> formation of [Ni(II)-H]⁺ intermediates involved in the coordination-insertion mechanism, as verified by H/D isotopic scrambling and H₂-D₂ exchange experiments that quantified the number of [Ni(II)-H]⁺ intermediates formed.</p> <p> The prevalence of the coordination-insertion cycles at Ni²⁺ cations provides a framework to interpret the kinetic consequences of the structure of Ni²⁺ sites that are precursors to the dimerization active sites. Beta zeotypes predominantly containing either exchanged Ni²⁺ cations or grafted Ni²⁺ cations show noteworthy differences for ethene dimerization catalysis. The deactivation transients for butene site-time yields on exchanged Ni²⁺ cations indicate two sites are involved in each deactivation event, while those for grafted Ni²⁺ cations indicate involvement of a single site. The site-time yields of butenes extrapolated to initial time, and then further extrapolated to zero ethene site-time, rigorously determined initial ethene dimerization rates (453 K, per Ni) that showed a first-order dependence in ethene pressure (0.05-1 kPa). This kinetic dependence implies the β-agostic [Ni(II)-ethyl]⁺complex to be the most abundant reactive intermediate for the Beta zeolites containing exchanged and grafted Ni²⁺ cations. Further, the apparent first-order dimerization rate constant was two orders of magnitude higher for exchanged Ni²⁺ cations than for grafted Ni²⁺ cations, reflecting differences in ethene adsorption or dimerization transition state free energies at these two types of Ni sites. </p> <p> The presence of residual H⁺ sites on aluminosilicate zeotypes, in addition to the Ni²⁺ sites, causes formation of saturated hydrocarbons and oligomers that are heavier than butenes and those containing odd numbers of carbon atoms. The reaction pathways on Ni²⁺ and H⁺ sites are systematically probed on a model Ni-exchanged Beta catalyst that forms a 1:1 composition of these sites <i>in-situ</i>. The quantitative determination of apparent deactivation orders for the decay of product space-time yields provides insights into the site origins of the products formed. Further, Delplot analysis systematically identifies the primary and secondary products in the reaction network. This strategy shows linear butene isomers to be primary products formed at Ni²⁺-derived sites, while isobutene is formed as a secondary product by skeletal isomerization at H⁺ sites. In addition, propene is formed as a secondary product, purportedly by cross-metathesis between linear butene isomers and the reactant ethene at Ni²⁺-derived sites. Also, ethane is a secondary product that forms by hydrogenation of ethene at H⁺ sites, with the requisite H₂ generated <i>in-situ</i> likely by dehydrogenation and aromatization of ethene at H⁺ sites.</p> <a>The predominance of the coordination-insertion mechanism at Ni²⁺-derived sites implies kinetic factors influence isomer distributions within the dimer products, providing an opportunity to influence the selectivity toward linear and terminal alkene products of dimerization. In the case of bifunctional materials, reaction pathways on the Ni²⁺ and H⁺sites dictate the interplay between kinetically-controlled product selectivity at Ni sites and thermodynamic preference of product isomers formed at the H⁺ sites. </a>In summary, through synthesis of control catalytic materials and rigorous treatment of transient kinetic data, this work presents a detailed mechanistic understanding of the reaction pathways at the Ni²⁺ and H⁺ sites, stipulating design parameters that have predictable consequences on the product composition of alkene dimerization and oligomerization.

The work focuses on new processes for the preparation of lactic acid derivatives. The main objective was to verify the feasibility of preparing high molecular weight polylactide using ethyl lactate as a precursor of lactide synthesis. Part of the work is devoted to the new ethyl lactate synthesis method. The experimental part of the thesis is divided into partial key steps on the way from the lactic acid to the high molecular polylactide. The preparation of anhydrous ethyl ester of lactic acid (EtLA) was solved in an innovative way using alcoholysis of the oligomeric lactic acid. A kinetic model for isothermal alcoholysis and equimolar reactants ratio was derived from this method. The ethyl lactate was oligomerized by transesterification into the low and high molecular weight oligomer with the help of newly found catalysts suitable for the reaction medium. Stannous lactate was used as catalyst for oligomerization of the low molecular weight polymer suitable for the preparation of lactide (Mn 1000 g.mol-1). Experimental polymerization of ethyl lactate into high molecular weight product in tens of kDa has been investigated with newly synthesized tetraethyllactoyl titanate, Ti(EtLA)4. The laboratory method was derived for the depolymerization of the oligomer into lactide. Optimal conditions found for lactidation are as follow: temperature 225 ° C, pressure 2 kPa, catalysis 0,05 mol% of stannous lactate (with respect to oligomer lactate units). The prepared lactide was refined to polymer grade purity by distillation and subsequent recrystallization from ethyl acetate and toluene. The method for the preparation of high molecular weight PLA through ROP polymerization of lactide has been optimized. By optimization, suitable catalyst concentration was found in combination with the polymerization temperature and the polymerization length. An equimolar mixture of Tin 2-ethylhexanoate and 1-decanol was used as the catalytic system. The optimal ROP conditions for achieving the maximum molecular weight and suppressed polymer coloration (yellowing) are: catalyst concentration 0,01 mol%, temperature 160 °C, and polymerization length 4 hours. PLA with molecular weight Mw= 447 ± 7,8 kg.mol-1 was prepared at these conditions and good repeatability of the result was achieved. The effect of naturally occurring lactide contaminants and their influence on the course and ROP result was experimentally verified on the optimized polymerization system. The purpose was to explore the effect of lactide contamination with water and ethanol as natural lactide contaminants. The results clearly confirm the orderly lower sensitivity of the polymerization system for the presence of ethanol as compared to water contamination. In the case of the presence of water, the course and the result of the polymerization in terms of both the conversion and achieved molecular weights are negatively affected, even when the water content is in the order of 0,001%. Conversely, the presence of ethanol has a positive effect on lactide conversion and polymer polydispersity. Lactides with an alcohol content of about one-tenth of a percent are suitable to reach molecular weights of PLA similar to commercially available products. This difference shoves a significant advantage in the proposed technology of preparing PLA from ethyl lactate, especially for easier purification of crude lactide into polymer grade purity.