Les protéines de la famille des AAA+ ATPases sont présentes dans de nombreux complexes moléculaires. Ces protéines sont capables de s’assembler en anneaux héxamériques (homomères ou hétéromères) pour former des moteurs moléculaires. Au cours de ma thèse, je me suis particulièrement intéressé à deux complexes macromoléculaires à AAA+ ATPases présentant un grand intérêt thérapeutique contre différents cancers : la particule régulatrice du protéasome 26S et l’hélicase du réplisome, Mcm2-7. Le protéasome 26S est la principale machinerie moléculaire impliquée dans la dégradation régulée des protéines poly-ubiquitinées tandis que l’hélicase mcm 2-7 est responsable du désappariement des brins de l’ADN chromosomique lors de la réplication de l’ADN. Ces deux complexes comprennent un anneau hétérohéxamérique de sous-unités AAA+ ATPases appelé Rpt1 à Rpt6 dans le cas du protéasome 26S et Mcm2 à Mcm7 dans le cas de l’hélicase mcm2-7. J’ai focalisé mes travaux sur l’étude du rôle du chaperon Hsm3/S5b dans l’assemblage du protéasome 26S d’une part, et le rôle spécifique de la sous-unité Mcm2 dans le transfert intergénérationnel des histones d’autre part. Le chaperon Hsm3/S5b se lie avec la sous-unité Rpt1. L’étude des complexes de levure Hsm3-Rpt1 et humain S5b-Rpt1 par cristallographie aux rayons X m’a permis de proposer que le chaperon d’Hsm3/S5b pourrait jouer un rôle de médiateur entre les sous-unités Rpt1, Rpt2 et Rpn1 lors de l’assemblage de la particule régulatrice. De plus, ce chaperon pourrait jouer également un rôle d’inhibiteur pour l’assemblage entre la particule régulatrice 19S et la particule cœur 20S du protéasome 26S. Certaines sous-unités AAA+ ATPase, telles que celles du réplisome, possèdent des domaines additionnels, leur conférant un rôle secondaire spécifique et indépendant de leur rôle principal de moteur moléculaire. C’est le cas de Mcm2, qui lie les histones H3-H4 par son domaine N-terminal. J’ai mis en évidence et caractériser cette interaction par différentes techniques biophysiques, en particulier la cristallographie aux rayons X, la RMN et le SEC-MALS. Ces résultats m’ont permis de proposer un modèle pour le transfert intergénérationnel des histones dans lequel Mcm2 joue un rôle crucial de chaperon moléculaire des histones directement intégré dans la machinerie de réplication. / AAA+ ATPases are involved in numerous molecular complexes. These proteins form homomeric or heteromeric hexamers and constitute molecular motors. During my Ph. D., I focused my work on two macromolecular complexes composed of AAA+ ATPases: the 26S proteasome regulatory particle and the Mcm2-7 helicase of the replisome. These complexes are implicated in the development of cancers and constitute interesting therapeutic targets. The 26S proteasome is the main machinery responsible for the regulated degradation of poly-ubiquitinated proteins and the helicase Mcm2-7 is responsible for the unwinding of the DNA during replication. These two complexes are composed of a heterohexameric ring of six AAA+ ATPases called Rpt1 to 6 for the 26S proteasome regulatory particle and Mcm2 to 7 for the replisome. I have studied the role of Hsm3/S5b in the assembly mechanism of the proteasome and the specific role of the subunit Mcm2 in the intergenerational transfer of the epigenetic information. X-ray structures of the complexes Hsm3-Rpt1 and S5b-Rpt1 allowed us to elucidate the dual functions of the assembly chaperone Hsm3/S5b which mediates the assembly of the subcomplex Rpt1-Rpt2-Rpn1 during the assembly of the regulatory particle. In addition, hsm3/S5b inhibits the association of a premature regulatory particle onto the core particle and protects the HbYX motif of Rpt1. Other AAA+ ATPases, like the replisome subunits, possess additional domains which confer specific roles. I also studied the interaction between the N-terminal domain of Mcm2 and the tetrameric form of histones H3-H4 by several methods like X-ray crystallography, NMR and SEC-MALS. I propose a model of the intergenerational transfer of histones H3-H4 in which Mcm2 plays a crucial role of molecular histones chaperone directly integrated in the replication machinery.

Dans les matériaux de batterie, le contrôle de la mobilité des cations lithium est la clé pour repousser les limites de la puissance et des vitesses de charge des batteries. La spectroscopie RMN permet de mesurer les coefficients d’auto-diffusion des espèces porteuses d’un spin nucléaire en utilisant des gradients de champ pulsés qui mesurent le déplacement atomique sur une échelle de 1-2 μm. La relaxation des spins nucléaires `a des champs magnétiques élevés, d’autre part, est régie par les fluctuations des interactions à des fréquences proches de la fréquence de Larmor (fluctuant donc à l’échelle de la nanoseconde), et celles-ci sont généralement liées à des mouvements atomiques sur 1 Å - 1 nm. Dans cette thèse, nous avons mesuré les coefficients d’auto-diffusion et les temps de relaxation du ⁷Li pour deux électrolytes polymères: LiTFSI dans de l’oxyde de polyéthylène (PEO) et dans un copolymère à blocs PS-PEO(LiTFSI)-PS. Les temps de relaxation ont été mesurées à trois champs magnétiques élevés (4.7, 9.4 et 17.6 Tesla) nous permettant d’effectuer une étude simple par relaxométrie des mouvements du Li+ à l’échelle de la nanoseconde. Afin de reproduire l’ensemble des vitesses de relaxation, il est apparu nécessaire d’introduire un modèle simple à deux temps de corrélation. Ceci a montré pour la première fois que la dynamique de lithium dans PS-PEO(LiTFSI)-PS est ralentie par la présence de domaines de PS par rapport au PEO pur avec des longueurs de chaîne similaires. Les résultats sont analysés et comparés à d’autres études basées sur des modèles de dynamique moléculaire ou de diffusion dans les polymères. Une deuxième série d’électrolytes polymères en gel à base de poly(fluorure de vinylidène-co-hexafluoropropylène (PVDF-HFP), PEGM (Poly-éther méthylique d’éthylèneglycol méthacrylate)/PEGDM (Poly diméthacrylate d’éthylèneglycol), et LiTFSI dans les liquides ioniques ont également été étudiés. L’ajout de polymères oxygénés permet d’augmenter la rétention des liquides ioniques, mais ralentit la diffusion expliquant ainsi la baisse de performance de ces batteries à vitesse de charge élevée. / In battery materials, the mobility of lithium cations is the key to the limitations in battery power and charging rates. NMR spectroscopy can give access to self-diffusion coefficients of spin bearing species using pulsed field gradients which measure atomic displacement over 1 − 2μm length scales. The relaxation of nuclear spins at high magnetic fields, on the other hand, is governed by fluctuations of NMR interactions resonant with the Larmor frequency, at the nanosecond timescale, and these are usually related to atomic motions over 1 Å - 1 nm. In this thesis, we recorded self-diffusion coefficients and ⁷Li relaxation rates for two polymer electrolytes: LiTFSI in polyethylene oxide (PEO) and in a block-copolymer PS-PEO(LiTFSI)-PS. We first investigated the effect of magic-angle spinning (MAS) on diffusion and relaxation, showing that MAS can help retrieve coefficient diffusion when relaxation is fast and diffusion is slow, and second, that lithium motion is not perturbed by the partial alignment of PEO under MAS induced pressure. The relaxation rates of 7Liwere measured at three high magnetic fields (4.7, 9.4 and 17.6 Tesla) allowing us to perform a simple relaxometry study of Li+motion at the nanosecond timescale. In order to reproduce the transverse and longitudinal relaxation behaviors, it proved necessary to introduce a simple model with two correlation times. It showed for the first time that the lithium dynamics in PS-PEO(LiTFSI)-PSis slowed down by the presence of PS domains compared to the pure PEO with similar chain lengths. The results are analyzed and compared to other studies based on molecular dynamics or physical models of diffusion in polymers. A second series of gel polymer electrolytes based on poly(vinylidene fluoride-co-hexafluoropropylene (PVdF-HFP), PEGM(Poly ethyleneglycol methyl ether methacrylate)/PEGDM (Poly ethyleneglycol dimethacrylate), and LiTFSI in ionic liquids were also studied. Adding oxygenated polymers to increase the retention of ionic liquids slowed the diffusion down and explained why the battery performance was degraded at higher charging rates.

La zone d'enrobage des aciers des structures en béton armé est soumise au cours de la vie de l'ouvrage à l'action de divers agents agressifs (ions chlorure, dioxyde de carbone, etc.) qui sont susceptibles d'entraîner la corrosion des armatures. Dans le cadre de la maintenance de l'ouvrage, une réparation du béton dégradé est généralement imposée afin d'assurer l'intégrité et la sécurité de la structure, et d'en allonger la pérennité. Cette étude s'attache à explorer les transferts hydriques qui limitent l'efficacité de la réparation au cours de l'hydratation du mortier de réparation. L'évolution microstructural et les profils hydriques sont obtenus par des techniques non-destructives comme l'imagerie résonance magnétique (IRM) et la gamma-densimétrie (GD) depuis très jeune âge à 28 jours. La durabilité du béton réparé est concernée. L'effet du séchage est évité dans cette étude. Le mortier isolé d'une même formulation est préparé pour la comparaison du comportement d'hydratation avec le mortier de réparation. Des diverses techniques classiques aident de compléter les résultats obtenus par IRM et GD. Donc le système de réparation est désigné. Les transferts hydriques à l'interface sont visualisés pendant la réparation (0 - 28 jours), afin de permettre une exploration en profondeur sur les mécanismes des couplages physico-chimiques. La réparation plus efficace donc est déterminée par cette méthodologie, en étudiant divers matériaux de réparation, divers états de support (saturé ou séché) et divers conditions environnementaux, etc. De plus, après la réparation (1 - 2 mois), la porosité totale est mesurée par GD ou la porosimétrie par l'intrusion de mercure (PIM). La distribution poreuse est aussi examinée par PIM selon la hauteur du système de réparation. Les indicateurs de durabilité (Cl- et CO2) après la réparation (> 2 mois) présentent l'empêchement de pénétration des ions chlorures et un effet non-évident pour la pénétration du dioxyde de carbone. L'évolution est suivie au fur et à mesure pendant la pénétration, afin d'explorer l'efficacité de la réparation qui s'adresse à une vie de service prolongée. Cette méthodologie pourra être appliquée aussi sur des autres systèmes, où il existe des transferts hydriques. Par exemple, une couche de protection en Béton Fibré à Ultra-haute Performance (BFUHP) sur le béton ordinaire. En conclusion, cette méthodologie en combinant des techniques non-destructifs et destructifs, est un outil d'étudier le système de réparation par un moyen systématique et quantitatif. C'est intéressant de comparer des divers cas. L'efficacité de réparation est étudiée afin d'assurer une durabilité à long terme / The coating area of steel reinforced concrete structures is subjected during service life time to various aggressive agents (carbon dioxide, chloride ions, etc.), which causes corrosion of steel rebars. Concerning the maintenance, repair works of degraded concrete cover are generally imposed to ensure its integrity and structural safety, and to extend long-term durability. This research aims in exploring moisture transfers which limit the efficiency of repair work during mortar hydration. Microstructure evolution and water profiles are obtained by non-destructive techniques such as Magnetic Resonance Imaging (MRI) and Gamma-Ray Attenuation (GRA) since casting until 28 days. The durability of repaired concrete is involved after the repair procedure. The effect of drying is avoided in this research. Sealed mortar of the same formula is prepared to compare its hydration performance with the repair mortar. Various conventional techniques help to supplement the results obtained by MRI and GRA. The configuration of designed repair systems is shown. Moisture transfers at the interface between the repair mortar and the old concrete are investigated during the repair procedure (0 - 28 days), which allows exploring the mechanisms of physico-chemical couplings. Efficient repair work can be determined for various repair materials, various substrates (initially-saturated or initially-dried), various environmental conditions, etc. Furthermore, total porosity is measured by GRA or by Mercury Intrusion Porosimetry (MIP) after the repair procedure (1 - 2 months). Pore size distribution (PSD) is also investigated by MIP at different positions within the repair systems. Durability indicators (Cl- and CO2) after the repair procedure (> 2 months) present a prevention of chloride penetration and a non-evident influence on carbonation. Evolution is followed during penetration, in order to explore repair efficiency during prolonged service life time. This methodology could also be applied on various systems where exist moisture transfers. For example, a protective layer of Ultra High Performance Fiber-Reinforced Concrete (UHPFRC) on conventional concrete. In conclusion, this methodology is a tool to investigate the repair systems in a systematic and quantitative way, by combining non-destructive and destructive techniques. It is interesting to compare aforementioned systems. Repair efficiency is investigated in order to ensure a long-term durability

La clausine est une bactériocine sécrétée par la souche probiotique Bacillus clausii. Elle est active contre des bactéries multi-résistantes aux antibiotiques, comme le MRSA (Staphylococcus aureus résistant à la méticilline) et le VRE (Enterococcus résistant à la Vancomycine). Nous avons produit, purifié, caractérisé la structure de la clausine et effectué des expériences d'interaction par Résonance Magnétique Nucléaire (RMN) du liquide. La clausine s'est révélée être un nouveau peptide maturé de la famille des lantibiotiques. Il est composé de trois ponts thioéthers ou lanthionines, d'un pont aminovinyle et de trois résidus deshydratés. La clausine possède une structure tri-dimensionnelle bi-modulaire, similaire à la fois à la nisine et à la mersacidine. Comme ces dernières, elle s'est révélée se lier aux lipides bactoprénols, tels que le lipide II (LII) et l'UndécaprénylPyroPhosphate (UPP), empêchant ainsi la formation de la paroi bactérienne. Nous nous sommes ensuite focalisés sur le mode d'action de la clausine sur des modèles de membranes bactériennes. Dans ce but, nous nous sommes basés sur la composition phospholipidique de trois bactéries affectées par la clausine : Staphylococcus aureus, Micrococcus Luteus et Bacillus Megaterium. Nous avons utilisé la RMN du solide du 2H et du 31P pour caractériser l'effet de la clausine sur la dynamique membranaire, ainsi que l'éllipsométrie et l'épifuorescence avec le LII-dansylé pour visualiser son effet sur des monocouches lipidiques. La clausine seule a entraîné une désorganisation membranaire à fort ratio peptide/lipide et formé des domaines. Nous avons souligné l'importance de la tête polaire des bactoprénols sur la dynamique lipidique. Nous avons finalement observé un effet synergique de la clausine avec les bactoprénols, suggèrant la formation de domaines « raft-like » organisés en réseau avec l'UPP ou en fibre avec lelipide II. Nos résultats in vitro permettent d'expliquer les divisions cellulaires aberrantes observées in vivo par un mécanisme de séquestration des bactoprénols par les peptides lantibiotiques. / Clausin is a bacteriocin secreted by the probiotic strain Bacillus clausii. It is active against multi-resistant bacteria to antibiotics, such as MRSA (Methicillin-Resistant Staphylococcus Aureus) and VRE (Vancomycin-Resistant Enterococcus).We have produced, purified, characterized the structure of Clausin and performed binding studies with Liquid-state Nuclear Magnetic Resonance (NMR). Clausin has been shown to be a new mature peptide from the lantibiotic family. It consists of three thioether rings called lanthionines, an aminovinyl ring and three dehydrated residues. Clausin displays a bi-modular three-dimensional structure similar to nisin as well as to mersacidin. It binds as well to bactoprenol lipids, such asLipid II (LII) and UndecaprenylPyroPhosphate (UPP), blocking then bacterial cell wall formation.We have then focused our attention to the action mode of Clausin on bacterial membrane models. For this purpose, we have used the phospholipid composition of three bacteria affected by Clausin: Staphylococcus aureus, Micrococcus Luteus and Bacillus Megaterium. We have used 2H and 31P Solid-state NMR for characterizing the Clausin effect on lipid dynamics, as well as ellipsometry and epifluorescence with dansylated-LII for visualizing its effect on lipid monolayer. Clausin alone has led to membrane desorganization at a high peptide/lipid ratio and formed domains. We have further underlined the importance of the polar head-group of bactoprenols on lipid dynamics. We have finally observed a synergic effect of Clausin with bactoprenols on lipid dynamics suggesting the formation of « raft-like » domains organized in network with UPP or evenfiber with LII. Our in vitro results allow us to explain the aberrant bacterial cell division observed in vivo through a mechanism of bactoprenol sequestration by lantibiotic peptides.

Le sujet de cette thèse concerne les études structurales d'une nouvelle génération d'oligomères, les foldamères, construits à partir de mimes pseudopeptidiques capables d'adopter des structures secondaires déterminées. Trois familles d'oligomères ont été étudiées. Plusieurs méthodes spectroscopiques ont été à chaque fois employées couplées à la modélisation moléculaire, afin de caractériser les propriétés structurales des foldamères. La première famille a été synthétisée par le groupe de Muriel Amblard et Monique Calmès de l'IBMM à Montpellier. Nous avons évalué les propriétés à se structurer du motif (S)-ABOC. Plusieurs oligomères de tailles variables ont été synthétisés et nous avons montré le motif (S)ABOC lorsqu'il est introduit dans une séquence peptidique est capable de générer des coudes beta. L'enchaînement de ces coudes conduits à la formation d'hélices qui en fonction des motifs pseudo-peptidiques associés présentent des caractéristiques différentes. La seconde famille étudiée comprenait des oligomères formés à partir de monomères contraints de type thiazole. Nous avons montré que ce motif, développé par Ludovic Maillard à l'IBMM à Montpellier forme des pseudocycles en C9 qui s'organisent ensuite en une hélice qui est observable dès le tétramère. La troisième famille d‘oligomères étudiée est issue du LCPM. Elle comprend des oligomères alternant des acides alpha-aminés et des acides aminés de type aza dans lesquels le carbone alpha a été remplacé par un atome d'azote. Nous avons montré que le motif aza induit un repliement en coude beta facilitant la cyclisation de la molécule. Dans ma thèse nous avons caractérisé les propriétés à s'auto-structurer de trois familles de foldamères pseudopeptidiques en combinant les résultats expérimentaux issus de la résonance magnétique nucléaire, la spectroscopie infrarouge, le dichroïsme circulaire et la diffraction des rayons X et en les intégrant à des calculs de modélisation moléculaire. Notre étude répertorie les propriétés de chaque motif à former des éléments de structure secondaire et à induire la formation de foldamères / The subject of this thesis deals with the structural studies of a new generation of oligomers, so called foldamers, constructed from pseudopeptidic mimics and able to adopt determined secondary structures. Three families of oligomers were investigated. For each oligomer several spectroscopic methods have been used combined with molecular modeling in order to characterize their structural properties. The first family was studied in collaration with the group of Muriel Amblard and Monique Calmès at IBMM in Montpellier. We have evaluated the structural properties of the motif (S)-ABOC. Several oligomers of varying sizes were synthesized and we have shown that the pattern (S)-ABOC, when introduced into a peptide sequence, is capable of generating beta turn. The sequence of these turn leads to the formation of helices, which, according to associated pseudo-peptide patterns shows different characteristics. The second project discussed the conformation of oligomers family formed from constrainted thiazole monomers. We have shown that this pattern developed by Ludovic Maillard at IBMM in Montpellier adopted pseudocycles C9 which led to helices. THis property was observed starting from the tetramer. The third family of oligomers comes from the LCPM, alternating oligomers of alpha amino acids and aza amino acids, in which the alpha carbon has been replaced by a nitrogen atom. We showed that the pattern aza induces folding of the beta turn, facilitating the cyclization of the molecule. In this thesis we have characterized the properties to self-structuring of three families of pseudopeptide foldamers by combining experimental results from nuclear magnetic resonance, infrared spectroscopy, circular dichroism and X-ray diffraction combined with molecular modeling calculations. Our study lists the properties of each pattern to form secondary structure elements and to induce the formation of foldamers

La thèse porte sur le développement d’une méthodologie IRM originale (sonde RMN de surface) permettant l’imagerie haute résolution et quantitative de l’eau dans la membrane polymère, un des constituants principaux des piles à combustible de type PEMFC. Les sondes radiofréquences développées sont dédiées à (1) l’étude du coefficient d’auto-diffusion de l’eau dans une membrane soumise à une contrainte de traction et à (2) l’imagerie en temps réel de la distribution de l’eau dans une membrane soumise à différentes conditions en humidité. Les mesures indiquent que l’anisotropie de diffusion de l’eau est intimement liée à l’anisotropie de la structure du polymère. L’observation de la progression des fronts d’humidités dans la membrane suggère que la limitation au transfert de masse est due aux résistances d’interfaces de la membrane mais surtout à la relaxation du polymère lors de l’adsorption de l’eau / This work focuses on the development of MRI/NMR methods dedicated to the characterization of water transport properties in a polymer membrane used in PEMFC. Radiofrequency coils are used to (1) study water self-diffusion coefficient in uniaxially stretched membranes and (2) spatially and temporally resolved measurements of the water distribution through the membrane. These measurements provide information about the influence of the polymer structure on water transport properties and also on real-time water distribution through the membrane in various humidity conditions. The water diffusion anisotropy is intimately linked to the structural order of the polymer. The steady-state and transient profiles of water content through the membrane indicate that mass transfer is limited by the interfacial resistances and mainly by the polymer relaxation during sorption

Le béton de chanvre est obtenu par le mélange d'un granulat végétal, constitué de bois de chanvre, et d'un liant. Il confère au mur de bonnes propriétés d'isolation thermique et acoustique, ainsi qu'une régulation de l'humidité. Cependant, des problèmes de non-prise, qui semblent fortement liés aux transferts d'eau dans le béton au cours des premières heures, peuvent parfois se produire. La RMN permet de quantifier l'eau dans le chanvre et dans le liant et donc de décrire et comprendre les transferts au cours de la prise. Nous montrons d'abord que, dans le liant constitué d'un mélange de chaux hydraté et de ciment, la chaux accélère la prise du ciment. Cette prise est d'autant plus inhibée que la quantité de chanvre au contact du ciment est importante. Nous montrons également que l'absorption d'eau par le granulat de chanvre s'étale sur trois jours, et qu'elle se produit en deux phases successives correspondant à l'imprégnation de deux zones du chanvre. L'étude des transferts dans le béton pendant la prise révèle une absorption d'eau rapide par le chanvre initialement, puis un transfert vers le liant. L'étude d'un béton de chanvre "modèle" nous permet d'associer ce transfert au retrait chimique du ciment au cours de la prise / Hemp concrete results from the mix of a vegetal aggregate (hemp shives) and a binder. It provides thermal and acoustic insulation to the wall, as well as a good moisture regulation. However, problems sometimes occur during setting, that seem strongly linked to transfers of water in the concrete in the first hours. NMR allows to quantify water in hemp and in the binder and thus to describe and understand the transfers during setting. We first show that, in the binder consisting of a mixture of hydrated lime and cement, lime accelerates cement hydration. This hydration is inhibited as the amount of hemp in contact with the cement increases. We also show that the absorption of water by hemp shives is takes place during three days, and it occurs in two successive phases corresponding to two areas of hemp that imbibe. The study of transfers in the concrete during the setting shows a rapid water absorption by the hemp initially, followed by a transfer to the binder. The study of a "model" hemp concrete allows us to associate this transfer to the chemical shrinkage of cement during hydration

Orientador: Jarbas José Rodrigues Rohwedder / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-22T12:58:01Z (GMT). No. of bitstreams: 1 Killner_MarioHenriqueMontazzolli_D.pdf: 3491134 bytes, checksum: 684c66e65646bff8d702f6275096bfa6 (MD5) Previous issue date: 2013 / Resumo: No presente trabalho foram desenvolvidos novos métodos analíticos empregando espectroscopia NIR e de RMN H, visando o monitoramento on-line da reação de transesterificação de óleos vegetais para a produção de biodiesel e determinação de parâmetros de qualidade de diesel combustível através da espectroscopia de RMN H. Empregando a espectroscopia NIR foi utilizada pela primeira vez a região espectral compreendida entre 5928 e 5959 cm para a determinação on-line, por meio de um modelo de regressão multivariada, das taxas de conversão dos triglicerídeos provenientes de óleo de soja em ésteres de ácidos graxos (biodiesel). O modelo desenvolvido foi avaliado no monitoramento de duas reações de transesterificação conduzidas a 20 ± 0,2 °C e 55 ± 0,2 °C que apresentaram erros médios de previsão para um conjunto de validação de 0,6 e 1,0% de conversão, respectivamente. De maneira similar foram desenvolvidos outros 3 métodos analíticos empregando um espectrômetro de RMN H de bancada de baixo campo para a determinação das taxas de conversão dos triglicerídeos provenientes do óleo de semente colza em ésteres metílicos de ácidos graxos. O primeiro método utiliza as áreas dos picos dos prótons metoxílicos dos ésteres de ácidos graxos e dos prótons etilênicos da cadeia de ácidos graxos para a determinação direta das taxas de conversão dos triglicerídeos. Um segundo método utiliza um modelo de regressão PLS para a determinação das taxas de conversão. Finalmente, o terceiro método emprega os desvios químicos dos prótons hidroxílicos do metanol e glicerol presentes na fase oleica para determinar as taxas de conversão dos triglicerídeos em ésteres. Outro importante resultado alcançado durante o desenvolvimento do trabalho foi o entendimento da partição das moléculas dos alcoóis (metanol e glicerol) entre as duas fases que constituem o meio reacional durante o processo de transesterificação. Este fato é importante para compreender melhor o mecanismo de reação de produção de biodiesel, uma vez que a taxa de reação depende da quantidade de metanol e catalisador dissolvidos inicialmente na fase oleica. A possibilidade de identificar os valores da partição dos alcoóis ao final da reação possibilita também o desenvolvimento de técnicas de separação que permitam otimizar a recuperação de biodiesel e metanol em uma planta de produção de biodiesel, refletindo diretamente na redução dos custos. Modelos de calibração PLS empregando espectros de RNM H de baixo campo também foram desenvolvidos para a determinação de diversos parâmetros de qualidade de diesel combustível B5, como: Massa Específica, Curva de Destilação para 10%, 50%, 85% e 90% de recuperação, Número de Cetano e Ponto de Fulgor. Os espectros foram adquiridos sem qualquer tipo de preparo de amostra ou utilização de padrão interno. Todos os modelos desenvolvidos apresentaram valores de RMSEP inferiores às reprodutibilidades das técnicas analíticas empregadas atualmente como referência pelas normas ASTM e ABNT NBR. A técnica de RMN H de baixo campo foi empregada também para a determinação do Teor de Biodiesel nas amostras de diesel combustível, apresentando um erro relativo médio de 2,5% em relação a técnica analítica de referência / Abstract: The present work reports on the development of new analytical methods, based on NIR and H NMR spectroscopy, aiming at on-line monitoring of the transesterification reaction of vegetable oils to produce biodiesel, and the determination of quality parameters of diesel fuel applying H NMR spectroscopy. The NIR spectral region between 5928 and 5959 cm-1 was used for the first time for on-line determination, employing a multivariate model, of the conversion ratio of triglycerides from the soybean oil into fatty acid methyl esters (biodiesel). The developed model was evaluated on the transesterification reaction monitoring of two new reactions carried out at 20 ± 0.2 °C and 55 ± 0.2 °C which showed RMSEP of 0.6 and 1.0 %, respectively. In a similar way 3 analytical methods were developed using a bench-size low-field NMR H spectrometer to determine the triglycerides (rapeseed oil) conversion ratios into fatty acid methyl esters. The first method applies the area of the methoxylic protons peak of fatty acid methyl esters and the ethylenic protons peak of fatty acid chain for direct determination of the triglycerides conversion ratios. A second method applies a PLS regression model to determine the conversion ratio. finally, a third method applies the hydroxylic protons chemical shift of methanol and glycerol present in the oleic phase to correlate the conversion ratio of triglycerides into esters. Another important result obtained during the development of this work was the comprehension of partitioning of alcohol molecules (methanol and glycerol) between the two phases that constitute the reactional medium during biodiesel production process. This fact is relevant to understand the reaction mechanism of the biodiesel production, because the reaction rate strongly depends on the methanol and catalyst amount initially dissolved in the oleic phase. Also, the possibility of identification of the alcohols partitioning values at the end of the reaction process helps in the development of new separation techniques to optimize the biodiesel and methanol recovering in a biodiesel producing plant, directly influencing the process costs. PLS regression models applying the low-field H NMR spectra were also developed for the determination of different quality parameters of diesel fuel (B5), such as: Specific Gravity, Distillation Curve (10%, 50%, 85% e 90% of recovering), Cetane Number and Flash Point. The spectra were acquired without adding any reference compound or sample pre-treatment. All regression models developed showed RMSEP values lower than the reproducibility of the analytical techniques used as reference by ASTM and ABNT NBR. The low-field H NMR technique were also used to determine the biodiesel content in diesel fuel samples, showing a mean relative error of 2.5% in comparison to the standard analytical technique / Doutorado / Quimica Analitica / Doutor em Quimica

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-07-13T14:09:49Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Zenaide S Monte - Tese Doutorado - Defesa 15-jul-2016.pdf: 10509605 bytes, checksum: 9a625f96607c0ccae6d58b896ba38772 (MD5) / Made available in DSpace on 2017-07-13T14:09:49Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Zenaide S Monte - Tese Doutorado - Defesa 15-jul-2016.pdf: 10509605 bytes, checksum: 9a625f96607c0ccae6d58b896ba38772 (MD5) Previous issue date: 2016-07-15 / CAPES / Compostos heterocíclicos do tipo pirimidina são conhecidos pela enorme riqueza de seus potenciais biológicos e farmacológicos. Numerosas reações de heterociclização que permitem a obtenção dessa classe de compostos são igualmente reconhecidas, devido à diversidade de atividades e do conhecimento prévio de inúmeras viabilidades sintéticas. As modificações estruturais obtidas na busca da otimização das rotas sintéticas conferem às novas moléculas diferentes propriedades físicas e alteram a reatividade das moléculas, acarretando em mudanças na distribuição nas células e nos tecidos. Essas discretas modificações estruturais podem revelar efeitos biológicos que haviam estado latentes ou eclipsados pelos efeitos colaterais do composto protótipo. Sendo assim, o presente trabalho apresenta a síntese por método convencional dos derivados da pirimidina 3a-q e 5o-q. Estes compostos foram avaliados quanto às propriedades duais: Antimicrobiana e Fotoluminescência, estas apresentaram resultados promissores, podem atuar como boas sondas fluorescentes. Foram realizados também estudos Espectrofotométricos em diferentes solventes além de determinarmos o Cálculo do Orbital Molecular de Fronteira nesta série. Realizou-se também a nanoencapsulação em lipossomas para o composto 3o. Obtivemos também p-arilamidinas 3a-j por método convencional e avaliamos as propriedades Antimicrobiana e Citotóxica, as quais apresentaram resultados significativos. Realizamos a síntese dos derivados pirimidínicos 3a-f e 6a-f pelo método em Irradiação em micro-ondas, em fase sólida e em solução, respectivamente. Estudamos também para estes as propriedades Antimicrobiana e Antioxidante, cujos os resultados foram satisfatórios. Foram obtidos bons rendimentos para todos os compostos obtidos. Um estudo inédito de RMN bidimensional, COSY, HSQC e HMBC foram aplicados às pirimidinas foi realizado, proporcionando a caracterização estrutural destes compostos. Os resultados obtidos apresentaram significativa potencialidades biológicas, farmacológicas e fotofísicas. / Heterocyclic compounds of pyrimidine are known for rich biological and pharmacological potential. A number of heterociclic reactions that enable the obtention of this class of compounds are also recognized, due to the diversity of activities and prior knowledge of their various synthetic viabilities. The structural modifications obtained in search for the best synthetic routes confered different physical properties on the new molecules and altered the reactivity of the molecules, resulting in changes in the distribution in cells and tissues. These discrete structural changes may reveal biological effects that may have been latent or eclipsed by the side effects of the compound prototype. This work presents the synthesis by conventional method of derivatives from pyrimidine 3a-q and 5o-q. These compounds were evaluated for dual properties: antimicrobial and photoluminescence. They showed promising results can act as fluorescent probes. Spectroscopic studies were carried out with different solvents and the Frontier Molecular Orbital calculation for this series was determined. Nanoencapsulation of liposomes for the compound 3o was prepared. The conventional method was used to obtain 3a-j p-arilamidinas. Results were significant for the antimicrobial and cytotoxic properties. Finally, the synthesis of pyrimidines derivatives 3a-f and 6a-f by a new method, solid phase micro-wave and in solution, respectively was carried out. We have studied both the Antimicrobial and Antioxidant properties with satisfactory results. Good yields were obtained for all compounds obtained. New studies of two-dimensional NMR, COSY, HSQC and HMBC were performed besides the characterization of the compounds by standard methods. The results show that the compounds obtained have great biological, pharmacological potential and Photophysical.

Novas abordagens terapêuticas vêm sendo introduzidas para doenças musculares genéticas como distrofias musculares e miopatias congênitas, distúrbios que permanecem sem cura até o momento. Estes recentes avanços motivaram um interesse renovado e crescente por métodos não invasivos para a caracterização e monitoramento do músculo afetado, particularmente durante e após intervenções terapêuticas. Neste contexto, modelos animais são essenciais para uma melhor compreensão dos mecanismos das doenças e para testar novas terapias. Recentemente, avanços significativos na avaliação não invasiva de modelos murinos para doenças musculares genéticas foram alcançados. Entretanto, diversas linhagens de camundongos ainda não foram caracterizadas de maneira não invasiva, e ainda é necessário o desenvolvimento de métodos sensíveis para a identificação precoce de alterações sutis no músculo de camundongos afetados. A proposta desta tese é aplicar técnicas não invasivas inovadoras no estudo do músculo de modelos murinos para doenças musculares genéticas com fenótipos variados. Três modelos murinos para distrofias musculares (mdx, Largemyd, mdx/ Largemyd) e um modelo murino para miopatia congênita (KI-Dnm2R465W) foram estudados com métodos de Ressonância Magnética Nuclear (RMN). Duas linhagens distróficas (Largemyd, mdx/ Largemyd) e camundongos normais após injúria foram estudados através de micro-Tomografia Computadorizada (micro-CT). Em RMN, todas as linhagens de camundongos afetados apresentaram aumento de T2 muscular, o que foi relacionado a diversas anomalias na análise histológica, como necrose e inflamação, mas também a conjuntos de fibras em regeneração ou a fibras com citoarquitetura alterada. A combinação de RMN com análise de textura permitiu a identificação não ambígua de todas as linhagens distróficas, sendo que apenas a comparação dos valores de T2 muscular não permitiu esta diferenciação. Camundongos mdx mostraram alterações funcionais e morfológicas na rede vascular do músculo. Estudo piloto em camundongos KI-Dnm2R465W revelou tendências de comprometimento da função muscular. Por fim, imagens de micro-CT não permitiram a detecção de diferenças na composição muscular em camundongos distróficos. Este conjunto de resultados não apenas enriquece o painel de modelos murinos para doenças musculares genéticas caracterizados de maneira não invasiva, mas também demonstra um certo grau de especificidade nas anomalias observadas nas imagens, como revelado pela análise de textura. Estes resultados também mostraram que métodos não invasivos de RMN podem ser suficientemente sensíveis para identificar alterações sutis no fenótipo muscular murino, mesmo em estágios precoces. Esta tese foi desenvolvida sob acordo de co-tutela internacional entre a França e o Brasil, e compreendeu uma importante transferência de conhecimento, com os primeiros estudos não invasivos de músculo murino realizados no Brasil. / Novel therapeutic approaches are being introduced for genetic muscle diseases such as muscle dystrophies and congenital myopathies, all of them having remained without cure so far. These recent developments have motivated a renewed and augmented interest in non-invasive methods for muscle characterization and monitoring, particularly during and after therapeutic intervention. In this context, animal models are essential to better understand the disease mechanisms and to test new therapies. Recently, significant advances in the non-invasive evaluation of mouse models for genetic muscle diseases have been achieved. Nevertheless, there were still several mouse strains not characterized non-invasively, and it was necessary to develop sensitive methods to identify subtle alterations in the murine affected muscle. The purpose of this thesis was to apply non-invasive techniques in the study of murine models for genetic muscle diseases with variable phenotypes. Three mouse models for muscle dystrophy (mdx, Largemyd, mdx/ Largemyd) and one mouse model for congenital myopathy (KI-Dnm2R465W) were studied with Nuclear Magnetic Resonance (NMR) methods. Two dystrophic strains (Largemyd, mdx/ Largemyd) and normal mice after injury were studied through micro-Computed Tomography (micro-CT). On NMR, all affected mouse strains presented increased muscle T2, which could be related to variable features in the histological evaluation, including necrosis and inflammation, but also to clusters of fibers under regeneration or with altered cytoarchitecture. The combination of NMR and texture analyses allowed the unambiguous differential identification of all the dystrophic strains, although it was not feasible when comparing the muscle T2 measurements only. Mdx mice showed functional and morphological alterations of vascular network. In the KI-Dnm2R465W mice, a pilot study revealed tendencies of functional impairment. Finally, micro-CT images were unable to detect differences in muscle´s content in dystrophic mice. Altogether, these results not only increased the number of murine models for genetic muscle diseases non-invasively characterized, it also demonstrated some degree of specificity of the imaging anomalies, as revealed by texture analysis. It also showed that non-invasive NMR methods can be sensitive enough to identify subtle alterations in murine muscle phenotype, even in early stages. This thesis was developed under an international joint supervision between France and Brazil, and comprised an important transfer of technology, with the first non-invasive studies of murine muscles performed in Brazil.