京都大学 / 新制・課程博士 / 博士(医学) / 甲第23366号 / 医博第4735号 / 新制||医||1051(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 岩田 想, 教授 萩原 正敏, 教授 稲垣 暢也 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The research herein presents an approach to stabilising reactive materials by engineering and designing strategies for forming multi-component materials containing the reactive molecules by use of their non-covalent intermolecular interactions. These interactions may be utilised as part of a design approach to create new materials of more beneficial physical and chemical properties for the desired application. The reactive materials focussed on in this research are organic peroxyacids, in particular peroxyacetic acid, meta-chloroperbenzoic acid and 6-phthalimidoperoxyhexanoic acid. The stabilities of these target materials under different conditions are probed to find a suitable environment for crystallisation experiments. Crystal structures of the materials were isolated and characterised and the peroxyacids were subsequently cocrystallised with materials chosen to interact with the target molecules to form new molecular complexes, including carboxylic acids, π stacking materials and metal salts. A hosting approach was also employed to form multi-component systems containing these materials, crystallising them with larger, stable, structure-generating compounds with the aim of intercalating the reactive molecules in their stable structure. To this end, urea based compounds, cyclodextrins and Montmorillonite clay were investigated as hosting materials. Candidate multi-component materials were synthesised which successfully retain peroxyacid reactivity. A second set of materials studied was agrichemicals, which also frequently have reactive character, in which a change in physical properties was pursued by the method of forming new crystalline complexes. Five new crystalline agrochemical molecular complexes were synthesised and tested for thermal stability in comparison to the original materials to assess for changes in properties of the multi-component materials.

No / The synthesis and crystal structure of the lithium hydrate salt of the charge transfer complex between 3,5-dinitrobenzoic acid and 4-(dimethylamino) benzoic acid is reported. It is the first crystal structure reported for such a class of hybrid inorganic/organic material. The design principles may have utility in the future creation of new ternary and higher complexes.

Toxoplasma gondii, parasite digestif des Félidés, est l'agent de la toxoplasmose, maladie pouvant être grave voire mortelle en cas d'infection fœtale ou chez l'immunodéprimé. Les traitements actuellement disponibles permettent de prévenir ou traiter la plupart des cas, mais peuvent présenter un risque d'effets indésirables relativement sévères et ne permettent pas de détruire les kystes responsables de l'infection chronique et du risque de réactivation chez l'immunodéprimé. Les aminoacyl-ARNt synthétases (aaRS) sont des enzymes essentielles au mécanisme de traduction, où elles participent au chargement d'un acide aminé sur une molécule dédiée d'ARN de transfert, une étape initiale du processus de synthèse protéique. Un gène codant une protéine homologue de p43, un partenaire protéique de certaines aaRS chez les Eucaryotes supérieurs, a été identifié dans le génome de T. gondii. La localisation subcellulaire post-invasion de Tg-p43 montre qu'il ne s'agit pas d'une cytokine sécrétée, contrairement à son homologue humaine ; au contraire, son immunopurification a révélé son association à quatre aaRS, les Méthionyl-, Glutamyl-, Glutaminyl- et Tyrosyl-ARNt synthétases, qui constituent donc le premier complexe multi-aaRS (MARS) décrit chez les parasites Apicomplexes, de localisation exclusivement cytoplasmique. La présence inattendue de la Tyrosyl-ARNt synthétase soulève plusieurs questions sur le plan de l'organisation et de l'assemblage du complexe. Des images de microscopie électronique et des analyses biochimiques soulignent l'hétérogénéité et la structure relâchée du complexe et confirment les récentes données issues des complexes MARS purifiés chez d'autres organismes. L'inactivation du gène Tg-p43 n'induit pas de modifications phénotypiques majeures (capacité d'invasion, prolifération) ni de diminution de la virulence ou de la kystogénèse en modèle murin. Les résultats sur la caractérisation du MARS ont été complétés par une approche thérapeutique. Un criblage in vitro de candidats-médicaments présélectionnés in silico pour inhiber la Glutaminyl-ARNt synthétase toxoplasmique a permis de mettre en évidence un composé parasitostatique, inhibiteur de la croissance des tachyzoïtes, avec une toxicité sur les cellules hôtes in vitro relativement faible. Ce travail de thèse pose les bases moléculaires et structurales du complexe MARS chez T. gondii. Il permet également d'aborder dans une certaine mesure l'histoire évolutive de ce complexe. Sa fonction biologique reste cependant un mystère ; le rôle de Tg-p43 dans le contrôle post-transcriptionnel, voire d'autres fonctions biologiques, est probablement trop subtil pour être mesuré dans nos conditions expérimentales. Le versant thérapeutique de ce travail constitue une étude préliminaire pouvant servir de point de départ pour la recherche de médicaments anti-toxoplasmiques ciblant les aaRS, qui constituent assurément des cibles thérapeutiques intéressantes. / Toxoplasma gondii, a parasite of felids gut, is responsible for toxoplasmosis, a disease that can induce severe sequelae or death in the foetus or immune-depressed patients. Currently available treatments can prevent or cure most of the cases, but are at risk for side effects and cannot suppress cysts, which cause the chronic disease and are responsible for disease when the immune status is altered. Aminoacyl-tRNA synthetases (aaRS) are essential for translation, by charging tRNA with cognate aminoacids, a preliminary step of the protein synthesis process.A gene coding for a protein homologous to p43 (which interacts with a subset of aaRSs in higher eukaryotes) was identified in the genome of T. gondii. Following its epitope tagging, we show that Tg-p43 is not secreted nor exported beyond the vacuole as a cytokine, as it is for its human counterpart; however, biochemical analysis of the Tg-p43 interactome reveals four aaRSs as interacting partners, namely Methionyl-, Glutamyl-, Glutaminyl- and Tyrosyl-tRNA synthetases. This is the first description of the multi-aaRS (MARS) complex in the Apicomplexa phylum; it is strictly localized in the parasite cytoplasm. The unexpected presence of the Tyrosyl-tRNA synthetase in the complex raises several questions about how the complex is organised and assembled, and also evolved. Electronic microscopy along with size exclusion chromatography shows heterogeneity and loose structure of the complex, similarly to recent data characterizing higher eukaryotic complexes. Disruption of the complex by knocking-out of the gene Tg-p43 does not induce detectable phenotypic modification, nor alterations of the virulence and cystogenesis in a murine model.Alongside the study on the MARS complex, we used an in silico approach to screen for new compounds to inhibit T. gondii Glutaminyl-tRNA synthetase. We thus identified one parasitostatic compound that was able to significantly slow down parasite growth while having a relatively low in vitro toxicity against the human host cell. The function of the MARS in T. gondii still remains unknown; the role of Tg-p43 in the post-transcriptional control or any other biological function is probably too subtle to be measured under our experimental conditions. However, our data help to some extent to better measure the evolutionary history of the MARS family. The therapeutic side of this work, although preliminary, may serve as a base for anti-T. gondii drug discovery focusing on aaRS inhibitors, which are obviously good candidate targets.

No / The multicomponent ternary complex of 4-dimethylaminobenzoic acid (4-DABA), 3,5-dinitrobenzoic acid (3,5-DNBA), and 4,40-bipyridine (BIPY) has been studied by variable temperature X-ray and neutron diffraction. Proton disorder is observed within the 4-DABA homodimers present and quantitatively evaluated from neutron data. The effect of the crystal environment and in particular the pyramidalization of the nitrogen atom within the 4-DABA molecule and the consequential effect on the presence of hydrogen atom disorder are discussed with reference to the previously determined pure 4-DABA structure and the binary cocrystal with 3,5-DNBA.