Ce manuscrit est consacré à l’élaboration de phases monolithiques à base de dioxyde de titane pour les techniques séparatives appliquées à l’analyse des produits phosphorylés. La partie bibliographique situe d’abord l’intérêt des monolithes pour le développement des techniques séparatives en portant une attention particulière à la problématique de miniaturisation. L’état de l’art sur l’utilisation du dioxyde de titane, dans les sciences séparatives, est ensuite établi. À ce niveau, les principales caractéristiques physico-chimiques et chromatographiques de ce matériau sont revues pour les lits particulaires. Un descriptif de la synthèse des monolithes de dioxyde de titane par le procédé sol-gel est ensuite détaillé avant d’illustrer la mise en oeuvre de ce type de colonnes dans les techniques séparatives. La partie expérimentale est axée d’abord sur la compréhension du comportement du dioxyde de titane particulaire en chromatographie liquide à interaction hydrophile (HILIC). Le travail expérimental est ensuite orienté vers l’élaboration d’un procédé sol-gel répétable, permettant d’obtenir le matériau sous le format monolithique. Dans un premier temps, le support est élaboré sous forme de barreau, dont l’utilisation potentielle après mise en colonne est illustrée dans le cadre de la purification et de l’enrichissement des acides aminés phosphorylés. Des adaptations de protocole sont apportées pour la synthèse in situ de ces monolithes à l’intérieur des capillaires afin de répondre aux contraintes des techniques séparatives miniaturisées. Ces monolithes sont caractérisés en HILIC, par comparaison avec les phases particulaires. Pour terminer, les propriétés chromatographiques originales de ces phases sont mises à profit pour la séparation et le traitement d’échantillons, contenant des produits phosphorylés / This manuscript is dedicated to the development of monolithic titania phases for chromatographic analysis of phosphorylated compounds. The bibliography section first summarizes the interest of monolithic phases for the development of separation techniques while emphasizing on the problem of miniaturization. The state of the art on the use of titanium dioxide in liquid chromatography techniques is established. In this subject, the physico-chemical and chromatographic behaviour of this material are reviewed for particle beds. Then a detailed description of the sol-gel synthesis of monolithic titania is presented with a final illustration of the potential use of this support in separation techniques. The experimental part concentrates first on the analysis of the chromatographic behaviour of particulate titania in the hydrophilic interaction mode (HILIC). The work is then focused on the development of a repeatable sol-gel process that enables the formation of monolithic titania. The monolithic phase is first synthesized at a large scale, and its potential use, after column cladding, is illustrated with the purification and enrichment of phosphorylated amino acids. The elaboration process is also adapted to meet with the miniaturized separation techniques by performing an in situ synthesis route within capillary columns. These columns are characterized in HILIC by comparison with the chromatographic properties observed for titania particulate beds. The original behaviour of native titania observed also for the synthesized monolithic phases is finally applied to the separation and sample treatment of mixtures of phosphorylated products

Symbiotic nitrogen (N) fixation (SNF) occurs in specialized organs called nodules after successful interactions between legume hosts and rhizobia. Within nodule cells, N-fixing rhizobia are surrounded by plant-derived symbiosome membranes, through which the exchange of nutrients and ammonium occurs between bacteria and the host legume. Phosphorus (P) is an essential macronutrient, and N2-fixing legumes have a higher requirement for P than legumes grown on mineral N. First, I investigated the impact of P deprivation on wild-type Medicago truncatula plants. My observations that plants had impaired SNF activity, reduced growth, and accumulated less phosphate in P-deficient tissues (leaves, roots and nodules) is consistent with those of similar previous studies. Galactolipids decreased with increase in phospholipids in all P-starved organs. Matrix-assisted laser desorption/ionization–mass spectrometry imaging (MALDI-MSI) of phosphatidylcholine (PC) species in nodules showed that under low P environments distributions of some PC species changed, indicating that membrane lipid remodeling during P stress is not uniform across the nodule. Secondly, a metabolomics study was carried out to test the alterations in the metabolic profile of the nodules in P-stress. GC-MS based untargeted metabolomics showed increased levels of amino acids and sugars and decline in amounts of organic acids in P deprived nodules. Subsequently, LC-MS/MS was used to quantify these compounds including phosphorylated metabolites in whole plant. My findings showed strong drop in levels of organic acids and phosphorylated compounds in P deprived leaves with moderate reduction in P deprived roots and nodules. Moreover, sugars and amino acids were elevated in whole plant under P deprivation. Finally, the last project of my thesis involved studying the response of PDIL3 (Phosphate Deficiency-Induced LncRNA-3) a long non-coding RNA (lncRNA) mutant under severe P stress. PDIL3 is known to regulate Pi-deficiency signaling and transport in M. truncatula (Wang et al., 2017). My results confirmed that in P starvation, pdil3 plants showed better shoot growth, accumulated more phosphate in shoots, had impaired SNF and less rhizobial occupancy in nodules than WT. Subsequently, MALDI–MS imaging was used to spatially map and compare the distribution of phosphatidylcholine (PC) species in nodules of pdil3 and WT in P-replete and P-depleted conditions. Several PC species showed changes in distributions in pdil3 nodules compared to WT in both P sufficient and P deprived conditions. These data suggest that PDIL3's role is not just suppression of the Pi transporter, but it may also influence P partitioning between shoots and nodulated roots, meriting further investigation.

Phospholipids are dominant components of bacterial cell membranes, where they create double layers. Bacteria differ in their phospholipid composition determination of which can help in identification of important groups of microorganisms. Phospholipid composition of bacteria is influenced by many environmental factors, therefore its variation can be observed within one bacterial stem also. Because of its simplicity, thin layer chromatography is usually applied to identification and determination of bacterial phospholipids. Disadvantage of this method are the high demands of time, carefulness and skills of the analytical personnel. The increasing interest in the phospholipid double-layer promotes the detailed investigation of their fatty acid composition because the more detailed analyses allows for more information yield about bacteria. Gas chromatography hyphenated with mass spectrometry seems to be the best choice for these purposes. Fatty acid identity and total fatty acid content in phospholipid molecules could be determined by this method. Additionally, number, position and isomerism of double bonds and presence of other functional groups on hydrocarbon chain could be determined. Whereas a suitable and...