Glucocorticoids are steroid hormones involved in the stress response, with a well-established role in promoting cardiovascular risk factors including obesity and diabetes. The focus of glucocorticoid research has shifted from understanding control of blood levels, to understanding the factors that control tissue steroid concentrations available for receptor activation; it is disruption of these tissue-specific factors that has emerged as underpinning pathophysiological mechanisms in cardiovascular risk, and revealed potential therapeutic targets. However, the field is hampered by the inability at present to measure concentrations of steroid within individual tissues and indeed within component cell types. This research project explores the potential for steroid measurements using mass spectrometry-based tissue imaging techniques combining matrix assisted laser desorption ionization with on-tissue derivatisation with Girard T and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (OTCD-MALDIFTICRMS). A mass spectrometry imaging (MSI) platform was developed and validated to quantify inert substrate and active product (11-dehydrocorticosterone (11DHC), corticosterone (CORT) respectively) of the glucocorticoid-amplifying enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in rodent tissues. A novel approach to derivatising keto-steroids in tissue sections using Girard T reagent was developed and validated. Signals were boosted (10⁴ fold) by formation of GirT hydrazones compared to non-derivatised neutral steroids. Active and inert glucocorticoids were detected in a variety of tissues, including adrenal gland and brain; in the latter, highest abundance was found in the cortex and hippocampus. The MSI platform was also applied to human biopsies and murine tissues for the analysis of other ketosterols such as androgens and oxysterols. Proof-of-principle validation that the MSI platform could be used to quantify differences in enzyme activity was carried out by following in vivo manipulation of 11β-HSD1. Regional steroid distribution of both substrate and product were imaged at 150-200μm resolution in mouse brain sections, and the identification confirmed by collision induced dissociation/liquid extraction surface analysis (CID-LESA). To validate the technique, the CORT/11DHC ratios (active/inert) were determined in 11β- HSD1 deficient mice and found to be reduced (KO vs WT; cortex (49 %*); hippocampus (46 %*); amygdala (57 %)). Following pharmacological inhibition by administration of UE2316, drug levels peaked at 1 h in tissue and at this time point, a reduction in CORT/11DHC ratios were also determined, although to a lesser degree than in KO mice, cortex (22%), hippocampus (25 %) and amygdala (33 %). The changes in ratios appeared driven by accumulation of DHC, the enzyme substrate. In brains of mice with 11β-HSD1 deficiency or inhibition, decreases in sub-regional CORT/11DHC ratio were quantified, as well as accumulation of an alternative 11β- HSD1 substrate, 7-ketocholesterol. MSI data correlated well with the standard liquid chromatography tandem mass spectrometry (LC-MS/MS) in whole brain homogenates. Subsequently, the MSI platform was also applied to measure the dynamic turnover of glucocorticoids by 11β-HSD1 in metabolic tissues using stable isotope tracers (Cortisol-D4 (9,11,12,12-D4) (D4F). D4F was detected in plasma, liver and brain after 6 h infusion and after 48 h in adipose. D3F generation was detected at 6 h in plasma and liver; at 24 h in brain specifically in cortex, hippocampus and amygdala; and at 48 h in adipose. The spatial distribution of d3F generation in brain by MSI closely matched enzyme localisation. In liver, an 11β-HSD1-riched tissue, substantial generation of d3F was detected, with a difference in d4F/d3F ratios compared with plasma (ᴧTTRᴧ 0.18± 0.03 (6 h), 0.27± 0.05 (24 h) and 0.38±0.04 (48 h)). A smaller difference in TTR was also detected between plasma and brain (ᴧTTR 0.09 ± 0.03 (24 h), 0.13±0.04 (48 h)), with no detectable regeneration in adipose. After genetic disruption of 11β-HSD1, d3F generation was not detected in plasma or any tissues, suggesting that 11β-HSD1 is the only enzyme carrying out this reaction. After pharmacological inhibition, a similar pattern was seen. The circulating concentration of drug peaked at 2 h and declined towards 4 h, with same pattern in liver and brain. The ᴧTTR ratios 2HPD between plasma and liver (0.27±0.08vs. 0.45± 0.04) and brain (0.11±0.2 vs. 0.19± 0.04) were smaller following drug administration than vehicle, indicating less d3F generation. Extent of enzyme inhibition in liver responded quickly to the declining drug, with ᴧTTR returning to normal by 4 h (0.38± 0.06). ᴧTTR had not normalised 4HPD in brain (0.12±0.02, suggesting buffering of this pool. In adipose, UE2316 was not detected and nor were rates of d3F altered by the drug. Two possible phase I CYP450 metabolites were identified in the brain differing in spatial distribution. In conclusion, MSI with on-tissue derivatisation is a powerful new tool to study the regional variation in abundance of steroids within tissues. We have demonstrated that keto-steroids can be studied by MALDI-MSI by using the chemical derivatisation method developed here and exemplified its utility for measuring pharmacodynamic effects of small molecule inhibitors of 11β-HSD1. This approach offers the prospect of many novel insights into tissue-specific steroid and sterol biology.

La découverte de nouveaux médicaments par le criblage biomoléculaire est au centre de la recherche pharmaceutique actuelle. La spectrométrie de masse, en tant que technique d’analyse fiable, reproductible, sensible, spécifique, compatible avec de nombreux types d’échantillons et permettant un débit d’analyse conséquent, trouve ainsi sa place dans les stratégies de recherche et développement de nouveaux médicaments. Le but de ce travail était de mettre en place et de valider une stratégie originale, impliquant la désorption/ionisation laser assistée par matrice couplée à la spectrométrie de masse (MALDI-MS) comme technique de détection, en vue du criblage de la liaison de composés à des cibles moléculaires applicable directement à des extraits de plantes. Le protocole que nous avons développé s’articule en trois étapes successives qui sont l’incubation des molécules à tester avec la cible moléculaire choisie (la tubuline ou la DHFR), l’élimination des composés non liés et enfin l’analyse par MALDI-TOFMS des composés liés. Notre démarche a fait l’objet d’une démarche de validation et les résultats pouvant être obtenus ont été discutés. Le débit pouvant être évalué à 60 échantillons en 1h50 à 3h30 soit de 18 à 32 échantillons à l’heure. Enfin, une démarche innovante nous a permis de prouver que notre approche pouvait être utile également en criblage secondaire. L’application de notre approche à des extraits bruts de plantes (Colchique d’Automne, Pervenche de Madagascar et Thé vert) à permis de mettre en évidence 20 molécules actives se liant à l’une ou l’autre des cibles moléculaires utilisées et d’évaluer l’affinité relative de l’une d’entre elles / Discovering new drugs by biomolecular screening is a central task of pharmaceutical research. Mass spectrometry, as a reliable, reproducible, sensitive and specific technique, compatible with a wide range of samples and offering an excellent throughput, shows its potential in different strategies of research and development. The aim of this work was to develop and validate a new strategy, involving matrix assisted laser desorption/ionization coupled to time of flight mass spectrometry (MALDI-TOFMS) to screen the ability of different compounds, including plant extracts, to bind to two biological targets (tubulin and DHFR). The protocol is therefore divided into three main steps : an incubation of the compounds to be tested with target, an elimination of all unbound compounds and the MALDI-TOFMS detection of target-bound compounds. Our protocol was validated and the results that can be obtained were discussed. The throughput offered by this technique was evaluated as 60 samples in 1h50 to 3h30, or 18 to 32 samples per hour. Finally, we developed a new approach to perform a secondary screening of active compounds. The protocol was applied to screen crude plants extracts (colchicum autumnale, catharanthus roseus and green tea) and allowed to find 20 tubulin-binding or DHFR-binding molecules, and the relative affinity of one of these was also evaluated

La fumée de cigarette est reconnue pour être un important polluant des espaces confinés. La matière particulaire qui lui est associée présente un intérêt toxicologique important, tant dans le cadre du tabagisme actif que passif, de par sa forte propension à être retenue dans les voies respiratoires. De plus, les molécules qui les constituent ou qui sont adsorbées à leur surface peuvent être, après leur rétention, transférées à l'organisme sur des périodes de temps importantes ce qui conduit à une exposition prolongée à ces molécules, certaines étant reconnues toxiques. Cependant, les méthodes d'analyses classiques ne permettent qu'une description partielle et ciblée. C'est dans ce contexte qu'a été entrepris ce travail de thèse sur l'étude détaillée de la composition des particules impliquées dans le tabagisme actif et passif. Dans un premier temps, une méthode de quantification par chromatographie gazeuse couplée à la spectrométrie de masse en tandem (GC-MS/MS) a été mise en place dans le but de valider le dispositif de fumage et de prélèvement. Ainsi, la reproductibilité des échantillons de fumées de cigarette soumis à l'étude a pu être assurée. En considérant l'évolution de traceurs comme la nicotine ou certains HAPs, un plan d'expérience a permis d'évaluer l'influence sur la nature des prélèvements, de différentes caractéristiques associées aux bouffées (durée, débit d'aspiration) et à la cigarette (taux d'humidité, perméabilité du filtre). Dans un second temps, les particules de fumées inspirées (MSS), expirées (EXS) et de fumées émises entre deux bouffées (SSS) ont été analysées par spectrométrie de masse à résonance cyclotronique des ions et à transformée de Fourier couplée à l'ionisation/désorption laser (LDI-FTICRMS) ou électrospray (ESI-FTICRMS). La comparaison MSS/EXS met en évidence une rétention préférentielle dans l'organisme des composés à forte polarité. Les SSS quant à elles révèlent des composés plus aromatiques et moins oxygénés que les MSS. Ce résultat a été associé à une différence de température et à une différence d'apport d'oxygène au niveau du foyer de combustion pendant et entre les bouffées. Ces paramètres influencent en effet la nature du phénomène majoritaire qui s'y produit (phases de pyrolyse et/ou de combustion). Un autre paramètre qui permet d'expliquer les différences observées est l'absence d'interactions des SSS après leur émission. Au contraire, les MSS, qui traversent le tabac encore non-consumé et le filtre de la cigarette, sont en mesure d'entrainer les molécules volatiles qu'ils contiennent telle que la nicotine par exemple. Ces résultats ont été, pour bon nombre, confirmés et complétés par des études réalisées en résonance magnétique nucléaire (RMN). L'ensemble des résultats obtenus dans l'étude des MSS, SSS et EXS montre que, pour une part importante, fumeur passif et fumeur actif sont exposés à des classes de molécules différentes qui seraient en mesure de conduire à des pathologies spécifiques / Cigarette smoke is one of the more significant indoor contaminant. Cigarette smoke is associated with gas phase and particulate matter phase components. The cigarette smoke particles are of major concern from a toxicological viewpoint, for both active and passive smoking. Indeed a large part of them is retained by the different levels of human respiratory system. As a consequence, the molecules adsorbed on their surface or the molecules, which compose the particle itself, can be slowly released in the organism. In this context, particles involved in active and passive smoking were analyzed and compared. In a first step, a quantification method by gas chromatography-tandem mass spectrometry (GC-MS/MS) for some tracers has been developed and validated to allow the validation of both the smoking and the sampling procedures to be achieved. This is important to be sure that for a given cigarette smoke types, the different samples obtained from different collection procedure are statistically identical. This also allowed the influence of some smoking variables (type and humidity of the cigarette, puff duration, and flow rate of the puff) on the composition of the particles to be evaluated. In a second step, mainstream cigarette smoke (MSS), sidestream cigarette smoke (SSS) and exhaled cigarette smoke (EXS) particles were analyzed by ion cyclotron resonance mass spectrometry coupled with laser desorption/ionisation (LDI-FTICRMS) or electrospray ion (ESI-FTICRMS). The comparison of MSS and EXS revealed the selective retention of the more polar molecules in the organism. In contrast to what it is obtained for MSS, it was found that the compounds detected in the analysis of SSS particles are highly unsaturated and content low amounts of oxygen and nitrogen atoms. This has to be linked to a lower dioxygen amounts and to a lower temperature between two puffs in the burning zone. Consequently, pyrolytic and combustion phenomena are the main process which induce the formation of SSS and MSS, respectively. Moreover, MSS cross unburned tobacco before to be collected and can induce distillation of some volatile compounds (e.g. nicotine) which are thought to be more saturated and more oxygenated that the compounds generated in the burning zone. These results were confirmed and completed by nuclear magnetic resonance (NMR) spectroscopy analysis. The results of the MSS, SSS and EXS particle analysis highlight the differences of chemical composition of these different cigarette smokes which is associated to active smoking for the first one and passive smoking for the latter ones. Consequently, differences of toxicological effect can be expected for each kinds of smoking behavior