Les glioblastomes (GBMs) sont des tumeurs cérébrales au pronostic défavorable. La résistance aux thérapies actuelles et la rechute des GBMs pourraient être due à l'existence de cellules aux propriétés souches. L'objectif de ma thèse a été la caractérisation des cellules souches de GBMs (CSGs) isolées à partir de tumeurs. L'analyse du profil souche et de pluripotence des CSGs a montré qu'elles sont maintenues dans un état souche par SOX2 et que COL1A1 et IFITM1 peuvent être des cibles thérapeutiques potentielles. L'étude de la radiosensibilité des CSGs à travers l'analyse des courbes de clonogénicité a mis en évidence deux groupes dont un «atypique» pouvant être composé de sous-populations de cellules aux radiosensibilités différentes qu'il conviendra de caractériser. L'étude de la réparation a mis en évidence deux autres groupes dont un ayant un fort potentiel de réparation qui exprime plus fortement le gène RAD51 après irradiations. Le traitement par un inhibiteur spécifique de RAD51 ralentirait la capacité de réparation de ces cellules. Malgré cette hétérogénéité, l'inhibition de la voie Hedgehog (HH) par un vecteur glucuronylé de la cyclopamine, activé par le microenvironnement tumoral, inhibe la prolifération et l'auto-renouvellement des CSGs in vitro et ralentit la croissance tumorale in vivo. La voie HH semble être une cible thérapeutique intéressante commune à toutes les CSGs. Néanmoins, il est nécessaire de prendre en compte l'hétérogénéité dans les populations tumorales pour le développement de la médecine personnalisée. / Glioblastomas (GBMs) are brain tumors with a poor prognosis. Their resistance to current therapies and the occurrence of tumor relapse may be related to the existence of cells bearing stem cell characteristics. The aim of this PhD research was to characterize glioblastoma stem cells (GSCs) having been isolated from tumors. Analysis of the stemness and pluripotency profiles of GSCs indicated that their stemness states are maintained by SOX2 and that COL1A1 and IFITM1 may be potential therapeutic targets. Clonogenic studies of GSC radiosensitivity underscored the presence of two groups, one of them composed of sub-populations of cells with different degrees of radiosensitivity that have yet to be fully characterized. Study of DNA repair capacity highlighted two additional groups including one with high repair potential overexpressing the RAD51 gene after 4Gy. However, treatment with RAD51 inhibitor is likely to slow down repair of GSC lesions. Notwithstanding GSC heterogeneity, in our study inhibition of the Hedgehog pathway (HH) by a cyclopamine glucuronid prodrug, activated by the tumor microenvironment, inhibited in vitro proliferation and self-renewal in all the GSCs tested and slowed down tumor growth in vivo. Hence, HH pathway appears to be conserved among GSCs and constitutes an interesting potential therapeutic target. With regard to the development of personalized medicine, it is nevertheless highly advisable to take into account the pronounded heterogeneity of tumor populations.

<p>This thesis presents novel approaches for structural investigation of glucuronides using chemical derivatization in combination with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MSⁿ).</p><p>Today, LC-ESI-MSⁿ is the dominant technique for quantitative as well as qualitative analyses of metabolites, due to its high sensitivity and selectivity. However, for compounds without an easily ionizable group, e.g., steroids, the sensitivity is limited. In the work presented in this thesis, a derivatization procedure forming a basic oxime significantly increased the detection sensitivity for the altrenogest glucuronide. </p><p>Furthermore, in structural evaluations of glucuronides, the limitation of LC-MSⁿ becomes evident due to the initial neutral loss of 176 u, i.e. monodehydrated glucuronic acid, which often makes it impossible to elucidate the structures of the conjugates. To solve this problem, the main part of the work described in this thesis was devoted to chemical derivatization as a means of facilitating the determination of the site of conjugation. </p><p>For the first time, the isomeric estriol glucuronides were evaluated using a combination of three reagents 2-chloro-1-methylpyridinium iodide (CMPI), 1-ethyl-3-(3-dimethyl- aminopropyl)-carbodiimide (EDC), and 2-picolylamine (PA). Interestingly, the derivatization gave a selective fragmentation pattern leading to differentiation of the isomers. </p><p>Another derivatization reagent, 1,2-dimethylimidazole-4-sulfonyl chloride (DMISC), was also tested for the first time in structural investigations. The isomeric glucuronides of morphine, formoterol, and hydroxypropranolol were evaluated. They can all be conjugated in aliphatic as well as aromatic positions. DMISC was proven to be useful in two ways. Firstly, the morphine and formoterol glucuronides that contained a free phenol could be differentiated from those that were conjugated in the aromatic position based on different reactivity. Secondly, for the aromatic <i>O</i>-glucuronide of 4’-hydroxypropranolol, DMISC was proven to react with the amine. This product gave a different fragmentation pattern compared to the corresponding derivative of the aliphatic glucuronide. </p>

This thesis presents novel approaches for structural investigation of glucuronides using chemical derivatization in combination with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MSn). Today, LC-ESI-MSn is the dominant technique for quantitative as well as qualitative analyses of metabolites, due to its high sensitivity and selectivity. However, for compounds without an easily ionizable group, e.g., steroids, the sensitivity is limited. In the work presented in this thesis, a derivatization procedure forming a basic oxime significantly increased the detection sensitivity for the altrenogest glucuronide. Furthermore, in structural evaluations of glucuronides, the limitation of LC-MSn becomes evident due to the initial neutral loss of 176 u, i.e. monodehydrated glucuronic acid, which often makes it impossible to elucidate the structures of the conjugates. To solve this problem, the main part of the work described in this thesis was devoted to chemical derivatization as a means of facilitating the determination of the site of conjugation. For the first time, the isomeric estriol glucuronides were evaluated using a combination of three reagents 2-chloro-1-methylpyridinium iodide (CMPI), 1-ethyl-3-(3-dimethyl- aminopropyl)-carbodiimide (EDC), and 2-picolylamine (PA). Interestingly, the derivatization gave a selective fragmentation pattern leading to differentiation of the isomers. Another derivatization reagent, 1,2-dimethylimidazole-4-sulfonyl chloride (DMISC), was also tested for the first time in structural investigations. The isomeric glucuronides of morphine, formoterol, and hydroxypropranolol were evaluated. They can all be conjugated in aliphatic as well as aromatic positions. DMISC was proven to be useful in two ways. Firstly, the morphine and formoterol glucuronides that contained a free phenol could be differentiated from those that were conjugated in the aromatic position based on different reactivity. Secondly, for the aromatic O-glucuronide of 4’-hydroxypropranolol, DMISC was proven to react with the amine. This product gave a different fragmentation pattern compared to the corresponding derivative of the aliphatic glucuronide.

Drug facilitated assault (DFA) is an increasing problem in the UK. The crime often occurs through the surreptitious administration of a drug into a victims drink, rendering the victim unable to resist the assault. The detection of these drugs in a biological specimen from the victim is one of the most challenging facets of forensic chemistry. Drug concentrations can be very low, as often only a single dose is administered, and the pharmacodynamics of commonly employed drugs further hinders the testing process. The research presented in this work shows the development of several new assays for the detection of flunitrazepam, gamma-hydroxybutyrate (GHB) and ethyl glucuronide (EtG) in a variety of biological matrices. New methods of drug testing in blood and urine are demonstrated, as well as interesting developments in the field of hair testing. Using hair to detect drug exposure allows a much wider window of detection than the more traditional matrices of blood and urine. New methods are presented in this work using gas chromatography-tandem mass spectrometry (GCMS/MS) to detect drugs in hair. Validation data is presented along with the results of authentic DFA testing. All aspects of the drug testing procedure have been evaluated, from new extraction techniques utilising water instead of solvents, to novel clean up stages involving the unique combination of SFE and SPME. Several confirmation techniques are explored including single quadrupole, triple quadrupole and ion trap mass spectrometry. In addition to developing assays for DFA cases, the versatility of this type of analytical chemistry is explored in two population studies. The first study evaluates alcohol consumption between two groups; drugs users and non drug users in medico-legal cases. There is an anecdotal belief amongst drug clinic staff that alcohol use is lower in drugs users than it is in non drug users. This study presents the first scientific confirmation of this belief through EtG (an alcohol metabolite) testing in hair of the two groups. The second study investigates whether there is a correlation between EtG and cocaethylene (a metabolite of cocaine only produced in the presence of alcohol) in cocaine users. Results f this study suggest that there is no positive correlation between the two compounds. The research presented in this thesis aims to further the analytical science surrounding FA investigation and provide accurate, sensitive and reliable methodology for drug esting in blood, urine and hair.