Grâce aux récents progrès en terme d'instrumentation analytique, la protéomique, en tant que science qui étudie le protéome d'un organisme ou d'un milieu, a connu un véritable tournant et a permis d'étendre le champ des connaissances sur le fonctionnement du vivant dans son ensemble (structure, fonction, métabolisme, dynamisme). Néanmoins, l'étude des protéomes représente un challenge pour de nombreux biologistes, chimistes et biochimistes, en raison notamment de la complexité des échantillons étudiés. De nombreux protocoles analytiques ont d'ores et déjà été développés. Cependant, dans leur ensemble, ces stratégies sont relativement longues et multi-étapes et le plus souvent ciblées sur une protéine donnée.Dans ce contexte, ce travail de thèse a pour objectif de simplifier les protocoles expérimentaux existants afin de limiter les pertes en protéines et ainsi amplifier leurs signaux au cours d'une analyse « Bottom-up » par LC-HRMS (analyse « hors gel »). Chaque étape du processus a été décortiquée et optimisée. Dans un premier temps, les paramètres UPLC-HRMS/MS ont été optimisés afin d'améliorer la détection et la quantification des protéines présentes à des concentrations très variables dans les milieux étudiés. Ensuite, une approche de purification simplifiée qui repose sur une seule et unique étape de lavage et solubilisation des protéines a été mise au point. La démonstration de son efficacité « chimique » et « biologique » a ensuite été réalisée via une étude mécanistique au cours de laquelle les changements de conformation des protéines ont été étudiés à chacune des étapes de purification proposées. Enfin, certains paramètres influençant l'extraction des protéines à partir de ces mêmes matrices ont été étudiés afin de proposer à terme un protocole d'extraction à la carte compatible avec une analyse directe par LC-HRMS/MS. / Recent advances in proteomics have been spurred by the rapid development of hybrid and/or high-resolution mass analysers (HRMS/MS). These powerful instrumentations have led to significant improvements in « Bottom-up » approach and have enabled to deepen our knowledge on the functionality of biological systems (structure, function, metabolism, dynamic, etc).Despite their high sensibilities, the potential of such instruments could be significantly lessened by an imperfect sample pre-treatment. In this context, current sample pretreatments follow multi-steps experimental workflows, which alternatively lead to low recoveries of proteins. In this line, this study aims at developing a simple and versatile strategy in order to reduce protein losses and enhance their detection in gel-free LC-MS analysis. First, an analytical method based on liquid chromatography and tandem mass spectrometry was developed to detect and quantify complex peptides mixture. Secondly, a universal, simple and fast purification approach was designed with the aim to purify protein extracts in only one-step. For this purpose, the molecular reactivity, dynamics and conformational changes of proteins at each development step were comprehensively investigated with a set of spectroscopic techniques, in order to select the best strategy. Finally, different factors influencing extraction of proteins were investigated with the goal in the long term to propose an on-demand extraction protocol for direct analysis of proteins by LC-HRMS/MS.

Einige der Polyzyklischen Aromatischen Kohlenwasserstoffe (PAK) weisen Krebs auslösende Eigenschaften auf. Die bekannteste karzinogene PAK-Verbindung ist das Benzo[a]pyren (BaP), welches bislang als Leitsubstanz verwendet wird. Mittlerweile bestehen jedoch Zweifel, ob BaP als alleinige Leitsubstanz geeignet ist. Daher empfiehlt die EU-Kommission die Untersuchung der PAK auf die so genannten 16 EFSA-PAK auszudehnen. Zur Überprüfung der Anwendbarkeit von BaP als Leitsubstanz war es notwendig, Erkenntnisse über das Verhältnis des Gehaltes an BaP zum PAKges-Gehalt sowie die einzelnen PAK-Profile zu gewinnen. Die zu untersuchenden Lebensmittelgruppen wurden eingeschränkt auf Fleischerzeugnisse, Rauchwürzer und Räuchersalze, Räucherdärme, Gewürze, Tee und Schokolade. Nach der Entwicklung und Überprüfung von Methoden zur Bestimmung der EFSA-PAK und der anschließenden Analytik einer Anzahl repräsentativer Proben verschiedener Lebensmittelgruppen konnte mit Hilfe einer Datensammlung zu den Gehalten der 16 EFSA-PAK die Beurteilung von BaP als Leitsubstanz erfolgen. Es konnte eine Abhängigkeit der PAKges-Gehalte vom BaP-Gehalt ermittelt werden, welche sich mit zunehmender Konzentration der PAK manifestierte. Aus analytischer Sicht eignet sich BaP am besten als Leitsubstanz, da sie chromatographisch mit den heutigen Methoden leicht von anderen möglichen coeluierenden Substanzen abgetrennt werden kann und in Konzentrationen vorkommt, die zuverlässiger quantifiziert werden können.

Water resources in South Africa are scarce, and should therefore be protected against pollutants, also from persistent organic pollutants (POPs). POPs are a global concern due to their ubiquitous presence, persistence and toxicity. This is emphasised by the Stockholm Convention on POPs, which aims at reducing and ultimately eliminating them. South Africa signed and ratified the treaty, and it became international law on 17 May 2004, but there is still a lack of information regarding POPs in South Africa. This study focussed on establishing the levels of POPs and other organic pollutants, which included various organochlorine pesticides (OCPs), polycyclic aromatic hydrocarbons (PAHs), dioxin-like compounds (DLCs), non-dioxin-like polychlorinated biphenyls (PCBs) and polybrominated diphenyl ether (PBDE). Sampling regions included the industrial cities – Cape Town, Richards Bay, Durban and Bloemfontein, and low-income, high density residential areas surrounding a wetland in Soweto/Lenasia and Botshabelo. Additionally, rivers flowing into neighbouring countries, rivers in the vicinity of paper and pulp producers and high altitude rivers were included. Sediment samples were firstly screened for the presence of DLCs by the H4IIE-luc bio-assay, whereafter sites eliciting quantifiable responses were selected for further chemical analysis by high resolution gas chromatography-high resolution mass spectrometry (HRGC/HRMS). Of the 96 sites, only 23 had quantifiable levels of DLCs. These sites were mainly of industrial, semi-industrial or low-income residential nature. PAHs were the predominant class of compounds at most of the sites, while OCPs and PCBs were present in moderate concentrations and PBDEs in minor concentrations. The concentration of pollutants measured in South African soils and sediments were intermediate when compared to the levels measured in some European, Asian and Scandinavian countries, with the exception of a few sites where exceptionally high levels of compounds were measured. The carbon content normalized concentrations of certain compounds at some of the sites exceeded the Canadian sediment quality guidelines. The estimated cancer risk associated with dermal absorption of OCPs measured in this study was negligible when compared to the background cancer risk expected for South Africans due to life style factors. However, it was estimated that dermal exposure to PCBs, DLCs and PAHs may lead to severe increases in cancer cases, and may seriously impact on human health. / Thesis (Ph.D. (Environmental Science))--North-West University, Potchefstroom Campus, 2011.

Water resources in South Africa are scarce, and should therefore be protected against pollutants, also from persistent organic pollutants (POPs). POPs are a global concern due to their ubiquitous presence, persistence and toxicity. This is emphasised by the Stockholm Convention on POPs, which aims at reducing and ultimately eliminating them. South Africa signed and ratified the treaty, and it became international law on 17 May 2004, but there is still a lack of information regarding POPs in South Africa. This study focussed on establishing the levels of POPs and other organic pollutants, which included various organochlorine pesticides (OCPs), polycyclic aromatic hydrocarbons (PAHs), dioxin-like compounds (DLCs), non-dioxin-like polychlorinated biphenyls (PCBs) and polybrominated diphenyl ether (PBDE). Sampling regions included the industrial cities – Cape Town, Richards Bay, Durban and Bloemfontein, and low-income, high density residential areas surrounding a wetland in Soweto/Lenasia and Botshabelo. Additionally, rivers flowing into neighbouring countries, rivers in the vicinity of paper and pulp producers and high altitude rivers were included. Sediment samples were firstly screened for the presence of DLCs by the H4IIE-luc bio-assay, whereafter sites eliciting quantifiable responses were selected for further chemical analysis by high resolution gas chromatography-high resolution mass spectrometry (HRGC/HRMS). Of the 96 sites, only 23 had quantifiable levels of DLCs. These sites were mainly of industrial, semi-industrial or low-income residential nature. PAHs were the predominant class of compounds at most of the sites, while OCPs and PCBs were present in moderate concentrations and PBDEs in minor concentrations. The concentration of pollutants measured in South African soils and sediments were intermediate when compared to the levels measured in some European, Asian and Scandinavian countries, with the exception of a few sites where exceptionally high levels of compounds were measured. The carbon content normalized concentrations of certain compounds at some of the sites exceeded the Canadian sediment quality guidelines. The estimated cancer risk associated with dermal absorption of OCPs measured in this study was negligible when compared to the background cancer risk expected for South Africans due to life style factors. However, it was estimated that dermal exposure to PCBs, DLCs and PAHs may lead to severe increases in cancer cases, and may seriously impact on human health. / Thesis (Ph.D. (Environmental Science))--North-West University, Potchefstroom Campus, 2011.

The object of this thesis was the study on the pharmacokinetics and biotransformation of selected abused new synthetic drugs, specifically belonging to cathinone and aminoindane groups, in experimental rats. This work consisted of two parts: a biotransformation study of mephedrone, methylone, 5,6-methylenedioxy-2- aminoindane (MDAI) and a distribution profile study of mephedrone, methylone, MDAI and naphyrone. This document is presented as a commentary to the original papers, which were published in peer reviewed journals. The samples of blood and tissue (brain, lungs, liver) were taken at specific times after a subcutaneous single bolus dose of the studied drugs. The urine samples for the biotransformation studies were collected over a 24-hour period following the sc. dose. The LC/HRMS method was used for metabolite detection and quantitative analysis. Our findings not only included testing metabolites of the aforementioneddrugs against the synthesized reference standards, but also discovering previously unknown metabolites of these drugs. Ten phase I and five phase II metabolites aside from the parent drug were identified in urine after mephedrone administration by comparison of their spectra against those of reference standards and/or MS2 spectra of previously identified metabolites. The main...

Novel psychoactive substances (NPS) represent a great challenge to toxicologists due to the ability of illicit drug manufacturers to alter NPS chemical structures quickly and with ease to circumvent legislation regulating their use. Each time a new structure is introduced, there is a possibility that it has not been previously recorded in law enforcement or scientific databases. Many toxicology laboratories use targeted analytical methods that rely on libraries of known compounds to identify drugs in samples. However, these libraries do not include large numbers of NPS which could result in non-identification or detection. High-resolution mass spectrometry (HRMS) has been suggested as a method for screening a wide variety of analytes due to its higher sensitivity and mass accuracy as compared to some other forms of mass spectrometry. This technique can generate characteristic MS/MS spectral data for use in compound identification. The main goal of this research was to create a high-resolution mass spectrometry (HRMS) library of NPS and metabolites, as well as validate a method for screening and confirmation of these substances. The study consisted of three main tasks which included; the development of a large high-resolution MS/MS spectral library and database, validation of a method for screening and confirmation of over 800 NPS and metabolites, and screening of blind-spiked and authentic urine specimens to determine real-world applicability of the HRMS library and method. During validation, several isomeric and structurally related NPS were observed which could not be adequately separated using traditional LC methods. A fourth task was therefore added to investigate improved separation using two-dimensional liquid chromatography (2D-LC). Increased resolving power is achieved in 2D-LC through the coupling of multiple orthogonal separation systems. Ultimately, an on-line, comprehensive method was developed using orthogonal reversed-phase columns in each dimension (RP x RP) for improved separation of co-eluting and isomeric synthetic cannabinoids. This work can aid laboratories in the identification of NPS through the use of a validated LC-QTOF-MS method for screening and confirmation and HRMS spectral library. In instances where isomeric and structurally related NPS are not sufficiently separated, RP x RP methods can be explored.

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No description available.

Voxelotor (GBT440) is a haemoglobin S polymerization inhibitor used to treat anaemia in sickle cell disease. Due to an increase of arterial oxygen saturation as well as serum erythropoietin and haemoglobin, the World Anti-Doping Agency included voxelotor in the list of prohibited substances and methods in 2023. The objective of the present study was to identify and characterize metabolites of voxelotor to detect a potential misuse by athletes. The biotransformation was studied in vitro using the human hepatocellular cell line HepG2 and pooled human liver microsomes. The metabolites were analysed using high-performance liquid chromatography (high-resolution) mass spectrometry. In total, three phase I metabolites and six phase II metabolites (resulting from glucuro-conjugation and O-methylation) were formed by the HepG2 cells in a time-dependent manner, and two phase I metabolites were generated by the liver microsomes, among them one also found in the HepG2 incubations. A reduced metabolite and the glucuro-conjugate of a reduced metabolite were the most abundant formed by HepG2 cells. In addition, metabolites resulting from mono-hydroxylation, reduction and O-methylation in different combinations were identified. Voxelotor was also found as glucuro-conjugate with a low abundance. With the spectrometric behaviour of voxelotor and its in vitro metabolites described herein, an implementation in doping control screening and, consequently, a detection of an abuse in an athlete urine sample might be possible.

Dans le but de valoriser la biodiversité végétale des Alpes-Maritimes, une plante commune dans cette région, Solidago virgaurea, a été sélectionnée pour son potentiel inhibiteur de la conversion levure-hyphe de Candida albicans, micro-organisme responsable d’infections bucco-dentaires de type candidose. Le fractionnement bioguidé de l’extrait aqueux a conduit à l’identification d’une famille de saponines particulièrement active. Parmi les onze saponines majoritaires caractérisées par RMN et HRMS, cinq se sont révélées être de nouvelles structures. Les tests biologiques ont néanmoins montré qu’elles n’étaient pas toutes actives contre la forme filamenteuse de C. albicans. Ces résultats ont conduit à une étude de la variabilité de la composition en saponines de plusieurs populations alpines de S. virgaurea. Trois méthodes de dosage des saponines ont été développées par HPLC et HPTLC. Les résultats ont démontré l'influence de différents facteurs sur la composition en saponines. Enfin, la composition globale de différents extraits de S. virgaurea a été étudiée dans le but d'identifier des activités biologiques complémentaires. Parmi les composés identifiés, trois nouveaux acides octulosoniques ont été caractérisés, aux côtés de trois composés phénoliques identifiés pour la première fois chez S. virgaurea. Les tests biologiques sur les extraits et fractions ont par ailleurs mis en évidence des activités antioxydante, anti-tyrosinase et inhibitrice de cellules cancéreuses in vitro. Ces tests devront être approfondis ultérieurement. / Toward the promotion of plant diversity of Maritime Alps, a common plant of the alpine area, Solidago virgaurea, was chosen to its inhibiting activity of Candida albicans yeast-hyphal conversion, a causal agent of opportunistic oral infections named candidiasis. In a first step, an aqueous extract of S. virgaurea was submitted to bioassay guided fractionation. This led to an active saponin-containing fraction from which eleven saponins were characterized by carrying out NMR experiments along with HRMS analyses. Five out of these were identified for the first time and bioassays showed that saponins activity varied according to the molecular structure of the compound. In a second step, the saponins composition of various S. virgaurea populations was studied qualitatively and quantitatively, using HPLC and HPTLC. Results demonstrated that saponins composition depends on various factors. Finally, the overall chemical composition of different S. virgaurea extracts was investigated searching for additional bioactivities. Among all the identified compounds, three new octulosonic acids were characterized and three phenolic compounds were found for the first time in S. virgaurea. Moreover, bioassays on extracts and fractions showed antioxidant, anti-tyrosinase activity and inhibition of cancer cell lines in vitro. Further bioassays have now to be completed. As a conclusion, this work was the starting point of an oral care product development and the setting-up of an innovative sector.