Avec la hausse mondiale de la fréquence des floraisons de cyanobactéries (CB), dont certaines produisent des cyanotoxines (CT), le développement d’une méthode de détection/quantification rapide d’un maximum de CT s’impose. Cette méthode permettrait de faire un suivi quotidien de la toxicité de plans d’eau contaminés par des CB et ainsi d’émettre rapidement des avis d’alerte appropriés afin de protéger la santé publique. Une nouvelle technologie utilisant la désorption thermique induite par diode laser (LDTD) couplée à l’ionisation chimique sous pression atmosphérique (APCI) et reliée à la spectrométrie de masse en tandem (MS/MS) a déjà fait ses preuves avec des temps d'analyse de l’ordre de quelques secondes. Les analytes sont désorbés par la LDTD, ionisés en phase gazeuse par APCI et détectés par la MS/MS. Il n’y a donc pas de séparation chromatographique, et la préparation de l’échantillon avant l’analyse est minimale selon la complexité de la matrice contenant les analytes. Parmi les quatre CT testées (microcystine-LR, cylindrospermopsine, saxitoxine et anatoxine-a (ANA-a)), seule l’ANA-a a généré une désorption significative nécessaire au développement d’une méthode analytique avec l’interface LDTD-APCI. La forte polarité ou le poids moléculaire élevé des autres CT empêche probablement leur désorption. L’optimisation des paramètres instrumentaux, tout en tenant compte de l’interférence isobarique de l’acide aminé phénylalanine (PHE) lors de la détection de l’ANA-a par MS/MS, a généré une limite de détection d’ANA-a de l’ordre de 1 ug/L. Celle-ci a été évaluée à partir d’une matrice apparentée à une matrice réelle, démontrant qu’il serait possible d’utiliser la LDTD pour effectuer le suivi de l’ANA-a dans les eaux naturelles selon les normes environnementales applicables (1 à 12 ug/L). Il a été possible d’éviter l’interférence isobarique de la PHE en raison de sa très faible désorption avec l’interface LDTD-APCI. En effet, il a été démontré qu’une concentration aussi élevée que 500 ug/L de PHE ne causait aucune interférence sur le signal de l’ANA-a. / Within the context of the worldwide increasing frequency of cyanobacterial (CB) blooms, some containing cyanotoxins (CT), the development of a detection/quantification method for the fast analysis a maximum of CT is necessary. This method would allow daily tracking of the toxicity of CB-contaminated water such that, as warranted, appropriate measures can be taken quickly to protect public health. A new technology using laser diode thermal desorption (LDTD) coupled to atmospheric pressure chemical ionization (APCI)-tandem mass spectrometry (MS/MS) has shown great potential to reduce analysis time to seconds. Analytes are desorbed by the LDTD, ionized in gas-phase by APCI and detected by MS/MS. Therefore, there is no chromatographic separation and sample treatment prior to analysis is minimal, depending on the complexity of the sample matrix. Among the four CT tested (microcystin-LR, cylindrospermopsin, saxitoxin and anatoxin-a (ANA-a)), only ANA-a exhibited sufficient desorption which is necessary to develop an analytical method with the LDTD-APCI interface. The strong polarity or high molecular weight of the other CT probably inhibited their efficient desorption. Optimization of instrumental parameters, while accounting for the isobaric interference caused by the acid amino phenylalanine (PHE) in the detection of ANA-a by MS/MS, generated a detection limit of the order of 1 ug/L ANA-a. This value was obtained in a simulated natural matrix, demonstrating that it would be possible to use LDTD to monitor ANA-a in natural waters within the range of current applicable environmental guidelines (1 to 12 ug/L). Because PHE desorption is limited with the LDTD-APCI interface, this method avoids its interference on ANA-a analysis, even at PHE concentrations as high as 500 ug/L.

Alors que le protéome de l'ovocyte isolé, aux stades VG et MII a déjà été étudié, celui du follicule en croissance n'a jamais été décrit.Dans cette étude, nous avons cherché à identifier, comparer et caractériser les profils protéiques de follicules ovariens de souris à trois stades de leur développement in vitro distincts morphologiquement : follicules secondaires en début de culture - stade initial (IS), follicules avec une rupture complète de la membrane de Slavjanski (RMS) et follicules avec une cavité similaire à l'antrum (FA).Après un préfractionnement par IEF et une analyse LC-MS/MS en deux configurations (1D et 2D), 1403 protéines ont pu être identifiées dans le follicule ovarien de souris. 43,4 % (609) des protéines identifiées étaient communes aux trois stades et d'autres ont été identifiées uniquement à un stade : 71 au stade IS, 182 au stade RMS et 193 au stade FA. De plus, on a identifié 365 protéines qui n'avaient pas été décrites antérieurement dans le protéome de l'ovocyte ce qui pourrais indiquer qu'elles sont exprimées dans les cellules somatiques du follicule. Des analyses qualitatives et quantitatives complémentaires ont démontré une surreprésentation pour 44 fonctions biologiques par rapport aux fonctions biologiques des gènes constituant le génome de Mus musculus et mis en évidence des différences d'expression et d'abondance des protéines liées au cycle cellulaire, à la fixation des ions de calcium et à la glycolyse selon le stade de développement. Ces résultats représentent un point de départ pour beaucoup d'autres études de caractérisation moléculaire du développement folliculaire.

Purpose: Schizophrenia is a progressive degenerative illness that has been causally linked to mitochondrial dysfunction, oxidative stress and a pro-inflammatory state. Social isolation rearing (SIR) in rats models the neurodevelopmental aspects of schizophrenia. The antioxidant and glutamate modulator, N-acetyl cysteine (NAC), has demonstrated therapeutic potential in schizophrenia as adjunctive treatment, although this has not been tested in the SIR model. The purpose of this study was to assess whether SIR induces changes in mitochondrial function (adenosine triphosphate (ATP)), pro- vs. anti-inflammatory cytokine balance, tryptophan metabolism, a disturbance in cortico-striatal monoamines and related metabolites, and associated alterations in behaviors akin to schizophrenia, viz. social interaction, object recognition memory and prepulse inhibition (PPI). Moreover, I evaluated whether these bio-behavioral alterations could be reversed with sub-chronic clozapine, or NAC, and whether NAC may bolster the response to clozapine treatment. Methods: The objectives of the study were pursued through separately conducted studies. Male Sprague-Dawley (SD) rats (10 rats/group) were used in this study (Ethics number: NWU-0035-08-S5). Rats were randomly allocated to either social rearing or SIR for 8 weeks receiving either no treatment, vehicle, NAC (150 mg/kg/day), clozapine (5 mg/kg/day) or a combination of clozapine + NAC (CLZ + NAC) during the last 11 or 14 days of social rearing or SIR. After the 8 weeks, rats were tested for social interactive behaviors, object recognition memory and prepulse inhibition (PPI). Peripheral tryptophan metabolites (determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS)) and pro- and anti-inflammatory cytokines (IL-4, IL-6, TNF-α, IFN-γ) (enzyme-linked immunosorbent assay (ELISA)) were determined. Cortico-striatal ATP (bioluminescence assay) and monoamines (high performance liquid chromatography (HPLC)) were also determined. Results: SIR-induced significant deficits in social interactive behaviours, object recognition memory and PPI, associated with increased peripheral kynurenine, quinolinic acid (QA), and pro-inflammatory cytokines, as well as a decrease in kynurenic acid (KYNA), neuroprotective ratio and anti-inflammatory cytokines. I also observed an increase in striatal, but reduced frontal cortical ATP, dopamine, serotonin as well as their metabolites and noradrenaline’s metabolite, with noradrenaline increased in both brain regions in SIR rats. A separate dose-response study of NAC (50, 150, 250 mg/kg/day) found 150 mg/kg to be the most appropriate dose for the NAC and CLZ + NAC studies. Clozapine, NAC as well as CLZ + NAC reversed all these changes, with NAC being less effective than CLZ alone. CLZ + NAC was found to be more effective than clozapine alone in reversing certain bio-behavioral alterations induced by SIR. In addition NAC alone dose dependently reversed most of the SIR induced alterations. Conclusion: SIR induces behavioral alterations, a pro-inflammatory state, mitochondrial dysfunction and cortico-striatal monoamine alterations, closely resembling evidence in schizophrenia. Importantly, all these bio-behavioral alterations were reversed with clozapine, NAC and CLZ + NAC treatment. However, CLZ + NAC was more effective than clozapine alone in reversing some bio-behavioral alterations, supporting the therapeutic application of NAC as adjunctive treatment in schizophrenia. In addition, NAC dose dependently reversed SIR-induced cortico-striatal serotonin, noradrenaline and metabolites, emphasizing NAC’s potential use in other anxiety and stress- related disorders. / Thesis (PhD (Pharmacology))--North-West University, Potchefstroom Campus, 2013

Chronic, noncommunicable diseases such as chronic fatigue syndrome (also known as myalgic encephalomyelitis) are rapidly becoming a worldwide epidemic that profoundly affects public health and productivity. Chronic fatigue syndrome (CFS) is characterised by severe and debilitating fatigue and although its etiology is still unknown, recent studies have found considerable evidence that mitochondrial dysfunction and oxidative stress might be responsible for the underlying energy deficit in these patients. Adenine and pyridine nucleotides could be used as potential biomarkers for energy related disorders such as chronic fatigue syndrome because of their various functions in the energy and redox pathways. The first part of this study focussed on developing a liquid chromatography electrosprayionisation tandem mass spectrometry (LC-ESI-MS/MS) method for the quantification of these nucleotides in blood samples. Due to the instability of nucleotides in biological matrices it was also necessary to find a suitable extraction method that would be able to stop enzymatic activity via protein precipitation. Out of the four extraction methods investigated during this study, deproteinisation of whole blood samples with perchloric acid produced the highest nucleotide abundances. Although nucleotide standards were found to be stable in perchloric acid, nucleotide levels in blood samples were not stabilised by addition of perchloric acid. The second part of this study consisted of measuring the nucleotide levels in blood samples of controls and possible CFS patients in order to test the proof of concept of the new LCESI- MS/MS method. Despite changes in the nucleotide levels due to perchloric acid and problems with nucleotide instability, it was still possible to distinguish between the two groups based on the results obtained with the new LC-ESI-MS/MS method. The newly developed LC-ESI-MS/MS method proved to be reliable and adequate for nucleotide quantification in whole blood samples, thus the aim of this study was achieved. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2014

Purpose: Schizophrenia is a progressive degenerative illness that has been causally linked to mitochondrial dysfunction, oxidative stress and a pro-inflammatory state. Social isolation rearing (SIR) in rats models the neurodevelopmental aspects of schizophrenia. The antioxidant and glutamate modulator, N-acetyl cysteine (NAC), has demonstrated therapeutic potential in schizophrenia as adjunctive treatment, although this has not been tested in the SIR model. The purpose of this study was to assess whether SIR induces changes in mitochondrial function (adenosine triphosphate (ATP)), pro- vs. anti-inflammatory cytokine balance, tryptophan metabolism, a disturbance in cortico-striatal monoamines and related metabolites, and associated alterations in behaviors akin to schizophrenia, viz. social interaction, object recognition memory and prepulse inhibition (PPI). Moreover, I evaluated whether these bio-behavioral alterations could be reversed with sub-chronic clozapine, or NAC, and whether NAC may bolster the response to clozapine treatment. Methods: The objectives of the study were pursued through separately conducted studies. Male Sprague-Dawley (SD) rats (10 rats/group) were used in this study (Ethics number: NWU-0035-08-S5). Rats were randomly allocated to either social rearing or SIR for 8 weeks receiving either no treatment, vehicle, NAC (150 mg/kg/day), clozapine (5 mg/kg/day) or a combination of clozapine + NAC (CLZ + NAC) during the last 11 or 14 days of social rearing or SIR. After the 8 weeks, rats were tested for social interactive behaviors, object recognition memory and prepulse inhibition (PPI). Peripheral tryptophan metabolites (determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS)) and pro- and anti-inflammatory cytokines (IL-4, IL-6, TNF-α, IFN-γ) (enzyme-linked immunosorbent assay (ELISA)) were determined. Cortico-striatal ATP (bioluminescence assay) and monoamines (high performance liquid chromatography (HPLC)) were also determined. Results: SIR-induced significant deficits in social interactive behaviours, object recognition memory and PPI, associated with increased peripheral kynurenine, quinolinic acid (QA), and pro-inflammatory cytokines, as well as a decrease in kynurenic acid (KYNA), neuroprotective ratio and anti-inflammatory cytokines. I also observed an increase in striatal, but reduced frontal cortical ATP, dopamine, serotonin as well as their metabolites and noradrenaline’s metabolite, with noradrenaline increased in both brain regions in SIR rats. A separate dose-response study of NAC (50, 150, 250 mg/kg/day) found 150 mg/kg to be the most appropriate dose for the NAC and CLZ + NAC studies. Clozapine, NAC as well as CLZ + NAC reversed all these changes, with NAC being less effective than CLZ alone. CLZ + NAC was found to be more effective than clozapine alone in reversing certain bio-behavioral alterations induced by SIR. In addition NAC alone dose dependently reversed most of the SIR induced alterations. Conclusion: SIR induces behavioral alterations, a pro-inflammatory state, mitochondrial dysfunction and cortico-striatal monoamine alterations, closely resembling evidence in schizophrenia. Importantly, all these bio-behavioral alterations were reversed with clozapine, NAC and CLZ + NAC treatment. However, CLZ + NAC was more effective than clozapine alone in reversing some bio-behavioral alterations, supporting the therapeutic application of NAC as adjunctive treatment in schizophrenia. In addition, NAC dose dependently reversed SIR-induced cortico-striatal serotonin, noradrenaline and metabolites, emphasizing NAC’s potential use in other anxiety and stress- related disorders. / Thesis (PhD (Pharmacology))--North-West University, Potchefstroom Campus, 2013

Les dernières décennies ont été marquées par une augmentation du nombre des cas de cancers, ce qui a subséquemment conduit à une augmentation dans la consommation des agents de chimiothérapie. La toxicité et le caractère cancérogène de ces molécules justifient l’intérêt crucial porté à leur égard. Quelques études ont fait l’objet de détection et de quantification des agents de chimiothérapie dans des matrices environnementales. Dans ce projet, une méthode utilisant la chromatographie liquide couplée à la spectrométrie de masse en tandem (LC-MS/MS) précédée d’une extraction sur phase solide (SPE) automatisée ou en ligne a été développée pour la détection et la quantification d’un groupe de six agents de chimiothérapie. Parmi ceux-ci figurent les plus utilisés au Québec (gemcitabine, méthotrexate, cyclophosphamide, ifosfamide, irinotécan, épirubicine) et présentant des propriétés physico-chimiques et des structures chimiques différentes. La méthode développée a été validée dans une matrice réelle représentant l’affluent d’une station d’épuration dans la région de Montréal. Deux des six composés cytotoxiques étudiés en l’occurrence (cyclophosphamide et méthotrexate) ont été détectés dans huit échantillons sur les neuf qui ont été recensés, essentiellement au niveau de l’affluent et l’effluent de quelques stations d’épuration de la région de Montréal. Les résultats des analyses effectuées sur les échantillons réels ont montré qu’il n’y avait pas de différence significative dans la concentration entre l’affluent et l’effluent, et donc que les systèmes d’épuration semblent inefficaces pour la dégradation de ces molécules. / The last few decades have been marked by an increase in the number of cancer cases, which subsequently led to an increase in the consumption of chemotherapeutic agents. The toxicity and the carcinogenicity of these molecules justify the increased interest. Few studies have been conducted to detect and quantify chemotherapeutic agents in environmental matrices. In this project, a method using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) preceded by an online solid-phase extraction (SPE) has been developed for the detection and quantification of a group of six chemotherapeutic agents, which are among the most commonly used in Quebec (gemcitabine, methotrexate, cyclophosphamide, ifosfamide, irinotecan, epirubicin) and having different physico-chemical properties and different chemical structures. The developed method was validated in a real water matrix representing the influent of a sewage treatment plant in the Montreal area. Two of the six studied cytotoxic agents (cyclophosphamide and methotrexate) were detected in eight samples of the nine taken mainly at the influent and effluent of some treatment plants in the Montreal area. The results of the analysis of real samples showed that there was no significant difference in concentration between the influent and effluent. This also demonstrates the inadequacy of the current wastewater treatment approaches to remove those compounds.

Disease-specific compounds (biomarkers) are analyzed in clinical laboratories to assist with diagnosing diseases. This thesis describes development and validation of liquid chromatography tandem mass spectrometry (LC-MS/MS) based tests for diagnosing a diverse group of endocrine and metabolic diseases. The analytical methods used on-line and off-line sample extraction and analytical derivatization as means of enhancing the analytical sensitivity, specificity and clinical utility. All developed methods were extensively validated and reference intervals for the biomarker concentrations were established in blood samples of healthy adults and children. Advantages of the LC-MS/MS as an analytical technique include possibility of simultaneous measurement of multiple analytes and ability of confirming their identity. In this thesis we proposed and evaluated approaches for the assessment of the specificity of analysis in the methods that use tandem mass spectrometry detection. To enhance throughput of the LC-MS/MS tests for the biomarkers that have endogenous or exogenous isomers an approach was developed for quantitation of isomers from unresolved chromatographic peaks. Using methods developed in this thesis we performed a study of the steroidogenesis in ovarian follicles of healthy women and women with polycystic ovary syndrome (PCOS). Obtained data on the steroid concentrations and associations between the steroid metabolites in the pathway would be helpful for better understanding of the ovarian pathophysiology. Potential biomarkers of PCOS were identified in the thesis; further studies will be necessary to confirm their clinical utility.

The developmental aspect of drug transport across the blood-brain barrier (BBB) was investigated. Microdialysis was used to study unbound morphine BBB transport at different ages in sheep. An in vitro study was performed to find differentially expressed genes in brain capillary-rich fractions of the brain in rats of different ages. Microdialysis and brain-to-plasma ratios were used to study the contribution of breast cancer resistance protein (Bcrp) to the transport of nitrofurantoin (NTF) across the BBB of rats during development as well as in adult rats and mice. A method of analysing morphine and its metabolites in plasma and microdialysis samples was developed and validated. The in vivo recovery of deuterated morphine, used as a calibrator in microdialysis experiments, was not affected by the presence of morphine in the tissue. A net influx of morphine was observed in premature lambs and adult sheep, in contrast to the efflux seen in other species. This influx decreased with age, indicating that the morphine transport across the BBB changes with age. In contrast, the transport of the morphine metabolite morphine-3-glucuronide (M3G) did not change with age. Microarray data indicated that several active transporters are differentially expressed with age. Moreover, the mRNA expression levels of Abcg2 (Bcrp) and Slc22a8 (organic anion transporter 3) changed with age when quantified using real-time polymerase chain reaction. In contrast, the expression of Abcb1 (P-glycoprotein) and occludin (a tight junction protein) did not change with age. In rats, the brain distribution of NTF decreased with age due to increased protein binding in plasma. The concentration ratio of unbound NTF across the BBB was low in the adult rat, due to intra-brain metabolism and/or efflux by other transporters. Bcrp did not appear to have a significant contribution in the developing rat or in knock-out mice compared to wild-type controls with regard to NTF BBB transport. In conclusion, in vitro studies showed that the expression levels of some genes changed with age, presumably affecting subsequent drug distribution to the brain. Further, in vivo studies showed that distribution across the BBB changed with age for morphine but not for M3G or NTF.

Perennial ryegrass (PRG, Lolium perenne) is a hardy cool-season grass that is infected with the endophytic fungus Neotyphodium lolii, which enables the plant to be insect repellant and drought resistant, lowering the use of insecticides and fertilizers. However, this fungus produces the compound lolitrem B (LB, m/z 686.4) which causes the tremorgenic neurotoxicity syndrome 'ryegrass staggers' in livestock consuming forage which contains <2000 ppb LB. Ergovaline (EV, m/z 534) is a vasoconstrictor normally associated with tall fescue (Festuca arudinacea), but has also been found in endophyte-infected PRG. Past research has shown a strong linear correlation between levels of LB and EV in PRG. The purpose of this study was to examine the linear relationship between EV and LB in feces and determine common metabolites. To accomplish this, four groups of steers (n=6/group) consumed endophyte- infected PRG over 70 days consumed the following averages of LB and EV: group I 2254ppb LB/633 ppb EV; group II 1554ppb LB/ 373ppb EV, group III 1011ppb LB/259ppb EV, and group IV 246ppb LB/<100ppb EV. Group I in week 4 was inadvertently given a washout period at which time the steers consumed the amount of LB and EV given to group IV (control). Both feed and feces samples were extracted using difference solid phase extraction methods and quantified by HPLC-fluorescence for LB and EV. Concentrations of EV and LB obtained through HPLC-fluorescence in both PRG and feces showed a linear relationship. Additional screening for metabolites was conducted LC-MS/MS and showed possible oxidation and reduction metabolites for both toxins. / Graduation date: 2013

A espectrometria de massas, acoplada ou não a outras técnicas, tem sido de grande utilidade na determinação de novos compostos, produtos de transformação/degradação, metabólitos e na quantificação em nível de traços nas mais diferentes matrizes, devido à grande versatilidade dessa técnica, especialmente em relação aos modos de análise. Neste trabalho foi sistematizada uma metodologia para a aplicação das técnicas de espectrometria de massas na identificação de produtos de transformação/degradação de fármacos de uso humano e veterinário. Ferramentas da química computacional foram utilizadas para auxiliar na elucidação das estruturas dos compostos formados. A metodologia foi empregada para a identificação e quantificação dos produtos de transformação/degradação da rosuvastatina, gerados durante o processo de fotocatálise heterogênea com ZnO, para a identificação dos produtos de transformação/degradação gerados durante a fotólise em meio aquoso do sulfamentoxazol, ciprofloxacino e norfloxacino, e para a quantificação da amoxicilina transferida ao leite bovino. A viabilidade de se realizar a identificação de metabólitos da amoxicilina presentes no leite bovino por meio do mesmo processo de preparo de amostra e de análise utilizado para a quantificação também foi avaliada. Foram identificados dez principais produtos de transformação/degradação da fotocatálise da rosuvstatina, dez principais produtos de transformação/degradação da fotólise em meio aquoso do sulfametoxazol, quinze principais produtos de transformação/degradação da fotólise em meio aquoso do ciprofloxacino e quinze principais produtos de transformação/degradação da fotólise em meio aquoso do norfloxacino. A metodologia empregada para a quantificação da amoxicilina em leite bovino se demostrou adequada, mas estudos adicionais são necessários para que seja possível identificar os seus metabólitos nessa matriz. Os resultados dos cálculos ab initio que foram utilizados para o estudo dos produtos de transformação/degradação da rosuvastatina e sulfametoxazol mostram que essa técnica pode ser bastante útil na elucidação estrutural dos compostos que são formados. / Mass spectrometry, coupled or not with other techniques, is very useful for the determination of new compounds, transformation/degradation products, metabolites and quantification at trace levels in many different matrices, due to the versatility of such a technique, specially regarding to analysis modes. In the present work a methodology was systematized for the application of mass spectrometry techniques in order to identify metabolites and transformation/degradation products for human and veterinary drugs usage. Computational chemistry tools were used to assist in the elucidation of the structure of the compounds formed during the process. The methodology was employed for identification and quantification of rosuvastatin transformation/degradation products generated during the heterogeneous photocatalysis process with ZnO, for the identification of sulfamethoxazole, ciprofloxacin and norfloxacion transformation/degradation products generated during the photolysis in aqueous medium and for quantifying amoxicillin transferred to bovine milk. The feasibility of identification of amoxicillin metabolites present in bovine milk using the same sample preparation procedure exploited for the quantification analysis was also assessed. Ten main transformation/degradation products of rosuvastatin photocatalysis were identified, as well as ten main transformation/degradation products of sulfamethoxaxole photolysis in aqueous medium, fifteen main transformation/degradation products of ciprofloxacin photolysis in aqueous medium and fifteen main transformation/degradation products of norfloxacin photolysis in aqueous medium. The methodology applied for the bovine milk amoxicillin quantification was demonstrated to be adequate, but additional studies are needed to identify amoxicillin metabolites in the milk matrix. The results of ab initio calculations utilised for the study of rosuvastatin and sulfamethoxazole transformation/degradation products show that this technique can be very useful for the structural elucidation of chemical compounds which are formed.