Since the discovery of Mycobacterium tuberculosis (Mtb) as the bacterial agent causing tuberculosis, the permanent eradication of this disease has proven challenging. Although a number of drugs exist for the treatment of tuberculosis, 1.7 million people still die every year from this infection. The current treatment regimen involves lengthy combination therapy with four different drugs in an effort to combat the development of resistance. However, multidrug-resistant and extensively drug-resistant strains are emerging in all parts of the world. Therefore, new drugs effective in the treatment of tuberculosis are much-needed. The work presented in this thesis was focused on the early stages of drug discovery by applying different hit identification and hit expansion strategies in the exploration of two new potential drug targets, glutamine synthetase (GS) and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR). A literature survey was first carried out to identify new Mtb GS inhibitors from compounds known to inhibit GS in other species. Three compounds, structurally unrelated to the typical amino acid derivatives of previously known GS inhibitors, were then discovered by virtual screening and found to be Mtb GS inhibitors, exhibiting activities in the millimolar range. Imidazo[1,2-a]pyridine analogues were also investigated as Mtb GS inhibitors. The chemical functionality, size requirements and position of the substituents in the imidazo[1,2-a]pyridine hit were investigated, and a chemical library was designed based on a focused hierarchical design of experiments approach. The X-ray structure of one of the inhibitors in complex with Mtb GS provided additional insight into the structure–activity relationships of this class of compounds. Finally, new α-arylated fosmidomycin analogues were synthesized as inhibitors of Mtb DXR, exhibiting IC50 values down to 0.8 µM. This work shows that a wide variety of aryl groups are tolerated by the enzyme. Cinnamaldehydes are important synthetic intermediates in the synthesis of fosmidomycin analogues. These were prepared by an oxidative Heck reaction from acrolein and various arylboronic acids. Electron-rich, electron-poor, heterocyclic and sterically hindered boronic acids could be employed, furnishing cinnamaldehydes in 43–92% yield.

Uncontrolled spreading of genetically modified (GM) plants is one of the main concerns about their cultivation. Inducible RNA interference against an essential gene could be a tool for control of GM plants. After spraying with a chemical inducer, the essential gene will be silenced so the treated GM plant will die. For testing this strategy we chose two key enzymes of nitrogen metabolism, glutamate synthase (GOGAT) and glutamine synthetase (GS). GS processes ammonium ions into glutamine, then GOGAT transfers the amide group from glutamine to 2-oxoglutarate to form two glutamates. GS/GOGAT cycle is the main pathway for assimilation of ammonium ions, which could be toxic to plants in a higher concentration. Disruption of ammonium assimilation during photorespiration causes a strong inhibition of photosynthesis. The aim of this work was to describe the effects of silencing GOGAT and GS genes in Arabidopsis thaliana. To induce silencing, RNAi hairpin constructs under a control of constitutive or estradiol-inducible promoter were prepared. In selected independent transformants with the inducible hairpin against GOGAT, chlorosis and reduced growth were observed after the estradiol treatment in in vitro conditions. However, the spraying with estradiol was tricky, at the whole plant level, the induction of...

Ruminant animals consuming low N-diet have been reported to have increased urea reabsorption with the Nguni being categorized as N-recycling ruminant. The enzymes associated with N-cycling are hypothesized to contribute to survival of the Nguni in harsh conditions. Enzymes responsible for such a function needed to be characterized in order to determine their effect in the functioning of the Nguni as opposed to Hereford breed. Crude enzymes from both breeds were separated from most or some contaminants by sephadex G-25, DEAE sephacel, and different affinity column chromatography. CPS and GDH were successfully purified and characterized by LC-MS/MS and further analysed by ProteinPilot™, blasted and matched >95% with those of Bos Taurus. Comparison of characterized enzymes and those which failed to ionise such as ARG, GS and GA was done using kinetics and graphs annotating specific activities. Partial purification and characterization was in part achieved. / Life and Consumer Sciences / M. Sc. (Life Sciences)

Contexte et objectifs : L'encéphalopathie hépatique (EH) est un trouble neuropsychiatrique, une complication majeure de la maladie hépatique chronique (MHC). L'EH se manifeste par un large éventail de symptômes, allant d'un léger manque d'attention et de troubles de la mémoire à une léthargie sévère et un coma. L'hyperammoniémie est centrale dans la pathogenèse de l'EH puisque l'ammoniac est neurotoxique et que l'ammoniac dérivé du sang traverse facilement la barrière hémato-encéphalique (BHE). Cependant, d'autres facteurs pathogènes sont également impliqués dans l'EH, notamment le stress oxydatif. Au cours de la MHC, le muscle joue un rôle compensatoire essentiel dans l'élimination de l'ammoniac par l'action de l'enzyme glutamine synthétase (GS), qui transforme le glutamate en glutamine. Étant donné que les cellules endothéliales de la BHE sont l'interface entre le sang et le cerveau, il est plausible qu'elles métabolisent l'ammoniac pour protéger le cerveau de la neurotoxicité induite par l'ammoniac. Cependant, cela n'a jamais fait l'objet d'études. Les thérapies de réduction de l'ammoniac sont les traitements courants de l'EH. Cependant, les réponses des patients aux traitements sont hétérogènes, et les différences de sexe pourraient en être la cause. Par conséquent, nos objectifs étaient 1) d'explorer le métabolisme de l'ammoniac dans les cellules endothéliales de la BHE par la présence de GS et 2) d'évaluer l'impact du sexe sur la MHC et ses complications, y compris la sarcopénie et l'EH. Méthodes : Pour le premier objectif, nous avons évalué l'expression et l'activité de la protéine GS in vitro et ex vivo chez des rats naïfs. Nous avons également évalué l'impact de l'ornithine, du glutamate et du α-kétoglutarate sur l'activité de la GS dans les cellules endothéliales de la BHE via la génération de glutamine 5-13C marquée. Pour le deuxième objectif, nous avons évalué l'impact du sexe sur le neurophénotype (anxiété, mémoire, coordination motrice et activité) chez des rats ligaturés des voies biliaires (BDL) (et contrôles respectifs) ainsi que sur le développement d'une EH sévère (léthargie/perte du réflexe de redressement). Nous avons également évalué les marqueurs des lésions hépatiques, l'hyperammoniémie, le stress oxydatif systémique, la masse et la fonction musculaire et la clairance de l'ammoniac musculaire. Résultats : Nous avons trouvé l'activité et l'expression de la GS in vivo et ex vivo dans les cellules endothéliales de la BHE. L'analyse au microscope confocal a montré que la GS dans les cellules endothéliales est moins abondante que dans les astrocytes. L'exposition de cellules endothéliales cultivées à des substrats marqués a révélé que l'ornithine est la plus efficace pour générer de la glutamine. Chez les femmes, la chirurgie BDL a provoqué une MHC (augmentation des enzymes hépatiques circulantes et de la bilirubine) et de l'EH (altération de la coordination motrice et de l'activité nocturne) par rapport aux rats contrôles respectifs. De plus, le degré d'hyperammoniémie et la clairance musculaire de l'ammoniac étaient similaires entre les sexes. Contrairement aux mâles, les rats femelles n'ont pas développé de perte musculaire, d'œdème cérébral et de perte de mémoire à court terme. De plus, les femelles présentaient un stress oxydatif plus faible et étaient complètement protégées contre les EH sévères précipitées par l'ammoniac par rapport aux mâles BDL. Conclusions : Nous concluons que la GS est exprimée dans les cellules endothéliales de la BHE, jouant peut-être un rôle dans l'atténuation ou le retard de l'entrée de l'ammoniac dans le cerveau et que la supplémentation en ornithine améliore l'activité de la GS en fournissant du glutamate pour la détoxification de l'ammoniac. De plus, nous concluons que le sexe a un impact sur les complications des maladies du foie, y compris la sarcopénie et l'EH, le stress oxydatif systémique jouant un rôle vital dans la susceptibilité à l'EH sévère induite par l'ammoniac. / Background and aims: Hepatic encephalopathy (HE) is a neuropsychiatric disorder and a major complication of chronic liver disease (CLD). HE manifests with a wide range of symptoms, from mild lack of attention and memory impairments to severe lethargy and coma. Hyperammonemia is central in the pathogenesis of HE since ammonia is neurotoxic, and blood-derived ammonia easily crosses the blood-brain barrier (BBB). However, other pathogenic factors are also implicated in HE, including oxidative stress. During CLD, muscle plays an essential compensatory role in removing ammonia by the action of the enzyme glutamine synthetase (GS), which amidates glutamate into glutamine. Since the endothelial cells of the BBB are the interface between the blood and the brain, it is plausible that they metabolize ammonia to protect the brain from ammonia-induced neurotoxicity. However, this has never been investigated. Ammonia lowering therapies are the mainstream treatments for HE. However, patients' response to treatments are heterogeneous, and sex differences might be the cause. Therefore, our aims were 1) To explore ammonia metabolism in BBB’s endothelial cells through the presence of GS and 2) to assess the impact of sex on CLD and its complications, including sarcopenia and HE. Methods: For the first aim, we assessed GS protein expression and activity in vitro and ex vivo in naïve rats. We also evaluated the impact of ornithine, glutamate, and α-ketoglutarate on GS activity in endothelial cells of the BBB via the generation of labeled 5-13C glutamine. For the second aim, we assessed the impact of sex on the neurophenotype (anxiety, memory, motor coordination, and activity) in bile-duct ligated (BDL) rats (and respective SHAMs) as well as on the development of an ammonia-precipitated severe HE (lethargy/loss of righting reflex). We also assessed liver injury markers, hyperammonemia, systemic oxidative stress, muscle mass and function, and muscle ammonia clearance. Results: We found GS activity and expression in vivo and ex vivo in endothelial BBB cells. The confocal microscope analysis showed that GS in endothelial cells is less abundant than astrocytes. Exposing cultured endothelial cells to labeled substrates revealed that ornithine is the most efficient in generating glutamine. In females, BDL surgery caused CLD (increased hepatic enzymes and bilirubin) and HE (impaired motor coordination and night activity) vs. respective SHAMs. Furthermore, the degree of hyperammonemia and muscle ammonia clearance was similar between sexes. Contrary to males, female rats did not develop muscle loss, brain edema, and short-term memory loss. In addition, females had lower oxidative stress and were completely protected against ammonia-precipitated severe HE compared to male BDLs. Conclusions: We conclude that GS is expressed in endothelial cells of the BBB, possibly playing a role in attenuating or delaying ammonia entry into the brain and, ornithine supplementation enhances GS activity by providing glutamate for ammonia detoxification. In addition, we conclude that sex impacts the complications of liver disease, including sarcopenia and HE, with systemic oxidative stress playing a vital role in the susceptibility to ammonia-induced overt HE.