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Au niveau mondial, la tuberculose est toujours l’une des dix premières causes de mortalité. L’un des principaux facteurs expliquant ce phénomène est l'émergence de souches du bacille tuberculeux, Mycobacterium tuberculosis, résistantes aux antibiotiques. Ainsi, l’OMS a déclaré prioritaire le développement d'une nouvelle génération d’antituberculeux qui soient efficaces contre les souches résistantes. Des études de criblage phénotypique récentes sur M. tuberculosis ont montré que des voies métaboliques de composés de l’enveloppe, telles que la biosynthèse des acides mycoliques, représentent des cibles thérapeutiques très pertinentes. Les acides mycoliques entrent dans la composition de facteurs de pathogénicité lipidiques du bacille tuberculeux. Ils portent divers types de fonctions chimiques déterminantes pour leurs rôles biologiques. Le système Fatty Acid Synthase de type II (FAS-II) impliqué dans leur biosynthèse constitue une cible thérapeutique validée. En effet, il est essentiel à la survie du bacille, possède des caractéristiques uniques, et a un rôle-clé dans sa virulence et sa persistance chez les organismes infectés. De plus, le système FAS-II est la cible de plusieurs médicaments antituberculeux tels que l’isoniazide et l’éthionamide. FAS-II est constitué d'un ensemble d'enzymes monofonctionnelles acyl carrier protein (ACP)-dépendantes. Une partie de ces protéines a été identifiée par des approches de génomique classique. Malgré cela, 20 ans après le séquençage du génome de M. tuberculosis, la composition complète de ce système ainsi que sa fonction précise restent encore à caractériser. L’objectif des travaux de recherche menés durant ma thèse est de caractériser le système FAS-II mycobactérien selon un nouvel angle d'attaque, à l'échelle du système entier. L'approche adoptée visait à i) développer et valider des méthodes expérimentales pour isoler le système FAS-II sous une forme la plus complète possible, ii) décrypter en profondeur la composition en protéines de FAS-II (collaboration équipe O. Schiltz, IPBS) ; iii) identifier de nouvelles enzymes partenaires potentielles. La 1ère partie de ce travail a consisté à mettre au point l'isolement du système FAS-II de deux mycobactéries, M. smegmatis, une espèce-modèle à croissance rapide, et M. bovis BCG, la souche vaccinale très proche de M. tuberculosis. Deux stratégies complémentaires ont été développées : la co-immunoprécipitation et la Single Step Affinity Purification (SSAP). Plusieurs enzymes du système FAS-II ont été utilisées alternativement comme appâts pour co-purifier des protéines partenaires. De nombreux paramètres ont été optimisés afin d'améliorer le taux d'enrichissement en FAS-II et de conserver le plus possible l’intégrité du système : nature de la protéine-appât, délétion du gène endogène (SSAP), conditions de lyse des bactéries, pontage chimique, étapes de purification... La 2ème partie de ma thèse a porté sur l'analyse par protéomique des fractions de purification obtenues et la 3ème partie a consisté à identifier de nouvelles enzymes partenaires. Le traitement des données a permis 1) de mettre en évidence des interactions physiques entre certaines enzymes du système FAS-II ; 2) de démontrer la présence d’une nouvelle enzyme partenaire du système : cette protéine, nommée HadD, est potentiellement impliquée dans l’une des étapes-clés de la biosynthèse des acides mycoliques nécessaire pour l’introduction des groupements fonctionnels sur ces lipides. / Globally, tuberculosis is still one of the top ten leading causes of death. In 2015, the World Health Organization (WHO) reported nearly 480,000 new cases of multidrug-resistant tuberculosis and called for the development of new antituberculosis agents. Recent phenotypic screening studies on Mycobacterium tuberculosis have shown that envelope metabolic pathways, such as mycolic acid biosynthesis, are highly relevant therapeutic targets. Mycolic acids constitute the lipid moiety of pathogenicity factors of the tubercle bacillus. They are essential mycobacterial fatty acids holding various chemical functions decisive for their biological roles. The Fatty Acid Synthase type II (FAS-II) system involved in their biosynthesis is a relevant and validated therapeutic target. Indeed, it is essential to the survival of the bacillus, has unique features, and plays a key role in its virulence and persistence in infected organisms. In addition, the FAS-II system is the target of several antituberculosis drugs such as isoniazid and ethionamide. FAS-II is made of discrete monofunctional acyl carrier protein (ACP)-dependent proteins. Some of these proteins have been identified by conventional genomic approaches. However, 20 years after the M. tuberculosis genome sequencing, the complete composition of this system and its precise function remain poorly known. The objective of my PhD project is to characterize the mycobacterial FAS-II multienzyme system with a completely new line of attack, at the scale of the entire system. The adopted approach was aimed at i) developing and validating experimental methods to isolate the FAS-II system in the most complete form, ii) deciphering the FAS-II protein composition (collaboration with O. Schiltz's team, IPBS); iii) identifying potential new partner enzymes. The first part of this work consisted of developing the isolation of the FAS-II system from two mycobacteria, M. smegmatis, a fast-growing model species, and M. bovis BCG, the vaccine strain very close to M. tuberculosis. Two complementary strategies were developed: co-immunoprecipitation and Single Step Affinity Purification (SSAP). Several enzymes of the FAS-II system were used alternatively as a bait in an attempt to co-purify partner proteins. To improve the level of FAS-II enrichment and to preserve as much as possible the integrity of the system, many parameters were optimized: nature of the bait protein, endogenous gene deletion (SSAP), bacteria lysis conditions, chemical cross-link, purification steps… The second part of my thesis focused on the proteomics analysis of the purification fractions obtained and the third part consisted in identifying new partner enzymes. The data processing allowed: 1) to demonstrate the presence of physical interactions between some FAS-II system enzymes; 2) to demonstrate the presence of a new partner enzyme of the system: this protein, called HadD, is potentially involved in one of the key steps of the mycolic acid biosynthesis pathway necessary for the introduction of functional groups on these lipids. The results obtained thanks to the optimized co-immunoprecipitation and SSAP protocols have contributed to the knowledge of the mycobacterial FAS-II system and bring promising leads for the discovery of new partner enzymes.

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lsoflurane interacts with cytochrome P-450 in rat and human hepatic microsomes and the Δ6- and Δ5-desaturases in rat hepatic microsomes. The interaction of isoflurane with cytochrome P-450 results in its metabolism to fluoride ion and organofluorine metabolites. The cytochrome P-450 isozymes catalysing the defluorination of isoflurane were assessed in hepatic microsomes from phenobarbital-, β-naphthoflavone- and pregnenolone-16α-carbonitrilepretreated and untreated rats. One or more of the cytochrome P-450 isozymes induced by phenobarbital and pregnenolone-16α-carbonitrile appear to defluorinate isoflurane, but those induced by β-naphthoflavone do not. From a comparison of the extent of defluorination of isoflurane in hepatic microsomes from phenobarbital- and pregnenolone-16α-carbonitrile-pretreated rats, and their Kₘ and Vₘₐₓ values, it appears that isoflurane is defluorinated by one or more isozymes induced by both phenobarbital and pregnenolone-16α-carbonitrile. The major isozyme is probably cytochrome P-450PCN1. The metabolites of isoflurane were identified in human and phenobarbital-induced rat hepatic microsomes. In microsomes from phenobarbital-pretreated rats, isoflurane is metabolised to fluoride ion and trifluoroacetaldehyde; trifluoroacetic acid is not produced in measureable amounts. The trifluoroacetaldehyde produced binds to microsomal constituents. In human hepatic microsomes, the organofluorine metabolite is identified as trifluoroacetic acid. It is proposed that isoflurane is metabolised by different pathways in human and phenobarbital-induced rat hepatic microsomes. The interaction of isoflurane with the cyanide-sensitive factors was assessed by several criteria. Firstly, using the reoxidation of cytochrome b₅ as an index of fatty acid desaturase activity, isoflurane appears to interact with the Δ6- and/or Δ5-desaturases, but not the Δ9-desaturase. Secondly, these results were confirmed and clarified by the use of direct assays to measure the fatty acid desaturase activity. Using the direct assay, we confirmed that isoflurane did not inhibit the Δ9-desaturase and inhibited Δ6-desaturation of linoleic acid, but not the Δ6-desaturation of α-linolenic acid. The inhibition of the Δ6-desaturation of linoleic acid occurred at low millimolar concentrations of isoflurane. lsoflurane inhibits the Δ5-desaturation of eicosa-8, 11, 14-trienoic acid to a small extent which is only apparent at much higher concentrations of isoflurane than that which inhibits the Δ6-desaturase. Further studies focussed on measurement of the activity of Δ6-desaturase in order to attempt to study the kinetics of the inhibition of the Δ6-desaturase by isoflurane: Δ6-desaturase activity was assessed using hepatic microsomes as the source of the enzyme and linoleic acid as substrate precursor. In the course of these studies, we identified a number of factors that affected the apparent activity of the Δ6-desaturase in hepatic microsomes. These included significant levels of endogenous substrate and competing reactions in the hepatic microsomes. Endogenous substrate levels were quantified and corrected for. We then resorted to computer modelling to extract the kinetics of the Δ6-desaturase free of contributions from acyl-CoA synthetase and lysophospholipid acyltransferase, as well as enzyme decay. The kinetics of isoflurane inhibition of the Δ6-desaturase were then superimposed and studied by computer modelling.

Volatile fatty acids (VFAs) have become attractive and gained high research interest due to its significance for the chemical industry and economical advantage. These acids can be produced by utilizing organic waste such as food waste as substrate through anaerobic digestion. Anaerobic digestion is an environmental process that occurs naturally and produces biogas as the main product. VFAs are intermediate products formed during anaerobic digestion where acetic acid, a type of VFA, is the primary product. The main objective of this study was to utilize acetic acid as carbon and energy source for production of edible fungi, Rhizopus ologisporus, Mucor indicus and Volvariella volvacea. The first step was to evaluate if acetic acid could be used as carbon and energy source for edible fungi production. The results showed, that acetic acid is suitable as carbon and energy source for fungal biomass production. The second step was to optimize growth in liquid media. The cultivations were carried out by using five different conditions, where the liquid media contained different combinations of acetic acid, yeast extract and minerals as well as comparing orbital and linear oscillations. Fungal cultivation was possible regardless of the medium composition and type of water shaking baths. However, a linear water shaking bath with a combination of acetic acid yeast extract and/or minerals seems to be the best. Finally, as step three, acetic acid concentrations, 0.2 g/l and 2.0 g/l were used under similar conditions as in step two to see whether a higher concentration of acetic acid would be beneficial. Although the cultivation containing 2.0 g/l gave a higher value of dry weight, the value of yield is questionable. Further studies are needed to confirm if a higher concentration is beneficial or if it might act as an inhibitor for fungal cultivation. / Flyktiga fettsyror (VFAs) har ekonomiska fördelar och kan användas inom kemiska industrier i olika sammanhang, detta har lett till ett stort forskningsintresse för att kunna nyttja VFAs. Organiskt avfall, såsom matavfall, kan användas som substrat för att producera fettsyror genom anaerob rötning. Anaerob rötning är en miljövänlig process och VFAs bildas som intermediära produkter under den anaeroba nedbrytningen där annars bildas biogas som slutprodukt. Syftet med denna studie var att använda ättiksyra, (den vanligaste typen av VFAs), som kol- och energikälla vid odling av tre olika ätbara svampar, som Rhizopus oligosporus, Mucor indicus, och Volvariella volvacea. Först odlades dessa ätbara svampar i odlingsmedium innehållande ättiksyra. Resultatet visade att ättiksyra kan användas som kol- och energikälla vid produktion av svampbiomassa. Målet i de nästkommande stegen var att optimera tillväxtförhållande för svampodlingen. Fem olika odlingsmedier som innehöll olika kombinationer av ättiksyra, jästextrakt och mineraler användes. Det undersöktes dessutom hur två olika skakmetoder, orbitalt, eller linjärt, skakbad påverkar odlingen. Svamptillväxt var möjligt vid alla olika förhållanden oavsett sammansättningen av medium och typ av skakbad, däremot verkar odlingsmedium som innehåller ättiksyra, jästextrakt och/eller mineraler i kombination med linjär skakning vara de bästa förutsättningar för tillväxt av biomassa. I det sista steget kultiverades svamp med olika koncentrationer av ättiksyra, 0,2 g/l och 2,0 g/l, under liknande optimerade förhållanden som ovan, för att undersöka om en högre koncentration av ättiksyra skulle vara fördelaktig. Det producerades mer svampbiomassa (som torrvikt) vid koncentration av 2,0 g/l ättiksyra jämfört med när 0,2 g/l ättiksyra användes, dock var det svårt att säkerställa utbytet. Det behövs därför ytterligare fortsatta studier för att kunna bevisa om en högre koncentration av ättiksyra är fördelaktig för odlingen, eller om en högre koncentration skulle verka inhiberande för tillväxten.

The regulation of short chain fatty acid metabolism has been examined. Metabolism of acetoacetate, and short chain fatty acids such as butyrate and valerate, is predicated upon the expression of genes of the ato operon. Acetoacetate induces expression of a CoA transferase (encoded by the atoDA genes) and expression of a thiolase (encoded by the atoB gene). Metabolism of saturated short chain fatty acids requires the activities of the transferase and thiolase and enzymes of 6-oxidation as well. Spontaneous mutant strains were isolated that were either constitutive or that were inducible by valerate or butyrate instead of acetoacetate.

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N-acylethanolamines (NAEs) are a diverse family of signaling lipids that occur in eukaryotes and their presence is specific to developmental stage and tissue type. In plants, NAEs with an acyl chain ranging from C12 to C18 are common with NAE 18:2 generally being the most abundant type, particularly in desiccated seeds. In Arabidopsis, NAEs negatively regulate growth and mediate stress responses via abscisic acid-dependent and -independent signaling pathway. The function of NAEs is terminated by a highly conserved fatty acid amide hydrolase (FAAH). Because of the significant role NAEs were shown to play in model plant Arabidopsis it is pertinent to elucidate this conserved metabolic pathway in crop species such as tomato. It is hypothesized that NAE pathway occurs in tomato and that there is a functional FAAH that hydrolyzes NAEs.To test this hypothesis, NAE content and composition will be determined in various tissues and developmental stages of tomato by selective lipidomic analysis. Furthermore, a functional homolog of AtFAAH has been identified in tomato and will be biochemically characterized.Thus far, full-length coding sequence of SlFAAH1 and SlFAAH2 were isolated and cloned into a heterologous expression system. The expressed protein will be characterized for its hydrolytic activity against radiolabelled NAE substrates. Temporal expression of SlFAAH1 and SlFAAH2 in different tissues will also be analyzed by quantitative PCR to correlate with the NAE levels. The molecular and biochemical characterization of FAAH in addition to determining the composition of NAEs in tomato will further validate the conserved nature of NAE metabolic pathway in plants.

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