Practical resistance of Erysiphe necator to quinone outside inhibitors (QoIs) is now widespread, and resistance to demethylation inhibitors (DMIs) has also developed.  The goal of this research was to characterize fungicide resistance by elucidating resistance mechanisms and determining its stability.  QoI resistance persisted for several years in a field population after QoI application ended.  Resistant isolates were highly competitive in mixed populations in competition assays under laboratory conditions, indicating a lack of fitness cost.  In one competition trial under field conditions, resistance frequency declined, possibly due to spore migration and influx of background inoculum, but in a second trial, it did not decline.  Double resistance to QoI and DMI was detected and DMI application may have been partially responsible for maintaining QoI resistance in the field.  One isolate with QoI resistance but an undetectable level of the major QoI mutation was shown to be heteroplasmic -- resistant strains could be selected from this isolate. DMI resistance mechanisms in E. necator included the Y136F mutation in CYP51 and cyp51 over-expression.  The first mechanism was present in almost all isolates with substantial levels of resistance, and cyp51 expression level was correlated with resistance level.  Three cyp51 genotypes were detected.  Wildtype isolates with the TAT genotype were sensitive to DMIs, while isolates with increased resistance had either a TTT or TWT genotype; TWT indicated the presence of both wildtype and mutant alleles.  Cyp51 was expressed 1.4 to 19 times more in mutants than in wildtype.  It is not known whether the significant differences in cyp51 expression level among isolates and among genotype groups are due to gene copy number variation.  DMI resistance was found to decline after years of subculturing, and the decline appeared to occur after a few culture transfers of field samples on fungicide-free host leaves.  The observed decline, together with the finding that isolates could be "trained" to increase resistance, and may be slightly induced in cyp51 expression when successively challenged to grow in increasing fungicide concentration, indicate instability of DMI resistance. / Ph. D.

L’accumulation de biopolymère de réserve (PolyHydroxyAlcanoates ou PHA) par la souche Cupriavidus necator, à partir de substrats de type acides gras volatils (acide butyrique, acide propionique et acide acétique) a été étudiée. Elle est induite par une limitation phosphore. Les performances atteintes lors des cultures se situent parmi les meilleures de la littérature pour ce type de substrat : jusqu’à 66 g.L-1 de biomasse totale avec un pourcentage d’accumulation massique de 88% en PHB –PolyHydroxyButyrate- ou en PHB-co-HV -PolyHydroxyButyrate-co-HydroxyValerate- comportant jusqu’à 52% de motifs d’HV.Pour chaque source carbonée, une caractérisation cinétique et stœchiométrique de la souche a été réalisée en l’absence d’effets inhibiteurs dus aux substrats acides grâce à des cultures de type Fed-Batch avec des apports non limitants et non inhibiteurs en carbone. Il a été dégagé :- un taux de croissance maximal de la souche de 0,33 h-1 pour les trois acides étudiés- une relation entre vitesse spécifique de production de PHA et taux de croissance fixée par la disponibilité et les flux de production de NADPH2 avec un découplage inverse pour les taux de croissance supérieurs à 0,05 h-1 et un couplage partiel pour les taux de croissance inférieurs- un optimum de 0,35 Cmole.Cmole-1.h-1, associé à un taux de croissance de l’ordre de 0,05 h-1.- une amélioration de la production de PHB en termes de vitesses spécifiques mais également en termes de rendements si une faible croissance résiduelle est maintenueLa réponse de la souche à un excès de substrat acide a été caractérisée via l’étude de régimes transitoires induits par des pulses sur des cultures continues préalablement stabilisées en régime permanent. Il a été montré qu’en excès de phosphore, face à un brusque excès de substrat, la souche est incapable d’adapter rapidement son taux de croissance. L’excès est donc dirigé vers la production de PHA dont les voies sont plus rapidement mobilisables. En conditions limitantes de phosphore, le substrat excédentaire est utilisé pour la production de PHA. L’inhibition par les acides se traduit par une diminution des capacités de biosynthèse de la biomasse et des PHA entrainant une réduction de l’assimilation du carbone puis une diminution des rendements de conversion. D’autre part la sensibilité d’un système continu à un excès de substrat dépend du point de fonctionnement choisi : plus il est optimal en termes de vitesse, moins le système est robuste. L’acide propionique est très inhibiteur comparé aux autres acides étudiés (dès 3-4 mM contre 30-40 mM). Il n’agit pas simplement via une accumulation excessive dans le cytoplasme mais il exerce également une inhibition spécifique des voies métaboliques.Un antagonisme entre les substrats (acide acétique et butyrique) a été constaté et expliqué grâce à une analyse des flux métaboliques. L’acide acétique est assimilé préférentiellement pour produire la biomasse, l’énergie et les cofacteurs nécessaires à la production de PHA, alors que l’acide butyrique est utilisé pour la synthèse de PHB. La proportion maximale d’acide acétique admise dans l’alimentation en fonction des conditions fixées en régime permanent est calculée et peut être limitée à 40% du carbone.Enfin il a été déterminé que si une croissance résiduelle est assurée grâce à un apport en phosphore, le pourcentage maximal d’HV dans le polymère dépend du taux d’acide propionique dans l’alimentation et ne peux dépasser 33 ± 5% sur acide propionique pur. Par contre, si aucune croissance résiduelle n’est assurée, il est possible de convertir l’acide propionique en motifs d’HV uniquement / Reserve Biopolymer (PolyHydroxyAlkanoates or PHA) accumulation by the strain Cupriavidus necator, from Volatile Fatty Acids (VFA, like butyric acid, propionic acid and acetic acid) was investigated. This production is induced by a phosphorus limitation. For this type of substrates, performances reached during cultures are among the best listed in the literature: up to 66 g.L-1 of total biomass with 88% (w/w) of PHB –PolyHydroxyButyrate- or PHB-co-HV -PolyHydroxyButyrate-co-HydroxyValerate- with a HV content up to 52 Mole%.For each carbon source, kinetic and stoechiometric characterization has been carried out thanks to Fed-Batch cultures with non-limiting and non-inhibitory carbon feed. It has been established:- a maximal growth rate of 0,33 h-1 for the three acid investigated- a relationship between specific PHA production rate and growth rate which is set by the availability and production flux of NADPH2. For growth rate above 0,05 h-1, there is an inverse coupling. For growth rate under 0,05 h-1, there is a partial coupling.- an optimum of 0,35 Cmole.Cmole-1.h-1 is associated with a growth rate of 0,05 h-1.- if a low residual growth rate is maintained, an improvement of PHB production is recorded in terms of specific production rate and yieldsThe response of the strain to an excess of acid substrate was characterized through the investigation of transient state induced by pulsed addition of substrate during continuous cultures stabilized in steady state. It was shown that in excess of phosphorus, when there is a substrate excess, the strain is unable to quickly adapt its growth rate, so the excess is directed to PHA production whose ways seem to be more easily mobilized. Under phosphorus limitation, an excess of substrate is used for PHA production. Acid inhibition results in a decrease in biomass and PHA production capacity which leads to a decrease in carbon assimilation and conversion yields. The sensitivity of a continuous system to an excess of substrate depends on the chosen operating point: the more it is optimal in terms of specific production rate, the less the system is robust. Propionic acid is highly inhibitory compared to the other acids studied (from 3-4 mM versus 30-40 mM). It does not act only via an excessive accumulation in the cytoplasm but also exerts a specific inhibition of metabolic pathways.An antagonism between substrates (acetic and butyric acid) has been established and explained thanks to the Metabolic Flux Analysis. Acetic acid is preferentially used to produce biomass, energy and cofactors for PHA synthesis, whereas butyric acid is used to product PHB. According to the conditions set during steady state, maximal content of acetic acid admitted in the feed can be calculated. It can be limited to 40% of the carbon in the feed.Finally if a growth rate is maintained thanks to a phosphorus supply, the maximal HV content in polymer is function of propionic acid in the feed and cannot exceed 33 ± 5 Mole% on pure propionic acid. Conversely, if there is no residual growth, a total conversion of propionic acid into HV is allowed

La vigne est une culture pérenne sensible à de nombreux bioagresseurs et sur laquelle il est nécessaire de réaliser de nombreux traitements pesticides, susceptibles de causer des problèmes environnementaux, de santé humaine et d’apparition de résistance au sein des populations de bioagresseurs. Aujourd’hui, il est indispensable de développer des stratégies nouvelles de lutte contre les bioagresseurs, plus raisonnées mais permettant de conserver une viticulture compétitive. L’utilisation de produits de biocontrôle semble, en ce sens, être une approche prometteuse permettant d’allier agriculture durable et intensive.Deux écoproduits généralistes à fort potentiel ont été identifiés, comme possédant des actions intéressantes sur les principales maladies cryptogamiques de la vigne. Le premier est un extrait naturel de plante, sans action fongicide directe mais capable de stimuler efficacement et de façon systémique les défenses de la plante. Le second, quant à lui, est un microorganisme qui possède une forte action antagoniste fongicide, mais aussi la capacité à stimuler les défenses de plante. Dans un premier temps, des études en conditions contrôlées ont mis en évidence l’efficacité des deux actifs pour inhiber le développement de diverses souches d’Erysiphe necator, Plasmopara viticola, Botrytis cinerea et Botryosphaeriaceae. En parallèle, des expérimentations au vignoble, ont confirmé le fort potentiel de ces produits de biocontrôle, avec des bonnes efficacités, particulièrement stable avec l’extrait naturel. Ces différentes études nous ont permis d’identifier et d’élaborer des stratégies d’utilisation pour ces deux produits de biocontrôle. / Grapevine is a perennial crop sensitive to many fungal pathogens that require numerous pesticide treatments. However, its uses lead to environmental, human health and fungicide resistance problems. Developing sustainable pest management strategies while keeping a good wine quality is of major importance. In this sense, the use of bio-pesticides products seems to be a promising approach to combine sustainable and intensive agriculture.Two generalist bio-pesticides of great potential have been preliminary identified, forits actions on major fungal diseases of grapevine. The first one is a natural plant extract, with no direct fungicide action but able to systemically stimulate plant defenses. The second one is a microorganism showing strong antagonist fungicide actions, and important ability to stimulate plant defenses. First, the studies conducted in controlled conditions have demonstrated the effectiveness of both products in the suppression of various isolates of Erysiphe necator, Plasmopara viticola, Botrytis cinerea and Botryosphaeriaceae. In parallel,the good efficiencies of these products have been confirmed during vineyard assays. This was especially well demonstrated for the natural extract. As a whole, these studies confirm thepotential of these two products as promising bio-pesticides, of which the strategy of application have been further defined.

La vigne est soumise à la pression de nombreux bioagresseurs dont des parasites obligatoires tels que l’oïdium et le mildiou. La lutte contre les maladies causées par les pathogènes biotrophes nécessite une utilisation souvent intensive de fongicides. Le vignoble consomme à lui seul 16% des fongicides commercialisés chaque année en France. Pour réduire leur impact environnemental qui conduit à l’acquisition de la résistance aux pesticides des pathogène et la présence de résidus dans les vins et dans l’atmosphère, des efforts doivent être entrepris pour développer des stratégies de protection innovante de remplacement ou complémentaire permettant de réduire les intrants pesticides.Les stimulateurs des défenses des plantes permettent de limiter le développement des bioagresseurs en conditions contrôlées. Toutefois, leurs efficacités in natura sont variables et souvent décevantes. Suite au grand nombre de produits potentiellement stimulateurs des défenses des plantes, et à l’intérêt que leur portent les viticulteurs, il est nécessaire de disposer de connaissances et d’outils qui permettent d’évaluer leus efficacités et mieux connaitre leurs potentiels de protection du vignoble. Pour ce faire, une méthode d’évaluation de l’efficacité de produits potentialisateurs ou éliciteurs a été développée au niveau biologique, moléculaire (expression de gènes impliqués dans les défenses) et biochimique (analyses qualitatives et quantitatives des polyphénols), nommée "BioMolChem". Cette méthode a permis d’évaluer l’efficacité de deux phosphonates et d’un analogue de l’acide salicylique, sur différents génotypes et phénotypes de mildiou de la vigne et d’oïdium. Cette approche méthodologique "BioMolChem" a permis d’établir des corrélations entre l’expression de gènes de défense, la présence de certains stilbènes et une efficacité des défenses de Vitis vinifera cv. Cabernet-Sauvignon vis-à-vis de l’oïdium et du mildiou. Les modifications des patrons d’expression des 19 gènes suivis dans les feuilles de vigne et les profils HPLC de polyphénols révèlent des mécanismes de défense multigéniques et complexes. Ainsi, les réactions de défense de la plante sont-elles modulées, en fonction de l’éliciteur considéré, mais aussi en fonction de la diversité phénotypique et génétique des agents pathogènes contre lesquels elle se défend. Ces défenses se caractérisent par une sur-expression d’un ensemble de gènes de défense et une accumulation de composés phénoliques spécifiques.Les marqueurs (gènes et molécules) ainsi identifiés, la méthode "BioMolChem" a été appliquée in natura et a conforté, pour partie, les résultats obtenus au laboratoire. Dans des conditions de fortes pressions parasitaires, il est donc possible de protéger les feuilles et les grappes, à l’aide de SDP et des essais d’association ou d’alternance avec des fongicides conventionnels montrent l’intérêt potentiel de l’emploi des SDP au vignoble. Chemin faisant, dans le cadre d’une viticulture innovante et durable, les SDP et la méthode "BioMolChem" ont été appliqués sur des génotypes hybrides (Vitis vinifera x Muscadinia rotundifolia). Nous révélons que selon le niveau de résistance intrinsèque des génotypes (plus ou moins résistants à l’oïdium et au mildiou), il est possible d’augmenter le niveau de la résistance exprimée par élicitation. Ainsi, les SDP pourraient-ils s’avérer des alliés d’intérêt pour l’utilisation de variétés partiellement résistantes et limiter potentiellement le contournement des QTL de résistance. L’ensemble de ce travail, à but appliqué, a conduit à l’obtention de résultats qui nous permettent de mieux comprendre comment la vigne réagit aux SDP dans son environnement agronomique. Leur exploitation et leur finalisation devraient nous permettre d’exploiter et de mettre en place une utilisation des éliciteurs mieux adaptée, à des stratégies alternatives ou complémentaires de la gestion des bioagresseurs de la vigne. / Powdery (Erysiphe necator) and downy mildew (Plasmopara viticola) are very important grapevine diseases (Vitis vinifera). These two biotrophic pathogens, which are native to the United States, infect green vine tissues and cause significant economic loss as well as environmental damage through the repetitive applications of fungicides. To reduce their environmental impact efforts should be made to develop strategies to protect innovative alternative or complementary to reduce pesticide inputs.In this study, the efficacy and the role of Benzothiadiazole (BTH), a salicylic acid analogue, and two phosphonate derivatives strengthen plant defence mechanisms against various isolates of downy and powdery mildews (Plasmopara viticola and Erysiphe necator). These compounds showed differences in their efficacy depending on the variability of mildews and highly dependent on plant genetics, environmental conditions and selection pressure. The plant defense stimulation could be an alternative or additional method to traditional pest management in the grapevine.Tools “BioMolChem” were developed to better assess the defence status of the plant defences in vitro and in natura. Transcript kinetics of selected defence-related genes and polyphenol contents profiles, during Vitis vinifera-biotrophic pathogen interaction, were characterized, and the impact of pathogen diversity was investigated in the absence or presence of elicitation. In vineyard, under strong pathogen pressures, it is thus possible to protect leaves and clusters, with SDP and assays of association or alternation with conventional fungicides show the potential interest of the use of these SDP in the vineyard.The grapevine defense mechanisms are complex, depending on the elicitor, leading to the coordinated accumulation of pathogenesis-related proteins (PR), the production of phytoalexins, and the reinforcement of plant cell walls.On the way, within the framework of an innovative and sustainable viticulture, the SDP was applied to hybrid genotypes (V. vinifera x M. rotundifolia). We reveal that according to the level of intrinsic resistance of the genotypes (more or less resistant to powdery and to downy mildew), it is possible to increase the level of the expressed resistance. The SDP could become allies of interest in the use of partially resistant grapevine varieties.The present findings provide insights into the potential use of transcripts and stilbenes as markers of the defense status of grapevine leaves with or without elicitation or infection, which should allow us to exploit and develop a better use of elicitors in alternative or complementary strategies in grapevine pest management.

Los parásitos nematodos producen diversas proteínas solubles que unen lípidos (LBPs) estructuralmente distintas a las del huésped. Las funciones que cumplen se desconocen pero se hipotetiza que estarían involucradas en las funciones típicas internas de organismos multicelulares, como la utilización y transporte de compuestos no solubles, y en externas especializadas. Algunas de estas proteínas participarían en la modificación del entorno local en el tejido del huésped, posibilitando la modulación y la evasión de la respuesta inmune. Entre las LBPs producidas por nematodos se encuentran las FAR (Fatty Acid and Retinol binding proteins), una clase novedosa de proteínas que unen ácidos grasos y retinol. Tienen un tamaño aproximado de 19 kDa y sus estructuras que parecen ser ricas en alfa-hélices aún no han sido completamente dilucidadas. La comprensión del rol que cumple esta familia de proteínas tiene gran interés fisiopatológico ya que podrían desempeñar funciones relevantes en la biología de los parásitos que las producen y dadas las diferencias estructurales que presentarían con respecto a las LBPs de sus huéspedes, servirían como potenciales blancos para el diseño de nuevas terapias antiparasitarias. Con la finalidad de contribuir a la caracterización de las proteínas FAR y avanzar de este modo en la determinación de su función biológica, se llevaron a cabo estudios biofísicos y estructurales que permitieron resolver la estructura de Na-FAR-1 en solución por espectroscopía de resonancia magnética nuclear. Determinándose que consta de once hélices que conforman una cavidad interna de gran tamaño, donde podrían ubicarse ligandos hidrofóbicos. La estequiometría de unión de los complejos formados por Na-FAR-1 estaría dada por cuatro moléculas de ácido oleico por molécula de proteína, pero se limitaría a una única molécula de ligando en el caso del retinol y de los análogos fluorescentes de ácidos grasos empleados para su estudio. A su vez se evidenció que además de los ligandos esperados como ácidos grasos y retinol, esta proteína es capaz de unir fosfolípidos y diacilglicéridos. La amplia diversidad de unión a ligandos, sumada a su localización en el intestino del nematodo, indicarían que podría participar en el direccionamiento hacia los distintos tejidos de los lípidos ingeridos.

With over a billion people infected world wide, hookworms are considered as important human pathogens, particularly in developing countries which have the highest rates of infections. Hookworms reside in the gastrointestinal tract of the host where they continuously feed on blood, leading to conditions such as chronic irondeficiency anaemia. The majority of blood-feeding parasites rely on proteins found in blood to provide many of their nutritional requirements for growth, reproduction and survival. Of the numerous proteins found in blood, haemoglobin (Hb) is one of the most abundant. In order to acquire amino acids for protein synthesis, it is thought that haematophagous parasites degrade Hb using various classes of endo- and exoproteases, in a manner similar to that which occurs in catabolism of proteins in mammalian cellular lysosomes. This study identified and characterised proteases involved in the Hb degradation process in the human hookworm, Necator americanus, in order to identify potential candidate antigens for a vaccine that interrupts blood-feeding. Red blood cells ingested by hookworms are lysed to release Hb, which is cleaved by various proteases into dipeptides or free amino acids and these are taken up through the gut membrane by amino acid transporters. Proteases expressed in the intestinal tract of hookworms are thought to play a major role in this process and would therefore make good targets for vaccine candidates aimed at interrupting blood-feeding. To identify these proteases, adult hookworms (both N. americanus and Ancylostoma caninum) were sectioned and intestinal tissue was dissected via laser microdissection microscopy. RNA extracted from the dissected tissue was used to generate gut-specific cDNA, which then was used to create plasmid libraries. Each library was subjected to shotgun sequencing, and of the 480 expressed sequence tags (ESTs) sequenced from each species, 268 and 276 contigs were assembled from the N. americanus and A. caninum libraries, respectively. Nine percent of N. americanus and 6.5% of A. caninum contigs were considered novel as no homologues were identified in any published/accessible database. The gene ontology (GO) classification system was used to categorise the contigs to predicted biological functions. Only 17% and 38% of N. americanus and A. caninum contigs, respectively, were assigned GO categories, while the rest were classified as being of unknown function. The most highly represented GO categories were molecular functions such as protein binding and catalytic activity. The most abundant transcripts encoded fatty acid binding proteins, C-type lectins and activation associated secreted proteins, indicative of the diversity of functions that occur in this complex organ. Of particular interest to this study were the contigs that encoded for cysteine and metalloproteases, expanding the list of potential N. americanus haemoglobinases. In the N. americanus cDNA library, four contigs encoding for cathepsin B cysteine proteases were identified. Three contigs from the A. caninum and one contig from the N. americanus cDNA libraries encoded for metalloproteases, including astacin-like and O-sialoglycoprotein endopeptidases, neither of which had previously been reported from adult hookworms. Apart from haemoglobinases, other mRNAs encoding potential vaccine candidate molecules were identified, including anti-clotting factors, defensins and membrane proteins. This study confirmed that the gut of hookworms encodes a diverse range of proteases, some of which are likely to be involved in Hb digestion and have the potential to be hidden (cryptic) vaccine antigens. Four cysteine proteases (Na-CP-2, -3, -4 and -5) were identified from the gut cDNA library of N. americanus. All four proteases belong to the clan CA, family C1, share homology with human cathepsin B and possess a modified occluding loop. Real-time PCR indicated that all transcripts were up-regulated in the adult stage of the hookworm parasite with high levels of mRNA expression detected in gut cDNA. All four proteases were expressed in recombinant form, but only Na-CP-3 was successfully expressed in soluble form in the yeast Pichia pastoris. Proteolytic activity for Na-CP-3 was detected on a gelatin zymogen gel, however no catalytic activity was detected against the class-specific fluorogenic peptides Z-Phe-Arg-AMC and Z-Arg-Arg-AMC. Mass spectrometry analysis of the purified protein suggested that the pro-region had not been processed in trans when the protein was secreted by yeast. Incubation of Na-CP-3 in salt buffers containing dextran sulfate resulted in autoprocessing of the pro-region as detected by Western blot and catalytic activity was detected against Z-Phe-Arg-AMC. Activated Na-CP-3 did not digest intact tetrameric human Hb. The other three cysteine proteases (Na-CP-2, -4, and -5) were expressed in insoluble form in Escherichia coli. Antibodies to all four proteins (Na- CP-2 to 5) immunolocalised to the gut region of the adult worm, supporting mRNA amplification results and strongly indicated that they might play a role in nutrient acquisition. Hb digestion in blood feeding parasites such as schistosomes and Plasmodium spp. occurs via a semi-ordered cascade of proteolysis involving numerous enzymes. In Plasmodium falciparum, at least three distinct mechanistic classes of endopeptidases have been implicated in this process, and at least two classes have been implicated in schistosomes. A similar process is thought to occur in hookworms. An aspartic protease, Na-APR-1, was expressed in P. pastoris and purified protein was shown to cleave the class-specific fluorogenic peptide 7- Methoxycoumarin-4-Acetyl-GKPILFFRLK(DNP)-D-Arg-Amide. Recombinant Na- APR-1 was able to cleave intact human Hb and completely degrade the 16 kDa monomer and 32 kDa dimer within one hour. Recombinant Na-CP-3 was not able to cleave intact Hb, but was able to further digest globin fragments that had previously been digested with Na-APR-1. A clan MA metalloprotease, Na-MEP-1, was identified in gut tissue of N. americanus and was expressed in recombinant form in Hi5 insect cells using the baculovirus expression system. Recombinant Na-MEP-1 displayed proteolytic activity when assessed by gelatin zymography, but was incapable of cleaving intact Hb. However, Na-MEP-1 did cleave globin fragments which had previously been incubated with Na-APR-1 and Na-CP-3. Hb digested with all three proteases was subjected to reverse phase HPLC and peptides were analysed using Liquid Chromatography-Mass Spectrometry (LC-MS). A total of 74 cleavage sites were identified within Hb ƒ¿ and ƒÀ chains. Na-APR-1 was responsible for cleavage of Hb at the hinge region, probably unravelling the molecule so that Na- CP-3 and Na-MEP-1 could gain access to globin peptides. All three proteases were promiscuous in their subsite specificities, but the most common P1-P1Œ residues were hydrophobic and/or bulky in nature, such as Phe, Leu and Ala. Antibodies to all three proteins (Na-APR-1, -CP-3, -MEP-1) immunolocalised to the gut region of the worm, further supporting their roles in Hb degradation. These results suggest that Hb degradation in N. americanus follows a similar pattern to that which has been described in Plasomdium falciparum. Studies conducted in this project have identified a number of potential haemoglobinases and have demonstrated that the gut region of the hookworm contains a multitude of proteases which could be targeted for production of new chemotherapies or as vaccine candidates. Results presented here also suggest that the Hb degradation process occurs in an ordered cascade, similar to those which have been reported in other haematophagous parasites. More importantly, it has been confirmed that Na-APR-1 plays a crucial role in the initiation of the Hb degradation process and therefore targeting this molecule as a vaccine candidate could provide high levels of protection against hookworm infection.

This diploma thesis focuses on the molecular characterization of selected PHA producers. Within this work, the PHA producing thermophilic isolates originating from the samples of activated sludge and compost were identified and characterized using molecular biological methods. By sequencing the 16S rRNA gene, the thermophilic isolates were identified and taxonomically classified into the Firmicutes bacterial phylum. In these bacterial isolates, the ability to produce PHA at the genotype level was determined by conventional PCR detection of the phaC gene encoding PHA synthase, which is a key enzyme in PHA biosynthesis. Class I, II and IV PHA synthases were detected in most of the isolated bacteria, wherein class I and II PHA synthases are not characteristic for these bacterial genera. The largest proportion of isolates was identified for the species of thermophilic bacterium Aneurinibacillus thermoaerophilus, in which class IV PHA synthase was detected. In the second part of the diploma thesis, the RT-qPCR method was implemented to study the expression of selected genes of the bacterium Cupriavidus necator H16 involved in PHA metabolism. As part of the implementation of this method, PCR-based detection of selected genes was optimized and quantification of genes using real-time PCR was performed. The tested method included steps of RNA isolation, cDNA synthesis and quantification of gene segments for which the critical points of the method were determined based on the obtained data.

The aim of this work is to study the possibility of microbial production of polyhydroxyalkanoates (PHA). PHA can be used as biodegradable materials. Bacterial strain Cupriavidus necator was used for laboratory production of PHA. This bacterium was cultivated in medium with various precursors to produce copolymers of 3HB with 3HV or 4HB. Another part of the work was aimed at cultivation of C. necator on different waste substrates, especially oils, with the aim to achieve the highest production of polymer. Another large part of the thesis is dedicated to isolation strategies of PHA using enzymes. Commercially used proteases – alcalase and pancreatin – can be used with advantages for digestion of bacterial cells. A number of optimization experiments showed that application of proteases leads to enhancement of PHA purity to about 13%. Purity increase up to 90 % was achieved by adding a surfactant, which promotes the solubility of non-PHA forming polymer. This surfactant increases the purity of 20 % when compared to control. The last part of presented work deals with the use of enzyme solution isolated from Bacillus subtilis medium. Its application to C. necator culture led to the yield of polymer at a purity exceeding 95 %. These results could represent the basis for new isolation strategies, which can lead to more efficient yield of PHA.

Předložená disertační práce se zabývá studiem produkce a degradace polymerních materiálů s využitím mikroorganismů. Hlavní pozornost je upřena ke studiu produkce polyesterů bakteriálního původu - polyhydroxyalkanoátů. Tyto materiály jsou akumulovány celou řadou bakterií jako zásobní zdroj uhlíku, energie a redukční síly. Díky svým mechanickým vlastnostem, kterými silně připomínají tradiční syntetické polymery jako jsou polyetylén nebo polypropylén, a také díky své snadné odbouratelnosti v přírodním prostředí, jsou polyhydroxyalkanoáty považovány za ekologickou alternativu k tradičním plastům vyráběným z ropy. Polyhydroxyalkanoáty mají potenciál najít řadu aplikací v průmyslu, zemědělství ale také v medicíně. Významná část předložené práce je zaměřena na produkci polyhydroxyalkanoátů z odpadních substrátů pocházejících především z potravinářských výrob. Testována byla odpadní syrovátka nebo odpadní oleje z různých zdrojů. Právě využití levných odpadních substrátů je strategií, která by mohla přispět ke snížení ceny polyhydroxyalkanoátů a tím usnadnit jejich masové rozšíření. Podle výsledků dosažených v této práci jsou právě odpadní olejové substráty velice perspektivní cestou k ekonomicky rentabilní biotechnologické produkci polyhydroxyalkanoátů. Další část předložené práce se zabývá studiu spojení metabolické role polyhydroxyalkanoátů a stresové odpovědi bakterií. V této práci bylo zjištěno, že expozice bakteriální kultury řízené dávce etanolu nebo peroxidu vodíku významně navýší dosažené výtěžky a to o přibližně 30 %. Po aplikaci výše zmíněných stresových faktorů došlo k aktivaci metabolických drah vedoucí k odbourání stresového faktoru z média. Výsledkem bylo navýšení poměru NAD(P)H/NAD(P)+, což vedlo k částečné inhibici Krebsova cyklu a naopak aktivaci biosyntetické dráhy polyhydroxyalkanoátů. Mimoto došlo k významnému navýšení molekulové hmotnosti výsledných materiálů. Podle těchto výsledků se regulovaná aplikace vhodně zvolených stresových podmínek zdá být zajímavou strategií, která vede nejen k navýšení celkových výtěžků, ale také významnému zlepšení vlastností polymeru. Poslední část disertační práce se zabývala studiem procesu biodegradace polyuretanových materiálů. Polyuretanové eleastomery byly modifikovány rozličnými biopolymery za účelem navýšení jejich biodegradability. Tyto materiály byly posléze vystaveny působení směsné termofilní kultury jako modelového systému, který simuluje přirozené konsorcium bakterií. Přítomnost testovaných materiálů v kultivačním médiu vedla k neobvyklým růstovým charakteristikám bakteriální kultury. V průběhu prvních několika dní byl růst kultury silně inhibován, nicméně po překonání této neobvykle dlouhé lag-fáze došlo k intenzivnímu nárůstu kultury. Hlavní podíl na hmotnostním úbytku testovaných materiálů během experimentů měl samovolný rozpad materiálů, nicméně byl pozorován i vliv bakteriální kultury, kdy míra biotické degradace závisela na použitém modifikačním činidle. Nejvyšší míra biotické degradace byla pozorována u polyuretanového materiálu modifikovaného acetylovanou celulózou. Lag-fáze byla způsobena uvolněním nezreagovaného katalyzátoru (dibutylcínlaurát) a polyolu do kultivačního média. Bakteriální kultura se však po čase dokázala na přítomnost toxických látek v médiu adaptovat nebo je dokázala eliminovat.

The Calvin – Benson cycle is the most widespread metabolic pathway capable of fixing CO2 in nature and a target of very high interest to metabolic engineers worldwide. In this study, 12 metabolites (ATP, AMP, NADP, NADPH, 2PG, 3PGA, FBP, RuBP, PEP, AKG, Ac-CoA and phenylalanine) were tested for protein – metabolite interactions against the proteome of Cupriavidus necator (previously Ralstonia eutropha) in the hopes of finding potential examples of allosteric regulation of the Calvin – Benson cycle. This is accomplished through the use of the LiP-SMap method, a recently developed shotgun proteomics method described by Piazza et al. capable of testing a metabolite of interest for interactions with the entire proteome of an organism at once. A functional protocol was developed and 234 protein – metabolite interactions between ATP and the proteome of C. necator are identified, 103 of which are potentially novel. Due to time constraints and setbacks in the lab, significant results were not produced for the other 11 metabolites tested. C. necator is an industrially relevant chemolithoautotroph that can be engineered to produce many valuable products and is capable of growth on CO2 and hydrogen gas. The bacteria were grown in continuous cultures after which the proteome was extracted while retaining its native state. Subsequently, the proteome was incubated with a metabolite of interest and subjected to limited, non-specific proteolysis. The resulting peptide mix was analyzed by liquid chromatography coupled tandem mass spectrometry (LC – MS/MS). / Calvin-Benson-cykeln är den mest utbredda metaboliska processen i naturen med vilken det är möjligt att fixera CO2 och en måltavla av högsta intresse för bioteknologer världen över. I den här studien testades 12 metaboliter (ATP, AMP, NADP, NADPH, 2PG, 3PGA, FBP, RuBP, PEP, AKG, Ac-CoA and phenylalanine) för interaktioner mot proteomet från Cupriavidus necator (tidigare Ralstonia eutropha) i hopp om att hitta potentiella exempel på allosterisk reglering av Calvin-Benson-cykeln. Detta uppnåddes genom användning av LiP-SMap-metoden, en nyligen utvecklad proteomikmetod beskriven av Piazza et al. kapabel av att testa en metabolit av intresse mot en organisms hela proteom simultant. Ett funktionellt protokoll utvecklades och 234 interaktioner mellan ATP och proteomet av C. necator identifierades, varav 103 potentiellt är nyupptäckta. På grund av tidsbrist och motgångar i labbet producerades inga signifikanta resultat för de resterande 11 metaboliterna som testades. C. necator är en industriellt relevant kemolitoautotrof som kan växa på CO2 och vätgas, samt manipuleras till att producera många värdefulla produkter. Bakterierna odlades i kemostater varefter proteomet extraherades i sitt naturliga tillstånd. Sedan inkuberades proteomet med en metabolit av intresse och utsattes för begränsad, icke-specifik proteolys. Den resulterande peptidblandningen analyserades via tandem masspektrometri kopplad till vätskekromatografi (LC – MS/MS).