Bacteroides fragilis is a member of the normal microbiota that resides in the human lower gastrointestinal tract. This bacterium is of clinical significance because it is the most frequently isolated Gram-negative obligate anaerobe from peritoneal abscesses and bloodstream infections. Human fibrinogen is a hexameric-glycoprotein that is important for fibrin-mediated abscess formation and limiting the spread of infection. B. fragilis can bind and degrade fibrinogen which may aid in its escape from abscesses into the bloodstream, thereby promoting bacteraemia. In addition to fibrinogen, binding of B. fragilis to fibronectin, a component of the extracellular matrix, found in association with fibrinogen at wound sites, has also been reported. An outer membrane protein, BF1705, expressed by B. fragilis was found to share homology with BspA from Tannerella forsythia which is known to bind fibrinogen. The gene encoding BF1705 was deleted from the B. fragilis NCTC 9343 genome in the present work using a markerless gene deletion technology. Proteins derived from the outer membranes of wild-type B. fragilis were able to bind fibronectin and fibrinogen in far-western blots. Similar protein extracts from the ΔBF1705 strain did not bind fibrinogen and fibronectin, which confirms the role of BF1705 in adhesive interactions with proteins of the host extracellular matrix. The possible involvement of BF1705 in fibrinogen degradation was ruled out because the ΔBF1705 strain still degraded fibrinogen. To identify the proteases involved in degradation of fibrinogen, four genes encoding putative extracellular metallo- and serine proteases in the size range 45-50 kDa were deleted from the NCTC 9343 genome. All of the single and multiple mutants defective in these selected proteases were still capable of degrading fibrinogen as determined by zymography. Expression of eight B. fragilis proteases in E. coli did not lead to detectable degradation of fibrinogen. These observations suggest that these proteases alone cannot degrade fibrinogen and either that an unidentified protease is responsible for degradation or that there is redundancy in the proteases involved. Under conditions of nitrogen limitation bacteria resort to scavenging nitrogen from the environment to replenish the depleting intracellular nitrogen content. By examining the differential regulation of the B. fragilis transcriptome under nitrogen replete and depleting conditions, a potential role for BF1705 and secreted proteases in nutrient binding and assimilation were studied. Growth on conventional glucose defined medium with ammonia as the nitrogen source was compared to growth in defined medium with glutamine as nitrogen source. A reduced doubling time and diauxic growth in the medium containing glutamine indicated nitrogen limitation. Comparison of the transcriptome derived from cultures of B. fragilis grown on either ammonia or glutamine by RNA-Seq did not reveal a significant upregulation of BF1705 in response to nitrogen limitation. This observation in conjunction with its inability to degrade fibrinogen suggests that the primary role of BF1705 might be as an adhesin and does not act directly in nutrient binding and degradation. Nevertheless, nitrogen limitation was found to induce the expression of four protease-encoding genes by over a 2-fold (adjusted p value < 0.05). The molecular weight of three of these proteases were identified to be within the size range of 45-55 kDa which corresponded to the lysis bands detected by fibrinogen zymography with wild-type B. fragilis protein extracts. Therefore the possible involvement of these three proteases in fibrinogen degradation could be assessed. A 155-fold upregulation (adjusted p value < 0.05) in asnB, encoding a homologue of asparagine synthetase B, under conditions of nitrogen limitation suggest a previously uncharacterised aspartate metabolism pathway for ammonia generation via arginine catabolism in B. fragilis. Ammonia thus formed might aid in sustaining B. fragilis growth under nitrogen deprived conditions. In addition to nitrogen assimilation, significant upregulation was observed in the expression of genes involved in regulation of oxidative stress and metronidazole resistance. The observed changes in the transcriptome will add to our understanding of the B. fragilis metabolism and potential assist with unravelling the mechanisms of infection mediated by this important opportunistic pathogen.

L’identification des réponses écophysiologiques des plantes est un préalable pour améliorer les défenses naturelles des plantes et réduire l’usage d’intrants chimiques dans les cultures. Les réponses des plantes vis-à-vis d’une agression herbivore fait intervenir des mécanismes de résistance et de tolérance. Les réponses à une agression d’une larve mineuse sont peu documentées contrairement aux insectes piqueurs ou broyeurs. L’objectif de ma thèse a été de caractériser les traits de réponse de la tomate vis-à-vis de Tuta absoluta afin de déterminer les réponses locales et systémiques en termes de composés solubles et volatils. Nous avons regardé les réponses de la tomate soumise à deux niveaux contrastés de la disponibilité en azote, sachant que l’azote est un levier sur lequel il est possible de s’appuyer pour modifier le niveau de défense des plantes. Nous avons travaillé sur la tomate, une solanacée cultivée en hydroponie et sujette à un ravageur Tuta absoluta. Notre stratégie de recherche s’est portée sur l’identification et la quantification des composés de défense solubles et volatils, et sur la détermination du pouvoir nutritionnel des tissus foliaires. Nous avons montré que la plante de tomate met en place une résistance induite par l’herbivore se traduisant par une augmentation de la concentration en acide chlorogénique, caffeoyl putrescine et tomatine localement au niveau de la zone parasitée et / ou de manière systémique dans les autres compartiments foliaires non infestés. Le rôle de ces molécules dans la défense de la tomate vis-à-vis de Tuta absoluta doit encore être confirmé. Nous avons montré aussi que la tomate met en place une défense indirecte contre Tuta absoluta, basée sur l’émission de composés volatils au voisinage des plantes. Nous avons identifié plusieurs nouveaux COVs synthétisés lors de l’herbivorie par Tuta absoluta (m-cymène, menthatriène, ß-cis-ocimène, ß-phellandrène, terpinolène, ß−caryophyllène, humulène). Parmi tous les COVs que nous avons identifiés, certains sont connus pour être émis en plus grande quantité lors d’une attaque herbivore et jouer un rôle dans l’attraction des insectes prédateurs ou la répulsion des femelles pondeuses. Ainsi, leur élicitation semble générique de l’herbivorie et pas spécifique de Tuta absoluta. La spécificité des interactions tomate - Tuta absoluta pourrait être renforcée par des analyses plus poussées en métabolomique, transcriptomique (expression de gènes RNAseq) et en s’intéressant aux voies de signalisation induite. Contrairement à ce qui était attendu, nos résultats montrent peu d’effets de la limitation en azote sur les réponses observées. Néanmoins, comme ce qui a été démontré précédemment, la limitation azotée impacte le développement larvaire. Mes travaux ont permis de renforcer l’hypothèse "trophique" du ralentissement du développement larvaire par la limitation en azote. Ainsi, la stœchiométrie du bol alimentaire est fortement modifiée et les larves doivent consommer plus de matières pour acquérir l’azote nécessaire à leur développement. Néanmoins nous ne pouvons pas écarter l’hypothèse "composés de défense". En effet, nous avons montré que les concentrations en composés phénoliques et tomatine et des émissions de COVs supposés être impliqués dans la défense constitutive, sont plus fortes en cas d’une limitation en azote. La diversité et la complémentarité des expérimentations permettront aux données acquises d’alimenter un modèle de fonctionnement de la plante agressée qui pourrait comporter une fonction de défense réactive à la nutrition azotée / The identification of ecophysiological responses of plants is a prerequisite for improving the natural defences of plants and reducing the use of chemical inputs in crops. Plant responses to herbivorous attack involve defence and tolerance mechanisms. Plant responses to a leafminer larva are pourly documented, in contrast to chewing and piercing insects. The objective of my Ph.D. was to characterize the tomato trait responses to Tuta absoluta herbivory in order to determine the local and systemic responses in terms of soluble and volatile compounds. We looked at the responses of tomatoes subjected to two contrasting levels of nitrogen availability, knowing that nitrogen is a lever to promote the basal plant defence level. We worked on tomato, a Solanaceae, grown in hydroponics and subjected to Tuta absoluta. Our research strategy has focused on identifying and quantifying soluble and volatile defence compounds and determining the nutritional value of foliar tissues. We have shown that tomatoes establish an induced- resistance against the herbivore, which results in a concentration increase of chlorogenic acid, caffeoyl putrescine and tomatine locally at the level of the parasite zone and / or systemically in the others non-infested foliar compartments. The defence role of these molecules on Tuta absoluta has yet to be confirmed. We have also shown that tomatoes set up an indirect defence against Tuta absoluta, based on the emission of volatile compounds in the vicinity of plants. We have identified several new herbivore-induced VOCs (m-cymene, menthatriene, ß-cis-ocimene, ß-phellandrene, terpinolene, ß−caryophyllene, humulene). Some of the identified VOCs are known to be emitted in greater quantities during a herbivorous attack and to play a role in attracting predatory insects or repelling egg-laying females. Thus, their elicitation seems generic to herbivory and not specific to Tuta absoluta. The specificity of tomato - Tuta absoluta interactions could be enhanced by further analysis in metabolomics, transcriptomics (expression of RNAseq genes) and by investigating the induced signaling pathways. Contrary to what was expected, our results show little effect of the nitrogen limitation on the observed responses. Nevertheless, as demonstrated previously, nitrogen restriction affects larval development. My work has reinforced the “trophic“ hypothesis of slowing larval development by a limited nitrogen availability. Thus, the stoichiometry of the food bowl is highly modified and the larvae have to consume more material to acquire the nitrogen necessary for their development. Nevertheless, we can not exclude the hypothesis “compounds of defence“. Indeed, we have shown that the concentrations of phenolic compounds and tomatine and of the VOC emission supposed to be involved in the constitutive defence are increased in the case of nitrogen limitation. The diversity and complementarity of the experiments will allow the acquired data to feed a functional model of the attacked plant. This model might include plant nitrogen regimes as a reactive plant defence factor

Thesis (MSc (Wine Biotechnology))--Stellenbosch University, 2008. / In the wine industry, the importance of selecting an appropriate yeast strain, generally of the species Saccharomyces cerevisiae, to ensure reliable fermentation and to achieve a desired level of quality has been well established. As a consequence, the demand for new starter cultures with improved or new oenological characteristics is increasing. Appropriately selected starter cultures can reduce the occurrence of stuck fermentations, impart specific aroma profiles and reduce the development of offflavours. Using standard breeding and selection procedures, several wine yeast strains that would be less likely than currently existing strains to experience stuck fermentation have previously been developed at the Institute for Wine Biotechnology. The target of these projects had been to develop strains with improved nitrogen efficiency [defined as the amount of fermented hexoses for a given amount of free amino nitrogen (FAN)], improved fructose utilization and ethanol tolerance. These three parameters are known contributors to stuck fermentation. Two of the strains that had been isolated in these projects, strain 116 for nitrogen efficiency and strain 38-1 for efficient fructose utilization, were chosen as parental strains for the current study. The aim was to further improve and possibly combine these traits in yeast strains by using hybridization followed by various enrichment and directed evolution procedures in a continuous fermentation setup. The strategy was to sequentially subject the population of mass-mated hybrids to a number of selective environments for a large number of generations. The yeasts were subjected to a high fructose/glucose ratio for 12 generations, followed by selection in an environment with a limited supply of nitrogen for 54 generations and finally to high ethanol stress. After each round of enrichment, individual strains were analysed to assess the results. For the hybrid strains selected after enrichment in a medium with a high fructose/glucose ratio, no general improvement could be discerned. However, one of the hybrids, hybrid strain 331, fermented fructose better than the parental strains and other hybrid strains. These results may suggest that the selection pressure was not applied for a sufficient number of generations and may not have been sufficiently strong. In addition, the parental strain may already performing at a rate that may render further improvement more difficult in this genetic background. The next aim of this study was to enhance fermentation performance of wine yeast hybrid strains in low nitrogen and high sugar conditions. Several hybrid strains 331, RR03 and 05R generated in this study showed improvement in efficiency of nitrogen utilization when compared to the parental strains, indicating a successful selection strategy. Several strains also showed higher ethanol tolerance, and some strains possessed] combinations of the traits to be improved. Future research will evaluate these hybrids regarding the production of aromatic compounds and of the sensory profile produced. Such strains would help the wine industry to control the occurrence of stuck fermentations and to produce quality wines.

L’objectif de notre travail était d’étudier les bascules métaboliques chez Yarrowia li-polytica d’un métabolisme purement oxydatif vers l’accumulation de lipides puis à l’excretion d’acide citrique.Le développement d’un procédé D-stat et d’un mode de conduite fed-batch nous a permis, dans un premier temps, de quantifier les ratios N/C caractéristiques pour chacune des bascules étudiées. Nos résultats montrent que les ratios rN/rC critiques aux bascules métaboliques sont de 0,085 molN.Cmol-1 et de 0,018-0,022 molN.Cmol-1 pour l’accumulation de lipides et production de citrate, respectivement.L’analyse systémique des cultures réalisées nous a permis de mettre en évidence des mécanismes de co-régulation de certaines enzymes du métabolisme lipidique ainsi qu’une prépondérance de mécanismes post-transcriptionnels dans l’établissement des bascules étudiées.Enfin, l’utilisation de souches génétiquement modifiées au niveau de l’ATP citrate lyase, la malate déshydrogénase et de la glycérol-3-phosphate déshydogénase a permis d’évaluer l’impact de ces enzymes sur le métabolisme lipidique / This thesis aimed at studying the metabolic shifts in Yarrowia lipolytica from the pure oxidative metabolism to lipid accumulation and citric acid excretion.The development of a D-stat culture and of a monitoring fed-batch strategy allowed us to determine the N/C ratio characteristic for each of metabolic shifts. rN/rC ratio were determined equal to 0,085 molN.Cmol-1 and 0,018-0,022 molN.Cmol-1 for the lipid accumu-lation and the citric acid production, respectively.Systemic analysis of the cultivations showed coregulation phenomena among some enzymes of the lipidic metabolism and post-transcriptional modifications in the onset of the metabolic shifts.Finally, the impact of enzymes (ATP citrate lyase, malate dehydrogenase and gly-cérol-3-phosphate dehydrogenase) on the lipidic metabolism was evaluated through systemic analysis of 3 genetically modified strains

Polyhydroxyalkanoates (PHA) are a family of biodegradable polyesters naturally synthesised by some bacteria and archaea. PHA have high industrial value as bioplastics for packaging and biomedical applications. However, their broader use is hindered by high production costs and uncontrolled variation of polymer properties. The extreme halophile Haloferax mediterranei shows bioprocess advantages that can be exploited for the low cost production of the PHA copolymer Poly(3-hydroxbutyrate-co-3-hydroxyvalterate) (PHBV). The focus of this thesis is to identify process variables responsible for the uncontrolled variation of PHA properties in order to progress towards the robust production of PHBV using H. mediterranei. The outcome of the investigation is a novel cultivation strategy for the reliable synthesis of PHBV copolymers with controlled composition and microstructure showing minor differences in material characteristics. Initially, growth kinetics and PHBV synthesis were characterised under nitrogen-excess and nitrogen-limiting conditions in ammonium and for the first time, nitrate. The nitrogen source and concentration influenced PHBV accumulation and variations in polymer composition were observed with ammonium, highlighting the importance of the control of cultivation conditions. Volatile fatty acids (VFA) were found to be a more direct approach to determine PHBV composition and for the first time were used as substrates in H. mediterranei cultures. When the cells were grown in C4:0/C5:0 mixtures, the 3HV fraction in the PHBV was proportional to the percentage of C5:0 in the feed mixture, allowing the synthesis of copolymers with a predefined composition ranging from pure PHB to pure PHV. The cultivation strategy proved effective for the synthesis of HV rich PHBV, which is not easily obtained due to the 3HV precursor toxicity. The polymer microstructure was controlled using different feeding strategies: co-feeding generated random copolymers, while sequential feeding created block and blend copolymers. The synthesis of block copolymers is of interest because the materials show enhanced yield strength and mechanical strength, making such materials more suitable for commodity uses. Bespoke random, block, and blend copolymers with 0−100 mol% 3HV were synthesized and their thermal and mechanical properties studied. Lastly, high temperature cultivation and several surfactants were tested to enhance the production of bespoke PHBV from VFA. PHBV productivity and accumulation was greatly improved in a fed-batch bioreactor fermentation at 37°C with Tween-80 and the maximum PHBV content 58.9% was obtained. The polymers from shake-flasks and from bioreactors showed minor variations in their material properties, demonstrating the scalability and the robustness of the process developed. Further understanding of the different process variables affecting polymer synthesis and composition was gained in this thesis. It is now possible to produce PHBV with controllable composition, microstructure and minor differences in material characteristics. The novel and robust production strategy developed address the bioprocess challenge of minimising the uncontrolled variation of polymer characteristics that is currently hindering the wider use of PHA hence allowing the production of high quality polymers for commodity goods, packaging and biomedical applications.

Made available in DSpace on 2015-03-26T13:52:02Z (GMT). No. of bitstreams: 1 texto completo.pdf: 445645 bytes, checksum: f448d5a3c851f1c181e95a80994ffb89 (MD5) Previous issue date: 2007-03-16 / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / In order to investigate the stress condition imposed by nitrogen limitation on kinetic of growth and aging of Kluyveromyces lactis, the kinetics constants of K. lactis culture as function of ammonium sulfate concentration were determined. The effect of aging of the culture on its specific initial growth rate was evaluated. Signals of aging such as cell wall chitin concentration and apoptosis markers were analyzed as function of growth rate on continuous culture. K. lactis was cultured in buffered YCB medium supplemented with ammonium sulfate at concentrations of 0.02 to 38.0 mmol.L-1. The growth kinetic followed the model proposed by Monod. The maximum specific growth rate (µmax) and saturation constant (Ks) were 0.44 h-1 and 0.15 mmol.L-1, respectively. The initial specific growth rates decrease as the aging of the culture increases. The cell wall chitin concentration increased with the ammonium concentrate and the aging of the cultures. The cell dimensions raised with aging in all the ammonium sulfate concentration analyzed. Apoptosis markers were only observed in the older cultures, after five days of recycling of the culture on the concentrations of 0.57 mmol.L-1, 3.80 mmol.L-1 and 7.60 mmol.L-1 and after eight days on the concentrations 38.0 mmol.L-1 of ammonium sulfate. Aging signals were investigated at K. lactis cells growing in nitrogen limiting continuous culture at growth rate 0.01, 0.06, 0.18 and 0.35 h-1. Chitin concentration of cell wall of cells growing at 0.01 h-1 and 0.06 h-1 was higher (27µg.mL-1 N-acetylglucosamine and 20µg.mL-1 N-acetylglucosamine) than of cells growing at 0.18 h-1 and 0.35 h-1 (6µg.mL-1 and 5µg.mL-1, respectively). The dimensions of K. lactis cells were largest on the growth rate of 0.01 h-1. It was detected apoptosis markers on the continuous culture under the nitrogen limiting growth rate of 0.01 h-1. The results demonstrate that nitrogen limitation and aging affect growth kinetic and suggest that nitrogen limiteted continuous culture conducted at growth rate 0.01 h-1 can still proven useful to investigate aging response of Kluyveromyces lactis. / Para investigar o estresse imposto por condições limitantes de nitrogênio na cinética de crescimento e na longevidade de culturas de Kluyveromyces lactis, determinaram-se as constantes cinéticas do crescimento de culturas de K. lactis em função da concentração de sulfato de amônio, avaliou-se o efeito da idade das culturas na velocidade específica inicial de crescimento e averiguaram-se os sinais de envelhecimento celular em função de diferentes velocidades de crescimento de culturas conduzidas em regime contínuo sob limitação por nitrogênio. Culturas de K. lactis foram cultivadas em meio YCB tamponado acrescido de sulfato de amônio, nas concentrações 0,02 a 38,0 mmol.L-1. A cinética de crescimento de K. lactis em função da concentração de sulfato de amônio, obedeceu o modelo cinético proposto por Monod. A velocidade específica máxima de crescimento (&#956;máx.) e a constante de saturação (Ks) foram de 0,44 h-1 e 0,15 mmol.L-1, respectivamente. A velocidade específica inicial de crescimento reduziu com a idade da cultura. Concentrações de quitina na parede celular aumentaram com a idade da cultura e com a concentração de sulfato de amônio. Dimensões celulares aumentaram com a idade independente da concentração de sulfato de amônio. A presença de características morfológicas de apoptose foi verificada a partir do quinto dia de transferência da cultura para meio novo nas concentrações de 0,57 mmol.L-1, 3,80 mmol.L-1, 7,60 mmol.L-1 e a partir do oitavo dia de repicagem na concentração de 38,0 mmol.L-1 de [NH4]2SO4. Culturas contínuas sob estresse por nitrogênio foram conduzidas nas velocidades de crescimento de 0,01, 0,06; 0,18 e 0,35 h-1. Culturas com velocidades de crescimento de 0,18 e 0,35 h-1 produziram maior rendimento em etanol e menor rendimento em glicerol. A concentração de quitina na parede celular de culturas de K. lactis conduzidas nas velocidades de crescimento de 0,01 h-1 e 0,06 h-1 foi maior (27 mg.mL-1 e 20 mg.mL-1 de N-acetilglicosamina) comparada com as culturas conduzidas nas velocidades de crescimento de 0,18 h-1 e 0,35 h-1, que apresentaram 6 &#956;g.mL-1 e 5 &#956;g.mL-1 de N-acetilglicosamina, respectivamente. As dimensões das células de K. lactis foram maiores na cultura conduzida com velocidade de crescimento de 0,01 h-1, se comparada com as demais velocidades de crescimento avaliadas: 0,06; 0,18 e 0,35 h-1. Apesar desses sinais de envelhecimento terem sido detectados, características morfológicas de apoptose só foram identificadas nas culturas de K. lactis conduzidas na velocidade de crescimento de 0,01 h-1. Os resultados mostraram que o estresse por nitrogênio e a idade afetam a cinética de crescimento de culturas de K. lactis e sugerem que culturas contínuas de Kluyveromyces lactis sob condições nitrogênio limitantes conduzidas com velocidade de crescimento correspondente a 0,01 h-1 podem ser úteis em estudos sobre sinais de envelhecimento celular.

Dans le cadre du changement climatique et de l'évolution de la réglementation concernant les intrants azotés, la culture du colza risque d'être fortement pénalisée dans la mesure où c'est une culture qui nécessite d'importants apports azotés pour atteindre son potentiel de rendement. Par ailleurs, comme chez le colza un déficit hydrique induit l'accumulation de certains composés azotés, il est vraisemblable que cela conduise au détournement d'une quantité importante d'azote vers les organes végétatifs aux dépens des organes reproducteurs et donc du rendement. Chez le colza, la réponse métabolique au déficit hydrique se traduit par une très forte accumulation de proline et dans une moindre mesure une augmentation de la teneur en GABA (acide γ-aminobutyrique), deux acides aminés connus chez la plupart des plantes pour leur réponse à de nombreux stress abiotiques. L'objectif de cette thèse est de déterminer comment le métabolisme de ces deux molécules contribue à l'allocation de l'azote au cours du développement de la plante en situation normale comme en condition de stress hydrique et/ou azoté. Pour répondre à cette question nous avons fait le choix de caractériser deux voies enzymatiques majeures impliquées dans le catabolisme de la proline et du GABA : la proline déshydrogénase (ProDH) et la GABA-transaminase (GABA-T) et d'évaluer l'impact de carences hydriques et/ou azotées sur ces voies. Cette étude nécessitait d'identifier au préalable les gènes codant ces enzymes afin d'aborder une approche fonctionnelle. Les résultats montrent l'existence de multiples copies de gènes ProDH et GABA-T dans le génome du colza. L'analyse de leurs profils d'expression suggère que des processus de sub-fonctionnalisation sont en cours conduisant à l'expression spécifique, de certaines copies en réponse aux stress, et d'autres dans les processus développementaux. La comparaison des profils métaboliques avec les profils spécifiques des transcrits a permis d'élaborer des hypothèses sur le rôle de ces voies dans la gestion de l'azote. L'étude conjointe des métabolismes de la proline et du GABA suggère l'existence de régulations connexes entre les deux. Enfin, l'utilisation de plantules a permis - d'approfondir la régulation des gènes étudiés à des stades précoces de développement - et de mettre en évidence les effets délétères de l'inhibition de la GABA-T par une approche pharmacologique. En conclusion ces résultats apportent des précisions sur la régulation de ces deux enzymes et fournissent des éléments de réponse quant au rôle fonctionnel des catabolismes de la proline et du GABA dans les processus de gestion de l'eau et de l'azote chez le colza. Ces travaux constituent donc une première étape dans une démarche de validation de ces gènes comme candidats pour des programmes d'amélioration du colza visant à sélectionner des génotypes mieux adaptés aux conditions environnementales futures. / In the context of climate change and recent regulation concerning nitrogen inputs, the oilseed rape yields may be severely decreased because its crop requires significant nitrogen supply to reach high yield performance. Moreover, as water deficit induces the accumulation of some nitrogen compounds in oilseed rape, it is likely that this could lead to diversion of significant amounts of nitrogen to the vegetative organs at the expense of the reproductive ones and therefore of the yield. In oilseed rape, the metabolic response to water deficit results in a very high proline accumulation and, to a lesser extent, an increased content of GABA (γ-aminobutyric acid), both these amino acids known for their response to many environmental stresses in most species. The objective of the work presented here was to determine how the metabolism of proline and GABA contributes to the nitrogen allocation during plant development under optimal conditions and under water stress and/or nitrogen depletion. To answer this question, we have chosen to characterize two major enzymatic pathways involved in the catabolism of proline and GABA, proline dehydrogenase (ProDH) and GABA transaminase (GABA-T), and assess the impact of water and/or nitrogen deficiency on these pathways. This study has required to preliminary identify the genes encoding these enzymes in order to initiate a functional approach. The results show the presence of multiple copies of ProDH and GABA-T genes in the oilseed rape genome. Analysis of their expression profiles suggests that sub-functionalization processes are occurring, leading to the specific expression of some copies in response to stress, and some in developmental processes. Comparison of metabolic profiles with specific profiles of transcripts allows us to hypothesize about the role of these pathways in management of nitrogen. The combined study of proline and GABA metabolisms suggests the existence of relationships between them. Finally, the use of seedlings allows - further studying the regulation of genes in the early stages of development - and highlighting the deleterious effects of the inhibition of GABA-T by a pharmacological approach. In conclusion these results supply information on the regulation of these two enzymes and provide answers about the functional roles of proline and GABA catabolisms in the management processes of water and nitrogen in oilseed rape. These works constitute a first step in validation process of these genes as putative candidates for oilseed rape breeding programs to select genotypes better adapted to future environmental conditions.

Cette thèse présente différents cas d'acclimatation d'une espèce de phytoplancton (Isochrysis sp.) à des modifications de son environnement physico-chimique avec une attention particulière portée sur la composante lipidique de la réponse aux changements. Dans ce contexte nous avons étudié les lipides à la fois comme des produits (triacylglycerol) et des acteurs (glycolipides) de la photosynthèse. Tout d'abord, nous avons étudié l’influence de plusieurs types de limitation par l'azote sur la teneur en lipides neutres des algues. Nous avons montré que la relation entre la disponibilité de l’azote et la teneur en lipide n'était pas monotone et qu'elle présentait un caractère hystérétique. Les cellules d’Isochrysis sp. n’accumulent des lipides neutres que transitoirement lors du passage d'une croissance forte vers une croissance faible. Nous nous sommes ensuite intéressé aux relations qui existent entre le cycle nycthéméral et l'accumulation de réserves énergétiques sous forme de lipides et de sucres. Les réserves sont accumulées pendant le jour en disproportion du reste du carbone et consommées pendant la nuit pour alimenter la division cellulaire et la croissance nocturne. La carence en azote dans ces conditions nycthémérales ne provoque pas d’accumulations significatives de lipides. Les variations diurnes de la teneur en lipides neutres sont plus importantes que les variations obtenues en carence. A travers ces différentes expériences nous avons pu mettre en évidences les relations qui existaient entre l'environnement, la croissance, et l'accumulation de réserves. La compréhension de ces mécanismes a permis de déterminer les conditions environnementales qui favorisent la production d’huile en vue d’une utilisation biocarburant. Nous avons aussi étudié l'influence des conditions de croissance sur la composition des glycolipides des membranes des chloroplastes. Les résultats montrent que les changements de la teneur en MGDG, DGDG et SQDG et de l’insaturation des acides gras de ces lipides accompagnent les modifications d’activité photosynthétique des cellules en limitation azotée et en cycle Jour/Nuit. / This thesis presents various cases of acclimation of a phytoplankton species (Isochrysis sp.) to changes in growth conditions, with special emphasis on the lipid response to those changes. In this context we have studied lipids as both products (triacylglycerol) and actors (glycolipids) of photosynthesis. First, we studied the influence of several types of nitrogen limitation on the neutral lipid content of algae. We showed that the relationship between nitrogen availability and lipid content was not monotonous and presented a hysteretic behaviour. Isochrysis sp. cells accumulated neutral lipids only transiently during the transition from high growth to low growth. We were also interested in the relationship between the circadian cycle and the accumulation of energy reserves as fats and sugars. Reserves were accumulated during the day and consumed during the night to provide energy for cell growth and division. Nitrogen deficiency under alternative light did not lead to significant accumulations of neutral lipids. Through these experiences we could understand the relationship between environment, growth, and accumulation of reserves. These results allowed us to identify the environmental conditions that enhance oil production for biofuel use. We also studied the influence of growth conditions on the composition of glycolipids that comprises chloroplast membranes. Changes in MGDG, DGDG and SQDG contents and in their fatty acids unsaturation were linked to changes in photosynthetic characteristics when growth conditions are modified (L/D cycle, N-limitation). Glycolipids are likely to be important actors of photosynthetic acclimation to environmental changes.

Les rivières reçoivent de l'azote de leurs bassins versants et elles constituent les derniers sites de transformations des nutriments avant leur livraison aux zones côtières. Les transformations de l’azote inorganique dissous en azote gazeux sont très variables et peuvent avoir un impact à la fois sur l’eutrophisation des côtes et les émissions de gaz à effet de serre à l’échelle globale. Avec l’augmentation de la charge en azote d’origine anthropique vers les écosystèmes aquatiques, les modèles d’émissions de gaz à effet de serre prédisent une augmentation des émissions d’oxyde nitreux (N2O) dans les rivières. Les mesures directes de N2O dans le Lac Saint-Pierre (LSP), un élargissement du Fleuve Saint-Laurent (SLR) indiquent que bien qu’étant une source nette de N2O vers l'atmosphère, les flux de N2O dans LSP sont faibles comparés à ceux des autres grandes rivières et fleuves du monde. Les émissions varient saisonnièrement et inter-annuellement à cause des changements hydrologiques. Les ratios d’émissions N2O: N2 sont également influencés par l’hydrologie et de faibles ratios sont observés dans des conditions de débit d'eau plus élevée et de charge en N élevé. Dans une analyse effectuée sur plusieurs grandes rivières, la charge hydraulique des systèmes semble moduler la relation entre les flux de N2O annuels et les concentrations de nitrate dans les rivières. Dans SLR, des tapis de cyanobactéries colonisant les zones à faible concentration de nitrate sont une source nette d’azote grâce à leur capacité de fixer l’azote atmosphérique (N2). Étant donné que la fixation a lieu pendant le jour alors que les concentrations d'oxygène dans la colonne d'eau sont sursaturées, nous supposons que la fixation de l’azote est effectuée dans des micro-zones d’anoxie et/ou possiblement par des diazotrophes hétérotrophes. La fixation de N dans les tapis explique le remplacement de près de 33 % de la perte de N par dénitrification dans tout l'écosystème au cours de la période d'étude. Dans la portion du fleuve Hudson soumis à la marée, la dénitrification et la production de N2 est très variable selon le type de végétation. La dénitrification est associée à la dynamique en oxygène dissous particulière à chaque espèce durant la marée descendante. La production de N2 est extrêmement élevée dans les zones occupées par les plantes envahissantes à feuilles flottantes (Trapa natans) mais elle est négligeable dans la végétation indigène submergée. Une estimation de la production de N2 dans les lits de Trapa durant l’été, suggère que ces lits représentent une zone très active d’élimination de l’azote. En effet, les grands lits de Trapa ne représentent que 2,7% de la superficie totale de la portion de fleuve étudiée, mais ils éliminent entre 70 et 100% de l'azote total retenu dans cette section pendant les mois d'été et contribuent à près de 25% de l’élimination annuelle d’azote. / Rivers receive nitrogen (N) from their watershed and are the final sites of nutrient processing before delivery to coastal waters. Transformations of dissolved inorganic N (DIN) to gaseous N are highly variable and can impact both coastal eutrophication and greenhouse gas emissions. With anthropogenic N loading to aquatic ecosystems on the rise, nitrous oxide (N2O) emission from rivers should increase. Direct measurements of N2O from lake St. Pierre (LSP), an enlargement of the St. Lawrence River (SLR) indicate that although LSP is a net atmospheric source of N2O to the atmosphere fluxes are low compared to others rivers. Emissions are seasonally and inter-annually highly variable due to changes in hydrological conditions. N2O: N2 is also influenced by hydrology and lower ratios are observed in conditions of higher water discharge and elevated N charge into the ecosystem. In a cross system analysis, hydraulic load mitigates the relation between annual N2O flux and nitrate concentrations in rivers. In SLR, cyanobacterial mats colonizing low nitrate areas are a net source of N with high negative di-nitrogen (N2) fluxes. Given that fixation occurred during daylight and that oxygen concentrations in the water column were supersaturated, we hypothesize that N2 fixation is performed by the dominant cyanobacteria in anoxic micro-zone of the mat and/ or possibly by heterotrophic diazotrophs. Our estimates indicate that N fixation in the mats account for the replacement of up to 33% of the N loss via denitrification in the entire ecosystem during the study period. In the tidal Hudson River N2 production is highly variable between vegetated shallows and was associated with species-driven differences in dissolved oxygen (DO) dynamics during the ebb tide. N2 production was extremely high in invasive floating-leaved plants (Trapa natans) but was insignificant in submersed native vegetation. An estimate of summertime N2 production in Trapa beds suggests that these beds are a major seasonal hotspot for N removal. Large Trapa beds represent only 2.7% of the total area of the tidal Hudson but they remove between 70 and 100% of the total N retained in this section of the river during summer months and contribute to as much as 25% of the annual N removal.

Les rivières reçoivent de l'azote de leurs bassins versants et elles constituent les derniers sites de transformations des nutriments avant leur livraison aux zones côtières. Les transformations de l’azote inorganique dissous en azote gazeux sont très variables et peuvent avoir un impact à la fois sur l’eutrophisation des côtes et les émissions de gaz à effet de serre à l’échelle globale. Avec l’augmentation de la charge en azote d’origine anthropique vers les écosystèmes aquatiques, les modèles d’émissions de gaz à effet de serre prédisent une augmentation des émissions d’oxyde nitreux (N2O) dans les rivières. Les mesures directes de N2O dans le Lac Saint-Pierre (LSP), un élargissement du Fleuve Saint-Laurent (SLR) indiquent que bien qu’étant une source nette de N2O vers l'atmosphère, les flux de N2O dans LSP sont faibles comparés à ceux des autres grandes rivières et fleuves du monde. Les émissions varient saisonnièrement et inter-annuellement à cause des changements hydrologiques. Les ratios d’émissions N2O: N2 sont également influencés par l’hydrologie et de faibles ratios sont observés dans des conditions de débit d'eau plus élevée et de charge en N élevé. Dans une analyse effectuée sur plusieurs grandes rivières, la charge hydraulique des systèmes semble moduler la relation entre les flux de N2O annuels et les concentrations de nitrate dans les rivières. Dans SLR, des tapis de cyanobactéries colonisant les zones à faible concentration de nitrate sont une source nette d’azote grâce à leur capacité de fixer l’azote atmosphérique (N2). Étant donné que la fixation a lieu pendant le jour alors que les concentrations d'oxygène dans la colonne d'eau sont sursaturées, nous supposons que la fixation de l’azote est effectuée dans des micro-zones d’anoxie et/ou possiblement par des diazotrophes hétérotrophes. La fixation de N dans les tapis explique le remplacement de près de 33 % de la perte de N par dénitrification dans tout l'écosystème au cours de la période d'étude. Dans la portion du fleuve Hudson soumis à la marée, la dénitrification et la production de N2 est très variable selon le type de végétation. La dénitrification est associée à la dynamique en oxygène dissous particulière à chaque espèce durant la marée descendante. La production de N2 est extrêmement élevée dans les zones occupées par les plantes envahissantes à feuilles flottantes (Trapa natans) mais elle est négligeable dans la végétation indigène submergée. Une estimation de la production de N2 dans les lits de Trapa durant l’été, suggère que ces lits représentent une zone très active d’élimination de l’azote. En effet, les grands lits de Trapa ne représentent que 2,7% de la superficie totale de la portion de fleuve étudiée, mais ils éliminent entre 70 et 100% de l'azote total retenu dans cette section pendant les mois d'été et contribuent à près de 25% de l’élimination annuelle d’azote. / Rivers receive nitrogen (N) from their watershed and are the final sites of nutrient processing before delivery to coastal waters. Transformations of dissolved inorganic N (DIN) to gaseous N are highly variable and can impact both coastal eutrophication and greenhouse gas emissions. With anthropogenic N loading to aquatic ecosystems on the rise, nitrous oxide (N2O) emission from rivers should increase. Direct measurements of N2O from lake St. Pierre (LSP), an enlargement of the St. Lawrence River (SLR) indicate that although LSP is a net atmospheric source of N2O to the atmosphere fluxes are low compared to others rivers. Emissions are seasonally and inter-annually highly variable due to changes in hydrological conditions. N2O: N2 is also influenced by hydrology and lower ratios are observed in conditions of higher water discharge and elevated N charge into the ecosystem. In a cross system analysis, hydraulic load mitigates the relation between annual N2O flux and nitrate concentrations in rivers. In SLR, cyanobacterial mats colonizing low nitrate areas are a net source of N with high negative di-nitrogen (N2) fluxes. Given that fixation occurred during daylight and that oxygen concentrations in the water column were supersaturated, we hypothesize that N2 fixation is performed by the dominant cyanobacteria in anoxic micro-zone of the mat and/ or possibly by heterotrophic diazotrophs. Our estimates indicate that N fixation in the mats account for the replacement of up to 33% of the N loss via denitrification in the entire ecosystem during the study period. In the tidal Hudson River N2 production is highly variable between vegetated shallows and was associated with species-driven differences in dissolved oxygen (DO) dynamics during the ebb tide. N2 production was extremely high in invasive floating-leaved plants (Trapa natans) but was insignificant in submersed native vegetation. An estimate of summertime N2 production in Trapa beds suggests that these beds are a major seasonal hotspot for N removal. Large Trapa beds represent only 2.7% of the total area of the tidal Hudson but they remove between 70 and 100% of the total N retained in this section of the river during summer months and contribute to as much as 25% of the annual N removal.