The aim of this thesis is to characterize metabolic changes in hemocytes during the immune response in D. melanogaster using in vivo markers as well as by measuring gene expression. The impact of the transcription factor HIF1 on the gene expression of glycolytic enzymes and its impact on the systemic metabolism was evaluated. The importance of HIF1 and LDH in the process of fighting against S. pneumoniae infection was tested as well.

<p>Triple Negative Breast Cancer (TNBC) is the most aggressive type of breast cancer. TNBC patients are resistant to virtually all target therapies and suffer a higher post-chemotherapy relapse with a worse overall survival compared with other types of breast cancers. Therefore, the development of an effective therapy is urgently needed. PKM2 plays a prominent role in mediating<b> </b>tumor glycolysis and PKM2 is often overexpressed in human cancers. However, whether PKM2 mediated glycolysis is necessary for cancer cell growth is questionable. Here, I have found that inhibition of PKM2 does not affect TNBC cell growth due to a metabolic switch from glycolysis to fatty acid oxidation (FAO). We show that PKM2 directly interacts with EZH2 to coordinately mediate epigenetic silencing of SLC16A9, transporter of a key player in FAO, Carnitine. Inhibition of either PKM2 or EZH2 increases levels of SLC16A9 and intracellular Carnitine to promote FAO and thereby sustains cancer cell growth. Direct inhibition of EZH2 using a clinically tested EZH2 inhibitor, GSK126, is able to elicit a previously unidentified vulnerability to a clinically tested FAO inhibitor, Etomoxir. As a result, combined GSK126-Etomoxir treatment synergistically abolishes TNBC xenograft tumor growth in vivo. Together, this study uncovers PKM2-EZH2 mediated metabolic reprogramming that leads to a new drug combination therapy by dual targeting of EZH2 and FAO for effective treatment of TNBC.<b> </b></p> <p> </p> <p>Furthermore, Dendritic Cell (DC) vaccination has shown promise in treating cancer patients. However, the <i>in vitro</i> generation of a fully functional DC remains a big challenge in this field. EZH2 inhibition has shown to be able to create an immunologically ‘hot’ tumors. Nonetheless, the role of EZH2 in regulation of DC function is still unclear. I found that the expression levels of EZH2 and its functional maker, H3K27Me3, are enhanced following maturation from immature DC (iDC) into two functional DCs, α-type 1-polarized-DC (αDC) and gold standard DC (sDC). Moreover, inhibition of EZH2 by GSK126 treatment elicits a dependency of sDC on FAO. These results suggest that EZH2 plays a role in maturation of DC through metabolic reprogramming, which may also provide new DC based immunotherapy of TNBC. </p>

The objective of this work is to better understand themetabolism and physiology ofEscherichiacoli(W3110) in defined medium cultures with thelong-term goal of improving cell yield and recombinant proteinproductivity. The order of amino acid utilization inE. colibatch cultures was investigated in a medium with16 amino acids and glucose. Ser, Pro, Asp, Gly, Thr, Glu andAla were rapidly consumed and depleted at the end of theexponential phase, while His, Arg, Val, Met, Ile, Leu, Phe, Lysand Tyr were consumed slowly during the following linear growthphase. The uptake order correlated to the maximum specificconsumption rate. Of the rapidly consumed amino acids onlyglyine and threonine improved growth when added individually.Serine was the first amino acid to be consumed, but inhibitedglucose uptake initially, which presumably is related to thefunction of PTS. Valine inhibited cell growth could be releasedby isoleucine. The critical medium concentration of valinetoxicity was 1.5 - 3 µmol L-1. Valine uptake was associated with exchange ofisoleucine out of the cells. Glycine significantly increased the cell yield,Yx/s,and growth rate ofE. coliin batch cultures in a glucose-mineral medium.Maximum effect occurred at pH 6.8, at 6 - 12 mmol L-1glycine, and below 1.15 g dw L-1.13C NMR technique was employed to identify [1-13C], [2-13C]and [1,2-13C]acetate in the cultures supplied with [2-13C]glycine. The NMR data revealed that littledegradation of added glycine occurred, and that serine/glycinebiosynthesis was repressed below 1.15 g dw L-1, implicating that glycine was a source ofglycine, serine, one-carbon units, and threonine. Above 1.15 gdw L-1, 53% of the consumed glycine carbon was excretedas acetate. Degradation of glycine was associated with anincreased uptake rate, cleavage by GCV, and degradation of bothglycine- and glucose-derived serine to pyruvate. This switch inmetabolism appears to be regulated by quorum sensing. A cell density-dependent metabolic switch occurred also inthe central metabolism. A 2 - 3 fold decrease in mostglycolytic and TCA cycle metabolites, but an increase inacetyl-CoA, occurred after the switch. The acetate productionrate decreased throughout the culture with a temporary increaseat the switch point, but the intracellular acetate poolremained relatively constant. Two mixtures of amino acids were fed together with glucosein fed-batch cultures ofE. coliW3110 pRIT44T2, expressing the recombinantprotein ZZT2. One mixture contained 20 amino acids and theother 5 so-called 'protein amino acids': Ala, Arg, Met, His andPhe. Although the amino aids increased the cell yield anddecreased the proteolysis rate in both cases, ZZT2 productionwas decreased. A decrease of ZZT2 synthesis rate is consideredto be the reason. Further studies of the 5 amino acidsindicated that a few amino acids disturb metabolism. Carbon mass balances were calculated in glucose limitedfed-batch cultures ofE. coli. In the end, the carbon recovery was ~90% basedon biomass, CO2and acetate, but ~100% if the all carbon in themedium was included. Outer membrane (OM) constituents,lipopolysaccharide, phospholipids, and carbohydratescontributed to 63% of the extracellular carbon. Little celllysis occurred and the unidentified (~30%) carbon was assumedto constitute complex carbohydrates. A novel cultivationtechnique Temperature-Limited Fed-Batch (TLFB) is developed toprevent OM shedding in high-cell density cultures. <b>Keywords</b>: Escherichia coli, amino acids, glycine, quorumsensing, metabolic switch, metabolite pools, carbon balance,outer membrane, lipopolysaccharide, batch culture, fed-batchculture

<p>The objective of this work is to better understand themetabolism and physiology of<i>Escherichiacoli</i>(W3110) in defined medium cultures with thelong-term goal of improving cell yield and recombinant proteinproductivity.</p><p>The order of amino acid utilization in<i>E. coli</i>batch cultures was investigated in a medium with16 amino acids and glucose. Ser, Pro, Asp, Gly, Thr, Glu andAla were rapidly consumed and depleted at the end of theexponential phase, while His, Arg, Val, Met, Ile, Leu, Phe, Lysand Tyr were consumed slowly during the following linear growthphase. The uptake order correlated to the maximum specificconsumption rate. Of the rapidly consumed amino acids onlyglyine and threonine improved growth when added individually.Serine was the first amino acid to be consumed, but inhibitedglucose uptake initially, which presumably is related to thefunction of PTS. Valine inhibited cell growth could be releasedby isoleucine. The critical medium concentration of valinetoxicity was 1.5 - 3 µmol L^-1. Valine uptake was associated with exchange ofisoleucine out of the cells.</p><p>Glycine significantly increased the cell yield,<i>Y</i>_x/s,and growth rate of<i>E. coli</i>in batch cultures in a glucose-mineral medium.Maximum effect occurred at pH 6.8, at 6 - 12 mmol L^-1glycine, and below 1.15 g dw L^-1.¹³C NMR technique was employed to identify [1-¹³C], [2-¹³C]and [1,2-¹³C]acetate in the cultures supplied with [2-¹³C]glycine. The NMR data revealed that littledegradation of added glycine occurred, and that serine/glycinebiosynthesis was repressed below 1.15 g dw L^-1, implicating that glycine was a source ofglycine, serine, one-carbon units, and threonine. Above 1.15 gdw L^-1, 53% of the consumed glycine carbon was excretedas acetate. Degradation of glycine was associated with anincreased uptake rate, cleavage by GCV, and degradation of bothglycine- and glucose-derived serine to pyruvate. This switch inmetabolism appears to be regulated by quorum sensing.</p><p>A cell density-dependent metabolic switch occurred also inthe central metabolism. A 2 - 3 fold decrease in mostglycolytic and TCA cycle metabolites, but an increase inacetyl-CoA, occurred after the switch. The acetate productionrate decreased throughout the culture with a temporary increaseat the switch point, but the intracellular acetate poolremained relatively constant.</p><p>Two mixtures of amino acids were fed together with glucosein fed-batch cultures of<i>E. coli</i>W3110 pRIT44T2, expressing the recombinantprotein ZZT2. One mixture contained 20 amino acids and theother 5 so-called 'protein amino acids': Ala, Arg, Met, His andPhe. Although the amino aids increased the cell yield anddecreased the proteolysis rate in both cases, ZZT2 productionwas decreased. A decrease of ZZT2 synthesis rate is consideredto be the reason. Further studies of the 5 amino acidsindicated that a few amino acids disturb metabolism.</p><p>Carbon mass balances were calculated in glucose limitedfed-batch cultures of<i>E. coli</i>. In the end, the carbon recovery was ~90% basedon biomass, CO₂and acetate, but ~100% if the all carbon in themedium was included. Outer membrane (OM) constituents,lipopolysaccharide, phospholipids, and carbohydratescontributed to 63% of the extracellular carbon. Little celllysis occurred and the unidentified (~30%) carbon was assumedto constitute complex carbohydrates. A novel cultivationtechnique Temperature-Limited Fed-Batch (TLFB) is developed toprevent OM shedding in high-cell density cultures.</p><p><b>Keywords</b>: Escherichia coli, amino acids, glycine, quorumsensing, metabolic switch, metabolite pools, carbon balance,outer membrane, lipopolysaccharide, batch culture, fed-batchculture</p>

L’objectif de ce projet de thèse est d’étudier et caractériser les mécanismes impliqués dans la bascule respiro-fermentaire chez des cellules eucaryotes dotées d’un métabolisme mitochondrial. Les cellules eucaryotes ont des besoins différents en oxygène pour la production d’énergie et leur survie dans un environnement donnée. Elles sont qualifiées de type aérobie stricte lorsque la présence d’oxygène leur est nécessaire ou aéro-anaérobie facultatif dans le cas où l’oxygène n’est pas indispensable à la production d’énergie. La levure Yarrowia lipolytica a été choisie comme modèle d’étude de par sa particularité à être un micro-organisme aérobie strict avec une grande capacité d’accumuler de lipides et de production d’acides organiques. Les études expérimentales et analytiques, par l’emploi de méthodes mathématiques de modélisation du métabolisme, ont permis d’identifier des contraintes métaboliques impliquées dans la transition respiro-fermentaire chez cette levure au métabolisme énergétique oxydatif. La production de l’acide citrique par Y. lipolytica, déjà rapportée dans la littérature, a été choisi comme un marqueur de cette transition respiro-fermentaire. Nous avons découvert que l’inhibition de la protéine oxydase alternative (AOX), impliquée dans la respiration mitochondriale, par la molécule n-propyl gallate (nPG) permet d’améliorer le rendement de production d’acide citrique par fermentation du glucose dans une culture de Y. lipolytica. Ces résultats montrent que la nPG, déjà utilisée dans l’industrie agro-alimentaire et pharmaceutique en tant que conservateur joue sur la bascule respiro-fermentaire par inhibition de la consommation d’oxygène et stimule ainsi la production d’acide citrique. La modélisation du réseau métabolique de Y. lipolytica, décrit à l’échelle du genome, par dynamic Flux Balance Analysis (dFBA) a permis d’identifier l’accumulation des espèces oxydantes dites ROS (Reactive Oxygen Species) comme un levier majeur de la bascule respiro-fermentaire et donc de la production d’acide citrique chez la levure Y. lipolytica. De plus, nos résultats préliminaires montrent que l’oxydation des lipides accumulés par Y. lipolytica pourrait être à l’origine de la génération des ROS. Cette étude doit être approfondie expérimentalement et constitue un apport important pour l’industrie agro-alimentaire et pharmaceutique.Mots clés : Bascule respiro-fermentaire, Acide citrique, lipides, Yarrowia lipolytica, n-propyl gallate, Reactive Oxygen Species, modélisation, dynamic Flux Balance Analysis / The main goal of this thesis project is to study and characterize mechanisms involved in respiratory to fermentative shift in eukaryotic cells endowed with mitochondrial metabolism. Eukaryotic cells have different oxygen requirements for energy production and survival in a given environment. They are described as strict aerobic when the presence of oxygen is necessary or optional aero-anaerobic in when oxygen is not essential for energy production. The yeast Yarrowia lipolytica was chosen as our study model thanks to its particularity since it is a strict aerobic microorganism with a high capacity to accumulate lipids and to produce organic acids. Experimental and analytical studies, using mathematical methods for modeling cell metabolism, allowed us to identify metabolic constraints involved in respiratory to fermentative transition in this yeast showing oxidative energy metabolism. Production of citric acid by Y. lipolytica, already reported in the literature, has been chosen as a marker for this in respiratory to fermentative shift. We found that the inhibition of the alternative oxidase protein (AOX) involved in mitochondrial respiration, by adding n-Propyl gallate (nPG) molecule improves the yield of citric acid production by fermentation of glucose in a Y. lipolytica culture. These results show that nPG, already used in food and pharmaceutical industry as a preservative, plays on respiratory to fermentative balance by inhibition of oxygen consumption and thus stimulates the production of citric acid. Modeling of the metabolic network of Y. lipolytica, described at genome-scale, by dynamic Flux Balance Analysis (FBA) has identified the accumulation of intracellular ROS (Reactive Oxygen Species) species as major levers for respiratory to fermentative shift and therefore the production of citric acid by Y. lipolytica. Therefore, our preliminary results show that oxidation of lipids accumulated by Y. lipolytica could be involved in generation of ROS species. This study must be experimentally deepened and constitutes an important contribution for the agri-food and pharmaceutical industry.Key words: Respiratory to fermentative shift, Citric acid, lipids, Yarrowia lipolytica, n-Propyl gallate, Reactive Oxygen Species, modeling, dynamic Flux Balance Analysis