L’étude des réseaux protéiques perturbés au cours de l’infection par les petits virus oncogéniques amena, vers la fin des années 80, à la découverte de nombreux régulateurs clés de la division et de la survie cellulaire. Parmi ceux-ci, la protéine E4F1 fût initialement identifiée comme une cible de l’oncoprotéine virale E1A. Originellement identifié comme un facteur de transcription, E4F1 est également une ubiquitine-E3 ligase atypique pour d'autres facteurs de transcription tel que le suppresseur de tumeurs p53. Au travers de ses multiples activités, E4F1 est nécessaire à la prolifération des cellules somatiques et souches, et à la survie des cellules cancéreuses. De plus, les travaux de différents laboratoires dont le mien suggèrent qu’E4F1 se situe au carrefour de plusieurs voies de signalisation qui sont fréquemment altérées au cours de l’oncogenèse, et notamment la voie impliquant le suppresseur de tumeurs p53. Afin d’étudier les fonctions physiologiques in vivo d’E4f1, mon laboratoire d’accueil a développé plusieurs modèles de souris génétiquement modifiées. La caractérisation de ces modèles a permis de mettre en évidence un rôle majeur d'E4F1 dans l'homéostasie de la peau. Plus précisément, E4F1 régule le pool de cellules souches de l'épiderme au travers de son rôle dans une voie de signalisation qui implique la protéine p53 et deux de ces régulateurs en amont: Arf et Bmi1. Cependant, il semble que les effets d'E4F1 dans le contrôle du maintien des cellules souches s'étendent au delà de son rôle sur cette voie de signalisation. En effet, j'ai récemment pu démontrer qu'E4F1, au travers de ces fonctions transcriptionnelles, régule directement l'expression d'un sous-groupe de gènes impliqués dans la régulation de l'activité de la pyruvate déshydrogénase (PDH). La PDH est un complexe multimérique situé dans la mitochondrie qui catalyse la décarboxylation du pyruvate (le produit final de la glycolyse) en acétyl coenzyme A (AcCoA), liant ainsi le métabolisme du pyruvate au cycle de Krebs. J’ai pu montrer que l’inactivation d’E4f1 spécifiquement dans l'épiderme conduisait à une diminution importante de l’activité de PDH et à une reprogrammation métabolique de ces cellules. Cette reprogrammation a pour conséquence d'altérer le micro-environnement des cellules souches qui conduit à leur détachement de leur niche et aboutit in fine à une absence du renouvellement de l'épiderme. Cette partie de mes travaux a donc permis d'illustrer pour la première fois l'importance du métabolisme du pyruvate dans l'homéostasie des cellules souches de la peau. Sur la base de ces résultats, je poursuis l'analyse des fonctions d’E4f1 dans l'homéostasie de la peau en étudiant son rôle dans d'autres types cellulaires tels que les mélanocytes. / The multifunctional protein E4F1 is an essential regulator of normal skin homeostasis. During my Phd, I demonstrated that E4f1 inactivation in adult skin results in stem cell autonomous defects causing exhaustion of the epidermal stem cell (ESC) pool. At the molecular level, I identified E4F1 as a new regulator of the pyruvate dehydrogenase complex (PDC) in keratinocytes, an essential mitochondrial complex that converts pyruvate into Acetyl-CoEnzyme A. Using genetically engineered mouse models, I showed that E4F1-mediated control of PDH activity is required to maintain normal skin homeostasis. Consistently, E4F1 deficiency in basal keratinocytes resulted in deregulated expression of dihydrolipoamide acetlytransferase (Dlat), a gene encoding the E2 subunit of the PDC, and impaired PDH activity. The metabolic reprogramming of E4f1 KO keratinocytes associated with the redirection of the glycolytic flux towards lactate production and increased lactate secretion in their microenvironment, leading to enhanced activity of extra-cellular-matrix remodelling proteases Finally, these defects ended in alterations of the basement membrane, ESC mislocalization and the exhaustion of the ESC pool. In the second part of my thesis, I have evaluated the role of E4F1-mediated control of the PDC in melanocytes and showed that the metabolic activities of E4F1 are important for melanocyte function. Consistently, mice with E4f1-deficient melanocytes exhibited hair graying and skin pigmentation defects. Altogether, my data demonstrate the importance of E4f1-mediated control of pyruvate metabolism for normal skin homeostasis.

Chez Bacillus subtilis comme pour toutes les bactéries, le métabolisme central du carbone est essentiel pour la croissance de la cellule. Elle utilise le glucose (source de carbone glycolytique) et le malate (source de carbone gluconéogenique) comme sources de carbone préférentielles. Ces deux sources de carbone sont capables d'induire la répression catabolique au travers de la protéine régulatrice CcpA et ainsi d'établir une hiérarchie dans l'utilisation des sources alternatives de carbone. Au centre du métabolisme du carbone se trouve le pyruvate que B. subtilis est capable d'utiliser comme seule source de carbone, mais son transporteur reste inconnu.Des analyses transcriptomiques ont montré que seul l'opéron ysbAB était spécifiquement induit en présence de pyruvate, et nous avons montré que sa délétion entraînait une perte de croissance presque totale sur pyruvate. En utilisant des protéines étiquetées, nous avons mis en évidence qu'YsbA et YsbB formaient un complexe se localisant à la membrane. Nous avons ensuite montré que ce complexe est le transporteur principal du pyruvate et fonctionne comme un transporteur par diffusion facilitée. A l'aide d'une fusion rapportrice, nous avons démontré que l'opéron lytST situé en amont d'ysbAB, et codant pour un système à deux composants, était responsable de l'induction d'ysbAB. Nous avons également montré qu'en plus d'une répression par CcpA en présence de glucose ou de malate, une régulation dépendante de l'activité enzyme malique de MaeA s'exerce sur ysbAB. Cette régulation est due à l'accumulation de pyruvate dans la cellule qui perturbe l'activation d'ysbAB par LytST.Nous avons aussi montré qu'une régulation indépendante de CcpA s'exerce sur dctP, le gène codant pour le transporteur du succinate et du fumarate en présence de malate, suggérant un mécanisme similaire à celui observé pour ysbAB. Enfin, nous avons montré que le flux métabolique traversant MaeA était également impliqué dans la régulation par CcpA de l'entrée des sources glycolytique par le malate. / In Bacillus subtilis like for all the bacteria, the central carbon metabolism is essential for growth. It uses glucose (a glycolytic carbon source) and malate (a gluconeogenic carbon source) as preferential carbon sources. These two carbon sources are able to induce carbon catabolite repression through the transcription factor CcpA and thus establishing a hierarchy in the use of alternative carbon sources. The pyruvate is in the middle of the carbon metabolism, and can be used by B. subtilis as sole carbon source; however its transporter remains unknown.Transcriptome analyses revealed that the only operon specifically expressed in cells grown on pyruvate is ysbAB, and we showed that its deletion led to a strong growth defect on pyruvate. Using tagged proteins, we highlighted that YsbA and YsbB formed a complex localized at the membrane. We next showed that this complex is the major pyruvate transporter, and operates as a facilitated transporter. Using a reporter fusion, we showed that the operon lytST located upstream of ysbAB, and coding for a two-component system, is responsible for the induction of ysbAB. We also showed that besides the CcpA-mediated repression by both glucose and malate, an additional regulation mechanism through the malic enzyme activity of MaeA is acting on ysbAB. This regulation is due to the accumulation of pyruvate in the cell which hinders the LytST-mediated induction of ysbAB.We also showed that a CcpA-independent repression is exerted on dctP, the gene coding for the succinate and fumarate transporter, in the presence of malate, suggesting a regulation mechanism similar to the one observed for ysbAB. Finally, we showed that the metabolic flux going through MaeA is also involved in the CcpA-dependent repression of the genes coding for glycolytic transporter in presence of malate.

Actinobacillus succinogenes cells were grown on Poraver® support particles in a packed-bed reactor. Dilution rates (D) of 0.054–0.72 h-1 were investigated. Glucose was used as substrate. CO2 (g) was bubbled into a complex medium to satisfy the fixation requirements and maintain anaerobic conditions. At D ≥ 0.31 h-1, an initial glucose concentration of 35 g.L-1 was used; at lower dilution rates, this was increased to 60 g.L-1 in order to avoid substrate limitations. By-product formation included acetic and formic acids. A maximum productivity of 10.7 g.L-1 was obtained at D = 0.7 h-1. It was found that the system provided repeatable results at a given D. The longest steady state period was maintained for about 97 h at D = 0.31 h-1. Steady state stability was maintained for > 72 h at D < 0.31 h-1. For periods longer than 75 h, however, inhibitory acid titres resulted in a gradual decline in productivity. At higher dilution rates, long-term stability could not be maintained. The low acid titres produced significant biofilm sloughing following aggressive biofilm growth, resulting in oscillatory system behaviour. For fermentation times < 115 h, the dilution rate was secondary to the attachment area in determining the total biomass at steady state. Total biomass values were then used to determine specific rates. A clear trend was observed, with the specific glucose consumption rate, and specific acid production rates, increasing with increasing D. This was explained by assuming a maintenance-driven system at all Ds studied. A product analysis indicated that at ΔS < 15 g.L-1, pyruvate formate lyase was the preferred oxidative route. A shift to the pyruvate dehydrogenase pathway occurred at higher ΔS values, so that the highest YSS values obtained exceeded 0.85 g.g-1. A decrease in C3 by-product formation resulted in high YSS values being maintained, indicating an additional, unknown source of nicotinamide adenine dinucleotide (NADH). It is recommended that any process utilising immobilised A. succinogenes cells should operate at an intermediate D, in order to maintain long-term reactor stability, high productivities and good yields. / Dissertation (MEng)--University of Pretoria, 2013. / gm2014 / Chemical Engineering / unrestricted

Ethyl pyruvate, a known ROS scavenger and anti-inflammatory drug was found to combat leukemia cells. Tumor cell killing was achieved by concerted action of necrosis/apoptosis induction, ATP depletion, and inhibition of glycolytic and para-glycolytic enzymes. Ethyl lactate was less harmful to leukemia cells but was found to arrest cell cycle in the G0/G1 phase. Both, ethyl pyruvate and ethyl lactate were identified as new inhibitors of GSK-3β. Despite the strong effect of ethyl pyruvate on leukemia cells, human cognate blood cells were only marginally affected. The data were compiled by immune blotting, flow cytometry, enzyme activity assay and gene array analysis. Our results inform new mechanisms of ethyl pyruvate-induced cell death, offering thereby a new treatment regime with a high therapeutic window for leukemic tumors.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 74-81).

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: This study aimed to assess the contribution of anaplerotic C prOVISIOn VIa phosphoenolpyruvate carboxylase (pEPc, EC 4.1.1.31), during Pi stress in the root and nodule components of Lupinus angustifolius. The role of PEPc in DIC metabolism in roots and nodules of phosphate-starved plants was studied. The symbioses involving leguminous plants and species of Rhizobium and Bradyrhizobium bacteria form an integral part of effective management ofN in the environment. In agricultural settings, roughly 80% of this biologically fixed N2 comes from this type of symbiotic relationship. Nitrogen-fixing bacteria in concert with legumes fix atmospheric nitrogen, which is then available to the infected plant. Worldwide, legumes are grown on approximately 250 Mha and they fix about 90 Tg (90 billion tons) of N, per year. The overall stoichiometry for nitrogen assimilation in the nodule requires one molecule of oxaloacetate to be converted to one molecule of asparagine per dinitrogen molecule fixed. One possible source for the required oxaloacetate is the reaction catalysed by PEPc. The reaction catalysed by PEPc is a major source of anaplerotic carbon for the plant and it is expected that this reaction will be even more important to plants under Pi stress, as the reaction is not ATP-dependent. Seeds of Lupinus angustifolius (cv. Wong a) were inoculated with Rhizobium sp. (Lupinus) bacteria and grown in hydroponic culture. Tanks were supplied with either 2 11MP04 (LP) or 2 mM P04 (HP) and air containing 360 ppm CO2. Roots experienced pronounced P stress with a greater decline in Pi, compared to nodules. Under P stress, PEPc activities increased in roots but not in nodules and these changes were not related to the expression of the enzyme. Root and nodular PEPc were not regulated by expression, but possibly by posttranslational control. LP roots also synthesised more pyruvate from malate than LP nodules. The role of pyruvate accumulation under Pi stress, was further highlighted by the metabolism of PEP via both the pyruvate kinase (PK, Ee 2.7.1.40) and PEPc routes. The enhanced PK activities supported these high pyruvate levels. The results show unequivocally that nodules do not experience P stress to the same extent as roots. Implications of the findings are that nodules require low P to function normally. Maintenance of phosphate levels in nodules may be at the expense of host. It can be suggested that when nodules are P-starved they can become aggressive scavengers for available P and even out-compete roots. / AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om die bydrae van anaplerotiese koolstof-voorsiening via fosfo-enolpirovaatkarboksilase (pEPc, EC 4.1.1.31), tydens fosfaatstremming in die wortels en wortelknoppies van Lupinus angustifolius te bepaal. Die rol van PEPc in die metabolisme van opgeloste anorganiese koolstofdioksied in fosfaat-beperkte wortels en wortelknoppies is ondersoek. Die simbiose tussen peulplante en spesies van Rhizobium en Bradyrhizobium bakterieë vorm 'n integrale deel van die doeltreffende bestuur van stikstof in die omgewing. In die landbou word ongever 80 %van biologies-gefikseerde stikstof deur hierde simbiotiese verhouding geproduseer. Stikstotbindende bakterieë, in simbiose met peulplante, fikseer atmosferiese stikstof, wat dan beskikbaar is vir die geïnfekteerde plant. Wêreldwyd fikseer peulplante ongeveer 90 biljoen ton stikstof per jaar. Die algehele stoïgiometrie vir stikstof-fiksering in wortelknoppies vereis dat een molekule oksaalsuur na een molekule asparagien omgesit word per stikstofmolekule wat gefikseer word. Een moontlike bron vir die benodigde oksaalsuur is die reaksie wat deur PEPc gekataliseer word. Die reaksie wat deur PEPc gekataliseer word is 'n belangrike bron van anaplerotiese koolstof vir die plant en dit word vermoed dat hierdie reaksie van nog groter belang sal wees vir plante onder fosfaatstremming, omdat die reaksie nie ATP-afhanklik is nie. Sade van Lupinus angustifolius (cv. Wonga) is geïnokuleer met Rhizobium sp. (Lupinus) bakterieë en gekweek in waterkultuur. Tenke is voorsien met óf 2 !lM P04 (LP), óf 2 mM P04 (HP) en lug wat 360 ppm CO2 bevat het. Wortels het skerp fosfaatstremming ervaar, met 'n groter afname in Pi, vergelykbaar met wortelknoppies. Tydens fosfaatstremming het die aktiwiteit van PEPc toegeneem in wortels, maar nie in wortelknoppies nie en hierdie veranderinge was nie verwant aan die uitdrukking van die ensiem nie. PEPc van wortels en wortelknoppies is nie gereguleer deur uitdrukking nie, maar moontlik deur post-translasie kontrole. Wortels onder 'n lae-fosfaat voorsiening het ook meer pirodruiwesuur vanaf malaat gesintetiseer as wortelknoppies. Die rol van pirodruiwesuur-akkumulering tydens fosfaatstremming is verder beklemtoon deur die metabolisme van PEP via beide die pirovaatkinase- (PK, EC 2.7.1.40) en PEPc- roetes. Die verhoogde PK-aktiwiteite verklaar hierdie hoër vlakke van pirodruiwesuur. Die resultate toon ondubbelsinnig dat wortelknoppies me tot dieselfde mate fosfaatstremming ervaar as wortels nie. Dit impliseer dat wortelknoppies min fosfaat benodig om normal te funksioneer. Handhawing van fosfaatvlakke in wortelknoppies mag ten koste van die wortel wees. Dit is moontlik dat, wanneer wortelknoppies fosfaatbeperk is, hulle aggressiewe opruimers word vir beskikbare fosfaat en selfs beter funksioneer as die wortels.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The microbiota has a strong influence on health and disease in humans. A causative shift favoring pathobionts is strongly linked to diseases. Therefore, anti-microbial agents selectively targeting potential pathogens as well as their biofilms are urgently demanded. Here we demonstrate the impact of ethyl pyruvate, so far known as ROS scavenger and antiinflammatory agent, on planktonic microbes and biofilms. Ethyl pyruvate combats preferably the growth of pathobionts belonging to bacteria and fungi independent of the genera and prevailing drug resistance. Surprisingly, this anti-microbial agent preserves symbionts like Lactobacillus species. Moreover, ethyl pyruvate prevents the formation of biofilms and promotes matured biofilms dissolution. This potentially new anti-microbial and anti-biofilm agent could have a tremendous positive impact on human, veterinary medicine and technical industry as well.

The primary focus of this dissertation is the metabolic enzyme pyruvate carboxylase (PC). The structure and function of this fascinating enzyme has been studies and characterized by many laboratories over many decades. This extensive background is reviewed in Chapter 1, with an overview of the biotin-dependent carboxylase family and a particular focus on PC. In this dissertation, we primarily use X-ray crystallography to study PC at a structural level. This dissertation is divided into two overarching sections, with the first section (Chapters 2-5) focusing on the bacterial second messenger cyclic-di-AMP (c-di-AMP). This project was initiated by our collaborators in the laboratory of Josh Woodward at the University of Washington, who performed the first screen to identify c-di-AMP binding proteins in the bacterium Listeria monocytogenes. In Chapters 2 and 3, the regulation of PC by c-di-AMP in L. monocytogenes as well as the bacterium Lactococcus lactis is discussed. Crystal structures of the PC from each of these species in complex with cyclic-di-AMP reveal the binding site and give insights into the molecular mechanisms of this regulation. In Chapters 4 and 5, structural studies of other c-di-AMP binding proteins identified in the screen are discussed. The second section (Chapters 6) focuses on a second class of PC enzymes called the two-subunit PCs, which are found in a subset of Gram-negative bacteria. In Chapter 6, the first crystal structure of a two-subunit PC from the bacterium Methylobacillus flagellatus is determined. In collaboration with the Lars Dietrich laboratory at Columbia University, we investigate the physiological function of the two-subunit PC in the pathogen Pseudomonas aeruginosa. A theme which emerges from these studies is that PC is an incredibly diverse enzyme which has been adapted for the peculiar physiological needs of each organism it inhabits. Because PC is found throughout nature in every kingdom of life, further studies of its unique properties and role in each organism are sure to provide more surprising insights in the years to come.