Les Leucémies Aigües Myéloïdes (LAM) sont des hémopathies malignes hétérogènes de mauvais pronostic qui se caractérisent par une expansion clonale de progéniteurs immatures. De nombreuses dérégulations de voies de signalisation sont retrouvées dans les cellules leucémiques et leur confèrent un avantage de prolifération et de survie. La voie de signalisation mTORC1, qui contrôle la traduction protéique, l’autophagie et plusieurs voies métaboliques, est ainsi constitutivement activée dans les cellules leucémiques. La reprogrammation métabolique notamment via « l’effet Warburg » est un phénomène bien décrit dans les cellules cancéreuses. L’augmentation de l’utilisation de la glycolyse, confère aux cellules tumorales un avantage de survie en favorisant une production rapide d’ATP et d’intermédiaires métaboliques nécessaires pour les biosynthèses de nucléotides, d’acides-aminés et de lipides. C’est donc dans ce contexte que j’ai étudié le métabolisme du glucose dans les cellules de LAM et l’implication de la voie de signalisation mTORC1 dans la dérégulation de ce métabolisme. J’ai tout d’abord identifié par une étude transcriptomique dans la lignée leucémique MOLM-14 que la signalisation mTORC1 contrôle plusieurs voies métaboliques notamment celles permettant l’utilisation du glucose. Ceci a été vérifié dans plusieurs lignées de LAM puisque l’inhibition ou la sur-activation de mTORC1 entrainent respectivement une diminution ou une augmentation de la consommation de glucose et de la production de lactate. De façon intéressante, le niveau d’activation de la voie mTORC1 détermine la sensibilité des cellules leucémiques à l’inhibition de la glycolyse. En effet, lorsque mTORC1 est activé, le blocage de la glycolyse induit de l’autophagie et l’apoptose des cellules leucémiques. A l’inverse, le blocage de mTORC1 induit une reprogrammation métabolique des cellules leucémiques qui utilisent alors principalement la phosphorylation oxydative pour produire l’ATP dont elles ont besoin. Leur survie devient alors indépendante du glucose. A l’inverse des cellules primaires de LAM, les cellules hématopoïétiques immatures normales CD34+ sont moins sensibles au blocage de la glycolyse. Le ciblage du métabolisme du glucose pourrait donc constituer une stratégie thérapeutique intéressante dans les LAM. Je me suis ensuite intéressée aux effets anti-leucémiques induits par l’inhibition de la voie des pentoses phosphates (PP) et plus particulièrement au ciblage de la G6PD (glucose-6-phosphate déshydrogénase) par le composé le 6-aminonicotinamide (6-AN). En effet, une étude de flux métabolique a permis de mettre en évidence qu’une proportion importante de glucose est dirigé vers la voie des PP, laissant suggérer que l’addiction des cellules leucémiques au glucose pourrait être liée à une utilisation augmentée de cette voie annexe. J’ai alors observé que le 6-AN induit une cytotoxicité in-vitro y compris dans les cellules primaires de patients, sans avoir d’effets sur les cellules hématopoïétiques normales et in-vivo dans un modèle de xénogreffe de la lignée MOLM-14 chez la souris NUDE. Cette étude a donc permis de montrer que l’activation constitutive de mTORC1 rend la survie des cellules de LAM dépendante de la glycolyse et crée une sensibilité spécifique à l’inhibition de la G6PD. La dérégulation de la signalisation mTORC1 étant quasi-constante dans les LAM, cibler la G6PD pourrait donc représenter une stratégie thérapeutique intéressante. / Acute Myeloid Leukemia (AML) are heterogeneous hematological diseases with poor prognosis characterized by a clonal expansion of immature progenitors. Many deregulation of signaling pathways are found in leukemic cells and give them an advantage of proliferation and survival. The MTORC1 signaling pathway, which controls protein translation, autophagy and several metabolic pathways, is constitutively activated in leukemic cells. Metabolic reprogramming in particular the "Warburg effect" is a phenomenon well described in cancer cells. High rate of glycolysis has been considered to give tumour cells advantages through rapid production of ATP and intermediates for the synthesis of nucleotides, amino acids, and lipids. In this context, I studied glucose metabolism in AML cells and the involvement of the mTORC1 signaling pathway in the deregulation of this metabolism. First, I identified by a transcriptomic analysis in the MOLM-14 cell line that mTORC1 signaling controls several metabolic pathways including those for glucose utilization. This has been verified in several AML cell lines, since inhibition or over-activation of mTORC1 respectively induces a decrease or an increase in glucose consumption and lactate production. Interestingly, the level of activation of the mTORC1 signaling pathway determines the sensitivity of AML cells to the inhibition of glycolysis. Indeed, when mTORC1 is activated, the blockade of glycolysis induces autophagy and apoptosis of leukemic cells. Conversely, blocking mTORC1 induces metabolic reprogramming of leukemic cells, which then mainly use oxidative phosphorylation to produce ATP for their needs. AML cell survival become independent of glucose. Unlike primary AML cells, survival of normal immature hematopoietic cells CD34+ is only barely affected by the blockade of glycolysis. Thus, targeting the glucose metabolism may constitute an attractive therapeutic strategy in AML. I then investigated the anti-leukemic activity induced by the inhibition of the pentose phosphate pathway (PPP) and more particularly by the specific blockade of G6PD (glucose 6-phosphate dehydrogenase) with the 6-aminonicotinamide (6- AN) compound. Indeed, a metabolic flux analysis demonstrated that a significant proportion of glucose was directed towards the PPP. This result suggested that the addiction of leukemic cells toward glucose might be related to an increased use of PPP. I then observed that the 6-AN induced in vitro cytotoxicity including in primary AML cells from patients without effect on normal immature hematopoietic cells CD34+ and in vivo in a xenograft model of MOLM-14 cell line in the NUDE mouse. This study therefore demonstrated that the constitutive activation of mTORC1 makes AML cells survival dependent on glycolysis, and creates a specific vulnerability to the inhibition of G6PD. Given that deregulation of the mTORC1 signaling pathway is almost constant in AML, targeting G6PD may therefore represent an interesting therapeutic strategy.

Ethanol produced from lignocellulosic biomass has the potential to become a promisingalternative to gasoline. In this work the simultaneous saccharification and fermentation (SSF)technology was applied for ethanol production from hardwood with focus on cell growth,ethanol production and contamination.The SSF was performed at PH 5.5 and 35°C for different suspended solid concentrations(8%, 10% and 12%) of pretreated birch slurry which contained 16 % total suspended solids.Two different hexose fermenting yeast strain (Ethanol Red) and pentose fermenting yeaststrain were used.Quantifying the concentration of chemical components and metabolites in the fermentationmedium demonstrated that glucose and xylose are the major fermentable sugars in the slurry.The higher load of slurry (12%) represents a higher content of carbohydrates and potentiallyhigher end concentration of ethanol. Moreover, more lactic acid is produced with the lowerload of slurry (8 % or 10 %), presumably due to a result of a less inhibitory environment forbacterial growth. In this context, acetic acid sticks out as the most important inhibitor withconcentrations of 15.2 and 12.5 and 9.7 g/l respectively in the 12 %, 10 % and 8 % (ofsuspended solids) trials. Using pentose fermenting yeast may lead to higher ethanolproduction, lower xylose uptake and lower lactic acid formation. Cell viability and cellvitality determination from fermentation media in all the trails represented a sharplydecreasing trend during the fermentation for both Ethanol Red yeast strain and the pentosefermenting strain yeast strain apparently due to cell decomposition. / Program: MSc in Resource Recovery - Industrial Biotechnology

<p>Apoptosis is a form of programmed cellular "suicide" which is activated in response to a variety of pro-death stimuli. Apoptotic cell death is orderly and energy-dependent, and cellular constituents are packaged into membrane-bound vesicles for consumption by phagocytes. Toxic intracellular signals are never exposed to neighboring cells or to the extracellular environment, and a host inflammatory response does not occur. Apoptosis is executed by the coordinated activation of caspase family proteins. Caspase-2 is an apical protease in this family, and promotes cell death after receipt of cues from intracellular stressor signals. Caspase-2 helps to initiate apoptosis by responding to cellular death stimuli and signaling for downstream cytochrome c release and executioner caspase activation.</p><p> Several years ago our lab determined that Xenopus laevis oocyte death is partly controlled by the activation of caspase-2. In the setting of oocyte or egg extract nutrient depletion, caspase-2 was observed to be activated upstream of mitochondrial cytochrome c. In fact, caspase-2 is suppressed in response to the nutrient status of the oocyte: nutrient-replete oocytes with healthy pentose phosphate pathway flux and abundant NADPH production are able to inhibit caspase-2 via S135 phosphorylation catalyzed by calcium/calmodulin-dependent protein kinase II. Phosphorylation of caspase-2 at S135 is critical in preventing oocyte cell death, and a caspase-2 mutant unable to be phosphorylated loses its ability to respond to suppressive NADPH signals. </p><p> In this dissertation we examine the converse mechanism of metabolically-regulated caspase-2 activation in the Xenopus egg extract. We now show that caspase-2 phosphorylated at S135 binds the interactor 14-3-3 zeta, thus preventing caspase-2 dephosphorylation. Moreover, we determined that S135 dephosphorylation is catalyzed by protein phosphatase-1, which directly binds caspase-2. Although caspase-2 dephosphorylation is responsive to metabolism, neither PP1 activity nor binding is metabolically regulated. Rather, release of 14-3-3 zeta from caspase-2 is the point of metabolic control and allows for caspase-2 dephosphorylation. Accordingly, a caspase-2 mutant unable to bind 14-3-3 zeta is highly susceptible to activation. Although this mechanism was initially established in Xenopus, we now demonstrate similar control of murine caspase-2 by phosphorylation and 14-3-3 binding in mouse eggs. </p><p> In the second part of this dissertation we examine the paradigm of caspase-2 metabolic regulation in a mammalian somatic cell context. We observed that mammalian caspase-2 is a metabolically-regulated phosphoprotein in somatic cells, and that the site of regulation is caspase-2 S164. Phosphorylation at S164 appears to inhibit mammalian caspase-2 by preventing its induced proximity oligomerization, thus also preventing procaspase-2 autocatalytic processing. We further identify some of the molecular machinery involved in S164 phosphorylation and demonstrate conservation with the validated Xenopus regulators. Interestingly, we extend the findings of caspase-2 phosphorylation to a study of ovarian cancer, and show that caspase-2 S164 phosphorylation might be involved in determining cancer cell chemosensitivity. We further provide evidence that chemosensitivity can be modulated by the cellular metabolic status in a caspase-2-dependent manner. Thus, we have identified a novel phosphorylation site on mammalian caspase-2 in somatic cells, and are working further to understand the implications of caspase-2 signaling in the context of cancer cell responsiveness to chemotherapeutic treatments.</p> / Dissertation

No description available.

Circadian rhythms are self-sustained endogenous biological oscillations with a period of approximately 24 hours. These rhythms are observed widely across kingdoms and at all levels of biological scale. Recent work has shown there to be circadian variation in metabolism, both at the organismal and cellular level. It has also been posited that rhythmic production of metabolites might be essential for maintenance of circadian rhythmicity within cells, even in the absence of nascent transcription. The first portion of this thesis investigates the contribution of primary carbohydrate metabolism to cellular timekeeping, with particular emphasis on the pentose phosphate pathway. I also describe and validate a new 13C labelling technique for accurate determination of the relative flux through early primary metabolic pathways. This is accompanied by the development and optimisation of a microfluidic system for long-term perfused tissue culture, which allows for longitudinal study of metabolic flux within the same population of cells with simultaneous recording of clock gene activity. This perfused system provides several advantages over static tissue culture. The second portion considers the effects of the metabolic hormone insulin on circadian rhythmicity, both at the level of the cell and of the whole organism. It shows that administration of insulin is sufficient to shift the phase of circadian gene expression and elicits induction of clock protein PER2. Strikingly, manipulation of insulin signalling is sufficient to determine all the essential parameters of the cellular clock (phase, period and amplitude) in a dose-dependent but glucose independent fashion. Using pharmacological and genetic approaches, a molecular explanation for this effect is determined. This data suggests that insulin is a primary determinant of rhythms in peripheral tissues and is most likely a major signal for circadian entrainment to feeding in mammals, for which I now propose a mechanistic basis.

Made available in DSpace on 2014-06-11T19:23:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-08-02Bitstream added on 2014-06-13T20:30:11Z : No. of bitstreams: 1 carrion_lm_me_sjrp.pdf: 381584 bytes, checksum: f7ac95eb0ed933b2a92b058b0e3faefb (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A efetiva produção de etanol lignocelulósico depende de leveduras capazes de fermentar a fração hemicelulósica da biomassa lignocelulósica que é composta em sua maioria por xilose. Há uma grande dificuldade em encontrar esses microrganismos já que a capacidade de utilizar xilose como fonte de carbono não é apresentada pela maioria das leveduras. Neste trabalho leveduras isoladas de diferentes materiais da zona rural,foram testadas quanto à assimilação de xilose e glicose, e fermentação de xilose a 30°C, onde observou-se diferentes comportamentos entre diferentes linhagens da mesma espécie. As linhagens de leveduras isoladas foram identificadas pela técnica molecular PCR-RFLP empregando-se as regiôes ITS 5,8 S e D1/D2 do DNA ribossômico. Três leveduras identificadas como espécies de Cândida tropicalis foram avaliadas quanto a capacidade de produção de xilitol e etanol a partir de meio basal com xilose como única fonte de carbono e do licor negro da extração da celulose hidrolisado por enzimas do fungo Thermoascus aurantiacus. A produção de xilitol foi semelhante nos dois substratos, entretanto a produção de etanol foi maior quando o licor negro foi empregado, esses resultados mostraram que a quantidade e a natureza dos açúcares presentes nos meios foram um interferente maior no metabolismo da levedura que os compostos tóxicos presentes no licor negro / The effective lignocellulosic ethanol production depends on yeasts that are able to ferment the hemicellulosic biomass fraction, which is mostly composed by xylose. There is a big difficulty in finding such microorganisms because most yeasts do not use xylose as a carbon source. In this work, yeasts isolated from different materials were tested for xylose and glucose assimilation and xylose fermentation at 30°C ,where it was observed different behaviors between strains of the same species. The strains isolated were identified by PCR-RFLP molecular technique employing the ITS 5,8 and D1/D2 regions of ribosomal DNA. Three yeasts were identified as Cândida tropicalis species and their ability to produce xylitol and ethanol from xylose and the black liquor from the cellulose extraction hydrolyzed by enzymes of the fungus Thermoascus aurantiacus was evaluated. The xylitol production was similar in both media, but the ethanol production was higher when the black liquor was used, these results shows that the quantity and nature of the sugars present in the media have a bigger interference in the metabolism of yeast to toxic compounds present in black liquor

Orientador: Eleni Gomes / Resumo: As leveduras são organismos quimiorganotróficos que utilizam principalmente glicose como fonte de energia e carbono. Além da glicose, outros açúcares fermentescíveis se encontram em abundância na natureza e têm sido subaproveitados na indústria, dos quais se destaca a xilose. Para algumas leveduras, como Saccharomyces cerevisiae, a utilização de pentoses é limitada pela carência de transportadores de membrana específicos e enzimas intracelulares para a metabolização deste açúcar. Entretanto, algumas leveduras são capazes de utilizar xilose como fonte de carbono e bioconverte-la em produtos como etanol, ácidos orgânicos ou peptídeos. Isso implica na existência de um sistema de transporte e enzimas intracelulares para metabolizala. Neste contexto, a prospecção de enzimas auxiliares despolimerizantes do material lignocelulósico, tais como β-glicosidases e α-L-arabinofuranosidases, também assume função importante para obtenção de açúcares fermentescíveis. Além disso, poucos estudos estão disponíveis a respeito da produção de peptídeos bioativos por leveduras, quais podem ser fontes promissoras de produção dos mesmos. Sendo assim, o presente trabalho buscou investigar o consumo de xilose, a produção de peptídeos com atividade biológica e a produção de βglicosidases pelas espécies Pichia ofunaensis e Trichosporon multisporon, assim como a produção de α-L-arabinofuranosidases por Aureobasidium pullulans e A. leucospermi. As enzimas foram prospectadas utilizando farelo de trigo como ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Yeasts are chemorganotrophic organisms that mainly use glucose as a source of energy and carbon. In addition to glucose, other fermentable sugars are found in abundance in nature and have been underutilized in the industry, of which xylose stands out. For some yeasts, such as Saccharomyces cerevisiae, the use of pentoses is limited by the lack of specific membrane transporters and intracellular enzymes for the metabolization of this sugar. However, some yeasts are able to use xylose as a carbon source and bioconvert it into products such as etanol, organic acids or peptides. This implies the existence of a transport system and intracellular enzymes to metabolize it. The fermentation of pentoses is an essential step to improve the yield in the production of ethanol and organic acids. In this context, the prospection of depolymerizing auxiliary enzymes of lignocellulosic material, such as β-glycosidases and α-Larabinofuranosidases, also plays an important role in obtaining fermentable sugars. In addition, few studies are available regarding the production of bioactive peptides by yeasts, which can be promising sources of their production. Thus, the present work sought to investigate the consumption of xylose, the production of peptides with biological activity and the production of β-glycosidases by the species Pichia ofunaensis and Trichosporon multisporon, as well as the production of α-L-arabinofuranosidases by Aureobasidium pullulans and A. leucospermi. The enzymes were pro... (Complete abstract click electronic access below) / Mestre

Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2020 / Production of bioethanol from lignocellulosic biomass has gained significant attention worldwide as an alternative fuel source for the transportation sector without affecting food supply. Efficient conversion of pentose sugars (L-arabinose and D-xylose) produced during hydrolysis of hemicellulose to ethanol can enhance the economic viability. In this study, a total of 390 yeasts isolated from Marula wine, the gut of dung beetles, herbal concoctions and banana residues were screened for the ability to ferment L-arabinose and D-xylose. Fourteen yeasts were able to ferment both pentose sugars and ten strains were subjected to an adaptation process in the presence of acetic acid using L-arabinose as carbon source. Four adapted strains of Meyerozyma caribbica were able to ferment L-arabinose to ethanol and arabitol in the presence of 3 g/L acetic acid at 35 °C. Meyerozyma caribbica Mu 2.2f fermented D-xylose, L-arabinose and a mixture of D-xylose and L-arabinose to produce 1.7, 3.0 and 1.9 g/L ethanol, respectively, compared to the parental strain with 1.5, 1.0 and 1.8 g/L ethanol, respectively, in the absence of acetic acid. The adapted strain of M. caribbica Mu 2.2f produced 3.6 and 0.8 g/L ethanol from L-arabinose and D-xylose, respectively in the presence of acetic acid while the parental strain failed to grow. In the bioreactor, the adapted strain of M. caribbica Mu 2.2f produced 5.7 g/L ethanol in the presence of 3 g/L acetic acid with an ethanol yield and productivity of 0.338 g/g and 0.158 g/L/h, respectively at a KLa value of 3.3 h-1. The adapted strain produced 26.7 g/L arabitol with a yield of 0.900 g/g at a KLa value of 4.9 h-1. Meyerozyma caribbica Mu 2.2f could potentially be used to produce ethanol and arabitol under stressed conditions. / National Research Foundation (NRF)

No description available.

This dissertation is a compilation of published works and a manuscript that seek to understand the possible role of polyamines in the regulation of capsule in Streptococcus pneumoniae (Spn, pneumococcus). Spn remains a major health risk worldwide while the capsule is widely recognized as the principal virulence factor. Polyamines on the other hand are small hydrocarbon molecules known to regulate a number of cellular processes in bacteria. This work investigates the impact of deletion of polyamine biosynthesis gene, SP_0916 (cadA, lysine decarboxylase at the time of first and second publication), on protein expression and the capsule biosynthesis of virulent pneumococcal serotype 4 (TIGR4). We identify loss of capsular polysaccharide (CPS) in the deletion strain and based on proteomics results, we hypothesized that a shift in metabolism that favors the pentose phosphate pathway (PPP) over glycolytic pathway, that could reduce the availability of precursors for CPS had occurred. Comparison of transcriptomic and untargeted metabolomics profile of ∆SP_0916 with TIGR4 shows impaired glycolysis and Leloir pathways that provide CPS precursors, in the mutant strain. Furthermore, gene expression changes indicate possible reduction of common polyamines (cadaverine, putrescine, spermidine and spermine). Targeted metabolomics analysis confirmed reduced levels of polyamines in SP_0916. However, the result suggests that SP_0916 encodes an arginine decarboxylase, contrary to its existing annotation as a lysine decarboxylase in many bioinformatics databases. Biochemical characterization of the purified protein encoded by SP_0916 confirms that it is indeed catalyzes arginine decarboxylation, and exogenous supplementation of agmatine, the product of the reaction, successfully restores capsule biosynthesis. This study fixes an error in annotation of the TIGR4 genome and further establishes the essentiality of agmatine, a product of arginine decarboxylation as the key polyamine molecule modulating pneumococcal capsule. We later compared the impact of deletion of polyamine synthesis by gene deletion (ΔSP_0916) with chemical inhibition of synthesis using α- difluoromethylornithine (DFMO), in multiple pneumococcal serotypes. Results of this dissertation confirmed that pneumococcal pathways impacted by the disruption of polyamine biosynthesis either by gene deletion or chemical intervention are conserved and could regulate capsule synthesis.