The gram-negative, gamma proteobacterium Pseudomonas aeruginosa demonstrates substantial metabolic flexibility, allowing it to survive and thrive in diverse environments. Indeed, its ability to carefully maintain a buffered intracellular oxidoreduction state permits it to maintain structural and functional stability in the face of both nutrient-poor and nutrient-rich conditions. It is clear that metabolism is simply central to the existence of this microbe, yet knowledge of the genetics and biochemistry underlying this deeply intricate world of metabolic regulation is fundamentally incomplete. Two critical metabolites, alpha-ketoglutarate and glutamine, appear at the intersection of carbon and nitrogen metabolism, and may represent the status of Pseudomonas’ intracellular carbon or nitrogen pools. However, the coordination of carbon and nitrogen assimilation, whether through PII proteins, the Ntr regulatory cascade, the CbrAB regulatory system, or the PTS-NTR system, as well as these pathways’ cross-talk and ability to control downstream processes based on nutrient availability, still remain to be elucidated. Further, a more comprehensive understanding of P. aeruginosa’s metabolic regulation could be significant for the development of new therapies that specifically target critical biochemical pathways involved in the metabolism of this organism and offer new hope to patients suffering from P. aeruginosa infections in the clinic. Thus, this review considers how, when, and why carbon and nitrogen metabolism may be regulated in Pseudomonas aeruginosa, and proposes there may be more interaction and cross-regulation between these two seemingly divergent metabolic arms than originally thought.

Le processus conduisant à la prolifération et différenciation des cellules souches hématopoïétiques (CSH) en cellules de toutes les lignées sanguines s’appelle l’hématopoïèse. Bien que l'engagement des CSH soit régi par les cytokines, les facteurs de transcription, les modificateurs épigénétiques et la niche des CSH, notre groupe a constaté que leur engagement vers la lignée érythroïde dépendait aussi du métabolisme de la glutamine. La glutaminolyse contribue à la biosynthèse des nucléotides de novo ainsi qu’à la production de l'alpha-kétoglutarate (αKG), intermédiaire métabolique du cycle TCA (Oburoglu et al. 2014). Il est cependant important de noter que la différenciation érythroïde est un processus unique, où chaque cellule fille est structurellement et fonctionnellement différente de sa cellule mère. Chaque division définit un stade de différenciation précis avec un dernier cycle de division produisant un réticulocyte énucléé. Ainsi, nous avons émis l'hypothèse que les réseaux métaboliques mobilisés dans les progéniteurs érythroïdes changent en fonction du stade de différenciation et que ces réseaux régulent la transition des progéniteurs d'un stade à l'autre.Au cours de ma thèse, j’ai caractérisé les états métaboliques associés aux différents stades de différenciation des progéniteurs érythroïdes. Nous avons ainsi montré qu'aux stades précoces de différenciation érythroïde, avant la différenciation terminale, les progéniteurs hématopoïétiques présentent une activité métabolique accrue avec un niveau de phosphorylation oxydative (OXPHOS) plus élevé. Ces données sont en corrélation avec l'augmentation de la génération de l’αKG à ces stades de différenciation. De plus, nous avons constaté une augmentation de l’OXPHOS de ces progéniteurs en présence d’αKG exogène. Cependant, la différenciation terminale des précurseurs érythroïdes, caractérisée par la perte de la masse mitochondriale et de leur potentiel membranaire, est associée à une diminution du niveau d'OXPHOS. Ainsi, l'administration exogène d’αKG, a fortement atténué la différenciation érythroïde terminale et l'énucléation, sans affecter la différenciation des pro-érythroblastes. Inversement, un antagoniste de l’αKG (diméthyloxalylglycine, DMOG) n'a pas altéré la différenciation terminale ou l'énucléation, malgré l'abrogation de l'OXPHOS dans les érythroblastes.Ces données suggèrent que la production d’αKG et sa contribution à l’OXPHOS perturbent l'énucléation des globules rouges. C'est pourquoi, dans le but de réduire les niveaux intracellulaires d’αKG, nous avons inhibé l’expression de l'isocitrate déshydrogénase I (IDH1), enzyme cytosolique catalysant la conversion de l'isocitrate en αKG. Cependant, comme IDH1 peut catalyser les réactions dans les deux sens, la diminution de son expression pourrait également augmenter les niveaux d’αKG. En effet, nous avons constaté que le knockdown d'IDH1 entraînait une forte atténuation de la différenciation terminale et de l'énucléation des précurseurs érythroïdes. Cet effet est probablement dû à un déséquilibre de la disponibilité des substrats ; ainsi l’administration ectopique de l’αKG ainsi que du citrate renforce l’altération de la différenciation terminale des précurseurs érythroïdes IDH1-/- ainsi que leur énucléation. Cette étude identifie donc un rôle crucial pour le métabolite αKG dans la régulation de la fonction mitochondriale et de l’OXPHOS, processus qui sont une condition sine qua non pour la différenciation des précurseurs érythroïdes au stade proérythroblaste. Nous montrons en outre que la suppression d’OXPHOS et la catalyse d’intermédiaires du TCA, substrats d’IDH1, sont requis pour les phases terminales de la différenciation érythroïde et l'énucléation.En conclusion, les résultats obtenus au cours de ma thèse mettent en évidence la nature dynamique des réseaux métaboliques qui régulent la progression des précurseurs érythroïdes tout au long des différents stades de la différenciation érythroïde. / Hematopoiesis is the process whereby hematopoietic stem cells (HSCs) proliferate and differentiate to all blood cell lineages. While HSC commitment is known to be regulated by cytokines, transcription factors, epigenetic modifiers and the HSC niche, our group found that specification of HSCs to the red cell lineage is dependent on glutamine metabolism. Glutaminolysis contributes to de novo nucleotide biosynthesis and to the generation of the alpha-ketoglutarate (αKG) TCA cycle metabolite (Oburoglu et al. 2014). Importantly though, erythroid differentiation is a unique process as each daughter cell is structurally and functionally different from its parent cell. Each division defines a stage of differentiation with the final division cycle resulting in the production of an enucleated reticulocyte which further matures to a biconcave erythrocyte. Thus, we hypothesized that progenitor metabolic networks change as a function of the erythroid differentiation stage and moreover, that they regulate the transition of progenitors from one stage of differentiation to the next.During my PhD, I assessed the metabolic alterations that occur as a function of the erythroid differentiation stage. We showed that at early stages of human red cell development, prior to terminal differentiation, hematopoietic progenitors exhibited an increased metabolic activity with a significantly higher level of oxidative phosphorylation (OXPHOS). This correlated with the increased generation of αKG and indeed, we found that ectopic αKG directly augmented OXPHOS in these progenitors. However, the terminal differentiation of erythroid precursors, characterized by the loss of mitochondrial mass and membrane potential, was associated with a decreased level of OXPHOS. Notably, ectopic αKG, which did not alter pro-erythroblast erythroid differentiation, severely attenuated terminal differentiation and enucleation. Conversely, an αKG antagonist (dimethyloxalyl glycine, DMOG) did not negatively impact on terminal differentiation or enucleation despite abrogating OXPHOS in erythroblasts.These data suggested that the production of αKG and its subsequent contribution to oxidative phosphorylation perturb red cell enucleation. We therefore downregulated isocitrate dehydrogenase I (IDH1), the cytosolic enzyme that catalyzes the conversion of isocitrate to αKG, by an shRNA approach in an attempt to decrease αKG levels. However, because IDH1 can catalyze both the forward and reverse reactions, its downregulation could also increase αKG levels. Indeed, we found that IDH1 knockdown resulted in a severe attenuation of terminal erythroid differentiation and enucleation. This effect was likely due to an imbalance in substrate availability––both ectopic αKG as well as citrate further decreased polychromatic to orthochromatic erythroblast differentiation and the subsequent enucleation of IDH1-knockdown erythroid precursors. Thus, the present study identifies a crucial role for the αKG metabolite in regulating mitochondrial function and oxidative phosphorylation, processes that are a sine qua non for erythroid precursors at the pro-erythroblast stage. We further show that terminal erythroid differentiation and enucleation requires OXPHOS suppression and the IDH1-mediated enzymatic catalysis of its TCA substrates.To conclude, the results generated during my PhD highlight the dynamic nature of the metabolic networks that regulate the progression of erythroid precursors through the distinct stages of erythroid differentiation.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Objetivo: Avaliar os efeito da ornitina &#945;-cetoglutarato (OKG) no sangue e tecido intestinal de ratos submetidos à isquemia/reperfusÃo intestinal atravÃs da determinaÃÃo das concentraÃÃes in vivo no sangue e no tecido do intestino delgado, submetido a isquemia/reperfusÃo, de glicose, G 6 PDH, piruvato, acetoacetato, lactato, 3 HBDH, glutationa, T-Bars, mieloperoxidase, CPK e LDH. MÃtodo: Sessenta ratos (Rattus norvergicus albinus, Rodentia Mammalia) foram distribuÃdos aleatoriamente em cinco grupos de 12 animais: Sham 0â (s0â), Sham 30â (s30â), Sham 60â (s60â), Isquemia (i30â), ReperfusÃo (r30â). Estes grupos foram distribuÃdos em subgrupos de acordo com o tempo e com o composto utilizado na gavagem. Todos os animais receberam gavagem de caseinato de cÃlcio ou OKG em dose Ãnica, trinta minutos antes da laparotomia exploradora (LE). Os subgrupos s0âCaCa s30âCaCa, s60âc, i30âCaCa e r30âCaCa receberam apenas caseinato, de cÃlcio. Os subgrupos s0âOKG, s30âOKG, s60âOKG, i30âOKG e r30âOKG receberam OKG na dose de 5g/kg de peso. As amostras foram colhidas em cinco momentos: imediatamente apÃs a LE; apÃs 30 minutos da LE; ApÃs 1h da LE; ApÃs 30 minutos de isquemia; ApÃs 30 minutos de reperfusÃo. A estatÃstica discritiva foi expressa atravÃs da mÃdia, erro e desvio padrÃo, acompanhando-se pelo intervalo de confianÃa da mÃdia a 95% . Para comparar os valores prà e pÃs-procedimentos nas concentraÃÃes das variÃveis estudadas foram empregados os teste âtâ de Student pareado (para variÃncia homogÃnea e heterogÃnea) e ANOVA apÃs anÃlise de normalidade atravÃs do teste Kolmogorov-Smirnov. Quando observou a nÃo normalidade aplicou-se o teste de Kruskal-Wallis. Resultados: Os resultados apontaram um aumento significativo na lactacemia (1.186 + 0,18 versus 0,794 + 0,06, p<0,01) nos animais que receberam OKG em relaÃÃo ao controle nos subgrupos isquemia trinta minutos (i30â). No tecido intestinal reperfundido (r30â) ocorreu reduÃÃo significativa da concentraÃÃo de lactato (0,107 + 0,01 versus 0,266 + 0,02, p<0,05) nos animais recipientes de OKG em relaÃÃo ao animais controle, O piruvato plasmÃtico e tecidual se mostrou significantemente reduzido (0,146 + 0,24 versus 0,156 + 0,17 e 0,094 + 0,02 versus 0,248 + 0,03, p<0,05) apÃs o perÃodo de reperfusÃo de trinta minutos nos animais recipientes da OKG em relaÃÃo aos animais controle. Houve reduÃÃo significativa da concentraÃÃo do acetoacetato no tecido intestinal nos tempos pÃs isquemia e pÃs reperfusÃo dos animais recipientes da OKG (0,57 + 0,01 versus 0,0685 + 0,01 e 0,128 + 0,04 versus 0,156 + 0,03,*p<0,05) quando comparados ao animais nÃo tratados. A glicose 6 PDH apresentou reduÃÃo significativa da sua concentraÃÃo plasmÃtica no tempo isquemia trinta minutos dos animais recipientes da OKG em relaÃÃo aos nÃo tratados ( 0,1442 + 0,048 versus 1,1098 + 0,0796, *p<0,05) , ocorrendo o mesmo na concentraÃÃo tecidual, no pÃs isquemia (0,1002 + 0,02 versus 0,147 + 0,0264, p<0,05). A LDH apresentou elevaÃÃo significativa da sua concentraÃÃo nos animais recipientes da OKG em relaÃÃo ao controle (278,01 + 51,52 versus 132,93 + 12,54, *p<0,05) no grupo isquemia (i30â) . Ocorreu reduÃÃo significativa da CPK no grupo reperfusÃo (r30â) dos animais recipientes da OKG em comparaÃÃo aos animais controle (115,13 + 11,77 versus 166,70+6,23,p<0,05). A glutationa tecidual apresentou elevaÃÃo significativa no sham OKG 30 minutos em relaÃÃo ao nimais controle (59,17 + 2,39 versus 25,09 + 1.53, p<0,05) e reduÃÃo significante no tempo isquemia, tanto nos animais OKG quanto CaCa. Durante o perÃodo de reperfusÃo a glutationa nÃo apresentou alteraÃÃes significativas entre os animais tratados e controle. A OKG influenciou de maneira significativa na reduÃÃo da concentraÃÃo da 3HDBH tecidual no tempo i30â (0,062 + 0,01 versus 0,075 + 0,02,p<0,01) Esta diferenÃa significativa foi mantida no sangue dos animais tratados no grupo reperfusÃo 30âOKG em relaÃÃo aos animais r30âCaCa (0,03 + 0,00 versus 0,0615 + 0,01, p<0,01). A T-bars tecidual apresentou reduÃÃo significante no grupo r30âOKG em comparaÃÃo ao r30âCaCa (0,0522 + 0,03 versus 0,0745 + 0,02, p<0,05), com elevaÃÃo significativa no grupo sham 60âCaCa em relaÃÃo aos animais tratados 0,0937 + 0,02 versus 0,020 + 0,01, p<0,01). A oferta exÃgena da alfa-cetoglutarato nÃo ocasionou nenhuma alteraÃÃo significante nas concentraÃÃes de glicose e mieloperoxidase (MPO) entre os animais do subgrupo experimento quando comparados aos do subgrupo controle. ConclusÃes: Os procedimentos realizados foram suficientes para desencadear alteraÃÃes significativas em alguns metabÃlitos estudados. O modelo de isquemia-reperfusÃo demonstrou efetividade. A oferta exÃgena OKG, em dose Ãnica por gavagem, sugere aumento na atividade prÃ-glicolÃticas aerÃbica a nÃvel tecidual e sistÃmico; proteÃÃo contra lesÃo celular do tecido intestinal, e efeito antioxidante tecidual e sistÃmico durante a lesÃo de isquemia e apÃs a lesÃo de reperfusÃo / Objective: To evaluate the effect of ornithine alpha-ketoglutarate (OKG) in the blood and intestinal tissue of rats submitted to intestinal ischemia/reperfusion, using the blood concentrations of glucose, G6PDH, pyruvate, acetoacetate, lactate, 3HBDH, glutathione, T-Bars, myeloperoxydase, CPK and LHD, evaluated in vivo on these tissues. Methods: Sixty rats (Rattus norvergicus albinus, Rodentia Mammalia) were selected and aleatorily distributed in five groups of twelve animals, which were: Sham0â(s0â), Sham30â(s30â), Sham60â(s60â), Isquemia(i30â), ReperfusÃo(r30â). These groups were distributed in subgroups according to the time and the compost used to the âgavagemâ. All de animals received the âgavagemâ with calcium caseinate or okg, only one dosage, thirty minutes before the exploratory laparotomy (EL). The subgroups s0âCa, s30âCa, s60âCa, i30âCa and r30âCa received only calcium caseinate. The subgroups s0âokg, s30âokg, s60âokg, i30âokg and r30âokg received 5g of okg par each kilogram. The samples were taken in five moments: immediately passed the EL; passed 30 minutes of the EL; passed 60 minutes of the EL; passed 30 minutes of the isquemia; passed 30 minutes of the reperfusion. The descriptive statistics were media, error and standard deviation. The values before and after the procedures were compared using the âtâ test (âStudent pareadoâ to homogeny and heterogeny variation) and ANOVA. Then, it was used Kolmogorov-Smirnov to compare the normal results. The results were not normal, it was used the Kruskal-Wallis test. Results: It was shown an improvement on the blood lactate(1.186 + 0,18 versus 0,794 + 0,06, p<0,01) to the animals that received okg from i30â. A reduction on blood lactate lactato (0,107 + 0,01 versus 0,266 + 0,02, p<0,05) was noticed in the group r30â that received okg. It occurred a reduction on plasmatic and tissue pyruvate reduzido (0,146 + 0,24 versus 0,156 + 0,17 and 0,094 + 0,02 versus 0,248 + 0,03, p<0,05) to the group r30â that received okg. The acetoacetate was reduced to both groups, isquemia and reperfusion, that received okg0,57 + 0,01 versus 0,0685 + 0,01 e 0,128 + 0,04 versus 0,156 + 0,03,*p<0,05). The plasmatic glucose was reduced to the group i30â( 0,1442 + 0,048 versus 1,1098 + 0,0796, *p<0,05) treated with okg and the same happened to the tissue glucose after isquemia (0,1002 + 0,02 versus 0,147 + 0,0264, p<0,05). The LDH had an improvement (0,1002 + 0,02 versus 0,147 + 0,0264, p<0,05) to the group i30â treated with okg. CPK was reduced (115,13 + 11,77 versus 166,70+6,23,p<0,05) to the group r30â treated with okg. The tissue glutathione had an improvement to sham okg 30â (59,17 + 2,39 versus 25,09 + 1.53, p<0,05) and a reduction on isquemia period to the animals treated with okg and CaCa . 3HBDH was reduced (0,062 + 0,01 versus 0,075 + 0,02,p<0,01) in the blood and in the tissues from i30â. This difference was kept to animalsâ blood from r30âokg when related to r30âCaCa(0,03 + 0,00 versus 0,0615 + 0,01, p<0,01). There was a reduction on tissue T-BARS to r30âOKG when compared to r30âCaCa(0,0522 + 0,03 versus 0,0745 + 0,02, p<0,05) and an improvement to sham60âCaCa(0,0937 + 0,02 versus 0,020 + 0,01, p<0,01). Glicose and Myeloperoxydase were not affected by the use of okg. All the results were compared to the respective control groups. Conclusion: The used procedures could bring useful results to metabolites in study. The isquemia/reperfusion showed efficiency, offering exogen okg leads to a rising on glicolitic and aerobic activity to tissues and systems. This offer protects yet from the tissue injury and has antioxidant effect during the isquemia/reperfusion injury.

No description available.

L’activation des cellules T est initiée suite à la rencontre avec un antigène spécifique. Les études réalisées pour mieux comprendre ce processus d’activation se sont principalement focalisées sur le rôle des cellules présentatrices d'antigènes et des cytokines. Toutefois, des données récentes soulignent également l'importance du microenvironnement métabolique pour soutenir l’augmentation des besoins énergétiques et biosynthétiques liés à la stimulation antigénique. Cette reprogrammation métabolique est conditionnée par la disponibilité en nutriments et la teneur en oxygène qui peuvent être altérés en conditions pathologiques, comme dans des tumeurs. En effet, plusieurs groupes dont le nôtre ont montré qu’en cas de faible disponibilité en nutriments, une compétition peut se créer entre les cellules tumorales et les cellules T, impactant de ce fait négativement leurs fonctions anti-tumorales. Cet effet est dû, du moins en partie, aux profils métaboliques distincts des sous-populations de cellules T; alors que les cellules T effectrices (dont les cellules Th1) sont fortement glycolytiques, les cellules T régulatrices suppressives (Treg) présentent un métabolisme plus mixte avec des niveaux accrus d'oxydation lipidique. Il est donc important de déterminer comment les changements métaboliques des cellules T anti-tumorales affectent leur persistance et leur fonctionnalité. Ainsi, j'ai entrepris des travaux afin d’évaluer si le niveau d’expression du transporteur de glucose Glut1 permettait d’identifier et de sélectionner des cellules T ayant des fonctions effectrices distinctes. Nous avons confirmé cette hypothèse et notamment montré que les cellules T exprimant un niveau élevé de Glut1 possèdent un potentiel de sécrétion d’IFNg accru.De plus, nos travaux montrent que la disponibilité en nutriments extracellulaires est un élément clé pour la différenciation terminale des cellules Th1. En effet, l'activation des cellules T CD4 naïves en conditions limitantes en glutamine induit leur différenciation en cellules Treg Foxp3+. Plus surprenant encore, cette carence induit un blocage de la différenciation Th1 même lors d’une polarisation vers ce lignage. De plus, en conditions de carence en glutamine, nous avons découvert que l'alpha-cétoglutarate (aKG), un métabolite dérivé de la glutamine, rétablit cette différenciation terminale Th1. J'ai ensuite évalué l'impact de l’aKG dans les processus de différenciation Th1/Treg en condition non limitante en glutamine. Mes données montrent que, dans des conditions de polarisation Th1, l’ajout d’aKG améliore la différenciation des cellules T CD4 naïfs en cellules Th1 et augmente la production d’IFNg. A l’inverse, l’ajout d’aKG s’accompagne d’une diminution des cellules Foxp3+ et d’une augmentation de la sécrétion de cytokines inflammatoires dans des conditions de polarisation Treg. L'altération de la différenciation des cellules T médiée par l'aKG est notamment associée à une phosphorylation oxydative (OXPHOS) accrue ; ainsi, l'ajout d’un inhibiteur du cycle de Krebs et du complexe mitochondrial II /succinate déshydrogénase, atténue le blocage de la différenciation Treg induit par l'aKG. De façon remarquable, ces modifications de l'équilibre Th1/Treg médiées par l'aKG sont maintenues in vivo et impactent le devenir de cellules T exprimant un récepteur chimérique anti-tumoral (CAR) injectées chez des souris porteuses de tumeurs. En résumé, nos données montrent qu'une faible teneur en aKG intracellulaire liée à une disponibilité limitée en glutamine, favorise un phénotype Treg, alors que des niveaux élevés d’aKG modifient l'équilibre vers un phénotype Th1.En conclusion, les données générées au cours de ma thèse devraient permettre le développement de stratégies permettant de sélectionner des cellules T ayant des propriétés effectrices anti-tumorales améliorées. / T cells are stimulated upon interaction with their cognate antigen. While much research has focused on the role of antigen presenting cells (APC) and cytokines as important components of the T cell microenvironment, recent data highlight the importance of the metabolic environment in sustaining the energetic and biosynthetic demands that are induced upon antigen stimulation. The subsequent metabolic reprogramming of the T cell is conditioned by the nutrient composition and oxygen levels. Notably, this environment can be altered by pathological conditions such as tumors and data from our group, as well as others, have shown that the competition of T cells and tumor cells for limiting amounts of nutrients has a negative impact on T cells, inhibiting their anti-tumor effector functions. This effect is due, at least in part, to the distinct metabolic profiles of T lymphocyte subsets; T effector cells (including Th1 cells) are highly glycolytic while suppressive Foxp3+ regulatory T cells (Tregs) display a mixed metabolism with increased levels of lipid oxidation. It is therefore important to determine how changes in the metabolic programming of anti-tumor T cells impacts on their persistence and function. Indeed, in the context of my PhD research, I found that high levels of the glucose transporter Glut1 was associated with a significantly increased level of IFNγ secretion by both CD4 and CD8 T cells. Furthermore, there was a bias of CD8 over CD4 lymphocytes in the Glut1-hi T cell subset. These data point to the importance of metabolic alterations in the fate and effector function of T lymphocytes and during my PhD, I focused on elucidating the metabolic parameters that regulate effector and regulatory T cells, with the goal of improving the efficacy of anti-tumor T cells. In this context, I contributed to initial studies from our group, revealing a critical role for extracellular nutrient availability in terminal CD4+ T cell differentiation. Activation of naïve CD4+ T cells under conditions of glutamine deprivation caused them to differentiate into induced Treg (iTreg). Moreover, the skewing of glutamine-deprived naive CD4+ T cells to a Foxp3+ fate occurred even under Th1-polarizing conditions, blocking terminal Th1 differentiation. Under glutamine-deprived conditions, we found that alpha-ketoglutarate (αKG), a glutamine-derived metabolite, rescued Th1 differentiation. I then evaluated the impact of aKG under glutamine-replete conditions in the Th1/iTreg differentiation processes. My studies showed that, under Th1-polarizing conditions, aKG markedly enhanced naïve CD4+ T cell differentiation into Th1 cells and increased IFNg secretion. Moreover, under Treg-polarizing conditions, αKG decreased Foxp3 expression and increased the secretion of inflammatory cytokines such as IFNg, GM-CSF and IL-17. Notably, the aKG-mediated alteration in T cell differentiation was associated with an augmented oxidative phosphorylation (OXPHOS), and inhibiting the citric acid cycle and the mitochondrial complex II with malonate, an inhibitor of succinate dehydrogenase (SDH), alleviated the αKG-mediated block in Treg differentiation. Impressively, these aKG-mediated changes in the Th1/Treg balance were maintained in vivo, promoting a Th1-like profile in T cells expressing an anti-tumor chimeric antigen receptor (CAR) in tumor-bearing mice. Thus, our data show that low intracellular aKG content, caused by limited external glutamine availability, imposes a Treg phenotype while high aKG levels shift the balance towards a Th1 phenotype.Altogether, the data generated during my PhD will promote the development of metabolic strategies aimed at modulating T cell function and foster the design of nutrient transporter-based approaches that can be used to select T lymphocytes with enhanced anti-tumor effector properties.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第25333号 / 農博第2599号 / 新制||農||1106(附属図書館) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 矢﨑 一史, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Pseudomonas aeruginosa is a Gram-negative, metabolically versatile, opportunistic pathogen that exhibits a multitude of virulence factors, and is extraordinarily resistant to a gamut of clinically significant antibiotics. This ability is in part mediated by two-component systems (TCS) that play a crucial role in regulating virulence mechanisms, metabolism and antibiotic resistance. Our sequence analysis of the P. aeruginosa PAO1 genome revealed the presence of two open reading frames, mifS and mifR, which encodes putative TCS proteins, a histidine sensor kinase MifS and a response regulator MifR, respectively. This two-gene operon was found immediately upstream of the poxAB operon, where poxB encodes a chromosomal ß-lactamase, hinting at the role of MifSR TCS in regulating antibiotic resistance. However, loss of mifSR had no effect on the antibiotic resistance profile when compared to P. aeruginosa parent PAO1 strain. Subsequently, our phenotypic microarray data (BioLOG) and growth profile studies indicated the inability of mifSR mutants to grow in α-ketoglutarate (α-KG), a key tricarboxylic acid (TCA) cycle intermediate, as a sole carbon source. To date, very little is known about the physiology of P. aeruginosa when provided with α-KG as its sole carbon source and the role of MifS and MifR TCS in virulence. Importantly, in the recent years, α-KG has gained notoriety for its newly identified role as a signaling molecule in addition to its conventional role in metabolism. This led us to hypothesize that MifSR TCS is involved in α-KG utilization and virulence in P. aeruginosa. Using mifS, mifR and mifSR clean in-frame deletion strains, our study demonstrates that the MifSR TCS modulates the expression P. aeruginosa kgtP (PA5530) and pcaT (PA0229) genes encoding putative α-KG permeases. In addition, our study shows that the MifSR-regulation of these transporters requires functional sigma factor RpoN (σ54). Loss of mifSR in the presence of α-KG, resulted in differential regulation of P. aeruginosa key virulence determinants including biofilm formation, motility, cell cytoxicity and the production of pyocyanin and pyoverdine. Involvement of multiple regulators and transporters suggests the presence of an intricate circuitry in the transport of α-KG and its importance in P. aeruginosa survival. This is further supported by the α-KG-dependent MifSR regulation of multiple virulence mechanisms. Simultaneous regulation of multiple mechanisms involved in P. aeruginosa pathogenesis suggests a complex mechanism of MifSR action. Understanding the physiological cues and regulation would provide a better stratagem to fight often indomitable P. aeruginosa infections.

Islet transplantation has found its niche in diabetes treatment. It has contributed to a better quality of life and better glycemic control of patients with diabetes suffering from severe hypoglycemia that are not eligible for vascularized pancreas transplantation. Islet isolation is a technically challenging procedure. The different studies within this doctoral thesis aim to improve and standardize different steps in the isolation procedure. They are in particular looking to improve human pancreas preservation during cold storage, to optimize islet release from the exocrine tissue and to assess whether the isolated islet yield can be predicted from a biopsy. We found that pancreas preservation with pre-oxygenated perfluorodecalin (two-layer method) did not improve the ischemic tolerance of the human pancreas as compared to cold storage with the University of Wisconsin (UW) solution. Furthermore, in pancreas with long cold ischemia time (CIT) (&gt;10 hours), Histidine-Tryptophan-Ketoglutarate (HTK) had a limited preservation capacity as compared with the UW solution with respect to isolation outcome. We also found that during enzymatic pancreas digestion, Vitacyte HA was able to provide a similar islet yield and quality as Serva NB1 with less collagenase activity and shorter digestion time. We further describe the first experience with a new GMP manufactured enzyme called Liberase MTF-S for successful human islet isolation. Finally, we found that the isolated islet yield could not be predicted from a biopsy taken from the head of the pancreas concerning solely morphological parameters of the islets tissue. The improvement of pancreas preservation will allow for marginal organs with prolonged cold ischemia time to expand the donor pool. Better knowledge of how the pancreatic extracellular matrix is digested by collagenase will lead to a fast and predictable islet release from the exocrine tissue. By standardizing the isolation procedure and improving organ selection we will increase the success rate in human islet isolation, thereby making islet transplantation available for more patients.

In the budding yeast Saccharomyces cerevisiae, the evolutionarily conserved Target of Rapamycin (TOR) signaling network regulates cell growth in accordance with nutrient and stress conditions. In this work, I present evidence that the TOR complex 1 (TORC1)-interacting proteins Nnk1, Fmp48, Mks1, and Sch9 link TOR to various facets of nitrogen metabolism and mitochondrial function. The Nnk1 kinase controlled nitrogen catabolite repression-sensitive gene expression via Ure2 and Gln3, and physically interacted with the NAD+-linked glutamate dehydrogenase Gdh2 that catalyzes deamination of glutamate to alpha-ketoglutarate and ammonia. In turn, Gdh2 modulated rapamycin sensitivity, was phosphorylated in Nnk1 immune complexes in vitro, and was relocalized to a discrete cytoplasmic focus in response to NNK1 overexpression or respiratory growth. The Fmp48 kinase regulated respiratory function and mitochondrial morphology, while Mks1 linked TORC1 to the mitochondria-to-nucleus retrograde signaling pathway. The Sch9 kinase appeared to act as both an upstream regulator and downstream sensor of mitochondrial function. Loss of Sch9 conferred a respiratory growth defect, a defect in mitochondrial DNA transmission, lower mitochondrial membrane potential, and decreased levels of reactive oxygen species. Conversely, loss of mitochondrial DNA caused loss of Sch9 enrichment at the vacuolar membrane, loss of Sch9 phospho-isoforms, and small cell size suggestive of reduced Sch9 activity. Sch9 also exhibited dynamic relocalization in response to stress, including enrichment at mitochondria under conditions that have previously been shown to induce apoptosis in yeast. Taken together, this work reveals intimate connections between TORC1, nitrogen metabolism, and mitochondrial function, and has implications for the role of TOR in regulating aging, cancer, and other human diseases.

In the budding yeast Saccharomyces cerevisiae, the evolutionarily conserved Target of Rapamycin (TOR) signaling network regulates cell growth in accordance with nutrient and stress conditions. In this work, I present evidence that the TOR complex 1 (TORC1)-interacting proteins Nnk1, Fmp48, Mks1, and Sch9 link TOR to various facets of nitrogen metabolism and mitochondrial function. The Nnk1 kinase controlled nitrogen catabolite repression-sensitive gene expression via Ure2 and Gln3, and physically interacted with the NAD+-linked glutamate dehydrogenase Gdh2 that catalyzes deamination of glutamate to alpha-ketoglutarate and ammonia. In turn, Gdh2 modulated rapamycin sensitivity, was phosphorylated in Nnk1 immune complexes in vitro, and was relocalized to a discrete cytoplasmic focus in response to NNK1 overexpression or respiratory growth. The Fmp48 kinase regulated respiratory function and mitochondrial morphology, while Mks1 linked TORC1 to the mitochondria-to-nucleus retrograde signaling pathway. The Sch9 kinase appeared to act as both an upstream regulator and downstream sensor of mitochondrial function. Loss of Sch9 conferred a respiratory growth defect, a defect in mitochondrial DNA transmission, lower mitochondrial membrane potential, and decreased levels of reactive oxygen species. Conversely, loss of mitochondrial DNA caused loss of Sch9 enrichment at the vacuolar membrane, loss of Sch9 phospho-isoforms, and small cell size suggestive of reduced Sch9 activity. Sch9 also exhibited dynamic relocalization in response to stress, including enrichment at mitochondria under conditions that have previously been shown to induce apoptosis in yeast. Taken together, this work reveals intimate connections between TORC1, nitrogen metabolism, and mitochondrial function, and has implications for the role of TOR in regulating aging, cancer, and other human diseases.