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Certaines enzymes impliquées dans le métabolisme intermédiaire sont moins spécifiques pour leur substrat que ce que l'on pense généralement. Ainsi, certaines d'entre-elles commettent des erreurs et forment de manière inappropriée des métabolites non-physiologiques, qui peuvent devenir délétères s'ils ne sont pas détru its. L'UDP-glucose pyrophosphorylase , une enzyme dont la fonction est d'activer le glucose en UDP-glucose, nécessaire pour la synthèse de glycogène, est décrite comme étant peu spécifique pour ses substrats.Le premier objectif de mon mémoire a été de tester la spécificité de l 'UDP-glucose pyrophosphorylase envers le mannose-1-phosphate. En effet, si cette enzyme forme de l'UDP­ mannose, ce métabolite non physiologique pourrait éventuellement être incorporé dans le glycogène et devenir une source de toxicité . Pour tester cette hypothèse, nous avons produit et purifié l'UDP-glucose pyrophosphorylase de souris. L'étude de sa spécificité a montré que l'enzyme recombinante de souris (ainsi que l'enzyme de S. cerevisae) ne forme pas d'UDP­ mannose, mais bien du CDP-glucose et du dTDP-glucose. Le second objectif de mon travail a consisté en la recherche d'une activité pyrophosphatase dans Je foie de rat, capable de réparer l 'erreur produite par l'UDP-glucose pyrophosphorylase, à l'aide d'un dosage radiochimique que nous avons mis au point. Nous avons pu identifier au moins deux types d'activités pyrophosphatase capables d'hydrolyser le CDP-glucose ainsi que le dTDP­ glucose. Une caractérisation plus approfondie de ces activités enzymatiques a montré qu'aucune des deux n'apparaît comme très intéressante, puisqu 'elles agissent également sur l'UDP-glucose, le produit spécifique de la réaction catalysée par l'UDP-glucose pyrophosphorylase. De plus, une d'entre elles est inhibée par plusieurs nucléotides et dépendante de la présence de Zn2 . Ses propriétés nous font penser qu'elle correspond vraisemblablement à une contamination de l'extrait par des ecto-enzymes membranaires qui ont leur site catalytique extracellulaire et non à une « enzyme de réparation » qui rendrait la synthèse de glycogène plus spécifique. L'autre activité enzymatique, qui semblait plus spécifique pour l'UDP-glucose, semble être contribuée par la galactose-1-phosphate uridylyltransferase, une enzyme clé du métabolisme du galactose très active dans le foie.Le troisième objectif de mon mémoire a été d'en savoir plus sur le rôle de NUDT14 , une UDP-glucose pyrophosphatase d'après la littérature, ainsi que sur celui de NU DT5, l 'enzyme mammalienne la plus proche de NUDT14. Nous avons montré que l'UDP-glucose n'est substrat de NUDT1 4 qu'à des pH alcalins, et qu'à pH physiologique, différents dérivés de I'ADP (normaux ou altérés) sont de bien meilleurs substrats que l'UDP-glucose. Ceci suggère que le substrat physiologique de NUDT14 est un nucléotide, peut-être non-classique ou abîmé, contenant de l'adénine, qui est métabolisé ou recyclé. Quant à NUDT5 , les deux meilleurs substrats que nous avons trouvés sont I'ADP-ribose et l'ADP-glucose. Puisque I'ADP-glucose n'est pas physiologique chez les mammifères et que nos cellules ont déjà une ADP-ribose pyrophosphatase (NUDT9) qui est très spécifique, il est aussi probable que NUDT5 ait une fonction qui soit autre que d'hydrolyser de I'ADP-ribose. En plus, nous avons utilisé le système CRISPR/CAS9 pour produire des lignées de cellules HCTI 16 déficientes en NUDT14 ou en NUDT5 qui pourront être utiles pour en savoir plus sur le rôle physiologique de ces deux protéines. It is becoming widely recognized that some enzymes involved in the intermediary metabolism are less specific than previously thought for their substrates. Indeed, some of them make mistakes and form non­ physiological metabolites that can accumulate and become toxic. The UDP-glucose pyrophosphorylase , the enzyme that converts glucose }-phosphate to UDP-glucose, required for the glycogen synthesis, is described in literature as lacking substrate specificity.The first objective of my research was to test the specificity of this enzyme for mannose 1-phosphate. Indeed, if UDP-glucose pyrophosphorylase could make UDP-mannose under physiological conditions, this non-physiological substrate might accumulate, be incorporated in glycogen and possibly become toxic for the cells. To validate this hypothesis, we produced and purified recombinant mouse UDP­ glucose pyrophosphorylase. The study of the substrate specificity for this enzyme showed that neither the mouse nor the S. cerevisae enzymes make UDP-mannose, but that they both produce small amounts of CDP-glucose and dTDP-glucose.This stimulated us to look in a rat liver extract, for a pyrophosphatase with an enzymatic activity that would be able to fix the metabolic mistake of UDP-glucose pyrophosphorylase. Using a radiochemical assay that we have developed, we identified at least two different pyrophosphatase activities hydrolyzing CDP-glucose and dTDP-glucose. However, their detailed characterization has shown that neither of them appeared to be very interesting, because they can also act on the UDP-glucose, the main product of the reaction catalysed by UDP-glucose pyrophosphorylase. Moreover, one of the enzymatic activities was inhibited by various nucleotides and was dependent on the presence of Zn2 . Its properties suggest that it probably corresponds to a contamination of the extract with ecto-enzymes that got detached from the membrane and which have an extracellular catalytic site. Therefore this activity does not correspond to a "repair enzyme" that would make the synthesis of the glycogen more specific. The other enzymatic activity, which seemed more specific for the UDP-glucose, appears to be contributed by galactose !­ phosphate uridyltransferase , an enzyme of galactose metabolism that is very active in the liver.In the third part of my work we addressed the function of NUDT14 , a pyrophosphatase from the NUDIX family of proteins that was previously described as an UDP-glucose pyrophosphorylase, as well as the role of NUDT5 that is its closest mammalian homologue. We could show that UDP-glucose is only a substrate of NUDT14 at alkaline pH and that at physiological pH various ADP-derivatives (normal or altered) are better substrates than the UDP-glucose. This suggests that the physiological substrate of NUDT14 is a nucleotide, but maybe a non-classical one or a damaged one, containing an adenine, which would need to be metabolized or recycled. Regarding NUDT5, the two bests substrates that we found are ADP-ribose and ADP-glucose, that have already been described. However, since ADP-glucose is not physiological in mammalian cells, and that they already appear to have a very specific ADP-ribose pyrophosphatase (NUDT9) it is possible that NUDT5 has another function different from the hydrolysis the ADP-ribose. In this context, we have used the CR1SPR/CAS9 system to produce HCTl 16 cell lines that are deficient in NUDTI 4 or in NUDT5, which might be useful to learn more about the physiological role of these proteins.

UTP-Glucose-1-Phosphate Uridylyltransferase --- Nudt14 protein, mouse --- Uridine Diphosphate Sugars

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Obesity and its co-morbidities, including atherosclerosis, insulin resistance and diabetes, are a world-wide epidemic. Inflammatory immune responses in metabolic tissues have emerged as a universal feature of these metabolic disorders. While initial work highlighted the contribution of macrophages to tissue inflammation and insulin resistance, recent studies demonstrate that cells of the adaptive immune compartment, including T and B lymphocytes and dendritic cells also participate in obesity-induced pathogenesis of these conditions. However, the molecular and cellular pathways by which the innate and adaptive branches of immunity control tissue and systemic metabolism remain poorly understood. To engage in growth and activation, cells need to increase their biomass and replicate their genome. This process presents a substantial bioenergetic challenge: growing and activated cells must increase ATP production and acquire or synthesize raw materials, including lipids, proteins and nucleic acids. To do so, they actively reprogram their intracellular metabolism from catabolic mitochondrial oxidative phosphorylation to glycolysis and other anabolic pathways. This metabolic reprogramming is under the control of specific signal transduction pathways whose underlying molecular mechanisms and relevance to physiology and disease are subject of considerable current interest and under intense study. Recent reports have elucidated the physiological role of metabolic reprogramming in macrophage and T cell activation and differentiation, B- and dendritic cell biology, as well as in the crosstalk of immune cells with endothelial and stem cells. It is also becoming increasingly evident that alterations of metabolic pathways play a major role in the pathogenesis of chronic inflammatory disorders. Due to the scientific distance between immunologists and experts in metabolism (e.g., clinicians and biochemists), however, there has been limited cross-talk between these communities. This collection of articles aims at promoting such cross-talk and accelerating discoveries in the emerging field of immunometabolism.

metabolic syndrome --- Obesity --- Immunometabolism --- TCA cycle --- Glycolysis --- macrophage --- Inflammation --- fatty acid oxidation --- Pentose Phosphate Pathway --- lymphocyte

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This book is based on a review of about 2,000 carefully selected articles about hydroxyapatite (HA) materials from about 150 peer-review journals in both engineering and medical areas and presents itself as a typical example of evidence-based learning (EBL). Evidence-based literature reviews can provide foundation skills in research-oriented bibliographic inquiry, with an emphasis on such review and synthesis of applicable literature. Information is gathered by surveying a broad array of multidisciplinary research publications written by scholars and researchers. HA is a very unique material which has been employed equally in both engineering and medical and dental fields. In addition, the name "apatite" comes from the Greek word áðáôù, which means to deceive. What is actually happening inside the apatite crystal structure is based on the unique characteristics of ion exchangeability. Because of this, versatility of HA has been recognized in wide ranges, including bone-grafting substitutes, various ways to fabricate HAs, HA-based coating materials, HA-based biocomposites, scaffold materials, and drug-delivery systems. This book covers all these interesting areas involved in HA materials science and technology.

Hydroxyapatite. --- Calcium phosphate hydroxide --- Hydroxylapatite --- Apatite --- animal tests --- biomimetic materials --- biowaste-origin HA --- biphasic biocomposites --- bone-graft substitute materials --- clinical reports --- crystallinity --- drug-delivery systems --- elemental substitutions --- hydroxyapatite coating materials --- hydroxyapatite-based biocomposites --- scaffolds materials and structures --- synthesis