Si la GAPDH est une enzyme bien caractérisée sur le plan biochimique et structural, la contribution de ses deux sites de reconnaissance anionique lors de la catalyse reste incomprise et nécessitait la détermination des structures de l'intermédiaire thioacylenzyme et du complexe enzyme-produit. Ce manuscrit présente la mise au point des conditions de cristallisation et d'accumulation de l'intermédiaire thioacylenzyme (suivi par microspectrophotométrie) et du complexe enzyme-produit, la résolution et l'analyse des structures cristallographiques correspondantes ainsi que les implications pour le mécanisme catalytique. Les résultats obtenus mettent notamment en évidence un mouvement de "va-et-vient" du substrat au cours de la catalyse entre les deux sites de reconnaissance anionique qui est lié à l'étape d'échange du cofacteur. Bien que structuralement proche des GAPDH, l’E4PDH est une enzyme pour laquelle peu de données sont disponibles. De nombreuses questions restent donc posées concernant notamment les déterminants de l'affinité pour le cofacteur, la nature du site de reconnaissance anionique, ou encore le mécanisme d'activation de la molécule d'eau nécessaire à une étape d'hydrolyse efficace. Sont présentées dans ce manuscrit trois structures cristallographiques de l'E4PDH d'E. coli, sous forme apoenzyme, en présence de phosphate ou en complexe avec un analogue de cofacteur. L'analyse de ces structures a notamment permis de caractériser l'unique site de reconnaissance anionique de l'enzyme, de proposer des hypothèses quant à la faible affinité de l'enzyme pour le cofacteur, et d'identifier un candidat possible pour l'activation de la molécule d'eau hydrolytique. / Even if GAPDH is well characterized from a biochemical and structural point of view, the contribution of its two anion recognition sites to catalysis is still matter of debate. This work presents the crystallization, the strategy for the accumulation of the thioacylenzyme intermediate and for the obtaining of the enzyme-product complex, the resolution and analysis of the corresponding crystallographic structures and the implications in terms of reaction mechanism. The results mainly shed light on the existence of a flip-flop movement of the substrate between the two anion recognition sites during catalysis which is related to the cofactor exchange step. Although structurally related to GAPDHs, the E4PDH is an enzyme fairly less characterized. Many interrogations thus remain on the determinants of cofactor affinity, on the nature of the anion recognition site or on the mechanism of activation of the water molecule that is needed for an efficient hydrolysis step. This dissertation presents the resolution of three crystallographic structures of the E4PDH from E. coli, under the apoenzyme form, in the presence of phosphate or in complex with a cofactor analog. The analysis f these structures allows the characterization of the unique anion recognition site of this enzyme, to propose structural hypotheses to explain the low affinity of this enzyme for its cofactor and also to identify possible candidates for the activation of the nucleophilic water molecule.

Dietary phosphate is essential for normal fracture healing and bone growth. Previous studies have established that mice given a phosphate deficient diet after a fracture demonstrate delayed cartilage maturation and callus mineralization, as well as changes in gene expression consistent with oxidative phosphorylation dysfunction. This study was undertaken in order to examine the role of inorganic and organic phosphate availability on chondrocyte differentiation and mineralization, and to define the relationship between these processes and changes in chondrocyte metabolic function. ATDC5 murine chondroprogenitor cell line, which has been shown to undergo in vitro differentiation and extracellular matrix mineralization, was cultured under both differentiating and non-differentiating media conditions under conditions in 1mM -0.25mM sodium phosphate monobasic (inorganic phosphate) in the presence or absence of 4mM β-glycerol phosphate (organic phosphate). In the first series of studies, overall cell growth (total DNA and protein contents), mineralization (calcium accumulation), and cell-normalized oxidative metabolism (basal respiration, maximal respiration, ATP turnover, spare capacity, proton leak, and non-mitochondrial respiration rates) were measured over a 28 day time course in cultures grown in differentiating (ascorbic acid, insulin-transferrin-selenium, and β-glycerol phosphate) conditions in 1mM phosphate. These studies found that when the cells were induced to differentiate, there was a measurable increase in protein content while DNA content decreased by 30%, indicating a fraction of the cells underwent cell death. Differentiation was further associated with an overall two-fold increase in oxidative respiration. Next we assessed how differentiation, the promotion of matrix mineralization, and inorganic phosphate availability affected oxidative respiration. When differentiation was not induced with ascorbic acid and β-glycerol phosphate, there was no over growth in the cultures nor any change in total extracellular matrix mineralization or oxidative respiration. In the absence only of β-glycerol phosphate, differentiation proceeded but matrix mineralization did not occur. However, overall protein content and oxidative respiration were statistically two- and 1.5-fold higher, respectively, independent of the inorganic phosphate contents of the growth media. These results suggest that both differentiation and overall protein accumulation are strongly associated with increased oxidative metabolism while mineralization of the matrix decreased oxidative function. Only at the lowest phosphate levels were changes in basal oxidative function observed. These results are consistent with previous in vivo findings suggesting that diminished expression of mitochondrial associated genes in callus tissues from hypophosphatemic mice were associated with an overall decrease in chondrocyte differentiation.

No description available.

En raison de l’augmentation de l'espérance de vie, le nombre de personnes âgées et par conséquent le taux de maladies chroniques augmente de plus en plus nécessitant le développement de nouveaux biomatériaux permettant la réparation osseuse. Le but de cette étude était d'évaluer le comportement in vitro de cellules ostéoprogénitrices cultivées sur des pastilles de phosphate tricalcique dopées (β-TCMP, Mg/Ca = 0,15) ou non (β-TCP) avec du magnésium dans un environnement statique et dynamique (débit de 0,3 mL/min), ainsi que la réponse de macrophages humains après contact avec des extraits des matériaux (ISO10993-12). Les pastilles de β-TCMP et β-TCP ont été obtenues par frittage respectivement d’une apatite calcium déficiente en calcium dopée au magnésium et de TCP (Merck). Les diffractogrammes et les spectres infrarouge ont confirmé la production de β-TCP et de β-TCMP, ratio Mg/Ca = 0,14. Des cellules osteoprogénitrices STRO+1A ont été cultivées sur des pastilles pendant 21 jours et leur comportement de prolifération et de différenciation cellulaire ont été vérifiés. Aucune différence entre les deux pastilles n’a été observée concernant le nombre de cellules après 21 jours de culture, ni sous condition statique ni dynamique. Cependant, il semble que l'environnement dynamique accélère la différenciation ostéoblastique. D'une façon générale, le β-TCMP n'a pas modifié la réponse des macrophages (le profil des cytokines) activés ou non par du lipopolysaccharide bactérien, cultivés pendant 72 heures avec des extraits de biomatériaux, par rapport au β-TCP. D’après ces résultats, les deux biomatériaux semblent être prometteurs pour le traitement des pertes osseuses. / Research on bone tissue regeneration is constantly expanding due to the improvement of life expectancy with a consequent increase in the rate of chronic diseases. The aim of this work was to evaluate the in vitro behavior of STRO+1A osteoprogenitor cells cultured onto dense tablets of tricalcium phosphate doped (β-TCMP, Mg/Ca = 0.15) or not (β-TCP) with magnesium under static and dynamic (flow rate of 0.3 mL/min), as well as the response of human macrophages after contact with the granules extracts of the materials (ISO10993-12: 2007). The β-TCMP and β-TCP tablets were obtained by sintering a calcium-deficient apatite doped with magnesium and commercial TCP (Merck), respectively. The diffractograms and infrared spectra confirmed the presence of β-TCP and β-TCMP with a Mg/Ca ratio of 0.14 (plasma spectrometry). A cell density of 5x103 was inoculated onto the tablets and then STRO+1A were cultured at 37°C/5% CO2 for up to 21 days, in which the proliferation and differentiation rate were assessed. There was no difference in cell density between the materials after 21 days of culture, neither under static nor dynamic conditions; however, it seems that the dynamic environment accelerated osteoblast differentiation. In a general way, β-TCMP did not change the response of macrophages (cytokine profile), activated or not, by bacterial lipopolysaccharide, cultured for up to 72 hours with the extracts of biomaterials, when compared to β-TCP. Based on these results, both materials appear to be adequate for bone loss therapy.

Disturbances in mineral metabolism contribute to vascular calcification and mortality risk in chronic kidney disease (CKD). Serum levels of fibroblast growth factor (FGF)23, a bone derived, phosphaturic peptide, are associated with cardiovascular mortality in CKD. Membrane bound klotho(KL) is an obligate co receptor for FGF23 signaling in the kidney. To study aspects of FGF23 in mild to moderate impairment of renal function we have analyzed FGF23, estimated glomerular filtration rate (eGFR), parathyroid hormone(PTH), 1,25 (OH)2 vitamin D (1,25D), calcium and phosphate in one patient with a FGF23 producing tumor, before and after tumor removal (study 1), in 72 CKD patients with varying degree of renal dysfunction (study 2), in 9 healthy kidney donors, before and after nephrectomy (study 3). We also analyzed FGF23 (study 4), and performed genotyping of 27 single nucleotide polymorphisms (SNP) of the KL gene (study 5) in 2838 elderly Swedish men (MrOs study) and examined the association with mortality. FGF23 normalizes in 30-45 minutes after removal of a FGF23 producing tumor (study 1). 1,25D increases in hours and remains elevated months, even when the other parameters have normalized. FGF23 increase early in CKD, initially slowly, in correlation with PTH, but exponentially when hyperphosphatemia ensues (study 2). After unilateral nephrectomy (study 3) mineral homeostasis remain stable, initially due to a rise in PTH and later to an increase in FGF23. FGF23 levels are not correlated with mortality in elderly men after adjustment for eGFR, but with mortality due to cardiovascular disease, even in persons with normal eGFR (study 4). Polymorphism of the KL gene do not correlate with increased mortality risk in elderly men (study 5), but there is a modulating effect on FGF23 levels. FGF23 is of importance in maintaining phosphate homeostasis as renal function declines. It is co regulated with PTH until advanced renal dysfunction, and adjust the 1,25D to the actual GFR. FGF23 is associated with cardiovascular mortality. Further studies are needed to determine the mechanism, and if reduction of FGF23 by reducing phosphate intake may be beneficial even in persons with mild to moderate renal function.

Mastitis is the most prevalent and major cause of economic losses in dairy farms. Bovine mastitis caused by strains of <i>S. aureus</i> is a major economically important disease affecting the dairy industry worldwide. <i>S. aureus</i> is one of the most common udder pathogens that cause either clinical or sub-clinical mammary gland infections. Different treatment regimes have failed to cure <i>S. aureus</i> intramammary infections. Most mastitis vaccination strategies have focused on the enhancement of systemic humoral immunity rather than strengthening local intramammary immunity. Vaccines aimed at enhancing intramammary immunity of dairy cows against <i>S. aureus</i> mastitis have had limited success. Commercially available vaccines show various degrees of success and work in research laboratories with experimental vaccines suggest that in part, the failure of these vaccines lies in the limited antigenic repertoire contained in the vaccine formulations. Moreover, not only does variation in the antigenic composition but also presence of capsular polysaccharide in most pathogenic strains and decreased activity of immune effectors in milk affect the success of vaccines. In addition to these, the ability of <i>S. aureus</i> to attach and internalize into mammary epithelial cells, enables bacteria to escape from the effect of immunity and antibiotics by being hidden in the intracellular niche and thereby causing chronic recurrent intramammary infection. <i>S. aureus</i> also has the ability to become electron-transport-defective and to form slow-growing small colonies that are non haemolytic and less virulent. These small colony variants might hide from the immune surveillance in the intracellular area and revert to the parental strain causing chronic recurrent infections. If immunization targets antigenic molecules that are conserved throughout all pathogenic strains, even the small colony variants can be controlled since the immune system will clear the parental strain which causes lethal infection. Thus, immunization trials should focus on conserved immunogenic antigen molecules among pathogenic strains formulated with an adjuvant and delivered by a route of immunization to induce maximum stimulation of the immune system. Moreover, immunization should focus on inducing Th1 responses, which is protective against <i>S. aureus</i> mastitis. It has been reported that proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity might be used as such antigens to induce protection against parasitic and microbial infections. Previous study in our laboratory on mastitis-causing streptococci indicates that GapC proteins of <i>S. uberis</i> and <i>S. dysgalactiae</i> have potential as vaccine antigens to protect dairy cows against mastitis caused by environmental streptococci. Two conserved cell wall associated proteins with iii glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, GapB and GapC have been identified from <i>S. aureus</i> isolates from bovine intramammary infections. The overall goal of this study was to improve our understanding on intramammary immunity using the GapC and GapB proteins of <i>S. aureus</i> as model antigens for mastitis and to determine the regulation of expression of <i>gapB</i> and <i>gapC</i> genes and their roles in the pathogenesis of bovine <i>S. aureus</i> mastitis. We hypothesized that strengthening local intramammary immunity using GapB and GapC proteins of <i>S. aureus</i> as antigens will protect against bovine <i>S. aureus</i> mastitis. To test this hypothesis we took the approach of using the <i>gapB</i> and <i>gapC</i> genes and constructed plasmids encoding GapB, GapC and GapB::GapC (GapC/B) chimeric proteins. We set six objectives to test our hypothesis using these proteins to enhance the intramammary immunity. In aim 1 we constructed plasmids encoding the GapB, GapC proteins and also constructed a chimeric gene encoding the GapC and GapB proteins as a single entity (GapC/B chimera) as the basis for a multivalent vaccine. In this objective the humoral and cellular immune responses to GapC/B were compared to the responses to the individual proteins alone or in combination in C57 BL/6 mice. Our results showed that the GapC/B protein elicited strong humoral and cellular immune responses as judged by the levels of total IgG, IgG1, IgG2a, IL-4 and IFN-ã secretion and lymphocyte proliferation. These results strongly suggest the potential of this chimeric protein as a target for vaccine production to control mastitis caused by <i>S. aureus</i>. In aim 2 we continued our studies on GapC/B by testing the effects of DNA vaccination with plasmids encoding the individual gapB and gapC genes as well as the gapC/B protein gene with or without a boost with the recombinant proteins. The results showed that DNA vaccination alone was unable to elicit a significant humoral response and barely able to elicit a detectable cell-mediated response to the recombinant antigens but subsequent immunization with the proteins elicited an excellent response. In addition, we found that DNA vaccination using a plasmid encoding the GapC/B chimera followed by a boost with the same protein, although successful, is less effective than priming with plasmids encoding GapB or GapC followed by a boost with the individual antigens. In aim 3 we optimized immune responses in cows by comparing route of vaccination (subcutaneous versus intradermal), site of vaccination (locally at the area drained by the supramammary lymph node versus distantly at area drained by parotid lymph node. Our results showed that both subcutaneous and intradermal immunizations with the GapC/B protein at the area drained by the supramammary and parotid lymph nodes resulted in significantly increased serum and milk titers of total IgG, IgG1, IgG2, iv and IgA in all vaccinated groups as compared to placebo. The anti-GapC/B IgG1 serum and milk titers were significantly higher in all vaccinated group as compared to the placebo group. These results indicated that vaccination at the area drained by the supramammary lymph node resulted in better immune responses. In aim 4 we tested different formulations of the GapC/B antigen with adjuvants such as PCPP, CpG, PCPP + CpG and VSA-3. We found that the VSA-3 formulation induced the best immune responses in cows. In this objective we also monitored immune responses longitudinally over one lactation cycle to determine the duration of immune responses by measuring IgG, IgG1, IgG2, and IgA on monthly blood and milk samples. We found that the duration of immune responses was about four months. In aim 5 we tested the role of GapC in the virulence of <i>S. aureus</i> mastitis using the <i>S. aureus</i> wild type strain RN6390 and its isogenic GapC mutant strain H330. Our results from both in vitro adhesion and invasion assays on MAC- T cells and in vivo infection of ovine mammary glands showed that GapC is an important virulence factor in <i>S. aureus</i> mastitis. In aim 6 we examined the role of sar and agr loci on the expression of <i>gapC</i> and <i>gapB</i> genes by qRT- PCR using <i>S. aureus</i> RN6390 and its isogenic mutants defective in agrA, sarA and sar/agr (double mutant) at exponential and stationary phases of growth. Our results showed that both <i>gapB</i> and <i>gapC</i> expression were down regulated in the mutant strains, indicating that the expression of the <i>gapB</i> and <i>gapC</i> genes is controlled by the universal virulence gene regulators, agr and sar. We also checked the role of environmental factors such as pH, growth media, and oxygen tension on the expression of <i>gapB</i> and <i>gapC</i> using q-RT-PCR. Our results showed that the expression of <i>gapB</i> and <i>gapC</i> genes in different strains of <i>S. aureus</i> was not consistent under the above-mentioned environmental conditions.

Low phosphate fertilizer efficiency in high pH soils is primarily due to the retardation of P movement in the soil-P fertilizer reaction zone. The objective of this study was to obtain fundamental information on the influence of multi-ionic interactions on the solubility and diffusion of P in columns containing a model soil system and two soil types. The study also aimed to identify the salt combinations and factors that have the potential to enhance the solubility and movement of P in calcareous soil condition. The results showed that the interaction of NH4+ and SO42- was consistent at enhancing the water solubility and movement of P under a high soil pH condition. This effect was attributed to the combination of various mechanistic factors associated with (NH4)2SO4 compound including significant pH reduction, cation exchange reaction of NH4+ with the exchangeable Ca2+, and anionic competition of SO42- with P for precipitation with Ca2+. / October 2006

Les glioblastomes sont, de par leur caractère invasif et infiltrant, des tumeurs cérébrales extrêmement résistantes aux thérapies classiques. Nous avons étudié les propriétés anti-cancérigènes de polyphénols extraits de produits naturels, notamment l'acide chlorogénique (CHL). Le CHL est un puissant inhibiteur fonctionnel du transporteur microsomale de glucose-6-phosphate (G6PT) responsable de l'étape limitante de la conversion du glucose-6-phosphate en glucose (Glc) et en phosphate inorganique par la glucose-6-phosphase (G6Pase) lors de la glyconéogenèse et de la glycogénolyse. En émettant l'hypothèse selon laquelle le système G6Pase se localise exclusivement dans les tissus glyconéogéniques tels que le foie et les reins, nos travaux ont alors pour objectif d'élucider le rôle de G6PT dans des tissus non producteurs de Glc tels les cellules gliales, et particulièrement dans la progression tumorale des glioblastomes. Au cours de notre étude, nous avons testé l'impact de l'inhibition de G6PT soit fonctionnelle par le CHL, soit génique par un siRNA spécifique, sur différents processus impliqués dans la survie et l'invasion tumorale tel que la migration cellulaire et la dégradation de la matrice extracellulaire (MEC) par les métalloprotéases (MMPs). Expérimentalement, les mesures des niveaux d'ARNm par RT-PCR, démontrent que l'expression génique de G6PT est plus élevée dans les glioblastomes U87, que dans toute autre lignée tumorale cérébrale testée. Le CHL inhibe la sécrétion de la MMP-2, et la migration cellulaire des U87, deux pré-requis pour l'invasion tumorale. La migration induite par G6PT recombinant suite à une transfection des cellules à l'aide du cDNA codant pour ce transporteur est aussi inhibée par le CHL. De plus, le CHL inhibe la migration cellulaire et la phosphorylation de ERK induite en réponse à la sphingosine-1-phosphate, un lysophospholipide abondant dans la MEC cérébrale. Nous démontrons, de plus, que le 2-deoxy-D-Glc et le 5-thio-Glc, deux analogues non métabolisables du Glc et menant à la déplétion de l'ATP intracellulaire, inhibent la sécrétion de MMP-2. Par ailleurs, l'inhibition de G6PT par un siRNA induit une mort cellulaire par apoptose détectée par cytométrie de flux. Une surexpression de la métalloprotéase membranaire de type 1 (MT1 -MMP) conduit à une diminution de l'expression génique de G6PT. De manière globale, nos résultats suggèrent que G6PT régulerait certaines fonctions invasives des cellules cancéreuses, et pourrait avoir une implication dans la signalisation intracellulaire de ces dernières, d'où son potentiel comme nouvelle cible pour les thérapies anti-cancer. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Glucose-6-phosphase, Acide chlorogénique, Glioblastomes.

β – Tricalcium phosphate (β-TCP, Ca3(PO4)2) is a commonly used bone substitute material due to its biocompatibility and resorption. This study focused on the production of almost fully dense β-TCP ceramics with varying degrees of impurities (Ca/PO4 ratio, addition of 5% Mg). Three methods were used to produce the β-TCP ceramics, uniaxial pressing, slip-casting and isostatic pressing. In this study the isostatic pressing and sintering at 1150ºC for 20h and 15min, resulted in the densest β-TCP ceramics (97.7-99.2%). No significant differences of grain size and density could be detected between the samples produced with various compositions. These isostatically pressed samples sintered at 1150ºC were then dissolved in 0.08M aceticacid solution to simulate the in vivo resorption. It was found that the samples containing extra Mg dissolved slower. Attempts to determine the chemical composition of the grain boundaries were made without success. However, SEM observations of partly dissolved β-TCP ceramics revealed that the grain boundaries dissolved faster than the grains. The study was performed at the RMS foundation in Switzerland.

The export of nitrogen (N) and phosphorus (P) from agricultural catchments is a major problem worldwide. The export of these nutrients is largely driven by storm events, and the hydrologic response of catchments varies within and between storm events. Antecedent soil moisture and rainfall rates have both been shown to affect the discharge and nutrient export from agricultural catchments, but their relationship to nutrient export is not fully understood. Currently, there are no studies that examine the leaching of both nitrate and phosphate from soil pools under the combined influence of differences in soil moisture and rainfall rates. The objectives of this study were to examine the combined effect of antecedent soil moisture and rainfall rates on the hydrologic response of soil and the export of nitrate and phosphate from the soil. The approach used intact soil monoliths in two experiments to first characterize the hydrologic response of the soil, and secondly to assess how the hydrologic response of the soil affects the leaching of nitrate and phosphate from soil pools. Differences in antecedent soil moisture and rainfall rates influenced both the amount of discharge and the hydrologic flow paths in the soil. As was expected, antecedent soil moisture governed the depth of discharge, with more discharge (runoff ratios= 0.89 to 0.91) produced by wet soil and the least runoff produced by dry soil (runoff ratios= 0.08 to 0.14) although this was not affected by the rainfall rate. Instead, rainfall rates predominantly affected hydrologic flow paths in the soil, with preferential flow at the beginning of the leaching period under high intensity rainfall (especially in wet soil), and predominantly matrix flow occurring under low intensity rainfall. The rainfall intensity did not appear to affect discharge volume. The mass of both nitrate and phosphate exported was higher under low intensity rainfall, ranging from 11.2 to 60.1mg/mU+00B2 and 77 to 4980μg/mU+00B2, respectively and from 0.9 to 34.4mg/mU+00B2 and 18.4 to 732μg/mU+00B2, respectively under high intensity rainfall. Antecedent soil moisture was significantly positively correlated with the depth of discharge produced, which also had a significant positive relationship with the mass of nitrate and phosphate exported (Spearman’s ρ= 0.75 to 0.81, p= <0.001), with greater masses of both nutrients exported from wet soil than dry soil. Soil moisture had contrasting influences on the nitrate concentrations in leachate, where nitrate concentrations and soil moisture were negatively related under low intensity rainfall and positively related under high intensity rainfall. Concentrations of phosphate in leachate were more variable, with no clear relationship to soil moisture, discharge, rainfall rate or soil phosphate pools. Antecedent soil moisture and the rainfall rate have a combined influence on the concentration of nitrate in leachate and an influence on the mass of both nitrate and phosphate exported. Although different hydrologic flow paths (matrix, preferential) were observed under the variable antecedent conditions and rainfall rates, this did not appear to affect nutrient fluxes from soil. This may be related to available nutrient pools and distributions in the soil in the current study. Understanding of the influence of flow types on the export of soil nutrient pools requires further study in a lab and a comparison of the breakthrough of nitrate and phosphate from soil pools with that of a conservative tracer (chloride). Nutrient and tracer breakthrough could then be compared to the hydraulic conductivity of the soil and the progression of the wetting front to fully understand the flow paths occurring and their effect on nutrient leaching.