Ce mémoire porte sur l'élaboration et la caractérisation d'un matériau composite chitosane/phosphate de calcium destiné à une utilisation dans le domaine de la substitution osseuse. Le procédé d'atomisation-séchage a été choisi pour élaborer ce composite sous forme d'une poudre susceptible d'être transformée en revêtement ou objet 3D. Une étude préliminaire a permis d'appréhender les mécanismes de synthèse du phosphate de calcium, la phase minérale, par atomisation-séchage. Une suspension de particules d'hydroxyapatite stœchiométrique ou d'apatite biomimétique dispersées dans une solution acide mène systématiquement à l'apparition d'une phase amorphe. La proportion de cette phase amorphe dans la poudre atomisée-séchée est dépendante de la taille et de la cristallinité du matériau d'origine. L'atomisation séchage d'une solution acide contenant les ions précurseurs de calcium et phosphate mène à la formation d'une phase principalement amorphe. Cette poudre a pu être décrite à différentes échelles : à une échelle de l'ordre du nanomètre apparaissent des clusters, à une échelle de l'ordre de la centaine de nanomètres des agrégats sphériques de clusters organisés en chapelet tortueux et imbriqués sont identifiés et enfin à une échelle micrométrique des agglomérats de nanoparticules ont été mises en évidence. Le phosphate de calcium ainsi synthétisé possède un rapport molaire Ca/P proche de 1.3. Au-delà de ce rapport dans la solution à atomiser, de l'acétate de calcium (utilisé ici comme précurseur) recristallise dans la poudre. Pour élaborer la poudre composite, le polymère a été solubilisé et ajouté d'abord dans une suspension acide d'hydroxyapatite avant atomisation. Cette première stratégie mène à la formation d'un composite qui présente de fortes inhomogénéités de répartition des phases organiques et minérales. Pour limiter ce problème lié à la distribution de tailles de grains de la phase minérale, une seconde stratégie a été développée. Une solution de polymère contenant des précurseurs de phosphate de calcium a été préparée pour favoriser l'association à l'échelle nanométrique des deux phases. Après atomisation-séchage, un matériau composite présentant une très bonne dispersion de la phase minérale dans la matrice organique est synthétisé. La structuration de la phase minérale est modifiée par la présence du polymère. Cette modification se traduit par une diminution de la fraction volumique des clusters et, à l'échelle supérieure, la phase minérale n'est plus présente sous forme de chapelet mais en particules sphériques isolées. Par ailleurs, une interaction chimique est envisagée en raison des liaisons de type hydrogène, ioniques ou de coordinations possibles entre les deux phases. La présence du polymère inhibe également la formation de l'acétate de calcium cristallin en favorisant la formation d'un sel d'acétate de chitosane. Deux techniques de mises en forme ont été étudiées (MAPLE, pour l'élaboration de revêtements minces et l'impression 3D de pâte pour l'obtention d'objet massif) et ont permis de mettre en avant le potentiel de transformation de la poudre préparée par atomisation séchage. Les études biologiques faites sur le revêtement ont de plus permis de démontrer les propriétés antibactériennes du matériau utilisé. / This thesis deals with the development and characterization of a chitosan/calcium phosphate composite material for use in the field of bone substitution. The spray-drying method was chosen to develop this composite in the form of a powder that could be transformed into a coating or 3D object. A preliminary study made it possible to understand the mechanisms of synthesis of calcium phosphate, the mineral phase, by spray-drying. A suspension of stoichiometric hydroxyapatite particles or biomimetic apatite dispersed in an acidic solution systematically leads to the appearance of an amorphous phase. The proportion of this amorphous phase in the spray-dried powder is dependent on the size and crystallinity of the original material. The spray drying of an acidic solution containing the precursor ions of calcium and phosphate leads to the formation of a mainly amorphous phase. This powder could be described at different scales: on a scale of about one nanometer appear clusters, on a scale of about one hundred nanometers spherical aggregates of clusters organized into tortuous and nested chaplets are identified and finally on a micrometric scale, agglomerates of nanoparticles were highlighted. The calcium phosphate thus synthesized has a molar ratio Ca/P close to 1.3. Beyond this ratio in the solution to be atomized, calcium acetate (used here as a precursor) recrystallizes in the powder. To develop the composite powder, the polymer was solubilized and added first into an acid suspension of hydroxyapatite before atomization. This first strategy leads to the formation of a composite that has strong in homogeneities in the distribution of organic and inorganic phases. To limit this problem related to the grain size distribution of the mineral phase, a second strategy has been developed. A polymer solution containing calcium phosphate precursors has been prepared to promote nanoscale association of the two phases. After spray-drying, a composite material having a very good dispersion of the mineral phase in the organic matrix is synthesized. The structuring of the mineral phase is modified by the presence of the polymer. This modification results in a reduction of the volume fraction of the clusters and, on the larger scale, the mineral phase is no longer present in the form of a string but in isolated spherical particles. Moreover, a chemical interaction is envisaged because of the hydrogen, ionic or possible coordination bonds between the two phases. The presence of the polymer also inhibits the formation of crystalline calcium acetate by promoting the formation of a salt of chitosan acetate. Two shaping techniques were studied (MAPLE, for the elaboration of thin coatings and the 3D printing of dough for obtaining massive objects) and made it possible to highlight the transformation potential of the prepared powder by spray drying. The biological studies made on the coating have also demonstrated the antibacterial properties of the material used.

Le récepteur cation-indépendant du mannose-6-phosphate (CI-RM6P) est une glycoprotéine transmembranaire impliquée dans de nombreux processus biologiques comme le transport des enzymes lysosomales vers les lysosomes et aussi dans le phénomène connu comme l’angiogenèse. Les analogues du M6P se sont avérés être des effecteurs de l’angiogenèse tumorale. La synthèse des nouveaux dérives bidentés, fonctionnalisés avec des analogues du M6P, ouvre la voie à une nouvelle méthode pour isoler le CI-RM6P.Ces “antennes biologiques” seront ainsi utilisées pour étudier leurs affinités vis-à-vis du CI-RM6P. En perspective, une nouvelle classe de dérivés de Sepharose, fonctionnalisée par nos ligands bidentés, vont être générée pour leurs emplois dans les traditionnels techniques de purifications des protéines. / The cation-indipendent mannose-6-phosphate receptor (CI-MPR) is a trans membrane glycoprotein implicated in numerous biological processes such as the transporting of the lysosomal enzymes to the lysosomes and in the phenomenon known as angiogenesis.The analogues of M6P have proven themselves to be effectors of tumour angiogenesis.The synthesis new bidentates derivatives, functionalised with analogues derivatives of M6P, opens the way to a new method to isolate the CI-MPR.These “biological antennae” will be used to study binding affinity with the receptor CI-MPR. In prospective, a new class of Sepharose derivatives, functionalised with ours bidentates ligands, will be generated to be used in the traditional technique of purifying proteins.

The wastewater treatment process has developed with the primary goals of protecting receiving water ecosystems and human health. Over time, there have been continuous innovations in process efficiencies, energy recovery, and nutrient removal. Wastewater offers opportunity for recovery of resources of various economic values, and recent research aims at process innovation to optimize resource recovery while still achieving the primary goals of the treatment process. The objective of this study is to assess the logistical and economic feasibility of recovery of nitrogen and phosphorus at two municipal treatment plants in the Tampa Bay area, one employing aerobic digestion and the other anaerobic digestion. The study is conducted using literature review of applicable processes, mass balance on the fate of nutrients (N and P) through the treatment plants and special attention to sludge handling. Based on the whole-plant mass balance conducted at the facilities, it is estimated that over 80% of the nutrient influent is routed to the solids handling side of the plant, warranting special attention to this area for nutrient recovery. Sludge digested through anaerobic and anaerobic processes have distinctly different characteristics and opportunities for resource recovery are specific to each process. Mass balances for nitrogen in the anaerobic digestion process show a high concentration of dissolved ammonia. The feasibility of struvite precipitation by addition of phosphate and magnesium compounds is evaluated through batch reaction using anaerobic sludge filtrate. Aerobic sludge contains most of the nutrient resources in the solid phase, ready for recovery if handled properly. Phosphorus release is a potential concern and specific phosphorus release rates are evaluated for a municipal scale aerobic digester.

Summary: A process is under investigation for the manufacture of dicalcium phosphate by the hydrolosis of concentrated superphosphate containing recycled monocalcium phosphate. The hydrolysis also results in the formation of an aqueous solution of monocalcium phosphate and free phosphoric acid. The phases are separated, followed by washing and drying of the solid dicalcium phosphate. The wash water is used in the hydrolyzer. The solution is returned to the superphosphate production step, where phosphate rock and additional phosphoric acid are added, and where water is evaporated to form the solid superphosphate. Pilot plant results have indicated that filtration rates vary widely under almost identical mixing conditions, presumably due to variations in particle size ranges. It was thought worthwhile, therefore, to study the effects of different mixing variables on the relative particle sizes as indicated by the settling rates of the mixture.

La contribution des cellules souches au développement tumoral est une percée conceptuelle récente dans notre compréhension des mécanismes moléculaires et cellulaires impliqués dans la carcinogenèse. En ce sens, il est reconnu depuis quelques années qu'une sous-population de cellules souches mésenchymateuses (MSC) mobilisables en réponse à des facteurs de croissance tumoraux pourrait contribuer au développement tumoral. Les recherches rapportées dans ce mémoire nous ont permis d'étudier certains partenaires clé dans la régulation de la migration et de la survie cellulaire des MSC. L'observation préalable d'une modulation conjointe de l'expression d'une métalloprotéase matricielle de type membranaire (MT1-MMP) et du transporteur microsomal de glucose-6-phosphate (G6PT) nous a permis d'évaluer la contribution respective de ces joueurs dans la signalisation affectant la chimiotaxie des cellules souches ainsi que des cellules tumorales cérébrales. De plus, nous avons évalué l'impact de certains « mannosides » synthétisés en vue de cibler spécifiquement les fonctions de surfaces de MT1-MMP et qui pourraient être à l'origine de nouvelles approches thérapeutiques anticancéreuses affectant le recrutement des cellules souches au foyer tumoral. Finalement, l'importance de l'axe de signalisation MT1-MMP/G6PT dans la mobilisation du calcium intracellulaire en réponse à la sphingosine-1-phosphate, un lipide bioactif synthétisé par des niveaux d'expression élevés de sphingosine-kinase retrouvé au niveau tumoral, permet également de concevoir le ciblage effectif de l'un ou l'autre de ces partenaires dans la progression tumorale. L'ensemble de nos résultats permettra de mieux comprendre les phénomènes régulant la survie et le recrutement des MSC aux sites de foyers tumoraux, en plus de fournir de précieux renseignements sur un nouvel axe original de signalisation liant les fonctions de MT1-MMP à celles, inattendues, de G6PT. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Cellules souches, Cancer, MT1-MMP, G6PT, Sphingosine-1-phosphate.

Carbamoyl phosphate synthetase (CPS) from E. coli catalyzes the formation of carbamoyl phosphate, an intermediate in the biosynthesis of pyrimidine nucleotides and arginine, from glutamine, bicarbonate and two molecules of MgATP. This reaction is catalyzed by three separate active sites that are separated in space by ~100 Å. The transfer of ammonia and carbamate through the two intramolecular tunnels was investigated by molecular dynamics simulations and experimental characterization of mutations within. The presence of an unstable reaction intermediate, carboxyphosphate, was established. A method for studying the synchronization of the two active sites on the large subunit of CPS was developed. The potential of mean force (PMF) calculations along the ammonia and carbamate transfer pathways indicate a low free-energy path for the translocation of ammonia. The highest barrier for ammonia is 7.2 kcal/mol which corresponds to a narrow turning gate surrounded by the side chains of Cys-232, Ala-251, and Ala-314 in the large subunit. A blockage in the passageway was introduced by the triple mutant C232V/A251V/A314V, which was unable to synthesize carbamoyl phosphate. The release of phosphate is necessary for the injection of carbamate into the carbamate tunnel. Two mutants, A23F and G575F, were designed to block the migration of carbamate through carbamate tunnel. The mutants retained only 1.7 percent and 3.8 percent of the catalytic activity for the synthesis of carbamoyl phosphate relative to the wild-type CPS, respectively. Formate can be utilized by CPS in the absence of bicarbonate to form formyl phosphate. This intermediate was observed by 31P, 13C, and 1H NMR. For the three NMR methods a peak corresponding to formyl phosphate was observed at 2.15 ppm (31P) , 162.4 ppm (13C), and 8.39 and 7.94 ppm (1H). The rate of formation of formyl phosphate is 0.025 ± 0.005 s-1. Formamide was not detected in the presence of an ammonia source. Fluorescence anisotropy measurements on the C551A/S171C and C551A/S717C mutants provided insight into a possible mechanism of synchronization between the two active sites on the large subunit. The biggest fluorescence anisotropy change was observed at the N-terminal domain in the presence of AMPPNP and ATP.

The constitutive studies of this thesis were achieved and presented in three parts. In the first part, the effects of solid state synthesis process parameters and the impurity content of primary calcium precursor on the cement-type hydration efficiency for the conversion of &alpha / -tricalcium phosphate (Ca3(PO4)2 or &alpha / -TCP) into hydroxyapatite (Ca10-xHPO4(PO4)6-x(OH)2-x x = 0&ndash / 1, or HAp) have been investigated (at 37&deg / C). &alpha / -TCP was synthesized by thermal processing of stoichiometric amounts of calcium carbonate (CaCO3) and monetite (CaHPO4) at 1150&ndash / 1350&deg / C for 2 h. Three commercial grade CaCO3 powders of different purity were used as starting materials for the synthesis process and the resultant &alpha / -TCP products for all synthesis routes were compared in terms of the material properties and their reactivities. In the second part of the studies, &alpha / -TCP and chitosan fiber (CF) composites were prepared as injectable bone cement systems which have a potential to degrade in time to be replaced by the natural bone tissue. &alpha / -TCP/CF composites were prepared in different compositions and the effect of CF addition on cement properties were examined by mechanical and injectability tests as well as microstructural and phase analysis studies. In the third part of the studies, metal chelating property of CFs was used on development of controlled zinc release systems that can be applied in local zinc deficiency therapies of bone tissue. For this purpose, CF scaffolds were prepared by wet-spinning technique and appropriate amount of zinc was loaded to these scaffolds in regard to the zinc content of a healthy human bone tissue. Zinc release studies were performed on calcium phosphate (CaP) covered and non-covered CF scaffolds and zinc ion concentrations of the release solutions were determined by ICP-MS.

Summary: A process is under investigation for the manufacture of dicalcium phosphate by the hydrolosis of concentrated superphosphate containing recycled monocalcium phosphate. The hydrolysis also results in the formation of an aqueous solution of monocalcium phosphate and free phosphoric acid. The phases are separated, followed by washing and drying of the solid dicalcium phosphate. The wash water is used in the hydrolyzer. The solution is returned to the superphosphate production step, where phosphate rock and additional phosphoric acid are added, and where water is evaporated to form the solid superphosphate.Pilot plant results have indicated that filtration rates vary widely under almost identical mixing conditions, presumably due to variations in particle size ranges. It was thought worthwhile, therefore, to study the effects of different mixing variables on the relative particle sizes as indicated by the settling rates of the mixture.

Thesis (M.S.)--Ohio State University, 2009. / Title from first page of PDF file. Includes vita. Includes bibliographical references (p. 35-41).

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