Phosphoryl transfers are essential chemical reactions in key life processes, including energy production, signal transduction and protein synthesis. They are known for having extremely low reaction rates in aqueous solution, reaching the scale of millions of years. In order to make life possible, enzymes that catalyse phosphoryl transfer, phosphoryl transferases, have evolved to be tremendously proficient catalysts, increasing reaction rates to the millisecond timescale. Due to the nature of the electronic structure of phosphorus atoms, understanding how hydrolysis of phosphate esters occurs is a complex task. Experimental studies on the hydrolysis of phosphate monoesters with acidic leaving groups suggest a concerted mechanism with a loose, metaphosphate-like transition state. Theoretical studies have suggested two possible concerted pathways, either with loose or tight transition state geometries, plus the possibility of a stepwise mechanism with the formation of a phosphorane intermediate. Different pathways were shown to be energetically preferable depending on the acidity of the leaving group. Here we performed computational studies to revisit how this mechanistic shift occurs along a series of aryl phosphate monoesters, suggesting possible factors leading to such change. The fact that distinct pathways can occur in solution could mean that the same is possible for an enzyme active site. We performed simulations on the catalytic activity of β-phosphoglucomutase, suggesting that it is possible for two mechanisms to occur at the same time for the phosphoryl transfer. Curiously, several phosphoryl transferases were shown to be able to catalyse not only phosphate ester hydrolysis, but also the cleavage of other compounds. We modeled the catalytic mechanism of two highly promiscuous members of the alkaline phosphatase superfamily. Our model reproduces key experimental observables and shows that these enzymes are electrostatically flexible, employing the same set of residues to enhance the rates of different reactions, with different electrostatic contributions per residue.

Breast cancer is one of the most common malignancy diagnosed in women and is the primary cause of cancer-related deaths in women worldwide. It is a heterogeneous group of diseases that have a different response, prognosis, and clinical outcomes. Estrogen, progesterone and HER2 negative breast cancer, known as triple negative breast cancer (TNBC), does not respond to hormonal therapy. Basal-like breast cancer (BLBC) has shorter overall survival rate among other subtypes. Tumors sharing both TNBC and BLBC are considered less responsive to currently available treatment. Chemoresistance to treatment has been a challenge in cancer biology and force investigation toward developing new targeted therapies, which selectively target specific subtypes. Sphingolipid metabolites have an important physiological role in determining cell fate. Sphingolipid metabolites, ceramide, sphingosine, and sphingosine-1-phosphate (S1P), are implicated in cancer. S1P exerts its functions via extracellular and intracellular targets. S1P synthesized inside the cell is exported outside and binds to G-protein coupled receptors, the sphingosine-1-phosphate receptors 1-5 (S1PR1-5). Although the intracellular function is not well defined, its suggested intracellular S1P promotes cell apoptosis. The S1P pathway has received great attention recently due its function in cell survival and death. This effect was reported to be concentration dependent. In this research, I focused on S1P effect on nine TNBC/BLBC cell lines. I examined the in-vitro effects of S1P on apoptosis, proliferation, and cytotoxicity in triple negative/ basal-like breast cancer cell lines. Moreover, I studied the co-administration of S1P with currently used chemotherapeutic agents in these cell lines. Data show that S1P can selectively induce cell death in TNBC/BLBC cell lines at a specific concentration. In this research, I found that the mechanism of cell death following treatment with different S1P concentrations was mainly due to apoptosis. Results show that S1P leads to cell shrinkage, rounding and detachment in the nine TNBC/BLBC cell lines. S1P combination with doxorubicin and docetaxel at different concentrations shows no beneficial effect of the combination compared to the chemotherapeuitc agent alone. In some cell lines, the combination showed a protective effect. Further studies are required to determine the mechanism by which S1P induces cell apoptosis, inhibits cell growth, and demonstrates lack of responsiveness in combination studies.

An increase in degenerative bone disease in an ageing population, combined with a rise in the number of patients suffering from bone defects caused by physical trauma, makes the repair of bone an issue of growing clinical relevance. Current treatments such as autografts and allografts have major drawbacks, including donor site morbidity, limited availability, disease transmission and immune rejection. To overcome these issues synthetic bone grafts have been developed to mimic the mineral phase of bone. Given the significant roles of silicon in bone growth and development there has been great interest in introducing silicon into synthetic bone grafts to enhance their bioactivity. Calcium phosphate based silicate containing grafts have demonstrated enhanced bioactivity, improved physical properties, enhanced protein adsorption and greater bone formation, when compared to non-silicated calcium phosphates such as hydroxyapatite. However, is not clear whether the increased bone formation associated with these materials is the result of greater osteoblast activity or a rise in numbers of osteoblasts resulting from activation and differentiation of stem/ progenitor cells. To answer this question, multipotent stem cells were cultured on silicate substituted calcium phosphate (Si-CaP) and hydroxyapatite (HA). Si-CaP promoted greater cell adhesion and enhanced proliferation when compared to HA. Cells differentiated along the osteogenic lineage on both substrates as evidenced by up regulation of osteoblast specific genes and proteins. However, cells on Si-CaP showed earlier and greater gene expression of all osteoblast genes examined, and greater protein production as detected by immunohistochemistry. Integrin gene expression analysis revealed up regulation of α an d β subunits on both substrates during differentiation. Integrins α5 and β1 expression were greater on Si-CaP than on HA, suggesting preferential binding of fibronectin. The implication of these findings for tissue engineering is clear, suggesting these substrates may be utilized to control stem cell fate in vivo and in vitro without the need for osteogenic supplementation. Furthermore, the increased rate of differentiation seen on Si-CaP may enable the development of novel substrates for osteogenic differentiation of MSC, which may have significant impact in regenerative medicine.

Oleic acid (18:1), a monounsaturated fatty acid (FA), is synthesized upon desaturation of stearic acid (18:0) and this reaction is catalyzed by the plastidal enzyme stearoyl-acyl carrier protein-desaturase (SACPD). A mutation in the SSI2/FAB2 encoded SACPD lowers 18:1 levels, which correlates with induction of various resistance (R) genes and increased resistance to pathogens. Genetic and molecular studies have identified several suppressors of ssi2 which restore altered defense signaling either by normalizing 18:1 levels or by affecting function(s) of a downstream component. Characterization of one such ssi2 suppressor mutant showed that it is required downstream of low 18:1-mediated constitutive signaling and partially restores altered defense signaling in the ssi2 mutant. Molecular and genetic studies showed that the second site mutation was in the Nitric Oxide Associated (NOA) 1 gene, which is thought to participate in NO biosynthesis. Consistent with this result, ssi2 plants accumulated high levels of NO and showed an altered transcriptional profile of NO-responsive genes. Interestingly, the partial defense phenotypes observed in ssi2 noa1 plants were completely restored by an additional mutation in either of the two nitrate reductases NIA1 or NIA2. This suggested that NOA1 and NIA proteins participated in NO biosynthesis in an additive manner. Biochemical studies showed that 18:1 physically bound NOA1, in turn leading to its degradation in a protease-dependent manner. In concurrence, overexpression of NOA1 did not promote NO-derived defense signaling in wild-type plants unless 18:1 levels were lowered. Subcellular localization showed that NOA1 and the 18:1-synthesizing SSI2 were present in close proximity within the nucleoids of chloroplasts. Indeed, pathogen- or low 18:1- induced accumulation of NO was primarily detected in the chloroplasts and their nucleoids. Together, these data suggested that 18:1 levels regulate NO synthesis and thereby NO-mediated retrograde signaling between the nucleoids and the nucleus. Since cellular pools of glycerol-3-phosphate (G3P) regulate 18:1 levels, I next analyzed the relationship between G3P and 18:1. Interestingly, unlike 18:1, an increased G3P pool was associated with enhanced systemic immunity in Arabidopsis. This was consistent with G3P-mediated transcriptional reprogramming in the distal tissues. To determine mechanism(s) underlying G3P-conferred systemic immunity, I analyzed the interaction between G3P and a lipid transfer protein (LTP), DIR1. In addition, I monitored localization of DIR1 in both Arabidopsis as well as tobacco. Contrary to its predicted apoplastic localization, DIR1 localized to endoplasmic reticulum and plasmodesmata. The symplastic localization of DIR1 was confirmed using several different assays, including co-localization with plasmodesmatal-localizing protein, plasmolysis and protoplast-based assays. Translocation assays showed that G3P increased DIR1 levels and translocated DIR1 to distal tissues. Together, these results showed that G3P and DIR1 are present in the symplast and their coordinated transport into distal tissues is likely essential for systemic immunity. In conclusion, this work showed that low 18:1-mediated signaling is mediated via NO, synthesis of which is likely initiated in the plastidal nucleoids. In addition, my work shows that G3P functions as an independent signal during systemic signaling by mediating translocation of the lipid transfer protein, DIR1.

The conserved metabolites, oleic acid (18:1), a major monounsaturated fatty acid (FA), and glycerol-3-phosphate (G3P) are obligatory precursors of glycerolipid biosynthesis in plants. In Arabidopsis, the SSI2-encoded SACPD is the major isoform that contributes to 18:1 biosynthesis. Signaling induced upon reduction in oleic acid (18:1) levels not only upregulates salicylic acid (SA)-mediated responses but also inhibits jasmonic acid (JA)- inducible defenses. I examined the transcription profile of ssi2 plants and identified two transcription factors, WRKY50 and WRKY51. Although the ssi2 wrky50 and ssi2 wrky51 plants were constitutively upregulated in SA-derived signaling, they were restored in JAdependent defense signaling. Not only did these plants show JA-inducible PDF1.2 expression, but they were also restored for basal resistance to the necrotrophic pathogen, Botrytis cinerea. Overall, my results show that the WRKY50 and WRKY51 proteins mediate both SA- and low 18:1-dependent repression of JA signaling in Arabidopsis plants. My studies also show that cellular G3P levels are important for plant defense to necrotrophic pathogens. I showed that G3P levels are induced in Arabidopsis in response to the necrotrophic fungal pathogen B. cinerea. G3P-dependant induction of basal defense is not via the activities of other defense-related hormones such as SA, JA or the phytoalexin camalexin. Arabidopsis mutants unable to accumulate G3P (gly1, gli1) showed enhanced susceptibility to B. cinerea. Previous studies in our lab identified acyl-carrier protein 4 (ACP4), a component of FA and lipid biosynthesis, as an important regulator of plant systemic immunity. ACP4 mutant plants were defective in systemic acquired resistance (SAR) because they contained a defective cuticle. I further investigated the role of the plant cuticle in SAR by studying the involvement of long-chain acyl-CoA synthetases (LACS), a gene family involved in long-chain FA and cuticle biosynthesis, in SAR. In all, eight lacs mutants (lacs1, lacs2, lacs3, lacs4, lacs6, lacs7, lacs8, lacs9) were isolated and characterized. Six mutants were compromised in SAR. Together, my studies show that the various LACS isoforms contribute differentially to both cuticle formation and systemic immunity in Arabidopsis.

Platelet exocytosis is essential for hemostasis and for many of its sequelae. Platelets release numerous bioactive molecules stored in their granules enabling them to exert a wide range of effects on the vascular microenvironment. Are these granule cargo released thematically in a context-specific pattern or via a stochastic, kinetically-controlled process? My work describes platelet exocytosis using a systematic examination of platelet secretion kinetics. Platelets were stimulated for increasing times with different agonists (i.e. thrombin, PAR1-agonist, PAR4-agonist, and convulxin) and micro-ELISA arrays were used to quantify the release of 28 distinct α-granule cargo molecules. Agonist potency directly correlated with the speed and extent of release. PAR4-agonist induced slower release of fewer molecules while thrombin rapidly induced the greatest release. Cargo with opposing actions (e.g. pro- and anti-angiogenic) had similar release profiles, suggesting limited thematic response to specific agonists. From the release time-course data, rate constants were calculated and used to probe for underlying patterns. Probability density function and operator variance analyses were consistent with three classes of release events, differing in their rates. The distribution of cargo into these three classes was heterogeneous suggesting that platelet secretion is a stochastic process potentially controlled by several factors such as cargo solubility, granule shape, and/or granule-plasma membrane fusion routes. Sphingosine 1 phosphate (S1P) is a bioactive lipid that is stored in platelets. S1P is essential for embryonic development, vascular integrity, and inflammation. Platelets are an abundant source of S1P due to the absence of the enzymes that degrade it. Platelets release S1P upon stimulation. My work attempts to determine how this bioactive lipid is released from platelets. Washed platelets were stimulated with agonists for defined periods of time and the supernatant and pellet fractions were separated by centrifugation. Lipids were separated by liquid phase extraction and S1P was quantified with a triple quadrapole mass spectrometer. A carrier molecule (BSA) is required to detect release of S1P. Further, there is a dose-dependent increase in total S1P with increasing BSA. S1P release shows characteristics similar to other platelet granule cargo e.g. platelet factor IV (PF4). Platelets from Unc13-d Jinx mice and VAMP8-/- mice, which are secretion-deficient (dense granule, alpha granule and lysosome), were utilized to understand the process of S1P release. S1P release was more affected in Unc13-d Jinx mice mirroring their dense granule secretion defect. Fluorescence microscopy and sub-cellular fractionation were used to examine localization of S1P in platelets. S1P was observed to be enriched in a granule population. These studies indicate the existence of two pools of S1P, a readily extractable agranular pool, sensitive to BSA, and a granular pool that requires the secretion machinery for release. The secretion machinery of platelets in addition to being involved in the release of normal granule cargo is thus proved to be involved in the release of bioactive lipid molecules like S1P.

<p>The general trend in gear industry, today, is an increased focus on gear transmission efficiency. Gear transmission efficiency losses arise from loaded and unloaded gear contacts, seals, lubricant and bearings. One way of minimising the losses is to lower the lubricant viscosity. This will reduce the speed dependent losses. However, the load dependent losses might increase. To avoid this, the ratio between lubricant film thickness and surface roughness must be maintained, which can be fulfilled by producing smoother gear surfaces. As a starting point for this realisation process, the present manufacturing processes, the design tools and the characteristics of the gear flank interface must be further investigated and developed. This must be achieved with an emphasis on economic production.</p><p>This thesis focuses on our understanding of how different gear manufacturing methods —particularly the contribution of the running-in process—affect the surface characteristics, with the view of increasing gearbox efficiency. The thesis consists of a summary and three appended papers.</p><p>Paper A and paper B discuss the relationship between design parameters and real gear wheel surfaces manufactured with different manufacturing methods. The research hypothesis was that the contact area ratio is a descriptive parameter for the contact condition. Paper A deals with the influence of manufacturing method on the initial contact conditions and also serves as a validation of the simulation program used. The emphasis in Paper B is the changes that occur during running-in, and to correlate these changes to design requirements. Paper C approaches the influences of manganese phosphate-coating and lubricants with respect to friction and the risk of scuffing at the initial contact.</p><p>The main conclusions of this thesis are that the contact area ratio presents a descriptive measure of how surface topography influences the contact, seen at both a global (form deviation) and local (roughness) level. The surface topography caused by the manufacturing method has a significant influence on the contact area ratio. This is an important result, since neither national standards nor commercially available gear evaluation programs handle surface topography on the local scale. Shaving was found to have the highest contact area ratio, and should therefore be the best choice if deviations from case hardening could be minimised. It is also confirmed that gear-like surfaces coated with manganese phosphate have a low coefficient of friction, and raise the limiting load for scuffing failure enormously compared to the ground equivalent.</p> / QC 20100518 / KUGG / Sustainable gear transmission realization

Les recherches de rendements agricoles de plus en plus élevés conduisent à l'utilisation des engrais chimiques et des produits phytosanitaires. L'utilisation non raisonnée de ces intrants agricoles a pour conséquences la pollution des sols, des eaux de surface et des eaux souterraines. Les recherches sur l'élaboration de nouveaux types de fertilisants écologiques et le devenir des polluants organiques dans l'environnement s'avèrent importantes. C'est dans ce cadre que se situe notre travail. La première étape de ce travail a été l'extraction et la caractérisation des substances humiques naturelles. Celles-ci ont été extraites d'un sol de Lomé (AHSL, AFSL), d'un sol de Badou (AHSB, AFSB) et d'un compost (AHC, AFC) élaboré à partir de déchets biodégradables. Les caractérisations de ces substances d'une part par analyse élémentaire et dosage du carbone organique total et d'autre part, par des méthodes physico-chimiques (ultraviolet-visible, Microscopie Electronique à Balayage, Infra Rouge) ont prouvé les caractères organique et humique de ces échantillons. Dans la deuxième partie, la dissolution des phosphates naturels de Hahotoé - Kpogamé (Togo) par des acides conventionnels (minéraux et organiques) et des substances humiques naturelles a été réalisée. Les résultats obtenus ont permis de montrer que : - la dissolution de l'apatite en milieu fortement acide est due essentiellement à l'attaque des ions hydronium. C'est d'ailleurs le seul effet de dissolution des acides minéraux; - la dissolution du minerai en milieux faiblement acide et alcalin par les solutions organiques est due à la complexation des ions métalliques (Ca2+) par les groupements fonctionnels. Le mécanisme des cette dissolution a été mis en évidence par des dosages conductimétriques et potentiométriques. Le pouvoir complexant de ces solutions augmente avec le pH. Ce mécanisme de complexation des substances humiques vis-à-vis des ions métalliques en général et du calcium en particulier peut donc être utilisé pour l'élaboration de nouveaux types d'engrais à base de la matière organique biodégradable et du phosphate naturel. Parallèlement aux études de dissolution, la dernière partie de notre travail a consisté à évaluer les capacités des substances humiques à photodégrader les polluants organiques. Les résultats obtenus montrent clairement que les acides humiques utilisés sont des inducteurs efficaces qui permettent la photodégradation de polluants organiques sous excitation solaire. Cette photodégradation est due à la formation par la matière organique des espèces réactives telles que l'oxygène singulet, les radicaux hydroxyles et les états excités à caractère triplet. En application de ces résultats, une étude de la photodégradation du profenofos (O(4-bromo-2- chlorophényl), O-éthyl, S-propylphosphorothioate) qui est un polluant organochloré utilisé dans la culture cotonnière au Togo par ces substances humiques a été réalisée. Les résultats montrent effectivement la transformation du polluant. Cette transformation est ralentie en milieu désoxygéné et en présence du propan-2-ol qui piège les radicaux hydroxyle issus des substances humiques. Ceci montre que ces radicaux sont à la base de la photodégradation du polluant organique. L'identification des intermédiaires dans les conditions diverses nous a conduit à proposer des structures des produits issus de la phototransformation.

Le polymère Nafion est l'électrolyte de référence de la pile à combustible. Lorsqu'il est hydraté, il présente une conductivité élevée (10^-2 S.cm^-1). Néanmoins cette conductivité chute à faible taux d'hydratation. L'ajout d'un composé hygroscopique dans la membrane, tel le phosphate de zirconium (ZrP), a été proposé dans la littérature pour répondre à ce problème. <br /><br />La conductivité est le fait de la structure du matériau, des mécanismes de diffusion du proton, et des interactions eau-polymère au sein de la membrane. Nous nous sommes intéressés à cette dernière partie du problème. Nous avons étudié les mécanismes d'hydratation à l'échelle moléculaire pour les membranes Nafion puis Nafion-ZrP par technique de spectrométrie infrarouge. Cette technique peut être couplée à une étude par dynamique moléculaire que nous avons initié sur le polymère Nafion. Les spectres infrarouges du Nafion et du Nafion-ZrP ont été mesurés sur toute la gamme d'hydratation.<br /><br />Les résultats obtenus font état de 5 mécanismes d'hydratation successifs pour la membrane Nafion. L'ionisation des groupes sulfoniques SO_3H est très rapide en début d'hydratation. Elle est suivie d'un éloignement des protons H^+ par rapport aux groupes sulfonates SO_3^- dont ils sont issus et d'une réorganisation du réseau de liaisons H autour de ces groupes ioniques. Enfin une eau de type « bulk » apparaît vers 40% d'hydratation. Nous avons ainsi une "photographie" de la membrane à chaque taux d'hydratation. L'ajout d'un composé inorganique ZrP n'influe pas sur les mécanismes d'hydratation. <br /><br />D'après la comparaison entre nos mécanismes et la courbe de conductivité, il est nécessaire de dissocier tous les groupes sulfoniques pour atteindre une diffusion optimale du proton, probablement assurée par le mécanisme de Grotthuss.

The mechanisms of the deterioration of cellulose acetate compounded with triphenyl phosphate were investigated. A key peak shift of 726cm⁻¹ to 718cm⁻¹ in the Raman spectrum of triphenyl phosphate (726cm-1 uncompounded) when compounded in cellulose acetate (718cm⁻¹) was tied to the action of C-O bonds in triphenyl phosphate. The molecular bonds responsible for the 726cm⁻¹ peak were identified by collecting and examining spectra of chemicals with functional groups similar to triphenyl phosphate. Initially it was hypothesized that triphenyl phosphate acts as nucleophilic catalyst of deacetylation. This mechanism was evaluated by dissolving triphenyl phosphate in solvents that served as functional group analogues of cellulose acetate. These liquid-solution systems have a faster rate of reaction and complete mixing with triphenyl phosphate compared with what is seen in cellulose acetate solid-solution systems. The results of the cellulose acetate analogue experiments did not support the hypothesis of triphenyl phosphate acting as a nucleophilic catalyst of deacetylation. The results instead support a new theory of deterioration induced by the recrystallization of triphenyl phosphate. Additionally, the prevailing theory of triphenyl phosphate induced deterioration as proposed by Shinagawa et al. in 1992 was reviewed. The experiments conducted here do not support Shinagawa's theory.