Les maladies inflammatoires du système nerveux central (SNC) sont caractérisées par l'altération de la barrière hémato-encéphalique induite par les cellules immunitaires et les cellules tumorales. Il est reconnu que les macrophages peuvent induire l'inflammation en infiltrant la BHE. Lors d'un dommage ou d'une infection du SNC, les macrophages dérivés du sang sont activés. Une fois activés, ceux-ci migrent au site infecté ou endommagé et libèrent des cytokines et médiateurs inflammatoires tels que IL1, TNFα, VEGF. Ces cytokines jouent un rôle essentiel dans l'inflammation du SNC puisqu'elles induisent des chimiokines tels que la sphingosine-1-phosphate (S1P), un sphingolipide fortement exprimé dans les glioblastomes et qui joue un rôle important dans la chimiotaxie et le trafic des cellules immunitaires. Nous avons étudié l'efficacité d'une molécule dérivée de notre diète possédant des propriétés chimiopréventives et anti-inflammatoires, l'épigallocatéchine gallate (EGCG), sur la régulation transcriptionnelle des récepteurs de la S1P à divers stades de différenciation des cellules promyélomonocytaires HL-60. Nous avons d'abord différencié les cellules promyélomonocytaires HL-60 en « macrophages-like » en utilisant un promoteur tumorigène et activateur de la protéine kinase C-Phorbol 12-myristate 13-acétate (PMA). Nous avons démontré que le PMA induit l'adhésion cellulaire et augmente l'expression des transcrits S1P1, S1P2 et S1P5. Nous avons ensuite constaté que les cellules adhérentes semblaient être sensibles à la S1P en induisant la phosphorylation d'ERK, de JNK et de P38 MAPK. Cependant, l'inclusion de l'EGCG avant la différenciation par le PMA inhibe l'induction de ces trois voies MAPK par la S1P. D'autre part, un traitement par l'EGCG au cours de la différenciation, c'est-à-dire simultanément avec le PMA, affecte seulement la voie P38 MAPK. De plus, les cellules différenciées « macrophages-like » devenaient insensibles à l'EGCG. Enfin, nous démontrons que seul le récepteur S1P2, parmi les récepteurs fortement induits par le PMA, diminue lors du prétraitement par l'EGCG. Nos résultats suggèrent que l'EGCG antagonise la réponse à la S1P dans les cellules prédifférenciées via l'inhibition de la signalisation induite par le PMA. Par conséquent, une réponse réduite à la S1P pourrait abroger la migration transendothéliale des monocytes vers le SNC, et prévenir la neuroinflammation, les infections cérébrales secondaires ou certaines pathologies cérébrales conséquentes à l'infiltration de cellules immunitaires. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Neuroinflammation, S1P, EGCG, macrophages, chimiotactisme, chimioprévention

No description available.

The mechanisms of plant response to nutrient limitation and utilisation are of great interest for agricultural purposes. Phosphate is a non-renewable resource and is one of the most important nutrients required for plant growth. Recently a new family of plant protein kinases, composed of 10 members, were discovered because of their involvement in stresses and their responses to the hormone abscisic acid (ABA). In Arabidopsis, all of these SnRK2 protein kinases have been shown to be activated by drought or hyperosmostic stress, with the exception of SnRK2.9. Five members are also activated by ABA treatment. Recently SnRK2.8 was linked to metabolic processes by being down regulated in low nutrient level conditions. In the present study, SnRK2.9 was investigated and shown to play a role in metabolic pathways, but in an opposite manner. Contrarily to SnRK2.8, transcripts level of SnRK2.9 is induced in response to phosphate, nitrogen, and sulphur deprivation. Interestingly, opposite to most phosphate-starvation inducible genes, sucrose decreases SnRK2.9's transcripts level. Transgenic plants that overexpress SnRK2.9 do not appear to be affected in terms of growth. On the other hand, overexpressing antisense SnRK2.9 or mutated snrk2.9 at residue Asp-123 by conversion to Glu (D123E), showed reduced plant growth. This phenotype was more pronounced in the absence of phosphate. A T-DNA knockout line for SnRK2.9 showed a 45% decrease in root and shoot biomass compared to wild-type Arabidopsis when grown under phosphate deprivation. Similar trends were observed when the Arabidopsis gene was introduced in Medicago sativa (alfalfa) under the control of the CaMV 35S promoter. Overexpressing D123E Atsnrk2.9 had a serious inhibitory effect on growth and the plants were no longer responsive to changes in phosphate levels. In Arabidopsis, the D123E snrk2.9 overexpressors had a 66% reduction in total seed yield when grown under +Pi conditions and a 33% reduction under -Pi conditions. These Arabidopsis transgenic lines do not share similar traits to the known phosphate metabolic mutants Pho1, Pho2, and Siz1. SnRK2.9 appears to play a key role in biomass and seed production. / Thesis (Ph.D, Biology) -- Queen's University, 2007-09-26 12:35:00.626

This thesis examines the influence of nutritional phosphate (Pi) deprivation on extracellular proteins secreted by the model plant Arabidopsis thaliana. Initial studies compared the secretome of Pi-sufficient (+Pi) versus Pi-deficient (-Pi) Arabidopsis cell cultures by 2-dimensional gel electrophoresis. Mass spectrometry identified 18 different secreted proteins that were upregulated by at least 2-fold by –Pi Arabidopsis. They were predicted to function in Pi scavenging, cell wall and ROS metabolism, proteolysis, and pathogen responses. The relationship between mRNA levels and relative amounts of selected secretome proteins was assessed. The results indicate that transcriptional control is but one of many factors contributing to Arabidopsis Pi starvation responses and highlight the importance of parallel biochemical and proteomic studies of –Pi plants. Three purple acid phosphatase (APase) isoforms were fully purified from the culture media of –Pi Arabidopsis cells and identified as AtPAP12 (At2g27190) and two AtPAP26 (At5g34850) glycoforms. As each purple APase exhibited broad substrate specificities and pH-activity profiles, it is hypothesized that their combined activities facilitate Pi scavenging from soil-localized organophosphates during nutritional Pi deprivation. AtPAP26 is dual-targeted during Pi stress since an earlier report demonstrated that it is also the principal intracellular (vacuolar) APase upregulated by -Pi Arabidopsis. The results indicate that differential glycosylation influences AtPAP26’s substrate specificity and subcellular targeting. An atpap26 T-DNA insertional mutant lacking AtPAP26 transcripts and immunoreactive AtPAP26 polypeptides exhibited: (i) 9- and 5-fold lower shoot and root APase activity, respectively, which did not change in response to Pi starvation, (ii) a 40% reduction in secreted APase activity during Pi deprivation, (iii) 35 and 50% reductions in free and total Pi concentration, respectively, in shoots of –Pi plants, and (iv) impaired shoot and root development when subjected to Pi deficiency. By contrast, no deleterious influence of AtPAP26 loss of function was apparent in +Pi plants. The results establish a firm role for AtPAP26 in the acclimation of Arabidopsis to Pi deficiency. The identification and functional characterization of secreted proteins upregulated by –Pi Arabidopsis is relevant to applied efforts to engineer Pi-efficient transgenic plants, needed to minimize the input of expensive, unsustainable, and polluting Pi fertilizers in crop production. / Thesis (Ph.D, Biology) -- Queen's University, 2010-04-28 17:20:46.892

Upregulation of intracellular and secreted acid phosphatases (APases) is a universal response of orthophosphate-starved (-Pi) plants. APases hydrolize Pi from a broad spectrum of phosphomonoesters at an acidic pH. Plant APases belong to a relatively large multigene family whose specific functions in Pi metabolism are poorly understood. This study focuses on the identification and characterization of cell wall (CW) localized purple acid APases (PAPs) upregulated by -Pi Arabidopsis thaliana. Three glycosylated PAP isozymes secreted into the CW of -Pi Arabidopsis suspension cells were purified and identified by peptide mass fingerprinting using mass spectrometry (MALDI-TOF MS) and N-terminal microsequencing as AtPAP12 (At2g27190; subunit size 60-kDa), AtPAP25 (At4g36350; subunit size 55-kDa) and AtPAP26 (At5g34850; subunit size 55-kDa). Both AtPAP12 and AtPAP26 were previously shown to be upregulated and secreted by –Pi Arabidopsis to scavenge Pi from extracellular organic-P. However, the novel AtPAP25 has never been suggested to be involved in the plant Pi-starvation response. Biochemical characterization of AtPAP25 revealed a monomeric 55 kDa protein. Similar to other PAPs it was purple-in-solution and insensitive to tartrate. Glycoprofiling via LC MS/MS revealed highly complex NXS/T glycosylation motifs at Asn172, Asn367 and Asn424. I hypothesize that these motifs play a role in AtPAP25 targeting and function. Kinetic characterization revealed a broad pH optimum centered at 5.6 and inhibition of activity by several common APase inhibitors. AtPAP25 exhibited broad substrate selectivity, low Vmax, and a Km (phosphoenolpyruvate) value of 0.52 mM. Immunoblot and semi-quantitative RT-PCR transcript analysis indicated that AtPAP25 is exclusively synthesized under –Pi conditions. Deduced amino acid sequences were compared using multiple sequence alignment and phylogenetic analysis. Growth of atpap25 T-DNA insertion mutant knockout seedlings was completely arrested when transferred to a soluble Pi deficient organic-P containing soil mix, pointing to a potential regulatory function of AtPAP25 during nutritional Pi stress. Overall, this research is helping to shed light on the functional importance of specific PAP isozymes in facilitating plant acclimation to nutritional Pi deficiency. This is important because there is an urgent need to engineer Pi-efficient transgenic crops to minimize the huge input of expensive, non-renewable, and polluting Pi fertilizers in agriculture. / Thesis (Master, Biology) -- Queen's University, 2012-09-10 08:28:21.631

No description available.

Froth flotation is the most efficient method for phosphate separation, which is a physic-chemical separation process based on the difference of surface properties between the valuable minerals and unwanted gangue minerals. However, the presence of clay slimes in the slurry after grinding consumes a large amount of reagents, decreases the collision probability between bubbles and minerals, prevents phosphate particle attachment to air bubbles, and thus considerably reduces flotation recovery and concentrate grade. Georgia Pacific Chemical, LLC has recently developed novel depressants, i.e., clay binders, which are a series of low molecular weight specialty polymers to help improve phosphate flotation performance by selectively agglomerating and depressing clay particles, thus lowering their surface area and reducing the adsorption of surfactants. This thesis addresses the effects of clay binders on phosphate flotation performance and their adsorption behavior on different minerals in a sedimentary phosphate ore. Quartz Crystal Microbalance with Dissipation technique (QCM-D) was used to study adsorption characteristics of clay binders and batch flotation tests were performed under different conditions to investigate phosphate flotation performance. The experimental results have shown that clay binders significantly improved phosphate flotation selectivity and reduced the dosages of collector and sodium silicate used as dispersant in the industry.

More than one million people worldwide receive some kind of bone graft each year. Grafts are often needed following bone tumour removal or traumatic fractures to fill voids in the bone and to aid in the healing process. The most common method involves bone transplantation, in which bone tissue is taken from one site to fill the defect in another site. The procedure thus involves two surgeries, which leads to an increased risk of complications. New, synthetic graft materials that can be used to fill defects and minimise the complications associated with bone tissue harvesting are therefore necessary. The synthetic materials available today lack the inherent biological factors of bone that stimulate the bone regeneration process. Much of today’s research concerning synthetic bone graft materials aims to solve this issue and researchers have suggested several different strategies. The purpose of this thesis is to improve the performance of acidic calcium phosphate cements, which are materials used as synthetic bone grafts. By combining these cements with drugs or ion additives, local delivery could be achieved with the potential to stimulate bone formation. Two different combinations were attempted in this thesis: cement in combination with simvastatin, or cement in combination with strontium halide salts. Both simvastatin and strontium are known to positively affect bone formation. The efficacy of the cements with the additives was evaluated using different bone cell cultures. The results regarding simvastatin showed that the cement’s mechanical property was not affected upon drug loading, and that the drug was released by a diffusion-controlled mechanism. Moreover, results showed that simvastatin stimulated the bone-forming cells (osteoblasts) to produce more bone tissue, while it inhibited bone-degrading cells (osteoclasts) from degrading the cement. These findings suggest that simvastatin could aid in the bone regeneration process in the local area surrounding the cement. The main purpose of the study using strontium halide salts was to increase the cement’s X-ray contrast, which is a property used to monitor cement during injection. In addition, strontium is believed to positively affect bone cells. The X-ray contrast did increase after the addition of 10 wt% strontium bromide or strontium iodide, while the cell study results did not indicate any significant effects on the bone-forming cells. In the last section of this thesis, zebrafish were used as a model to evaluate bone formation upon treatment with degradation products from synthetic bone grafts. The zebrafish is a small organism with 70 % gene homology to humans; due to its transparency, fast development and ease of handling, it is an interesting model for high-throughput studies. Silicate, which is an ionic degradation product of many different bone substitute materials, was used as a proof-of-concept to visualise bone formation in these fish. The results showed an increased bone formation upon treatment with 0.625 μM silicate ions. The results suggest that this model could be used as a complement to bone cell culture studies in pre-clinical evaluations of the degradation products of bone substitute materials, thus helping researchers to design materials with degradation products that could stimulate bone formation.

Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1974. / Bibliography: leaves 155-164. / viii, 164 leaves ill., map

The studies described in this thesis have used principally the rat neonatal cardiomyocyte (NCM) model to investigate previously unresolved questions regarding inositol phosphate signalling in the heart. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) is known to be an arrhythmogenic molecule in the setting of cardiacischaemia and subsequent reperfusion, but the mechanisms responsible for its enhanced generation in pathological circumstances, as well as those suppressing its generation during phospholipase C (PLC)-coupled receptor stimulation under physiological conditions, have not been characterised. [3H]Inositol-labelling in combination with anion-exchange high performance liquid chromatography (HPLC)was used to gain an accurate picture of the changes in various [3H]InsP isomers induced by PLC stimulation.