Wear of PEEK-Ti6Al4V systems under micro-abrasion and linear sliding conditions

Disserta����o de mestrado integrado em Engenharia Biom��dica

Solid polymer electrolytes based on chitosan and europium triflate

Solid polymer electrolytes (SPEs) were obtained from chitosan plasticized with glycerol and contained europium (III) trifluoromethanesulfonate salt. The transparent samples were characterized by thermal analysis (DSC and TGA), impedance spectroscopy and electron paramagnetic resonance (EPR). The sample with 55.34 wt.% of europium triflate showed the best ionic conductivity of 1.52 �� 10���6 and 7.66 �� 10���5 S cm���1 at 30��C and 80��C, respectively. The thermal analysis revealed that the degradation started at around 130���145��C and the weight loss ranged from 20 to 40%. The DSC of the samples showed no Tg, but only a large endothermic peak that was centered between 160 and 200 ��C. The EPR analysis showed a broadening of the EPR resonance lines with increasing europium contents in the chitosan membranes due to the magnetic dipole���dipole coupling and spin���spin exchange between the Eu2+ ions. Moreover, the electrolytes based on chitosan and europium triflate presented good flexibility, homogeneity, and transparency.

Ionic, paramagnetic and photophysical properties of a new biohybrid material incorporating copper perchlorate

The sol-gel method was employed in the synthesis of di-urethane cross-linked poly(���-caprolactone) (d-PCL(530)/siloxane biohybrid ormolytes incorporating copper perchlorate, (Cu(ClO4)2). The highest ionic conductivity of the d PCL(530)/siloxanenCu(ClO4)2 system is that with n = 10 (1.4 x 10-7 and 1.4 x 10-5 S cm-1, at 25 and 100 ��C, respectively). In an attempt to understand the ionic conductivity/ionic association relationship, we decide to inspect the chemical environment experienced by the Cu2+ ions in the d-PCL(530)/siloxane medium. The observed EPR spectra are typical of isolated monomeric Cu2+ ions in axially distorted sites. The molecular orbital coefficients obtained from the EPR spin Hamiltonian parameters and the optical absorption band suggests that bonding between the Cu2+ and its ligand in the ormolytes are moderately ionic. Investigation by photoluminescence spectroscopy did not evidence or allow selective excitation of transitions corresponding to complexed Cu2+ species.

Eco-friendly luminescent hybrid materials based on EuIII and LiI co-doped chitosan

Biopolymer-based materials have been of particular interest as alternatives do synthetic polymers due to their low toxicity, biodegradability and biocompatibility. Among them, chitosan is one of the most studied ones and has recently been investigated for the application as solid state polymer electrolytes. Furthermore, it can serve as a host for luminescent species such as rare earth ions, giving rise to materials with increased functionality, of particular interest for electrochemical devices. In this study, we investigate chitosan based luminescent materials doped wit Eu3+ and Li+ triflate salts from the structural, photophysical and conductivity points of view. Because the host presents a broad emission band in the blue to green, while Eu3+ emits in the red, fine tuning of emission colour and/or generation of white light is possible by optimizing composition and excitation scheme. Europium lifetimes (5D0) are in the range 270 ��� 350 ��s and quantum yields are as high as 2%. Although Li+ does not interfere with the luminescent properties, it grants ion-conducting properties to the material suggesting that a combination of both properties could be further explored in multifunctional device.

Imidazo-benzocrown ether functionalised amino acids: synthesis and fluorimetric chemosensing studies with metal cations

Comunica����o em painel - P59

The spatial analysis and speciation of uranium and thorium series radionuclides in the soil of the Cronamuck Valley, County Donegal, Ireland

This thesis details the findings of a study into the spatial distribution and speciation of 238U, 226Ra and 228Ra in the soils of the Cronamuck valley, County Donegal . The region lies on the north-eastern edge of the Barnesmore granite and has been the subject of uranium prospecting efforts in the past. The results of the project provide information on the practicability of geostatistical techniques as a means of estimating the spatial distribution of natural radionuclides and provide insight into the behaviour of these nuclides and their modes of occurrence and enrichment in an upland bog environment. The results of the geostatistical survey conducted on the area indicate that the primary control over the levels of the studied nuclides in the soil of the valley is the underlying geology. Isopleth maps of nuclide levels in the valley indicate a predominance of elevated nuclide levels in the samples drawn from the granite region, statistical analysis of the data indicating that levels of the nuclides in samples drawn from the granite are greater than levels drawn from the non-granite region by up to a factor of 4.6 for 238U and 4.9 for 226Ra. Redistribution of the nuclides occurs via drainage systems within the valley, this process being responsible for transport of nuclides away from the granite region resulting in enrichment of nuclides in soils not underlain by the granite. Distribution of the nuclides within the valley is erratic, the effect of drainage f lows on the nuclides resulting in localized enriched areas within the valley. Speciation of the nuclides within one of the enriched areas encountered in the study indicates that enrichment is as a result of saturation of the soil with drainage water containing trace amounts of radionuclides. 238U is primarily held within the labile fractions (exchangeable cat ions + easily oxidisable organics + amorphous iron oxides ) of the soil , 226Ra being associated with the non- labile fractions, most probably the resistant organic material. 228Ra displays a significant occurrence in both the labile and non- labile fractions. The ability of the soil to retain uranium appears to be affected largely by the redox status of the soil, samples drawn from oxidizing environments tending to have little or no uranium in the easily oxidisable and amorphous iron oxide fractions. This loss of uranium from oxidised soil samples is responsible for the elevated 226Ra /238U disequilibrium encountered in the enriched areas of the valley. Analysis of the data indicates that samples displaying elevated 226Ra/238U ratios also exhibit elevated 228Ra/238U ratios indicating a loss of uranium from the samples as opposed to an enrichment of 226Ra.

The Feedback Effect of Intensity of Ionizing Radiation With the Light Ions Content in Atmosphere. Paradox (the Tbilisi Type of a Smog), or Usual Phenomenon for the Strongly Pollution Cities?.

������������������������ ��������������������� ���������������������, ��������������������� ������ ��������������������������������� ��������������������������� ������������������������ ������������������������������ ��������������������������������� ������������������������������������ 2009-2010 ������������������ ��������������������������������� ������������������������������������ ��������������������������������� ������������������������ ������������������������������������ ������������������������������������ ������������������������������������ ��������������������������������������� ������ ��������������������������������� ��������������������� ��������������������� ��������������������������������� ��������������������������������� ������������������.

Expression analysis of the AtPHO1 gene family in Arabidopsis thaliana and the involvement of AtPHO1 in the regulation of stomatal movements

R��sum�� Le transfert du phosphate des racines vers les feuilles s'effectue par la voie du xyl��me. Il a ��t�� pr��c��demment d��montr�� que la prot��ine AtPHO1 ��tait indispensable au transfert du phosphate dans les vaisseaux du xyl��me des racines chez la plante mod��le Arabidopsis thaliana. Le s��quen��age et l'annotation du g��nome d'Arabidopsis ont permis d'identifier dix s��quences pr��sentant un niveau de similarit�� significatif avec le g��ne AtPHO1 et constituant une nouvelle famille de g��ne appel�� la famille de AtPHO1. Bas��e sur une ��tude mol��culaire et g��n��tique, cette th��se apporte des ��l��ments de r��ponse pour d��terminer le r��le des membres de ia famille de AtPHO1 chez Arabidopsis, inconnue �� ce jour. Dans un premier temps, une analyse bioinformatique des s��quences prot��iques des membres de la famille de AtPHO1 a r��v��l�� la pr��sence dans leur r��gion N-terminale d'un domaine nomm�� SPX. Ce dernier est conserv�� parmi de nombreuses prot��ines impliqu��es dans l'hom��ostasie du phosphate chez la levure, renfor��ant ainsi l'hypoth��se que les membres de la famille de AtPHO1 auraient comme AtPHO1 un r��le dans l'��quilibre du phosphate dans la plante. En parall��le, la localisation tissulaire de l'expression des g��nes AtPHO dans Arabidopsis a ��t�� identifi��e par l'analyse de plantes transg��niques exprimant le g��ne rapporteur uidA sous le contr��le des promoteurs respectifs des g��nes AtPHO. Un profil d'expression de chaque g��ne AtPHO au cours du d��veloppement de la plante a ��t�� obtenu. Une expression pr��dominante au niveau des tissus vasculaires des racines, des feuilles, des tiges et des fleurs a ��t�� observ��e, sugg��rant que les g��nes AtPHO pourraient avoir des fonctions redondantes au niveau du transfert de phosphate dans le cylindre vasculaire de ces diff��rents organes. Toutefois, plusieurs r��gions promotrices des g��nes AtPHO contr��lent ��galement un profil d'expression GUS non-vasculaire, indiquant un r��le putatif des g��nes AtPHO dans l'acquisition ou le recyclage de phosphate dans la plante. Dans un deuxi��me temps, l'analyse de l'expression des g��nes AtPHO durant une carence en phosphate a ��tabli que seule l'expression des g��nes AtPHO1, AtPHO1; H1 et AtPHO1; H10 est r��gul��e par cette carence. Une ��tude approfondie de leur expression en r��ponse �� des traitements affectant l'hom��ostasie du phosphate dans la plante a ensuite d��montr�� leur r��gulation par diff��rentes voies de signalisation. Ensuite, une analyse d��taill��e de la r��gulation de l'expression du g��ne AtPHO1; H1O dans des feuilles d'Arabidopsis bless��es ou d��shydrat��es a r��v��l�� que ce g��ne constitue le prem��er g��ne marqueur d'une nouvelle voie de signalisation induite par l'OPDA, pas par le JA et d��pendante de la prot��ine COI1. Ces r��sultats d��montrent pour la premi��re fois que l'OPDA et le JA peuvent activer diff��rents g��nes via des voies de signalisation d��pendantes de COI1. Enfin, cette th��se r��v��le l'identification d'un nouveau r��le de la prot��ine AtPHO1 dans la r��gulation de l'action de l'ABA au cours des processus de fermeture stomatique et de germination des graines chez Arabidopsis. Bien que les fonctions exactes des prot��ines AtPHO restent �� ��tre d��termin��es, ce travail de th��se sugg��re leur implication dans la propagation de diff��rents signaux dans la plante via la modulation du potentiel membranaire et/ou l'affectation de la composition en ions des cellules comme le font de nombreux transporteurs ou r��gulateur du transport d'ions. Summary Phosphate is transferred from the roots to the shoot via the xylem. The requirement for AtPHO1 protein to transfer phosphate to the xylem vessels of the root has been previously demonstrated in Arabidopsis thaliana. The sequencing and the annotation of the Arabidopsis genome had allowed the identification of ten sequences that show a significant level of similarity with the AtPHO1 gene. These 10 genes, of unknown functions, constitute a new gene family called the AtPHO1 gene family. Based on a molecular and genetics study, this thesis reveals some information needed to understand the role of the AtPHO1 family members in the plant Arabidopsis. First, a bioinformatics study revealed that the AtPHO sequences contained, in the N-terminal hydrophilic region, a motif called SPX and conserved among multiple proteins involved in phosphate homeostasis in yeast. This finding reinforces the hypothesis that all AtPHO1 family members have, as AtPHO1, a role in phosphate homeostasis. In parallel, we identified the pattern of expression of AtPHO genes in Arabidopsis via analysis of transgenic plants expressing the uidA reporter gene under the control of respective AtPHO promoter regions. The results exhibit a predominant expression of AtPHO genes in vascular tissues of all organs of the plant, implying that these AtPHO genes could have redundant functions in the transfer of phosphate to the vascular cylinder of various organs. The GUS expression pattern for several AtPHO promoter regions was also detected in non-vascular tissue indicating a broad role of AtPHO genes in the acquisition or in the recycling of phosphate in the plant. In a second step, the analysis of the expression of AtPHO genes during phosphate starvation established that only the expression of the AtPHO1, AtPHO1; H1 and AtPHO1; H10 genes were regulated by Pi starvation. Interestingly, different signalling pathways appeared to regulate these three genes during various treatments affecting Pi homeostasis in the plant. The third chapter presents a detailed analysis of the signalling pathways regulating the expression of the AtPHO1; H10 gene in Arabidopsis leaves during wound and dehydrated stresses. Surprisingly, the expression of AtPHO1; H10 was found to be regulated by OPDA (the precursor of JA) but not by JA itself and via the COI1 protein (the central regulator of the JA signalling pathway). These results demonstrated for the first time that OPDA and JA could activate distinct genes via COI1-dependent pathways. Finally, this thesis presents the identification of a novel role of the AtPHO1 protein in the regulation of ABA action in Arabidopsis guard cells and during seed germination. Although the exact role and function of AtPHO1 still need to be determined, these last findings suggest that AtPHO1 and by extension other AtPHO proteins could mediate the propagation of various signals in the plant by modulating the membrane potential and/or by affecting cellular ion composition, as it is the case for many ion transporters or regulators of ion transport.

onductivitat t��rmica i ��ndex de refracci�� no lineal dels compostos KRE(WO4)2 (RE=Gd,Y, Yb and Lu)

Projecte de recerca elaborat a partir d���una estada al Max Born Institute for Nonlinear Optics and Short Pulse Spectroscopy entre setembre i desembre 2007. Els materials monocristal���lins tungstats dobles de potassi i terra rara, KRE(WO4)2, a partir d'ara KREW, s��n en l'actualitat un material competitiu com a material actiu per sistemes de l��ser d'estat s��lid. Aquests materials monocl��nics s��n f��cils de dopar amb altres densitats de ions lant��nid, Ln3+, i a m��s presenten unes seccions eficaces d'absorci�� i d'emissi��, molt elevades. Dins d���aquesta fam��lia, destaca el KLuW; degut als seus millors resultats com a material l��ser. Durant aquesta estada d���un mes al laboratori Max Born de Berlin, s���han realitzat les mesures de conductivitat t��rmica d���aquest material, per tal de obtenir el seu tensor de segon ordre de conductivitat t��rmica. El bombeig ��ptic dels materials l��ser d���estat s��lid genera calor com a resultat de la termalitzaci�� en els multiplets, de les relaxacions no-radiatives i de les absorcions residuals (defectes, impureses). Per tant, el coneixement de les propietats t��rmiques de qualsevol material actiu ��s essencial pel disseny de la cavitat l��ser i l���avaluaci�� de la funci�� l��ser, especialment en r��gims d���altes pot��ncies(...)

Development of advanced silicon radiation detectors for high energy physics and medical imaging

The 1st chapter of this work presents the different experiments and collaborations in which I am involved during my PhD studies of Physics. Following those descriptions, the 2nd chapter is dedicated to how the radiation affects the silicon sensors, as well as some experimental measurements carried out at CERN (Geneve, Schwitzerland) and IFIC (Valencia, Spain) laboratories. Besides the previous investigation results, this chapter includes the most recent scientific papers appeared in the latest RD50 (Research & Development #50) Status Report, published in January 2007, as well as some others published this year. The 3rd and 4th are dedicated to the simulation of the electrical behavior of solid state detectors. In chapter 3 are reported the results obtained for the illumination of edgeless detectors irradiated at different fluences, in the framework of the TOSTER Collaboration. The 4th chapter reports about simulation design, simulation and fabrication of a novel 3D detector developed at CNM for ions detection in the future ITER fusion reactor. This chapter will be extended with irradiation simulations and experimental measurements in my PhD Thesis.

Fast screening and confirmation of doping agents by UHPLC-QTOF-MS/MS

Introduction: The general strategy to perform anti-doping analysis starts with a screening followed by a confirmatory step when a sample is suspected to be positive. The screening step should be fast, generic and able to highlight any sample that may contain a prohibited substance by avoiding false negative and reducing false positive results. The confirmatory step is a dedicated procedure comprising a selective sample preparation and detection mode. Aim: The purpose of the study is to develop rapid screening and selective confirmatory strategies to detect and identify 103 doping agents in urine. Methods: For the screening, urine samples were simply diluted by a factor 2 with ultra-pure water and directly injected ("dilute and shoot") in the ultrahigh- pressure liquid chromatography (UHPLC). The UHPLC separation was performed in two gradients (ESI positive and negative) from 5/95 to 95/5% of MeCN/Water containing 0.1% formic acid. The gradient analysis time is 9 min including 3 min reequilibration. Analytes detection was performed in full scan mode on a quadrupole time-of-flight (QTOF) mass spectrometer by acquiring the exact mass of the protonated (ESI positive) or deprotonated (ESI negative) molecular ion. For the confirmatory analysis, urine samples were extracted on SPE 96-well plate with mixed-mode cation (MCX) for basic and neutral compounds or anion exchange (MAX) sorbents for acidic molecules. The analytes were eluted in 3 min (including 1.5 min reequilibration) with a S1-25 Ann Toxicol Anal. 2009; 21(S1) Abstracts gradient from 5/95 to 95/5% of MeCN/Water containing 0.1% formic acid. Analytes confirmation was performed in MS and MS/MS mode on a QTOF mass spectrometer. Results: In the screening and confirmatory analysis, basic and neutral analytes were analysed in the positive ESI mode, whereas acidic compounds were analysed in the negative mode. The analyte identification was based on retention time (tR) and exact mass measurement. "Dilute and shoot" was used as a generic sample treatment in the screening procedure, but matrix effect (e.g., ion suppression) cannot be avoided. However, the sensitivity was sufficient for all analytes to reach the minimal required performance limit (MRPL) required by the World Anti Doping Agency (WADA). To avoid time-consuming confirmatory analysis of false positive samples, a pre-confirmatory step was added. It consists of the sample re-injection, the acquisition of MS/MS spectra and the comparison to reference material. For the confirmatory analysis, urine samples were extracted by SPE allowing a pre-concentration of the analyte. A fast chromatographic separation was developed as a single analyte has to be confirmed. A dedicated QTOF-MS and MS/MS acquisition was performed to acquire within the same run a parallel scanning of two functions. Low collision energy was applied in the first channel to obtain the protonated molecular ion (QTOF-MS), while dedicated collision energy was set in the second channel to obtain fragmented ions (QTOF-MS/MS). Enough identification points were obtained to compare the spectra with reference material and negative urine sample. Finally, the entire process was validated and matrix effects quantified. Conclusion: Thanks to the coupling of UHPLC with the QTOF mass spectrometer, high tR repeatability, sensitivity, mass accuracy and mass resolution over a broad mass range were obtained. The method was sensitive, robust and reliable enough to detect and identify doping agents in urine. Keywords: screening, confirmatory analysis, UHPLC, QTOF, doping agents

Towards the validation of "in silico" models and physicochemical filters to identify and characterize new chemical entities

Summary: Lipophilicity plays an important role in the determination and the comprehension of the pharmacokinetic behavior of drugs. It is usually expressed by the partition coefficient (log P) in the n-octanol/water system. The use of an additional solvent system (1,2-dichlorethane/water) is necessary to obtain complementary information, as the log Poct values alone are not sufficient to explain ail biological properties. The aim of this thesis is to develop tools allowing to predict lipophilicity of new drugs and to analyze the information yielded by those log P values. Part I presents the development of theoretical models used to predict lipophilicity. Chapter 2 shows the necessity to extend the existing solvatochromic analyses in order to predict correctly the lipophilicity of new and complex neutral compounds. In Chapter 3, solvatochromic analyses are used to develop a model for the prediction of the lipophilicity of ions. A global model was obtained allowing to estimate the lipophilicity of neutral, anionic and cationic solutes. Part II presents the detailed study of two physicochemical filters. Chapter 4 shows that the Discovery RP Amide C16 stationary phase allows to estimate lipophilicity of the neutral form of basic and acidic solutes, except of lipophilic acidic solutes. Those solutes present additional interactions with this particular stationary phase. In Chapter 5, 4 different IANI stationary phases are investigated. For neutral solutes, linear data are obtained whatever the IANI column used. For the ionized solutes, their retention is due to a balance of electrostatic and hydrophobie interactions. Thus no discrimination is observed between different series of solutes bearing the same charge, from one column to an other. Part III presents two examples illustrating the information obtained thanks to Structure-Properties Relationships (SPR). Comparing graphically lipophilicity values obtained in two different solvent systems allows to reveal the presence of intramolecular effects .such as internai H-bond (Chapter 6). SPR is used to study the partitioning of ionizable groups encountered in Medicinal Chemistry (Chapter7). R��sum�� La lipophilie joue un .r��le important dans la d��termination et la compr��hension du comportement pharmacocin��tique des m��dicaments. Elle est g��n��ralement exprim��e par le coefficient de partage (log P) d'un compos�� dans le syst��me de solvants n-octanol/eau. L'utilisation d'un deuxi��me syst��me de solvants (1,2-dichloro��thane/eau) s'est av��r��e n��cessaire afin d'obtenir des informations compl��mentaires, les valeurs de log Poct seules n'��tant pas suffisantes pour expliquer toutes les propri��t��s biologiques. Le but de cette th��se est de d��velopper des outils permettant de pr��dire la lipophilie de nouveaux candidats m��dicaments et d'analyser l'information fournie par les valeurs de log P. La Partie I pr��sente le d��veloppement de mod��les th��oriques utilis��s pour pr��dire la lipophilie. Le chapitre 2 montre la n��cessit�� de mettre �� jour les analyses solvatochromiques existantes mais inadapt��es �� la pr��diction de la lipophilie de nouveaux compos��s neutres. Dans le chapitre 3, la m��me m��thodologie des analyses solvatochromiques est utilis��e pour d��velopper un mod��le permettant de pr��dire la lipophilie des ions. Le mod��le global obtenu permet la pr��diction de la lipophilie de compos��s neutres, anioniques et cationiques. La Partie II pr��sente l'��tude approfondie de deux filtres physicochimiques. Le Chapitre 4 montre que la phase stationnaire Discovery RP Amide C16 permet la d��termination de la lipophilie de la forme neutre de compos��s basiques et acides, �� l'exception des acides tr��s lipophiles. Ces derniers pr��sentent des interactions suppl��mentaires avec cette phase stationnaire. Dans le Chapitre 5, 4 phases stationnaires IAM sont ��tudi��es. Pour les compos��s neutres ��tudi��s, des valeurs de r��tention lin��aires sont obtenues, quelque que soit la colonne IAM utilis��e. Pour les compos��s ionisables, leur r��tention est due �� une balance entre des interactions ��lectrostatiques et hydrophobes. Donc aucune discrimination n'est observ��e entre les diff��rentes s��ries de compos��s portant la m��me charge d'une colonne �� l'autre. La Partie III pr��sente deux exemples illustrant les informations obtenues par l'utilisation des relations structures-propri��t��s. Comparer graphiquement la lipophilie mesur��e dans deux diff��rents syst��mes de solvants permet de mettre en ��vidence la pr��sence d'effets intramol��culaires tels que les liaisons hydrog��ne intramol��culaires (Chapitre 6). Cette approche des relations structures-propri��t��s est aussi appliqu��e �� l'��tude du partage de fonctions ionisables rencontr��es en Chimie Th��rapeutique (Chapitre 7) R��sum�� large public Pour exercer son effet th��rapeutique, un m��dicament doit atteindre son site d'action en quantit�� suffisante. La quantit�� effective de m��dicament atteignant le site d'action d��pend du nombre d'interactions entre le m��dicament et de nombreux constituants de l'organisme comme, par exemple, les enzymes du m��tabolisme ou les membranes biologiques. Le passage du m��dicament �� travers ces membranes, appel�� perm��ation, est un param��tre important �� optimiser pour d��velopper des m��dicaments plus puissants. La lipophilie joue un r��le cl�� dans la compr��hension de la perm��ation passive des m��dicaments. La lipophilie est g��n��ralement exprim��e par le coefficient de partage (log P) dans le syst��me de solvants (non miscibles) n-octanol/eau. Les valeurs de log Poct seules se sont av��r��es insuffisantes pour expliquer la perm��ation �� travers toutes les diff��rentes membranes biologiques du corps humain. L'utilisation d'un syst��me de solvants additionnel (le syst��me 1,2-dichloro��thane/eau) a permis d'obtenir les informations compl��mentaires indispensables �� une bonne compr��hension du processus de perm��ation. Un grand nombre d'outils exp��rimentaux et th��oriques sont �� disposition pour ��tudier la lipophilie. Ce travail de th��se se focalise principalement sur le d��veloppement ou l'am��lioration de certains de ces outils pour permettre leur application �� un champ plus large de compos��s. Voici une br��ve description de deux de ces outils: 1)La factorisation de la lipophilie en fonction de certaines propri��t��s structurelles (telle que le volume) propres aux compos��s permet de d��velopper des mod��les th��oriques utilisables pour la pr��diction de la lipophilie de nouveaux compos��s ou m��dicaments. Cette approche est appliqu��e �� l'analyse de la lipophilie de compos��s neutres ainsi qu'�� la lipophilie de compos��s charg��s. 2)La chromatographie liquide �� haute pression sur phase inverse (RP-HPLC) est une m��thode couramment utilis��e pour la d��termination exp��rimentale des valeurs de log Poct.

Oxidative potential of a panel of carbonaceous and metallic nanoparticles

Characterisation of nanoparticles (NP) based on size distribution, surface area, reactivity, and aggregation status of nanoparticles (NP) are of prime importance because they are usually closely related to toxicity. To date, most of the toxicity studies are quite time and money consuming. In the present study we report the oxidative properties of a panel of various NP (four Carbonaceous, nine Metal oxides, and one Metal as showed in Table 1) assessed with an acellular reactivity test measuring dithiothreitol (DTT) consumption (Sauvain et al. 2008). Such a test allows determining the ability of NP to catalyse the transfer of electrons from DTT to oxygen. DTT is used as a reductant species. NP were diluted and sonicated in Tween 80�� to a final concentration of 50 g/mL. Printex 90 was diluted 5 times before doing the DTT assay because of its expected higher activity. Suspensions were characterised for NP size distribution by Nanoparticle Tracking Analysis (Nanosight��). Fresh solutions were incubated with DTT (100 μM). Aliquots were taken every 5 min and the remaining DTT was determined by reacting it with DTNB. The reaction rate was determined for NP suspensions and blank in parallel. The mean Brownian size distribution of NP agglomerates in suspension is presented in Table 1. D values correspond to 10th, and 50th percentiles of the particle diameters. All the NP agglomerated in Tween 80 with a D50 size corresponding to at least twice their primary size, except for Al2O3 (300 nm). The DTT test showed Printex 90 sample to be the most reactive one, followed by Diesel EPA and Nanotubes. Most of the metallic NP was nonresponding toward this test, except for NiO and Ag which reacted positively and ZnO which presented the most negative reactivity (see Figure 1). This last observation suggests that electron transfer between DTT and oxygen is hindered in presence of ZnO compared with the blank. Such "stabilization" could be attributable to ZnO dissolution and complexation between Zn2+ ions and DTT.

Cross-talk between glutamate and potassium regulation at the astrocyte membrane

Astrocytes play a central role in the brain by regulating glutamate and extracellular potassium concentrations ([K+]0), both released by neurons into the extracellular space during neuronal activity. Glutamate uptake is driven by the inwardly directed sodium gradient across the astrocyte membrane and involves the influx of three sodium ions and one proton and the efflux of one K+ ion per glutamate molecule. The glutamate transport induced rise in intracellular sodium stimulates the Na+/K+-ATPase which leads to significant energetic costs in astrocytes. To evaluate how these two fundamental functions of astrocytes, namely glutamate transport and K+ buffering, which are directly associated with neuronal activity, coexist and if they influence each other, in this thesis work we examined different cellular parameters of astrocytes. We therefore investigated the impact of altered [K+]0 on glutamate transporter activity. To assess this question we measured intracellular sodium fluctuations in mouse primary cultured astrocytes using dynamic fluorescence imaging. We found that glutamate uptake was tightly modulated both in amplitude and kinetics by [K+]0. Elevated [K+]0 strongly decreased glutamate transporter activity, with significant consequences on the cells energy metabolism. To ultimately evaluate potential effects of [K+]0 and glutamate on the astrocyte mitochondrial energy production we extended these studies by investigating their impact on the cytosolic and mitochondrial pH. We found that both [K+],, and glutamate strongly influenced cytosolic and mitochondrial pH, but in opposite directions. The effect of a simultaneous application of K+ and glutamate, however, did not fit with the arithmetical sum of each individual effects, suggesting that an additional non��linear process is involved. We also investigated the impact of [K+]0 and glutamate transport, respectively, on intracellular potassium concentrations ([K+]0 in cultured astrocytes by characterizing and applying a newly developed Insensitive fluorescent dye. We observed that [K+]i followed [K+]0 changes in a nearly proportional way and that glutamate superfusion caused a reversible, glutamate-concentration dependent drop of [K+],, Our study shows the powerful influence of [K+]u on glutamate capture. These findings have strong implications for our understanding of the tightly regulated interplay between astrocytes and neurons in situations where [K+]0 undergoes large activity-dependent fluctuations. However, depending on the extent of K+ versus glutamate extracellular rise, energy metabolism in astrocytes will be differently regulated. Moreover, the novel insights obtained during this thesis work help understanding some of the underlying processes that prevail in certain pathologies of central nervous system, such as epilepsy and stroke. These results will possibly provide a basis for the development of novel therapeutic strategies. -- Les astrocytes jouent un r��le central dans le cerveau en r��gulant les concentrations de potassium (K+) et de glutamate, qui sont rel��ch��s par les neurones dans l'espace extracellulaire lorsque ceux- ci sont actifs. La capture par les astrocytes du glutamate est un processus secondairement actif qui implique l'influx d'ions sodium (Na+) et d'un proton, ainsi que l'efflux d'ions K+, ce processus entra��ne un co��t m��tabolique important. Nous avons ��valu�� comment ces fonctions fondamentales des astrocytes, la r��gulation du glutamate et du K+ extracellulaire, qui sont directement associ��s �� l'activit�� neuronale, coexistent et si elles interagissent, en examinant diff��rents param��tres cellulaires. Dans ce projet de th��se nous avons ��valu�� l'impact des modifications de la concentration de potassium extracellulaire ([K+],,) sur le transport du glutamate. Nous avons mesur�� le transport du glutamate par le biais des fluctuations internes de Na+ gr��ce �� un colorant fluorescent en utilisant de l'imagerie �� fluorescence dynamique sur des cultures primaires d'astrocytes. Nous avons trouv�� que la capture du glutamate ��tait ��troitement r��gul��e par [K+]0 aussi bien dans son amplitude que dans sa cin��tique. Par la suite, nous avons port�� notre attention sur l'impact de [K+]0 et du glutamate sur le pH cytosolique et mitochondrial de l'astrocyte dans le but, in fine, d'��valuer les effets potentiels sur la production d'��nergie par la mitochondrie. Nous avons trouv�� qu'autant le K+ que le glutamate, de mani��re individuelle, influen��aient fortement le pH, cependant dans des directions oppos��es. Leurs effets individuels, ne peuvent toutefois pas ��tre additionn��s ce qui sugg��re qu'un processus additionnel non-lin��aire est impliqu��. En appliquant une nouvelle approche pour suivre et quantifier la concentration intracellulaire de potassium ([K+]0 par imagerie �� fluorescence, nous avons observ�� que [K+]i suivait les changements de [K+]0 de mani��re quasiment proportionnelle et que la superfusion de glutamate induisait un d��croissement rapide et r��versible de [K+]i, qui d��pend de la concentration de glutamate. Notre ��tude d��montre l'influence de [K+]0 sur la capture du glutamate. Ces r��sultats permettent d'am��liorer notre compr��hension de l'interaction entre astrocytes et neurones dans des situations o�� [K+]0 fluctue en fonction de l'activit�� neuronale. Cependant, en fonction de l'importance de l'augmentation extracellulaire du K+ versus le glutamate, le m��tabolisme ��nerg��tique des astrocytes va ��tre r��gul�� de mani��re diff��rente. De plus, les informations nouvelles que nous avons obtenues durant ce travail de th��se nous aident �� comprendre quelques- uns des processus sous-jacents qui pr��valent dans certaines pathologies du syst��me nerveux central, comme par exemple l'��pilepsie ou l'accident vasculaire c��r��bral. Ces informations pourront ��tre importantes �� int��grer dans la cadre du d��veloppement de nouvelles strat��gies th��rapeutiques.

A new life for an old pump: V-ATPase and neurotransmitter release.

Neurons fire by releasing neurotransmitters via fusion of synaptic vesicles with the plasma membrane. Fusion can be evoked by an incoming signal from a preceding neuron or can occur spontaneously. Synaptic vesicle fusion requires the formation of trans complexes between SNAREs as well as Ca(2+) ions. Wang et al. (2014. J. Cell Biol. http://dx.doi.org/jcb.201312109) now find that the Ca(2+)-binding protein Calmodulin promotes spontaneous release and SNARE complex formation via its interaction with the V0 sector of the V-ATPase.