Thèse de doctorat : Chimie et Physico-chimie Moléculaire : Nancy 1 : 2008. / Titre provenant de l'écran-titre.

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<p>Chlorite is a ferrous silicate mineral occurring as afracture filling mineral in the Swedish granite bedrock thatsurrounds the area of the candidate Swedish geologicalrepositories for nuclear waste.</p><p>To investigate the importance of chlorites as a naturalbarrier for radionuclide migration sorption of Ni(II) onto anatural chlorite was chosen as the studied system. The sorptionbehaviour was studied using batch technique under differentexperimental conditions by varying the concentration ofbackground electrolyte, pH and initial nickel concentration.Our experiments showed that sorption is dependent of pH; belowpH 4 no sorption occurs and in the pH range 7-11 the sorptionmaxima was found. The sorption shows no dependence of ionicstrength in our experiments and together with pH dependency weconclude that the sorption of nickel to chlorite is mostlyoccurring through surface complexation. The distributioncoefficient, K<i>d</i>, was determined for our results and in the range formaximal sorption the K<i>d</i>is approximately 10³cm³/g.</p><p>Our experimental data were described using a diffuse doublelayer model, including strong and weak surface sites, in thesoftware PHREECQ and the results obtained from PHREEQC werealso used for a fit of the data in the software FITEQL. Thesurface complexes that dominate the sorption of nickel tochlorite from our model are Chl_ONi⁺(weak) and for pH above 9 Chl_ONi(OH)^-(weak). Flow-through technique was used in ourinvestigations regarding the dissolution rate of a naturalchlorite and the experimental results show that the dissolutionrate of chlorite is strongly pH dependent and at pH 2 thehighest dissolution rate (R_Si) could be determined to 7• 10^-11mol/(m²s) based on silica data.</p><p><b>Keywords:</b>chlorite, nickel, sorption, surfacecomplexation, dissolution rate.</p>

Student Number : 0009716H - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / Positive charges play an important role in the active site of a variety of proteins including alpha class GST. The presence of Arg15 at the subunit interface between the H- and Gsite of hGST A1-1 was shown to be important for the catalytic function of the enzyme, thus providing an electrostatic potential in the G-site. This electrostatic potential favours ionisation and activation of GSH bound to hGST A1-1. Although much is known regarding the catalytic function of GSTs, little is known of the effect of Arg15 on the binding of glutathione conjugates and nonsubstrate ligands. Others (Matulis and Lovrien, 1998) have pointed out that binding of the nonsubstrate ligand ANS is accomplished via ion pair formation with a charged residue such as Arg. Therefore, in order to evaluate the importance of the positive charge provided by the side chain of Arg15 in the active site of hGST A1-1 protein engineering techniques were employed to generate an R15L variant. The variant does not display a detectable difference in the secondary or tertiary structural content relative to the wild type hGST A1-1 protein. The catalytic activity of the R15L variant is, however, substantially reduced indicating local tertiary structural changes at and possibly near the active site. In the presence of ANS the fluorescence spectra of the variant R15L protein had a significantly lower intensity relative to wild type protein indicating either increased exposure of hydrophobic surface area at the Hsite and/or reduced number of ANS molecules bound (i.e., reduced affinity). Although ANS is displaced by GSO3 - in the wild type protein, no such displacement is observed for the R15L variant protein. Isothermal titration calorimetry experiments for GSO3 - and ANS binding to R15L indicated that the variant protein binds GSO3 - more tightly (higher affinity) than ANS (lower affinity). The above information, taken together, argues in favour of both molecules (ANS and GSO3 -) occupying the active site of R15L simultaneously. Despite the fact that there is no experimental crystal structure of hGST A1-1 complexed with either of the ligands, the placement of ANS was argued (Dirr et al., 2005) to be at the H-site and of GSO3 - at the G-site of the protein. The binding of ANS is both enthalpically and entropically favourable over the temperature range investigated (5 to 25°C) indicating expulsion of water molecules from the active site as well as the lack of a localised C-terminal #1;9 helix. The hydrophobic character of both the ligand and the site which accommodates it (H-site) contribute favourably towards the reaction enthalpy. ANS is conformationaly more constrained than GSO3 - when found free in solution, thus exhibiting more favourable conformational entropy change upon binding to R15L variant. The binding of GSO3 - is enthalpically favourable but entropically opposed. The expulsion of water molecules from the G-site, which accommodates GSO3 -, is insufficient to compensate for the loss of conformational entropy of both the ligand and C-terminal #1;9 helix, which becomes localised upon binding. However, favourable enthalpic contributions arise from the formation of hydrogen bonds, salt links and van der Waals contacts between the R15L variant and the ligand and the C-terminal #1;9 helix. Arg15 forms a salt link with Glu104 of the neighbouring domain and therefore provided an ideal opportunity to evaluate the role of Arg15 and its impact on interdomain stability. Conformational stability and unfolding studies of the R15L variant, monitored via fluorescence, indicated that the variant deviates from the model of a two-state transition. There are no stable thermodynamic intermediate(s) observed, but the increase in the mvalue is an indicator of a transition state which is more stable against urea relative to the wild type protein. Kinetically (Wallace et al., 1998b), the wild type hGST A1-1 follows a three-state unfolding transition where the kinetic intermediate is defined as having undergone conformational changes at the domain-domain interface and the C-terminal region. The substitution of Arg15 with Leu may have impacted on the packing of the native form of the variant R15L relative to the wild type GST A1-1, thus rendering the R15L protein more stable against urea. Arg15, therefore, may have an important role at the interdomain interface of GSTs.

La compréhension des mécanismes de transport et du comportement des radionucléides dans la biogéosphère sont nécessaires à l’évaluation des risques sanitaires et environnementaux de l’industrie nucléaire. Ces mécanismes sont contrôlés par la spéciation des radioéléments, c’est-à-dire leur répartition entre leurs différentes formes physico-chimiques dans l’environnement. Dans cette optique, cette thèse traite de la spéciation de l’uranium en milieu naturel. La biogéochimie de l’uranium a fait l’objet d’une synthèse bibliographique détaillée, qui a permis de restreindre le sujet à la complexation de l’uranium par l’acide oxalique, un acide organique hydrophile fréquemment rencontré dans les sols et doté de bonnes propriétés de complexation. L’établissement de diagrammes de spéciation à partir des constantes de complexation de la littérature a permis de définir les conditions analytiques de formation des complexes. Le choix de la technique analytique s’est porté sur le couplage d’une technique séparative (chromatographie liquide LC ou électrophorèse capillaire EC) à la spectrométrie de masse (ICPMS). La présence des complexes étudiés dans les échantillons synthétiques a été vérifiée par spectrophotométrie UV/visible. Les analyses menées par LC-ICPMS ont prouvé la labilité des complexes uranyle-acides organiques, c’est-à-dire leur propension à se dissocier pendant l’analyse, ce qui empêche une détermination fiable de la spéciation de l’uranium. En revanche, l’étude des complexes labiles d’un système métal-ligand par ECICPMS a été rendue possible par l’emploi de la méthode d’affinité, permettant de déterminer les constantes de complexations et les mobilités électrophorétiques. Cette thèse a permis de comparer les différentes méthodes de traitement mathématique de l’isotherme de complexation et de prendre en compte la force ionique et la concentration réelle du ligand. La méthode d’affinité a été employée avec succès sur les systèmes lanthane-oxalate, pris comme modèle, et uranyle-oxalate. Les résultats obtenus ont été validés sur un système naturel (site du Bouchet). Ceci montre l’apport de la méthode mise au point dans la modélisation de la spéciation de l’uranium. / To understand the transport mechanisms and the radionuclides behaviour in the biogeosphere is necessary to evaluate healthy and environmental risks of nuclear industry. These mechanisms are monitored by radioelements speciation, namely the distribution between their different physico-chemical forms in the environment. From this perspective, this PhD thesis deals with uranium speciation in a natural background. A detailed summary of uranium biogeochemistry has been written, which enables to restrict the PhD issue to uranium complexation with oxalic acid, a hydrophilic organic acid with good binding properties, ubiquitous in soil waters. Analytical conditions have been established by means of speciation diagrams. The speciation diagrams building by means of literature stability constants has allowed to define the analytical conditions of complex formation. The chosen analytical technique is the hyphenation of a separative technique (liquid chromatography LC or capillary electrophoresis CE) with mass spectrometry (ICPMS). The studied complexes presence in the synthetic samples has been confirmed with UV/visible spectrophotometry. LC-ICPMS analyses have proved the lability of the uranyl-organic acid complexes, namely their tendency to dissociate during analysis, which prevents from studying uranium speciation. CE-ICPMS study of labile complexes from a metal-ligand system has been made possible by employing affinity capillary electrophoresis, which enables to determine stability constants and electrophoretic mobilities. This PhD thesis has allowed to compare the different mathematical treatments of binding isotherm and to take into account ionic strength and real ligand concentration. Affinity CE has been applied successfully to lanthanum-oxalate (model system) and uranium-oxalate systems. The obtained results have been applied to a real system (situated in Le Bouchet). This shows the contribution of the developed method to the modelling of uranium speciation.

Carboxylated macromolecules such as alginate and glycoproteins are abundant components of modern shallow marine sediments where they are secreted by bacteria and marine infauna. Both organic compounds are proton and metal reactive; hence, they have the potential to facilitate metal sorption and biomineralization reactions. In this study, lab experiments were coupled to field-based sampling to assess the role that these compounds play in microbial mats, with particular emphasis on the hypersaline lagoons of Los Roques, Venezuela. Here I applied a surface complexation approach to model proton and Cd adsorption behaviour of both uronic acid-rich alginate and mucin. Measured total site concentrations, available for metal adsorption, demonstrate that these compounds have the potential to induce metal partitioning in early diagenetic microenvironments. Field results from Venezuela are consistent with Mg- and trace metal- enrichment that follows a likely correlation with the degradation states of microbial biomass trapped during accretion of modern microbialites.

<p>In general, the research focuses on sequestration of naphthenic acids (NAs) from simulated oil sands process water (OSPW) conditions using engineered copolymers known as &beta;-cyclodextrin-based polyurethanes. The thesis research is divided into two main parts; <i>i)</i> synthesis and characterization of &beta;-cyclodextrin-based polyurethanes, and <i>ii)</i> sorption studies of the copolymer materials with NAs from aqueous solutions.</p> <o>In the first part, &beta;-cyclodextrin (&beta;-CD) was crosslinked with five types of linker molecules, respectively, under various synthetic conditions. The various diisocyanates investigated as linkers include the following: 1,6-hexamethylene diisocyanate (HDI), 4,4'-dicyclohexylmethane diisocyanate (CDI), 4,4'-diphenylmethane diisocyanate (MDI), 1,4-phenylene diisocyanate (PDI) and 1,5-naphthalene diisocyanate (NDI). The polyurethanes (PUs) were systematically designed at different mole ratios of monomers by maintaining &beta;-CD and varying the relative mole ratio of diisocyanate monomer from unity to greater values. Diffuse Reflectance Infrared Fourier Transform spectroscopy (DRIFTS), solid state ¹³C CP-MAS NMR, solution state ¹H/¹³C NMR spectroscopy, thermogravimetric analysis (TGA), elemental analysis (CHN), nitrogen porosimetry, and a dye-based (p-nitrophenol; PNP, and phenolphthalein; phth) sorption method were used to characterize the copolymer materials. In general, the &beta;-CD PUs were insoluble in water except for &beta;-CD crosslinked with HDI at the 1:1 mole ratio which was moderately water soluble. All techniques show complementary results about the structural and compositional characterization, particularly for the estimation of the ratios between the co-monomers. The optimal preparation of copolymer materials for sorption-based applications occurs for &beta;-CD/linker monomer mole ratios from 1:1 to 1:3. There is a maximum limit of the crosslinking density which is ~ 1:7 (&beta;-CD:linker) according to the steric effects of the substituents in the annular region of &beta;-CD. Also, the copolymers were generally found to be mesoporous with relatively low surface area (BET; ~10¹ m²/g) and they appear to exhibit swelling in aqueous solution due to hydration as observed from the estimation of the dye-based surface areas using PNP. The surface accessibilities of the &beta;-CD inclusion sites ranged between 1-100%, as evidenced by the decolourization of phenolphthalein (phth) due to the formation of 1:1 &beta;-CD/phth inclusion complexes.</p> <p>In the second part, the inclusion of NAs and its surrogates with three well known types of cyclodextrin (&alpha;-, &beta;-, and &gamma;-CD) was confirmed using negative ion mode electrospray ionization (ESI). The CDs were found to form well-defined 1:1 inclusion complexes. The binding constant (K2) of NAs and its surrogates inclusion with &beta;-CD was determined indirectly using the spectral displacement technique and were found to be 10³-10⁴ M^-1 (surrogates) and ~ 10⁴ M^-1 (NAs), respectively. Furthermore, the binding constants were found to increase with an increase of the lipophilic surface area (LSA) of the surrogates. The sorption results of NAs with three different types of &beta;-CD materials (i.e. &beta;-CD PU, &beta;-CD crosslinked with epichlorohydrin (EP) and a silica-based mesoporous material containing &beta;-CD), showed &beta;-CD PU had a greater affinity. The sorption capacity (~ 0 - 75.5 mg NAs/g copolymer) and binding affinity (~10³ - 10⁴ M^-1) of &beta;-CD PUs varied due to the nature of linker monomer and the mole ratio of the co-monomers. Aromatic-based copolymers showed high sorption binding affinity while aliphatic-based copolymers showed a relatively high sorption capacity at the co-monomer ratio. Finally, Syncrude-Derived NAs showed fluorescent characteristics which contradict the classic definition of NAs. Further studies using UV-Vis and fluorescence emission spectroscopy showed potential development of an analytical method that can be used to quantify NAs in OSPW for <i>in-situ</i> field applications.

Short interfering RNAs (siRNAs) trigger RNA interference (RNAi) both in vitro and in vivo, where the expression of the encoded protein is silenced. Its potential use as a therapeutic agent is limited by its rapid enzymatic degradation and low cellular uptake. Therefore, a delivery carrier is desired to increase its solution stability and improve cellular uptake. In this study, Arginine-9 (R9), a cell penetrating peptide derived from the HIV 1 Tat protein, was investigated as a potential carrier for siRNAs at pH 7.3. The optical activity of siRNA decreased with increasing R9 concentration, with only 7.8±3.8% of the initial absorbance at 260nm remained at siRNA saturation. The highest binding ratio of R9 to siRNA determined from the UV/Vis spectra was 10.3:1 (corresponds to a charge ratio of 2.2:1 (+/-)). The measured hydrodynamic diameter increased with increasing R9, with a maximum value of ~1um at siRNA saturation. At R9 to siRNA charge ratios below 5.74:1, the surface charge of the complexes increased rapidly with the addition of R9. However, the rate of increase of Zeta potential decreased significantly with subsequent addition of R9. At charge ratio above 1.43:1, the complexes expressed low surface charge which led to the formation of aggregates in solution. A new peptide library was designed which utilized several properties of known cell penetrating peptides. In vitro siRNA transfection of eGFP siRNA with C166-GFP cells was used to determine the transfection efficiency of the new peptides. Preliminary results of the newly designed peptides showed that some of them are not as effective when compared to Lipofectamine 2000 (Invitrogen). However, this experimental protocol can be extended to study transfection efficiency of the remaining peptides in the library.

<p>In general, the research focuses on sequestration of naphthenic acids (NAs) from simulated oil sands process water (OSPW) conditions using engineered copolymers known as &beta;-cyclodextrin-based polyurethanes. The thesis research is divided into two main parts; <i>i)</i> synthesis and characterization of &beta;-cyclodextrin-based polyurethanes, and <i>ii)</i> sorption studies of the copolymer materials with NAs from aqueous solutions.</p> <o>In the first part, &beta;-cyclodextrin (&beta;-CD) was crosslinked with five types of linker molecules, respectively, under various synthetic conditions. The various diisocyanates investigated as linkers include the following: 1,6-hexamethylene diisocyanate (HDI), 4,4'-dicyclohexylmethane diisocyanate (CDI), 4,4'-diphenylmethane diisocyanate (MDI), 1,4-phenylene diisocyanate (PDI) and 1,5-naphthalene diisocyanate (NDI). The polyurethanes (PUs) were systematically designed at different mole ratios of monomers by maintaining &beta;-CD and varying the relative mole ratio of diisocyanate monomer from unity to greater values. Diffuse Reflectance Infrared Fourier Transform spectroscopy (DRIFTS), solid state ¹³C CP-MAS NMR, solution state ¹H/¹³C NMR spectroscopy, thermogravimetric analysis (TGA), elemental analysis (CHN), nitrogen porosimetry, and a dye-based (p-nitrophenol; PNP, and phenolphthalein; phth) sorption method were used to characterize the copolymer materials. In general, the &beta;-CD PUs were insoluble in water except for &beta;-CD crosslinked with HDI at the 1:1 mole ratio which was moderately water soluble. All techniques show complementary results about the structural and compositional characterization, particularly for the estimation of the ratios between the co-monomers. The optimal preparation of copolymer materials for sorption-based applications occurs for &beta;-CD/linker monomer mole ratios from 1:1 to 1:3. There is a maximum limit of the crosslinking density which is ~ 1:7 (&beta;-CD:linker) according to the steric effects of the substituents in the annular region of &beta;-CD. Also, the copolymers were generally found to be mesoporous with relatively low surface area (BET; ~10¹ m²/g) and they appear to exhibit swelling in aqueous solution due to hydration as observed from the estimation of the dye-based surface areas using PNP. The surface accessibilities of the &beta;-CD inclusion sites ranged between 1-100%, as evidenced by the decolourization of phenolphthalein (phth) due to the formation of 1:1 &beta;-CD/phth inclusion complexes.</p> <p>In the second part, the inclusion of NAs and its surrogates with three well known types of cyclodextrin (&alpha;-, &beta;-, and &gamma;-CD) was confirmed using negative ion mode electrospray ionization (ESI). The CDs were found to form well-defined 1:1 inclusion complexes. The binding constant (K2) of NAs and its surrogates inclusion with &beta;-CD was determined indirectly using the spectral displacement technique and were found to be 10³-10⁴ M^-1 (surrogates) and ~ 10⁴ M^-1 (NAs), respectively. Furthermore, the binding constants were found to increase with an increase of the lipophilic surface area (LSA) of the surrogates. The sorption results of NAs with three different types of &beta;-CD materials (i.e. &beta;-CD PU, &beta;-CD crosslinked with epichlorohydrin (EP) and a silica-based mesoporous material containing &beta;-CD), showed &beta;-CD PU had a greater affinity. The sorption capacity (~ 0 - 75.5 mg NAs/g copolymer) and binding affinity (~10³ - 10⁴ M^-1) of &beta;-CD PUs varied due to the nature of linker monomer and the mole ratio of the co-monomers. Aromatic-based copolymers showed high sorption binding affinity while aliphatic-based copolymers showed a relatively high sorption capacity at the co-monomer ratio. Finally, Syncrude-Derived NAs showed fluorescent characteristics which contradict the classic definition of NAs. Further studies using UV-Vis and fluorescence emission spectroscopy showed potential development of an analytical method that can be used to quantify NAs in OSPW for <i>in-situ</i> field applications.

Short interfering RNAs (siRNAs) trigger RNA interference (RNAi) both in vitro and in vivo, where the expression of the encoded protein is silenced. Its potential use as a therapeutic agent is limited by its rapid enzymatic degradation and low cellular uptake. Therefore, a delivery carrier is desired to increase its solution stability and improve cellular uptake. In this study, Arginine-9 (R9), a cell penetrating peptide derived from the HIV 1 Tat protein, was investigated as a potential carrier for siRNAs at pH 7.3. The optical activity of siRNA decreased with increasing R9 concentration, with only 7.8±3.8% of the initial absorbance at 260nm remained at siRNA saturation. The highest binding ratio of R9 to siRNA determined from the UV/Vis spectra was 10.3:1 (corresponds to a charge ratio of 2.2:1 (+/-)). The measured hydrodynamic diameter increased with increasing R9, with a maximum value of ~1um at siRNA saturation. At R9 to siRNA charge ratios below 5.74:1, the surface charge of the complexes increased rapidly with the addition of R9. However, the rate of increase of Zeta potential decreased significantly with subsequent addition of R9. At charge ratio above 1.43:1, the complexes expressed low surface charge which led to the formation of aggregates in solution. A new peptide library was designed which utilized several properties of known cell penetrating peptides. In vitro siRNA transfection of eGFP siRNA with C166-GFP cells was used to determine the transfection efficiency of the new peptides. Preliminary results of the newly designed peptides showed that some of them are not as effective when compared to Lipofectamine 2000 (Invitrogen). However, this experimental protocol can be extended to study transfection efficiency of the remaining peptides in the library.