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Insects accumulate various xenobiotics and toxic molecules through feeding and environmental exposure. This study examines the toxicology and regulation of a class of toxic molecules, organic cations, in Drosophila melanogaster. The results of this thesis demonstrate that transepithelial tetraethylammonium (TEA) secretion across the main segment of the Malpighian tubules is increased in response to diuretic factors. Both cAMP and cGMP, which increase transepithelial potential (TEP), as well as tyramine and LK-1, which decrease TEP, all enhanced TEA secretion. Both inc~eases and decreases ofTEP may enhance proton transport into the lumen of the tubule thus increasing the rate of organic cation/proton exchange across the apical membrane. These findings suggest that factors previously referred to as diuretic factors may in fact :let primarily or secondarily as stimulants of organic cation excretion. Haemolymrh concentrations of TEA increased linearly with the concentration of TEA in the diet and declined rapidly upon transfer of the larvae to TEA-free diet. The rate of decline was reduced by slowing the metabolic rate or by the addition of cimetidine to a diet containing TEA. Although larvae tolerated high levels of TEA in the diet, mortality increased when TEA was combined with either quinidine or cimetidine. It is suggested that inhibition of TEA transport by cimetidine or quinidine results in prolonged exposure to higher levels of TEA in the haemolymph and a consequent increase in toxicity. Surprisingly, TEA flux and fluid secretion rate were both reduced in Malpighian tubules isolated from adult flies raised on TEA-enriched diet. This suggests that the high concentration of TEA in the diet produced a non-lethal yet deleterious effect on the Malpighian tubules of Drosophila. / Thesis / Master of Science (MS)

Organic cations (OCs) make up ~40% of all prescribed drugs and renal secretion plays a major role in clearing these (and other OCs), from the plasma. The active and rate-limiting step of renal OC secretion is mediated by luminal OC/H+ exchange, the molecular basis of which is suspected to involve two homologous transport proteins, Multidrug And Toxin Extruders 1&2-K (MATE1 and MATE2-K). This study has two aims to resolve outstanding issues dealing with the mechanism of MATE-mediated OC transport: (Aim 1) develop predictive models of ligand interaction with hMATE1; (Aim 2) establish the kinetic mechanism(s) of ligand interaction with MATE transporters and the extent to which inhibitory ligands serve as transported substrates of MATE transporters. Transport was measured using human MATE1 and MATE2-K stably expressed in Chinese Hamster Ovary cells. Both MATEs had similar affinities for the prototypic OC substrate, 1-methyl-4-phenylpyridinium (MPP), and had overlapping selectivity for most of the test inhibitors. The IC50 values for 59 structurally diverse inhibitory ligands were used to generate a common features (HIPHOP) pharmacophore and three quantitative pharmacophores for hMATE1 (each displaying a significant correlation between predicted and measured IC50 values). The models identified (i) structural features that influence ligand interaction with hMATE1, including hydrophobic regions, H-bond donor and acceptor sites and an ionizable feature; and (ii) novel high affinity inhibitors of MATE-mediated transport from 13 new drug classes. Whereas metformin and creatinine were shown to be competitive inhibitors of MPP, the inhibition of MATE1-mediated MPP transport produced by pyrimethamine (PYR) and related analogs was not competitive but, instead, had a "linear, mixed-type" inhibitory profile suggestive of a MATE binding surface rather than a singular binding site. "Competitive exchange diffusion" showed that selected inhibitory ligands (including quinidine, caffeine, and the organic anion, PAH) also serve as transported substrates for MATE1. In conclusion, these data are consistent with the presence of a MATE binding surface with multiple, non-overlapping binding sites that can display different kinetic interactions with structurally distinct substrates. The creation of hMATE1 pharmacophores offers insight into development and interpretation of predictive models of drug-drug interaction in the kidney.

L'objectif général de ce projet est d'apporter des données pertinentes sur le devenir de produits pharmaceutiques dans l'environnement, en particulier aux interfaces eau/sol, afin de mieux suivre les conséquences de leurs utilisations et de leurs rejets. A long terme, les enjeux concernent la protection de l'environnement et de la santé publique. Ces travaux portent sur la caractérisation des propriétés de rétention de cinq médicaments dans des sols calcaires de la région Champagne-Ardenne afin d'évaluer notamment leur potentiel transfert vers les milieux aquatiques. Différents paramètres ont été identifiés comme ayant une influence sur leur rétention. La rétention de l'enrofloxacine dépend fortement du pH et de la force ionique, celle du diclofénac des teneurs en CaCO3 et en matière organique des sols qui ont un effet antagoniste sur son adsorption, celle de la carbamazépine et du sulfaméthoxazole est faible sur un sol calcaire, et enfin la rétention du iopamidol est négligeable. Par ailleurs, l'influence des cations métalliques, polluants ubiquistes des sols, sur la rétention des médicaments a également été étudiée. Les expériences de co-adsorption ont montré que la présence de cuivre et de zinc influence significativement la rétention de l'enrofloxacine, conduisant à une augmentation des quantités adsorbées sur le sol via la formation d'un complexe ternaire de surface, et met ainsi en évidence l'importance de prendre en compte l'interaction des médicaments avec les métaux pour une meilleure compréhension de leur comportement dans les sols. Toutefois, aucune influence notable de la présence de cuivre sur la rétention des autres produits pharmaceutiques n'a été observée. / The overall objective of this project is to provide a better knowledge of pharmaceuticals fate in the environment, more particularly at water/soil interfaces, in order to follow the consequences of their use and disposal. The long-term issues concern the protection of the environment and public health. This work focuses on the characterization of retention properties of five pharmaceuticals in calcareous soils of the Champagne-Ardenne region in order to evaluate their potential transfer to water compartments. Various parameters having an influence on their retention were identified. The retention of enrofloxacin is highly affected by pH and ionic strength, diclofenac retention by CaCO3 and organic matter contents of soils which have antagonistic effect on its adsorption, the retention of sulfamethoxazole and carbamazepine is low on a calcareous soil, and iopamidol adsorption is negligible. Moreover, the influence of metal cations, ubiquitous pollutants in soils, on pharmaceuticals retention was also studied. Co-adsorption experiments indicated that the presence of copper and zinc modifies significantly enrofloxacin retention, leading to an increase of adsorbed amounts on the soil via the formation of a ternary surface complex, thus highlighting the importance to take into account the interaction between metals and pharmaceuticals for a better understanding of their behavior in soils. However, no noticeable impact of the presence of copper on other pharmaceuticals retention has been noticed.

Ion pumps establish homeostasis across the membranes of living cells. Hearing is a mechanotransduction event that takes place in a closed compartment containing a fluid high in K+ concentrations. In Drosophila melanogaster, this closed compartment is formed by a scolopale cell that wraps around the dendrite of sensory neurons. The receptor lymph is maintained by the scolopale cell. The lumenal membrane of the scolopale cell is the wall of the compartment containing the receptor lymph, the scolopale space. The ablumenal membrane of the scolopale cell creates the border of the scolopidium. The Na/K pump is located on the ablumenal membrane of the scolopale cell, bringing K+ into the scolopale cell cytoplasm and extruding K electrogenically (Roy et al, 2013). We explored other primary and secondary ion pumps that are involved in creating a K+-rich lumen in the Malpighian tubule (Day et al, 2008; Rodan et al, 2012). We used RNAi technology to knockdown one gene at a time and electrophysiology to measure a sound evoked potential (SEP) that reflects the fly’s ability to hear. We found that knocking down V-ATPase, a proton pump, subunits involved in proton extrusion significantly reduces the SEP of knockdown flies. The involvement of cation chloride cotransporters (CCCs) and cation proton antiporter (CPAs), both secondary ion pumps that use the gradients created by the Na/K pump and V-ATPase respectively to pump other ions up their gradient, is less clear. We found that knocking down Nhe3, a CPA, significantly reduced the SEP when knocked down in the scolopale cell, suggesting it as a partner to the V-ATPase. Knocking down CG31547, a CCC, statistically increased the SEP, possibly a type1 statistical error.

Although attractive interactions between π systems (π-π interaction) have been known for many years, understanding of its origin is still incomplete. Quantitative measuring of π-stacking is challenging due to the weak nature of the π-π interaction. This dissertation aims at elucidating a quantitative conformational analysis by NMR ring current anisotropy of an organic compound capable of intramolecular π-stacking in solution and studying charge effects on the stacking of π-systems. This dissertation offers four contributions to the area. (1) A general approach to four-state, conformational analysis based on the magnetic anisotropy of molecules undergoing fast dynamic exchange is described. (2) Study unveiled the importance of charges in the conformation of a dication in the solution. (3) Novel aromatic salt pairs of triangulene derivatives with the delocalized cation-anion interaction were synthesized and studied. (4) Study unveiled ionic π-systems preferred face-to-face stacking due to strong cation-π and anion-cation attractions. A general protocol for the application of magnetic anisotropy to quantitative multi-state conformational analysis of molecules undergoing fast conformational exchange was suggested in the current study. The reliability of this method of conformational analysis was checked by the mass balance. VT-NMR was also conducted to study the enthalpic parameters. This technique can be further used to study canonical interactions such as ion pairing, hydrogen boning, and molecular recognition. In the current study, dependence of the probe conformations on the dispersive interactions at the aromatic edges between solvent and probes was tested by conformational distributions of the fluorinated derivatives (2b and 2c) of the probe molecule (1a). Solution and solid studies of these molecules put the previous conclusion drawn by the Cammers group in question. Current studies show that the dispersive interaction at the aromatic edge could not be the predominant force on the conformational changes in the probe molecule 1a during the fluoroalkanol perturbation. This study indicated that charges might be important in the formation of the folding conformations in the solution and solid state of 1a, 2b, and 2c. A contribution of this thesis was to prepare and study a conformational model that lacked charges. The previous molecules were charged. The solid-state structures of pyridinium-derived aromatic rings from the CSD (Cambridge Structural Database) were studied to investigate the π-π interaction between cationic π-systems in solid state. Novel aromatic salt pairs of triangulene derivatives with the delocalized cation-anion interaction were synthesized to study the π-π interaction between two aromatic rings that carried opposite charges. This study showed that the interaction between ionic π-systems can be enhanced by cation-π and anion-cation attractions. The stackings of these π-systems introduce more overlap, closer packing and stronger atomic contact than that of the solid states of comparable neutral species. Cation-π and anion-cation attractions are synergistic in aromatic salts.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (p. 73-82).

L’AE1 (SLC4A1) est un échangeur chlorure/bicarbonate. Cette protéine est la protéine membranaire la plus abondante à la surface des globules rouges des vertébrés. Elle est participe au transport du CO2 et à l’ancrage du cytosquelette à la membrane plasmique. Des mutations ponctuelles dans la partie membranaire de l’AE1, liées à des pathologies humaines, convertissent l’échange électroneutre en voie de conductance pour le sodium et le potassium ou induisent une fuite de cations dans un échangeur d’anions toujours fonctionnel. Les déterminants moléculaires qui induisent les mouvements d’ions au travers de cet échangeur sont encore inconnus. Le travail présenté a eu pour but d’identifier et de cartographier le site de transport de la protéine normale ou « pathologique ». Nous avons adapté à l’AE1 des outils basés sur la chimie des sulfhydriles capable de donner des informations sur le rôle d’acides aminés dans le site de transport de la protéine. Cette stratégie combinée à l’élaboration d’un modèle tridimensionnel de la protéine in silico basé sur le symporteur uracile/proton nous a permis de définir le site de transport de l’AE1. Nos résultats démontrent qu’un site de transport unique dans l’AE1 peut basculer entre 3 conformations différentes : échange chlorure/bicarbonate, fuite de cation et échange anionique ou fuite de cation uniquement. Ce site met en jeu les segments transmembranaires (TM) 3, 5 et 8 ainsi qu’une boucle intracellulaire très conservée située entre les TM 8 et 9. Le site de transport se structure autour des acides aminés L468, F471, L530, I533 et L673 se terminant au niveau du E681. La boucle intracellulaire 690 à 705 agissant comme un filtre à cations. / AE1 (SLC4A1, band 3) is a member of the SLC4 bicarbonate transporter family. This protein is the most abundant membrane protein on the surface of vertebrate red blood cells. The AE1 exchanges chloride and bicarbonate ions in physiological conditions. In red blood cells, it is essential to many tasks including CO2 transport and cytoskeleton anchoring in the plasma membrane. Point mutations in the membrane spanning domain of AE1 convert the electroneutral exchange into a conductance for sodium and potassium cations or induce a cation leak in a still functional anionic exchanger.The molecular determinants that induce the movement of ions through the exchanger are still unknown. This work aims at identifying and mapping the transport site of AE1 protein in normal and pathological conditions. We modified a sulfhydryl-based chemistry to AE1. This provided information on the role of amino acids in the transport site of the protein. This strategy combined with the development of a three-dimensional model of the protein in silico, based on the uracil/proton symporter, allowed us to define the transport site of AE1. Analysis of our results showed that a single transport site in AE1 can switch between three different conformations depending on protein mutation: classical chloride/bicarbonate exchange, cation leak and anion exchange, cation leak only. The transport site involves the transmembrane segments 3, 5 and 8 and a highly conserved intracellular loop between transmembrane segments 8 and 9. The transport site is centered around the amino acids L468, F471, L530, L673, I533 and ends at glutamic acid 681. The intracellular loop 690-705 acts as a cation filter.

Lanthanide-based nanoparticles can be used in a variety of applications, including biomedical work such as imaging and cancer therapies, and in solar cells. This thesis presents two different potential applications for lanthanide-based nanoparticles and a possible new method for single nanoparticle analysis. Each of the projects presented in this thesis starts from the colloidal synthesis of the nanoparticles and then explores their varying properties, such as size and size distribution, crystallinity, elemental composition, and optical properties. Chapter 1 presents a short introduction to lanthanides and explores their ability to luminesce and upconvert. These optical properties make lanthanide-based nanoparticles attractive in both the visible and near-infrared (NIR) range. Chapter 2 explores the possibility of using β-LaF3:Nd3+ (5%) nanoparticles in a colloidal laser to overcome some issues that solid state lasers face due to thermal effects. A colloidal laser requires small nanoparticles that can emit a useful wavelength and that are dispersed in a high boiling point liquid. In Chapter 3, a cation exchange of ytterbium for yttrium and erbium in water-dispersible β-NaYF4:Er3+ nanoparticles across a polyvinylpyrrolidone (PVP) surface coating was tested as a possible synthesis route for radioactive nanoparticles. Incorporating radioactive materials at the end of a therapy preparation would limit the number of synthesis steps in an isotope laboratory. Chapter 4 presents single-particle analysis of β-NaYF4:Er3+ (50%) nanoparticles using X-ray absorption spectroscopy (XAS) at the Canadian Light Source (CLS). Electron beams in scanning electron transmission microscopes (STEM) can damage the samples, making quantification of nanoparticles challenging. Finally, Chapter 5 discusses some conclusions and suggests possible future work. / Graduate / 0494

Tourmaline is an aluminoborocyclosilicate mineral with a complex arrangement of atoms. With highly variable chemistry and multiple cation sites, tourmaline is one of the last complex minerals whose structure was unraveled, and its response to changes in Pressure-Temperature-Time (P-T-X) are not well understood. Due to its stability at high temperature and pressure, tourmaline has the potential to be an informative mineral in terms of petrogenetic indicators and could be used in assessing provenance, thermobarometry and geochronology. Three reactions were proposed to understand the cation exchange and disordering between the Y- and Z-sites in the tourmaline structure. These reactions include: 1. YFe2+ + ZAl + OH ; ZFe3+ + YAl + O + H ; in two samples with varying Fe2+ content. 2. YMg + ZAl ; ZMg + YAl. 3. YFe3+ + ZAl ; ZFe3+ + YAl. Using single crystal X-ray diffraction and stepwise heating, the extent and effect of the exchange between the Y- and Z-sites in response to changes in temperature was described. In response to increased temperature, equivalent amounts of Fe2+, Fe3+, Mg2+ of the Y-sites exchange with Al of the Z-sites. This leads to decreases in Y-site average bond length, increases in Z-site average bond length, shortening of a lattice parameters, lengthening of c lattice parameters and decreases in quadratic elongation. Additionally, the T-site experienced an increased in tetrahedral rotation and ditrigonality and changes to the crimping of the tetrahedral ring upon heating. The cation exchange and disordering in these samples relates to the stability of tourmaline at elevated temperatures in that tourmaline will undergo cation exchange and disordering to maintain the stability of the mineral. This has implications on the conditions in which tourmaline is formed as well as stability of tourmaline and other minerals and materials in different P-T-X conditions.