Les systèmes d’efflux tripartite de type Resistance, Nodulation and cell-Division (RND) sont essentiels dans le maintien de phénotypes de résistance multidrogues et contre les métaux lourds dans nombreuses bactéries Gram-négatives. Le transport de ces composés toxiques hors de la cellule est permis par l’assemblage d’une protéine de type antiporteur cation/proton (unité RND) insérée dans la membrane interne, connectée à une protéine insérée dans la membrane externe, pour former un canal de sorti qui traverse l’entièreté de l’enveloppe cellulaire. Le troisième composant du système, la protéine de type membrane fusion protein (MFP) qui est aussi appelée periplasmic adaptor protein (PAP), est requis pour permettre l’assemblage de tout ce complexe à trois composants. Cependant, les MFPs sont supposées jouer un rôle important et actif dans le mécanisme d’efflux du substrat. Pour mieux comprendre le rôle des MFPs au sein des systèmes d’efflux de type RND, nous avons étudié les protéines ZneB (précédemment appelée HmxB) et SilB, les composants périplasmiques des systèmes ZneCBA et SilABC responsables de la résistance aux métaux lourds chez Cupriavidus metallidurans CH34. Nous avons identifié la spécificité de liaison au substrat de ces protéines, montrant leur capacité à fixer le zinc (ZneB), ou le cuivre et l’argent (SilB). De plus, nous avons résolu la structure cristalline de ZneB à une résolution de 2.8 Å dans la forme apo- et avec un ion zinc fixé. La structure de ZneB possède une architecture générale composée de quatre domaines caractéristiques des MFPs, et la présence du site de coordination au zinc dans une région très flexible à l’interface des domaines β-barrel et membrane proximal. Les modifications structurales que la protéine subit lors de la fixation du zinc on été observée dans le cristal mais aussi en solution, ce qui suggère un rôle actif des MFPs dans le mécanisme d’efflux des métaux, vraisemblablement via la fixation et le relargage de l’ion à l’antiporteur. Les études de sélectivité de transport des antiporteurs ZneA et SilA montre que ces dernières et leurs protéines périplasmiques respectives ont des affinités similaires pour les métaux lourds. De plus, les études de transport ont apportés des arguments en faveur de l’hypothèse de capture cytoplasmique du substrat par l’antiporteur, tandis que la capacité des protéines périplasmiques à fixer les métaux lourds a apporté des arguments en faveur de l’hypothèse de capture périplasmique du substrat par l’antiporteur. Les deux modes de capture pourraient en réalité coexister ; cependant, le débat autour du compartiment cellulaire de capture du substrat par l’antiporteur est complexe et requiert de plus amples efforts afin d’être cerné. / Tripartite resistance nodulation cell division (RND)-based efflux complexes are paramount for multidrug and heavy metal resistance in numerous Gram-negative bacteria. The transport of these toxic compounds out of the cell is driven by the inner membrane proton/substrate antiporter (RND protein) connected to an outer membrane protein to form an exit duct that spans the entire cell envelope. The third component, a membrane fusion protein (MFP) also called periplasmic adaptor protein, is required for the assembly of this complex. However, MFPs are also proposed to play an important active role in substrate efflux. To better understand the role of MFPs in RND-driven efflux systems, we studied ZneB (formerly HmxB) and SilB, the MFP components of the ZneCAB and SilABC heavy metal RND-driven efflux complexes from Cupriavidus metallidurans CH34. We have identified the substrate binding specificity of the proteins, showing their ability to selectively bind zinc (ZneB), or copper and silver cations (SilB). Moreover, we have solved the crystal structure of the apo- and the metal-bound forms of ZneB to 2.8 Å resolution. The structure of ZneB displays a general architecture composed of four domains characteristic of MFPs, and it reveals the metal coordination site at the very flexible interface between the β-barrel and the membrane proximal domains. Structural modifications of the protein upon zinc binding were observed in both the crystal structure and in solution, suggesting an active role of MFPs in substrate efflux possibly through binding and release. The selectivity assays of the antiporter proteins ZneA and SilA demonstrated similar specificities in relation to their cognate MFPs toward heavy metal cations. Moreover, antiporter transport assays provide evidence for cytoplasmic substrate capture by this protein, whereas MFP substrate binding provides evidence for periplasmic substrate capture. Therefore, both modes of capture might co-exist; nevertheless, the substrate capture issue is a complex topic still needing consequent efforts to understand it.

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Protein based contrast agents (ProCAs) developed by the Yang lab exhibit unique capabilities in enabling magnetic resonance imaging (MRI) with significantly improved sensitivity and targeting capabilities by utilizing biomarkers which can target various carcinomas in animals. Further clinical in vivo human applications require modifications of these designed contrast agents to further improve organ and tissue biodistribution, biomarker and cell targeting capabilities, and reduction of immunogenicity. The aim of this thesis is to develop a novel protein modification on ProCA by glycosylation to improve liver distribution by targeting liver receptor, asialoglycoprotein receptor (ASGPR). Rat and humanized first generation and humanized third generation ProCA were expressed and purified using either glutathione s-transferase (GST) tagged or taggless methods. Rat ProCA1, rProCA1, was then used to optimize glycan modification with glycosylation achieved at the highest level using a 100:1 molar ratio and three lysine residues. Similar to non-modified rProCA1 and PEGylated rProCA1, metal binding affinity of gadolinium for glycan modified rProCA1, Glyco-rProCA1, was found to be 9.49 x 10-12 M, and relaxivity was found to be greater than clinically available contrast agents with 34.08 and 42.67 mM-1s-1 for r1 and r2 respectively. Glycosylation of rProCA1 has significantly increased human serum stability, and we have achieved significant liver MRI enhancement via tail vein injection due to high ASGPR expression in the liver altering biodistribution of glycan modified ProCA, and we have also imaged uptake in the secretory glands. These biodistribution changes were noted by immunohistochemistry (IHC) staining which was found to stain liver sinusoid with spaces in between. The distribution to the liver was further confirmed via inductively coupled plasma optical emission spectrometry (ICP-OES) which shows Glyco-rProCA1 has significant uptake of gadolinium in the liver tissue. This study represents the first achievement of in vivo liver imaging by glycosylation using a lactose targeting moiety covalently bonded to protein contrast agents for MRI showing promise for future more specific targeting or whole body imaging capabilities.

Carbonic anhydrase (CA) is a well-studied, zinc dependent metalloenzyme that catalyzes hydrolysis of carbon dioxide to the bicarbonate ion. In the past, metal binding studies related to CA have continually relied on equilibrium dialysis measurements to ascertain an extremely strong association constant (Ka= approx. 1.2 x 1012) for Zn2+. However, new methodology has allowed us to collect data using isothermal titration calorimetry (ITC), which calls that number and the association constants for many other first row transition metal ions into question. Thermodynamic parameters associated with Zn2+, Cu2+, Ni2+, and Co2+ binding to apoCA are unraveled from a series of complex equilibria associated with the in vitro metal binding event. This in-depth analysis adds clarity to the complex ion chemistry associated with metal ion binding to carbonic anhydrase and validates thermochemical methods that accurately measure association constants and thermodynamic parameters for complex-ion and coordination chemistry observed in vitro. Additionally, the as-isolated and the reconstituted ZnCA and other metalsubstituted CAs were probed using X-ray absorption spectroscopy. Both X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) analyses indicate the metal centers in the reconstituted carbonic anhydrases offer new metal binding coordination sites that can be used as models to understand nonheme metal sites in vivo.

<P> Natural organic matter (NOM) on the surface of resuspended particles influences the partition of metal ions between free dissolved organic matter (DOM), and the particulate species. It also affects the size distribution of floes under agitation. Equilibrium between dissolved organic and particulate organic matter should, therefore, be an important indicator for predicting metal ion exchange. </p> <p> We examined the exchange of cadmium along a pH edge and compared aqueous, organic, and particulate forms of the metal ion in reactors containing either settled or resuspended sediments. A pH edge profile between 3 and 8 shows two regions of speciation: above and below pH 6.2, which corresponds to a 1 : 1, H : Cd exchange. At low pH, cadmium remains in the free form when sediments are settled, but, upon resuspension, about half of it transfers to the dissolved organic and particulate forms. On the other hand, at high pH, all cadmium is particulate in both cases. In both cases there is also a release of DOM centred at pH 6.2, followed by readsorption at higher pH values. </p> <p> The interesting finding is that cadmium binding on particles appears independent of the surge in DOM, as though cadmium is weakly bound to DOM and strongly bound to particles. We can assume that two types of organic matter exist, one dissolved, one particulate. </p> <p> To fix size distribution, the sediment/metal ion mixture was put in a Couette flocculator and subjected to a uniform shear stress. Then, the partitioning of the metal ions between the solution and sediment phases was determined. A distribution coefficient, KD was calculated for each metal ion. A correlation between log KD and log K (hydrolysis constant) was apparent, and our data was consistent with the reported metal ion binding to both sediments and artificial surfaces. We envision that organic matter accumulating on the particle creates a polyelectrolyte domain responsible for greater binding capacity. We propose that an increase in metal ion binding with increasing particle size is due to this polyelectrolyte effect, where binding is governed by particle volume, and not surface area. </p> / Thesis / Master of Science (MSc)

This thesis describes the synthesis of functionalized spiroligomers and their applications in metal binding, metal-mediated catalysis, and organocatalysis. By synthesizing a family of functionalized bis-amino acids achieved from reductive alkylation, the Schafmeister group has developed access to highly functionalized and shape programmable structures named “spiroligomers.” The rigid backbones of spiroligomers are good at organizing the orientations of functional groups on their side chains. This property enables them as promising candidates for catalysts. Firstly we synthesized a few spiroligomer dimers presenting metal-binding groups such as terpys and bipys. With the right orientation of metal binding groups controlled by adjusting the stereocenter of the spiroligomer, macrocyclic “square” complexes with metals were obtained. The crystal structures of these intriguing complexes were solved. This work rendered the first structurally, spectroscopically and electronically characterized metal-spiroligomer complexes as well as the first crystal structure of spiroligomer. Secondly, the question of whether metal-binding spiroligomers are able to catalyze certain reactions became our major concern. We developed a binuclear copper catalyst that could accelerate a phosphate ester rearrangement, and that demonstrated that when the two copper binding terpyridine groups were best able to approach each other, they accelerated the rearrangement more than 1,000 times faster than the background reaction. Other molecules that did not properly organize the two copper atoms demonstrate considerably slower reaction rates. At last, catalysts based on spiroligomers without metals are also of interests. By displaying two hydrophobic groups in various directions on a monomeric spiroligomer (also can be regarded as a proline derivative), we observed variable activities and enantioselectivities in the catalysis of asymmetric Michael addition (up to 94% ee at -40 °C for one organocatalyst). / Chemistry

This thesis focuses on development of new regents which are suitable for recovering nickel, cobalt and copper from laterite leach solutions, specifically focusing on reagent requirements for novel base metal flowsheets developed by Anglo American. The work aims to design reagents which can extract nickel(II), cobalt(II) and copper(II) from a highly acidic aqueous sulfate solutions whilst showing selectivity over iron(II) and iron(III). Chapter 1 reviews current extractive metallurgy processes for separating and concentrating metals in laterite ores and describes new flowsheets proposed by Anglo American. Chapter 2 considers whether single reagent molecules with sets of tridentate donor atoms can generate sufficiently stable nickel(II) complexes to allow selective extraction of nickel from an aqueous sulfate solution. The salicylaldimines, 3-X-4-alkyl-6-(quinolin-8-imino)phenol, 3-X-4-alkyl-6-(2- methoxyphenylimino)phenol and 3-X-4-alkyl-6-(2-thiomethoxyphenylimino)phenol (alkyl = tert-butyl or tert-octyl; X = H, Br or NO2), were selected for study. The synthesis and characterisation of these proligands and their nickel(II) complexes are reported. XRD structures of Br-substituted salicylaldimines and their nickel(II) complexes are compared and discussed. The 4-tert-octylsalicylaldimines were used to extract nickel(II) from an aqueous sulfate solution with a pH > 2.8 and 3- nitro-4-tert-octyl-6-(quinolin-8-imino)phenol was found to be the strongest extractant in the series with a pH0.5 of 3.5. Computational studies of an analogous series of salicylaldimine proligands in the gas phase calculated the formation energies of their nickel(II) complexes and the predicted trend follows the experimentally determined solvent extraction results. Chapter 3 investigates modifications to phenolic pyrazoles, which are known copper(II) extractants. A series of 6-X-4-methyl-2-(5-alkyl-1H-pyrazol-3-yl)-phenols (X = H, OMe, Br and NO2) was synthesised and characterised. Varying the 6-X-substituent of the phenolic pyrazole altered the strength of copper extraction and 6-nitro-4-methyl-2-(5-(1,3,5-tri-methyl-pentyl)-1H-pyrazol-3-yl)-phenol was found to be the strongest extractant in the series. Analysis of XRD structures of related phenolic pyrazoles and their copper(II) complexes showed evidence of inter- and intra-molecular hydrogen bonding. Computational DFT studies in the gas phase were carried out to calculate the formation energies of analogous phenolic pyrazole copper complexes. The predicted order of these energies followed the same trend shown by experimental solvent extraction studies. The double deprotonation of 4-tert-butyl-(pyrazol-3-yl)-phenol at high pH forms a polynuclear complex in the organic phase with a copper(II) to ligand ratio of 1:1, thereby increasing the mass transport efficiency of copper by the reagent. The synthesis and characterisation of the [Cu16(4-tert-butyl-(pyrazol-3-yl)- phenolate)16(EtOH)4(H2O)2] wheel complex was carried out to demonstrate how such polynuclear copper(II) complexes could be formed under solvent extraction conditions. Chapter 4 explores the solvent extraction of nickel(II) and cobalt(II) by novel combinations of neutral nitrogen-donor heterocyclic ligands with organic acids, such as dinonylnaphthelenesulfonic acid (DNNSAH). The synthesis and characterisation of 2,6-bis(5-alkyl-1H-pyrazol-3-yl)-pyridine, 2-(5- alkyl-1H-pyrazol-3-yl)-pyridine and 5,5'-alkyl-3,3'-bi-1H-pyrazole (alkyl = tert-butyl or nonyl) and their nickel(II) complexes were reported. Also reported are synthesis and 6-N-alkyl-2-(2-pyridinyl)- benzothiazole (alkyl = n-butyl or n-decyl) and 2-(1-Isopropyl-benzimidazol-2-yl)-pyridine. The extraction of nickel(II) from highly acidic mixed metal aqueous sulfate solutions by some of these ligands was studied. These synergistic mixtures demonstrated remarkable strength and selectivity for nickel(II), and cobalt(II) over iron(II). XRD structures of nickel(II) complexes of 2,6-bis(5-tert-butyl- 1H-pyrazol-3-yl)-pyridine, 2-(5-tert-butyl-1H-pyrazol-3-yl)-pyridine and 5,5'-tert-butyl-3,3'-bi-1Hpyrazole with sulfonates or perchlorates as ion-pairs have intermolecular hydrogen bonding interactions between the inner-sphere ligands and the counterions.

Chitosan films are an excellent media for binding metal ions due to the electrostatic nature of the chitosan molecules. Addition of cross-linking or plasticizing agents alters texture of the films, but their effect on metal-binding capacity has not been fully characterized. The objective of this research was to determine effects of plasticizers and cross-linkers on physical and metal-binding properties of chitosan films and coatings prepared by casting and by spincoating. Chitosan films were prepared using 1% w/w chitosan in 1% acetic acid with or without (control) additives. Plasticizing agents were tetraethylene glycol (TEG) and glycerol while citric acid, ethylenediamine tetraacetic acid (EDTA), and tetraethylene glycol diacrylate (TEGDA) were used as cross-linkers. The additives were applied in concentrations of 0.10%, 0.25%, and 0.50% w/w of film-forming solution. The films were prepared by casting and by spincoating. Films were cast at ambient conditions for tests within one week (fresh films) and eight weeks (aged) after casting. The cast films were evaluated for thickness, residual moisture (by the Karl Fischer method), Cr(VI) binding capacity, puncture strength, and puncture deformation while the chitosan coatings were tested for thickness, Cr(VI) binding capacity, solubility in aqueous solution, and surface morphology (using atomic force microscopy). Cast films with cross-linkers showed an increase in resistance to puncture while plasticized films become more elastomeric. Control films bound 97.2% Cr(VI) ions from solution (0.56 mg Cr(VI)/g film), and addition of plasticizers did not affect chromium binding, tying up to 96.7% Cr(VI) ions from solution (0.56 mg Cr(VI)/g film). Films containing cross-linkers yielded binding capabilities ranging from 42.3% to 94.3% bound Cr(VI) ions (0.26-0.52 mg Cr(VI)/g film). Ultrathin coatings also possess the ability to bind Cr(VI) from solution, though only a maximum of 7.4% of Cr(VI) ions could be bound from solution, the thin films had the ability to bind up to 224 mg Cr(VI)/g ultrathin film. These coatings use less chitosan, but they display greater binding per mass. Overall, plasticizers do not alter, while cross-linkers may reduce, the binding capacity of chitosan films, but physical properties of the films can be controlled by inclusion of additives.