"September 2002" / Includes as appendix: a list of publications by the author arising from this work; and, copies of some published journal articles / Includes bibliographical references. / [12], 209 leaves, [35] pages : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Describes the synthesis, properties and reactions of transition metal complexes containing poly-ynyl ligands / Thesis (Ph.D.)--University of Adelaide, Dept. of Chemistry, 2002

Ce travail de thèse porte sur l'étude des excitations élémentaires de la matière dans des nanoparticules de ZnO: phonons et excitons.<br /> Nous montrons que les modes de vibration permettent d'illustrer des effets de surface, lesquels sont de deux types. Premièrement vient la réduction de la taille. L'élasticité linéaire ne décrit plus correctement les modes propres acoustiques pour des tailles inférieures à 2.5 nm. Les expériences que nous avons menées sont en accord avec ce point. Deuxièmement, nous montrons que les ligands qui habillent les nanoparticules sont préférentiellement localisés sur la surface latérale de ces dernières; et qu'ils étirent légèrement la maille cristalline de ZnO.<br /> Concernant les propriétés excitoniques, l'exciton est piégé à la surface des nanoparticules. En outre, de part la dynamique de recombinaison très rapide, le processus de recombinaison de l'exciton est dominé par des mécanismes non-radiatifs.

Neuronal nicotinic acetylcholine receptors can have their function modulated by zinc. Depending on concentration and subunit composition, zinc either inhibits or potentiates receptor function. The zinc ion potentiates the alpha4beta4 receptor at non-agonist binding interfaces or "pseudo sites." Zinc potentiation is reduced if certain residues are mutated or spatially interfered with. The residue contributing most to this potentiation reduction effect is histidine 162 on the alpha4 subunit. The anthelmintic compound levamisole potentiates acetylcholine response of certain neuronal nicotinic receptors. Levamisole and its functional analogues morantel, oxantel, and pyrantel all were found to potentiate alpha4beta4 receptors at low (µM) concentrations and inhibit them at high (mM) concentrations. Oxantel showed the greatest degree of potentiation, about a third of the maximal zinc potentiation measured. Oxantel was screened using the substituted cysteine accessibility method (SCAM) against the residue histidine 162 as well as nearby alpha4 residues histidine 61 and glutamate 59 and the beta4 residue aspartate 195. Screening was carried out by mutating said residues into cysteine, followed by covalent linkage with a disulfide bridge of that residue with a methanethiosulfonate compound. SCAM experiments allowed testing of the effects of a single residue and the area immediately adjacent to it. Receptors that lost zinc potentiation capacity from site-directed mutagenesis at the his 162 residue and subsequent methanethiosulfonate reaction still showed regular potentiation following oxantel treatment. Although these compounds exhibit similar biphasic potentiation dose-response curves as zinc, their mechanism for potentiation is not through the same mechanism.

Infrared spectroscopy detects molecular vibrations and assesses the properties of molecules and their environment. It is a powerful technique to detect ligand induced changes in biomolecules as it has distinct signals and provides different levels of structural information. An addition of a dialysis accessory to attenuated total reflection infrared spectroscopy makes this technique more universal for ligand binding studies. It facilitates to study ligand binding of substrates, activators, inhibitors and ions to macromolecules as well as effect of pH, ionic strength or denaturants on the structure of macromolecules, which play an important role in drug development. This method was tested with two proteins cyt c and calcium ATPase. We studied phosphoenol pyruvate (PEP) in different ionization states by infrared spectroscopy combined with theoretical analysis. Theoretical calculations helped to assign the bands. The infrared spectrum of labeled PEP and infrared measurement in D2O also helped in band assignment. We used the method dialysis accessory to attenuated total reflection infrared spectroscopy to investigate the binding of PEP and Mg2+ to pyruvate kinase (PK), where conformational changes of PK were revealed upon binding of PEP and Mg2+. Isotopic labeled PEP helped to assign and evaluate the infrared absorption bands. The difference spectrum of bound and free PEP indicates specific interactions between ligand and protein. The quantitative evaluation revealed that the enzyme environment has little influence on the P-O bond strengths, which are weakened by less than 3% upon binding. The carboxylate absorption bands indicate shortening of the C-O bond by as little as 1.3 pm. The binding of PEP to PK in presence of monovalent cations K+ and Na+ showed that the binding interactions are very similar. / doctoral

This thesis describes the development and use ofnon-immunoglobulin affinity proteins denoted affibodies asalternatives to antibodies in different immunotechnologyapplications. A 58 aa IgG Fc binding three-helix bundle domainZ, derived from staphylococcal protein A has been used asframework for library constructions, in which the face of themolecule involved in the native binding activity has beenengineered by combinatorial protein engineering. Recruting 13surface-located positions for simultanenous substitutionmutagenesis, using degenerated oligonucleotides for libraryassembly at the genetic level, two libraries differing in thechoice of codons were constructed to serve as general sourcesof novel affinity proteins. The libraries were adapted fordisplay onE. colifilamentous phage particles allowingin vitroselection of desired variants capable ofbinding a given target molecule. In selections using human IgAas target, several new IgA specific affibodies could beidentified. One variant ZIgA1, was further investigated and showed binding toboth IgA1 and IgA2 human subclasses as well as to secretoryIgA. This variant was further demonstrated uesful as ligand inaffinity chromatography purification for recovery of IgA fromdifferent samples including unconditioned human plasma.Affibodies of different specificities were also fused to otherprotein domains to construct fusion proteins of relevance forimmunotechnology applications. Using Fc of human IgG as genefusion partner, "artificial antbodies" could be produced inE. colias homodimeic proteins, where the antigenbinding was confered by N-terminally positioned affibodymoieties of different valencies. One area of application forthis type of constructs was demonstrated through specificdetection of the target protein by Western blotting. Exploitingthe uncomplicated structure of affibody affinity proteins, genefusions between affibodies and the homotetrameric reporterenzyme β-galactosidase were constructed, which could beproduced as soluble proteins intracellularly inE. coli. The potential use of such recombinantimmunoconjugates in immunotechnology was demonstrated in ELISAdot-blot and immunohistochemistry, where in the latter case IgAdepositions in the glomeruli of a human kidney biopsy could bespecfically detected with low background staining ofsurrounding tissues. In a novel format for sandwich ELISA, thepossible advantage of the bacterial origin of the affibodyclass of affinity proteins was investigated. As a means tocircumvent problems associated with the presence of humanheterophilic antibodies in serum, causing bakground signals dueto analyte-independent crosslinking of standard capture anddetection antibody reagents, assay formats based oncombinations of antibody and affibody reagents for capture anddetection were investigated and found to be of potentialuse. <b>Keywords:</b>phage display, combinatorial, affinity, IgAligand, immunohistochemistry, affibody-fusions

Dissolution dynamic nuclear polarization (DNP) provides several orders of magnitude of NMR signal enhancement by converting the much larger electron spin polarization to nuclear spin polarization. Polarization occurs at low temperature (1.4K) and is followed by quickly dissolving the sample for room temperature NMR detection. DNP is generally applicable to almost any small molecules and can polarize various nuclei including 1H, 19F and 13C. The large signal from DNP enhancement reduces the limit of detection to micromolar or sub-micromolar concentration in a single scan. Since DNP enhancement often provides the only source for the observable signal, it enables tracking of the polarization flow. Therefore, DNP is ideal for studying chemical processes. Here, quantitative tools are developed to separate kinetics and spin relaxation, as well as to obtain structural information from these measurements. Techniques needed for analyzing DNP polarized sample are different from those used in conventional NMR because a large, yet non-renewable hyperpolarization is available. Using small flip angle pulse excitation, the hyperpolarization can still be divided into multiple scans. Based on this principle, a scheme is presented that allows reconstruction of indirect spectral dimensions similarly to conventional 2D NMR. Additionally, small flip angle pulses can be used to obtain a succession of scans separated in time. A model describing the combined effects of the evolution of a chemical process and of spin-lattice relaxation is shown. Applied to a Diels-Alder reaction, it permitted measuring kinetics along with the effects of auto- and cross-relaxation. DNP polarization of small molecules also shows significant promise for studying protein-ligand interaction. The binding of fluorinated ligands to the protease trypsin was studied through the observation of various NMR parameter changes, such as line width, signal intensity and chemical shift of the ligands. Intermolecular polarization transfer from hyperpolarized ligand to protein can further provide information about the binding pocket of the protein. As an alternative to direct observation of protein signal, a model is presented to describe a two-step intermolecular polarization transfer between competitively binding ligands mediated through the common binding pocket of the protein. The solutions of this model relate the evolution of signal intensities to the intermolecular cross relaxation rates, which depend on individual distances in the binding epitope. In summary, DNP provides incomparable sensitivity, speed and selectivity to NMR. Quantitative models such as those discussed here enable taking full advantage of these benefits for the study of chemical processes.

The major pathway for O 2 binding to mammalian myoglobins (Mbs) and hemoglobins (Hbs) involves transient outward movements of the distal histidine (HisE7), which allows ligand migration into the distal portion of the heme pocket. This E7 gate pathway appears dominant in vertebrate Hbs and Mbs. However, a number of invertebrate globins, including the dimeric hemoglobin from the blood clam Scapharca inaequivalvis (ScHbI), have an inverted quaternary structure in which an EF:FE dimer interface appears to block the HisE7 gate. Another set of globins, including the mini-hemoglobin from the Nemertean sea worm Cerebratulus lacteus (CerHb), are missing the N-terminal A-helix, which results in an internal tunnel between the E- and H-helices. This apolar channel has been suggested to represent an alternative to the HisE7 gate pathway. To determine the roles of the E7 gate and alternative pathways, we have systematically examined the effects of mutations at the E7 position in ScHbI and CerHb and at 21 other positions along the polar channel in CerHb. As was observed for SwMb and HbA, there is a progressive decrease in the bimolecular rate constants for O 2 binding to ScHbI as the size of the amino acid at position E7 is increased from Ala to Trp. This pattern is unaffected when ScHbI is completely converted to the R- or high affinity quaternary state by the F97Y mutation or when the dimer interface is completely disrupted by the K30D mutation. In contrast, E7 mutations have little affect on the rates of ligand entry and escape in CerHb. Instead, ligands diffuse through the apolar channel between the E- and H-helices as judged by decreases in both overall association and dissociation rate constants and increases in the extent of geminate recombination when the channel is blocked by small to large amino acid mutations. In SwMb, these trends are only observed when the small to large mutations are constructed at or near the E7 gate or directly in the distal pocket where ligands are captured. Thus, it is clear that globins have evolved more than one pathway for rapid O 2 uptake and release.

Haemonchus contortus is a parasitic nematode that is controlled in large part by nematocidal drugs that target receptors of the parasitic nervous system. Hco-UNC-49 is a nematode GABA receptor that has a relatively low overall sequence homology to mammalian GABA receptors but is very similar to the UNC-49 receptor found in the free living nematode Caenorhabditis elegans. However, the nematode receptors do exhibit different sensitivities to GABA which may be linked to differences in the putative GABA binding domains. Mutational analysis conducted in this study identified at least one amino acid, positioned near the GABA binding domain, which may partially account for differences in nematode GABA sensitivity. In addition, positions reported to be crucial for GABA sensitivity in mammalian receptors also affect GABA sensitivity in Hco- UNC-49 suggesting that the GABA binding domains of the mammalian and nematode GABA receptors share some pharmacological similarities. However, there were some differences observed. For example, in mammalian GABAA receptors amino acids from both  and  subunits appear to be important for GABA sensitivity. For residues examined in this study, only those on the UNC-49B subunit, and not UNC-49C, appear important for GABA sensitivity. / UOIT

Metal dithiolenes [M(S2C2R2)n] have been studied for decades because of their interesting chemical and spectroscopic properties, which are related to the unusual electronic properties of the dithiolene ligand. The ligand-based reactivity of metal bisdithiolenes [M(S2C2R2)2] toward alkenes has been proposed for use in alkene purification schemes. According to the proposal, compounds Ni(S2C2R2)2 (R=CF3,CN) react with simple alkenes to form stable S,S-interligand adducts and the alkene can be released from the adduct by reduction. We showed that Ni(S2C2(CF3)2)2 reacts with ethylene and 1-hexene to form, preferentially, S,S-intraligand adducts, which rapidly decompose to inactive metal-containing materials and dihydrodithiins. However, the product selectivity can be significantly modified so that stable S,S-interligand adducts are obtained as dominant products by adding [Ni(S2C2(CF3)2)2]- to Ni(S2C2(CF3)2)2/alkene reaction mixtures. Mechanistic implications are discussed. Next, the reactions of Pt(S2C2(CF3)2)2 with 2,3-dimethyl-1,3-butadiene are addressed. Prior to our report, only symmetry-allowed S,S-interligand adducts had been observed as products in the reactions between conjugated dienes and metal bisdithiolenes. We discovered a novel mode of diene binding, where two dienes bind to one dithiolene ligand of Pt(S2C2(CF3)2)2, in an C,S-intraligand fashion, forming a new chiral bisthioether ligand. From bisdithiolenes, the focus shifts to new mixed-ligand molybdenum trisdithiolenes [Mo(S2C2(CF3)2)2(S2C6H4) and Mo(S2C6H4)2(S2C2(CF3)2)]. These complexes rapidly and cleanly bind ethylene, in an S,S-intraligand fashion, as predicted by MO arguments. The resulting intraligand adducts are sufficiently stable to be characterized, in contrast to the nickel bisdithiolene case. The metal-chelated dihydrobenzodithiin, formed upon ethylene addition, can be substituted with a variety of donor ligands, allowing access to new types of molybdenum dithiolenes. We have recently extended these studies to catalytic reactions: Mo(S2C2(CF3)2)2(S2C6H4) was used as a catalyst to form dihydrobenzodithiins from (S2C6H4)2 and a variety of alkenes, in the first example of dithiolene-based reactivity being exploited for carbon-heteroatom bond-forming catalysis. Finally, the synthesis, characterization and redox reactivity of a new Fe2Ni bis-double-decker complex is described, demonstrating for the first time a sandwich complex of a metal bisdithiolene with both NiS2C2 rings in an η5 π-donating mode. For the radical cation, experimental and computation evidence indicates that the lone electron is delocalized over the entire molecule.

Nuclear receptors (NR’s) comprise a large, ancient, superfamily of eukaryotic transcription factors that govern a wide range of metabolic, homeostatic, and developmental pathways, and which have been implicated in disease states including cancer, inflammation, and diabetes. The ability of NRs to activate or repress gene transcription is modulated through direct binding of small lipophilic ligands which induce conformational changes in their cognate receptor. These changes are structural in nature and lead to the recruitment of coactivator or corepressor complexes, ultimately regulating the expression of target genes to whose response elements NRs are bound. In Drosophila 18 NRs have been identified which have representative members belonging to each of the six major NR subfamilies, and which show a high degree of homology to their vertebrate counterparts. This fact, in addition to the power and ease of genetic manipulation, make Drosophila an excellent model system in which to study NR function. When I began my project, 17 of the 18 NRs in Drosophila were ‘orphan’ receptors for which no cognate ligand had been identified. As a first step in an effort to identify potential ligands for these 17 receptors I first set out to determine how, where and when nuclear receptors are regulated by small chemical ligands and/or their protein partners. In order to do so I contributed to developing a ‘ligand sensor’ system to visualize spatial activity patterns for each of the 18 Drosophila nuclear receptors in live, developing animals. This system is based upon transgenic lines that express the ligand binding domain of each Drosophila NR fused to the DNA-binding domain of yeast GAL4. When combined with a GAL4-responsive reporter gene, these fusion proteins show tissue- and stage-specific patterns of activation. Analysis using this system has revealed the stage and tissue specificity of NR activation for each of the fly NRs. The amnioserosa, yolk, midgut and fat body, which play major roles in lipid storage, metabolism and developmental timing, were identified as frequent sites of nuclear receptor activity. Dynamic changes in activation that are indicative of sweeping changes in ligand and/or co-factor production are also a prominent feature that has been revealed using this approach. In addition, I went on to characterize the ligand regulated function of a single Drosophila nuclear receptor, Ecdysone inducible protein 75 (E75). Previous work from our lab has demonstrated that E75 binds to heme, and that its function as a transcriptional repressor is regulated in vitro by binding of the small diatomic gases nitric oxide (NO) and carbon monoxide (CO) to its heme moiety. In an effort to validate and to further understand the in vivo relevance of E75 regulation by NO I used gain and loss of function transgenes, as well as tissues manipulated in culture to show that NO acts directly on the Drosophila nuclear receptor E75, reversing its ability to block the activity of its heterodimer partner Drosophila Hormone Receptor 3 (DHR3). By specifically focusing on the Drosophila larval ring gland, the principal endocrine organ responsible for the production of the metamorphosis-inducing hormone, ecdysone, I have shown that failure to produce NO and to inactivate E75 results in failure to recognize the signals that normally trigger metamorphosis.