Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Lucie Sedlářová Supervisor: Dr. Přemysl Mladěnka, Ph.D. Title of thesis: Standardization of method for in vitro assessment of copper chelation. Cooper is a trace element, which is essential for many biochemical processes in the human body. Disruption of copper homeostasis can cause a variety of diseases. There are primarily two hereditary diseases associated with copper dyshomeostasis: Wilson's disease, which is linked with the excess of copper in the liver and other tissues, and Menkes disease, which is associated with a lack of systemic copper. In addition, local disturbances of copper play a role also in other diseases (neurodegenerative diseases, tumors, myocardial infarction). The main objective of this experimental work was the standardization of a simple but accurate method for rapid screening of copper chelators, which could be applied for the therapy of systemic or local copper excess in the future. The methodology is based on the spectrophotometric determination of copper in microplates using appropriate indicators, bathocuproindisulfonic acid disodium salt (BCS) and hematoxylin. This thesis has shown that the BCS methodology is able to determine chelation of cupric as...

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Pei, Congjian = 銅銦硒/硫和銅鋅錫硒/硫一維納米結構的合成與表徵 / 裴聰健. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 76-78). / Abstracts in English and Chinese. / Pei, Congjian = Tong yin xi/liu he tong xin xi xi/liu yi wei na mi jie gou de he cheng yu biao zheng / Pei Congjian. / Abstract --- p.i / 論文摘要 --- p.ii / Acknowledge: --- p.iii / Contents: --- p.iv / List of Figures: --- p.vii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Background --- p.3 / Chapter 2.1. --- Overview --- p.3 / Chapter 2.2 --- Methodology --- p.3 / Chapter 2.2.1 --- General growth strategies for synthesizing nanowires (NWs) --- p.3 / Chapter 2.2.2 --- Synthesis NWs from vapor phase (VLS mechanism) --- p.4 / Chapter 2.2.3 --- Synthesis NWs from solution phase --- p.7 / Chapter 2.2.4 --- Synthesis NWs assist by template --- p.8 / Chapter 2.3 --- Instrumentation --- p.10 / Chapter 2.3.1 --- XRD (X-Ray Diffraction) --- p.10 / Chapter 2.3.2 --- SEM (Scanning Electron Microscopy) --- p.11 / Chapter 2.3.3 --- TEM (Transmission Electron Microscopy) --- p.13 / Chapter Chapter 3 --- Synthesis and characterization of In2Se3 nanowires --- p.17 / Chapter 3.1 --- Overview: --- p.17 / Chapter 3.2 --- Experimental Section: --- p.17 / Chapter 3.3 --- Results and Discussions: --- p.19 / Chapter 3.3.1 --- Results of high temperature (~800oC) synthesized sample --- p.20 / Chapter 3.3.2 --- Results of the low temperature (~600oC) synthesized sample --- p.26 / Chapter 3.3.3 --- Results of thermal evaporate CuInSe2 source: --- p.30 / Chapter 3.4 --- Conclusion: --- p.31 / Chapter Chapter 4 --- Synthesis and Characterization of ID CuInSe2 and CuInS2 structures via template assist method --- p.33 / Chapter 4.1 --- Overview: --- p.33 / Chapter 4.2 --- Experimental: --- p.33 / Chapter 4.2.1 --- Fabrication of CuInSe2 nanowire arrays --- p.34 / Chapter 4.2.1 --- Fabrication of CuInS2 nanowire arrays --- p.35 / Chapter 4.3 --- CuInSe2 nanowire arrays: --- p.36 / Chapter 4.4 --- CuInS2 nanotube &nanowire array: --- p.42 / Chapter 4.5 --- Discussion of the formation mechanisms: --- p.45 / Chapter 4.6 --- Conclusion: --- p.50 / Chapter Chapter 5 --- Synthesis of ordered single-crystalline nanowires arrays of Cu2ZnSnS4 and Cu2ZnSnSe4 --- p.51 / Chapter 5.1 --- Overview: --- p.51 / Chapter 5.2 --- Experimental: --- p.52 / Chapter 5.3 --- Results and discussion: --- p.54 / Chapter 5.4 --- Discussion of the formation mechanisms: --- p.68 / Chapter 5.5 --- Conclusion: --- p.72 / Chapter Chapter 6 --- Summary --- p.73 / Reference: --- p.76

Copper toxicity was investigated using the green alga, Selenastrum capricornutum. Two major areas were investigated: the forms of copper which are toxic to growth of the alga and factors that can change the concentration of free copper, Cu2+ (aq), in the growth medium during the logarithmic growth of the organism. This research has shown that S. capricornutum is sensitive to the free Copper ion, Cu 2+ (aq), irrespective of the total copper concentration.

The research described in this thesis has led to an understanding of the geochemical conditions controlling the formation, paragenesis and distribution of oxide zoner copper species in the Eastern Fold Belt of the Mt. Isa Inlier. This area is also known as the Cloncurry Complex. The regional geology and genesis of the copper deposits is reviewed and the deposits of particular interest to the study are described. Oxidation of pyrite and chalcopyrite by oxygen-bearing groundwater and the sources and mechanisms by which anions are carried by groundwater to reaction sites to form secondary copper species are discussed. Physical and chemical conditions control the development of particular species. Equilibrium phase diagrams have been constructed to represent stability fields. An explanation for the relative abundance and spatial distribution of the basic copper phosphates is provided. Stability field data supported by observations made on deposits in the Cloncurry district and elsewhere provides a basis for assessing the paragenesis and distribution of secondary copper species in this and similar environments. This is discussed and illustrated using the Great Australia mine as a model / Master of Science (Hons)

Acid leaching of different natural chrysocolla samples under ambient conditions demonstrated that the chemical and chrystallographic inhomogenity significantly affects the rate and that it is virtually impossible to use the mineral as a model for the kinetics of hydrometallurgical processing of copper ores.Dissolution experiments, stability diagrams,and X-ray powder diffraction studies are undertaken and studied in this research and results are given.It is found that a complete solid solution series exists in Nature with most tungstenian wulfenites and molybdenum stolzites containing a range of compositions. The first occurrence of a molybdenum stolzite from Broken Hill, Australia, which also contains small amounts of chromium is noted.Raman microprobe spectroscopy has proven to be a very quick and effective non-destructive method for semi-quantitative analysis of the wulfenite-stolzite and powellite-scheelite solid-solution series and in detecting very small amounts of substituted chromate in wulfenite, stolzite and anglesite / Doctor of Philosophy (PhD)

Copper is one of the most important trace elements in ruminant nutrition. Its deficiency causes certain pathologies that can be cured by supplementation, by either five ("inorganic") or complexed ("chelated") forms. With the objective of being able to quantitatively compare the distribution of copper between the two forms of supplements, the speciation of copper in ruminant fluids was studied here. For this study, copper lysine supplement was used. It was necessary to first study the acid-base and complexation chemistry of lysine with copper (II). In addition, the complexation chemistry of glycine and histidine, were investigated. Mass balance equations were used to describe the distribution of copper (II) amongst different ligands. The results of the distribution of copper (II) ions in McDougall's solution (a simulated form of bovine saliva), indicate no significant differences in the distribution of copper using the different form of supplements. 1H NMR was used to validate the results from the computer model. Using a combination of the results from the saliva simulation model and the chemical shifts from the NMR studies, the chemical shift changes that would accompany the addition of copper (II) to McDougall's solution were predicted. Results from the models do not show any appreciable differences from experimental values. Rumen samples were collected. Important peaks in the 1H NMR spectrum were assigned. The spectrum indicated that acetic acid, resulting from the fermentation in the rumen, was a good probe for monitoring the speciation pattern. Speciation calculation indicated that the bulk of the copper would be bound to ammonia in the rumen. Changes in chemical shifts that result from the introduction of copper (II) to the rumen contents were predicted. Results were compared with experimental values. Agreement between the two sets of results was found to be satisfactory. The study shows that any advantages that result from the use of copper lysine supplement are not as a result of its remaining intact. Though metal bioavailabilities are hard to predict this approach could help better our understanding of this process. The methods developed here could be extended to other metal complexation problems in biological fluids. (Abstract shortened by UMI.)

<p>Copper was first released into the environment as the result of the mass generation of oxygen from photosynthetic bacteria roughly 2.7 billion years ago. While it proved to be poisonous to early life on Earth, those that met the evolutionary challenge utilized the metal as a cofactor in enzymes to perform biochemically significant functions, while controlling intracellular levels of copper with a sophisticated network of trafficking proteins. Proteins and enzymes that utilize copper as a cofactor have evolved significantly different coordination environments than copper trafficking proteins, as a result of the different functions they perform. Of particular interest was characterizing the unique Cu(I) binding events observed in some of these proteins, the extracellular N-terminal regions of eukaryotic high affinity copper transport proteins (Ctr), and the bacterial periplamsic CusF protein of the CusFBCA Cu(I)/Ag(I) efflux pathway. </p><p>Model peptides corresponding to the methionine rich binding motifs (Mets motifs) were characterized in terms of Cu(I) binding affinity, stoichiometry, and metal specificity, via an ascorbic acid oxidation assay and electrospray ionization mass spectrometry. Metal induced structural features and coordination environments were elucidated with NMR, CD and X-ray spectroscopy. A series of peptides was also examined to infer the relative Cu(I) binding affinities, and susceptibility to oxidation, of methionine, histidine, cysteine residues found in copper binding motifs. The resistance of Cu+ specific peptides to metal catalyzed oxidation is also described. Attempts were also made to model the Cu(I)/Ag(I) tryptophan cation-&#960; interaction observed in CusF.</p> / Dissertation

The purpose of this research was to identify and characterize novel molecular mechanisms in copper homeostasis. Pseudomonas putida KT2440 is a soil bacterium studied for its potential use in bioremediation of soils contaminated with aromatic organic contaminants. The cinAQ operon was analyzed. cinAQ is transcribed in presence of copper. The product of cinA is a periplasmic azurin-like protein with a methionine and histidine rich region, characterized by a high redox potential (456 ±4 mV). CinQ was shown to be a pyridine nucleotide-dependent nitrile oxidoreductase that catalyzes the reduction of preQ₀ to preQ₁, the first committed step in the biosynthetic pathway leading to the production of the unusual nucleotide queuosine. Gene disruption of cinQ in Pseudomonas putida KT2440 and in Pseudomonas aeruginosa PAO1 did not result in a significant increase in copper sensitivity on disk assays. Furthermore, a P. putida KT2440 cinA mutant also did not present a greater sensitivity to copper on disk assays while cinA mutants in Pseudomonas aeruginosa PAO1 presented increased toxicity to copper compared to the wild-type. CinA is by sequence similarity proposed to be an electron shuttle, and was shown to be upregulated in the presence of copper. Increasing CinA levels in the periplasm after copper stress may represent a mechanism used to regenerate the multicopper oxidase CopA (involved in Cu(I) to Cu(II) oxidation). Alternatively, CinA could act as an electron shuttle that takes part in an alternative electron transport chain once redox active copper is available, or it could represent a periplasmic copper chaperon. CinQ is involved in the biosynthesis of the rare hyper-modified nucleotide queuosine, found in the wobble position of several tRNAs, and required to avoid the readthrough of the stop codon UAG. Transcription of cinAQ was shown to be under the control of the two component system CinR-CinS. CinS is a histidine kinase, with a sensor domain located in the periplasm. CinR is the cognate response regulator that activates transcription of genes upon phosphorylation from CinS. The CinR-CinS two component system was shown to be responsive to 0.5 LM copper. CinS displayed very high metal specificity and elicited a response only in the presence of copper and silver, but not other metals. Modeling of the CinS protein structure, performed using Swiss Model and using the periplasmic sensor DcuS from Escherichia coli as a template, identified a potential copper binding site, containing H37 and H147. Sequence alignment of copper sensing histidine kinases further identified other conserved residues in the periplasmic domain. Site-Directed Mutagenesis was used to generate CinS mutants that were tested for their ability to activate the cinAQ promoter in presence of Cu. When challenged with copper CinS mutant H37R and H147R had an almost 10 fold reduction in copper sensitivity compared to the wild-type, indicating a possible role in Cu coordination. Other CinS mutants responded similarly to the wild-type in the presence of 10 μM of Cu.

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