Graduation date: 1994

Copper ions (Cu⁺, Cu²⁺) play important roles in many biological processes (i.e., oxidation, dioxygen transport, and electron transfer); many of the functions in these processes result from copper ions interacting with proteins and peptides. Previous studies using matrix assisted laser desorption/ionization (MALDI) mass spectrometry (MS) have shown that Cu⁺ ions preferentially bind to electron rich groups in gas phase (i.e., N-terminal amino group, the side-chains of lysine, histidine and arginine). For peptides with more than one Cu⁺ ligand, the interaction between Cu⁺ ions and ligands is described in terms of competitive binding; however, Cu⁺ coordination chemistry for multiple Cu⁺-containing proteins and peptides in gas phase is still not fully understood. In addition, no studies on the fragmentation chemistry for multiple Cu⁺-binding peptides, such as [M + 2Cu - H]⁺ ions, have been reported. The synthesized dinuclear copper complex (alpha-cyano-4-hydroxycinnamic acid (CHCA) copper salt (CHCA)₄Cu₂) enhances the ion abundances for [M + xCu - (x-1)H]⁺ (x = 1-6) ions in gas-phase when used as a MALDI matrix. Using this matrix we have investigated site-specific copper binding of several peptides using fragmentation chemistry of [M + Cu]⁺ and [M + 2Cu - H]⁺ ions. The fragmentation studies reveal that the binding of a single Cu⁺ ion and two Cu⁺ ions are different, and these differences are explained in terms of intramolecular interactions of the peptide-Cu ionic complex. The competitive Cu⁺ binding to C-terminus (i.e., amide, carboxyl, methyl ester) versus lysine, as well as cysteine (SH/SO₃H) versus arginine (guanidino), was also examined by MALDI MS and theoretical calculations (Density Functional Theory (DFT)). For example, results from theoretical and experimental (fragmentation reactions) studies on [M + Cu]⁺ and [M + 2Cu - H]⁺ ions suggest that cysteine side chains (SH/SO₃H) are important Cu⁺ ligands. Note that, the proton of the SH/SO₃H group is mobile and can be transferred to the arginine guanidino group. For [M + 2Cu - H]⁺ ions, deprotonation of the -SH/SO₃H group is energetically more favorable than that of the carboxyl group, and the resulting thiolate/sulfonate group plays an important role in the coordination structure of [M + 2Cu - H]⁺ ions.

O efeito bactericida de íons cobre é bem conhecido; no entanto a atividade inibitória depende diretamente da concentração desses íons no material base. Contudo, essa concentração deve ser controlada, pois o excesso destes íons pode ser tóxico e além disso, a implantação de cobre pode comprometer a resistência a corrosão do substrato metálico. A propriedade bactericida é desejável para aplicação em ligas metálicas empregadas em diversos setores, na assepsia de materiais que tenham contato direto com subprodutos que necessitam alta pureza e com baixíssimos índices de contaminação. O aço inoxidável austenítico AISI 304 é amplamente utilizado devido às suas propriedades mecânicas e de resistência à corrosão. Considerando isso, o aço AISI 304 é o foco do presente estudo, mesclando as características anticorrosivas naturais com a assepsia oligodinâmica proveniente da dopagem com íons de cobre. Nesse sentido, avaliou a resistência à corrosão do aço inoxidável AISI 304 com diferentes doses de cobre implantado (1015 íons.cm-2 e 1016 íons.cm-2) por monitoramento do potencial de circuito aberto e voltametria cíclica em uma solução eletrolítica de NaCl 3,5 % em peso. Através de simulações computacionais, previu-se que nos parâmetros escolhidos, a profundidade de implantação dos íons no substrato atingiu até 40 nm da superfície, com uma concentração de pico, maior teor de cobre, na profundidade de 12 nm. As amostras com doses de 1015 íons.cm-2 de cobre apresentaram o potencial de pites semelhante ao aço inoxidável austenítico AISI 304 sem implantação de Cu. Na análise de EDS observou-se que as áreas que não sofreram corrosão, apresentaram melhor distribuição dos elementos de liga e do cobre implantado em comparação com as regiões com maior incidência de pites. A maior intensidade de pites foi encontrada para a amostra com maior dose de cobre implantado (1016 íons.cm-2 de cobre). / The bactericidal effect of copper ions is well known; however, the inhibitory activity depends directly on the concentration of these ions in the base material. However, this concentration should be controlled because the excess of these ions can be toxic and the implantation of copper may compromise the corrosion resistance of the metal substrate. The bactericidal property is desirable for application in metal alloys used in the several sectors, in the asepsis of materials that have direct contact with by-products that require high purity and with very low contamination rates. The AISI 304 austenitic stainless steel is widely used because of its mechanical properties and corrosion resistance. Considering this, the AISI 304 is the focus of the present study, mixing the natural anticorrosive characteristics with the oligodynamic aseptic from the copper ions doping. The corrosion resistance of AISI 304 stainless steel with different doses of implanted copper (1015 ions.cm-2 and 1016 ions.cm-2) was evaluated by monitoring the open circuit potential and cyclic voltammetry in an electrolytic solution of NaCl 3,5% by weight. Through computational simulations, it was predicted that in the chosen parameters, the implantation depth of the ions in the substrate reached up to 40 nm from the surface, with a peak concentration, higher copper content, at the depth of 12 nm. The samples with doses of 1015 ions.cm-2 of copper had the pit potential similar to the austenitic stainless steel AISI 304 without implantation of Cu. In EDS analysis, it was observed that the areas that did not undergo corrosion had better distribution of the alloying elements and of the implanted copper compared to the regions with a higher incidence of pitting. The highest pitting intensity was found for the sample with the highest dose of implanted copper (1016 ions.cm-2 copper).

O efeito bactericida de íons cobre é bem conhecido; no entanto a atividade inibitória depende diretamente da concentração desses íons no material base. Contudo, essa concentração deve ser controlada, pois o excesso destes íons pode ser tóxico e além disso, a implantação de cobre pode comprometer a resistência a corrosão do substrato metálico. A propriedade bactericida é desejável para aplicação em ligas metálicas empregadas em diversos setores, na assepsia de materiais que tenham contato direto com subprodutos que necessitam alta pureza e com baixíssimos índices de contaminação. O aço inoxidável austenítico AISI 304 é amplamente utilizado devido às suas propriedades mecânicas e de resistência à corrosão. Considerando isso, o aço AISI 304 é o foco do presente estudo, mesclando as características anticorrosivas naturais com a assepsia oligodinâmica proveniente da dopagem com íons de cobre. Nesse sentido, avaliou a resistência à corrosão do aço inoxidável AISI 304 com diferentes doses de cobre implantado (1015 íons.cm-2 e 1016 íons.cm-2) por monitoramento do potencial de circuito aberto e voltametria cíclica em uma solução eletrolítica de NaCl 3,5 % em peso. Através de simulações computacionais, previu-se que nos parâmetros escolhidos, a profundidade de implantação dos íons no substrato atingiu até 40 nm da superfície, com uma concentração de pico, maior teor de cobre, na profundidade de 12 nm. As amostras com doses de 1015 íons.cm-2 de cobre apresentaram o potencial de pites semelhante ao aço inoxidável austenítico AISI 304 sem implantação de Cu. Na análise de EDS observou-se que as áreas que não sofreram corrosão, apresentaram melhor distribuição dos elementos de liga e do cobre implantado em comparação com as regiões com maior incidência de pites. A maior intensidade de pites foi encontrada para a amostra com maior dose de cobre implantado (1016 íons.cm-2 de cobre). / The bactericidal effect of copper ions is well known; however, the inhibitory activity depends directly on the concentration of these ions in the base material. However, this concentration should be controlled because the excess of these ions can be toxic and the implantation of copper may compromise the corrosion resistance of the metal substrate. The bactericidal property is desirable for application in metal alloys used in the several sectors, in the asepsis of materials that have direct contact with by-products that require high purity and with very low contamination rates. The AISI 304 austenitic stainless steel is widely used because of its mechanical properties and corrosion resistance. Considering this, the AISI 304 is the focus of the present study, mixing the natural anticorrosive characteristics with the oligodynamic aseptic from the copper ions doping. The corrosion resistance of AISI 304 stainless steel with different doses of implanted copper (1015 ions.cm-2 and 1016 ions.cm-2) was evaluated by monitoring the open circuit potential and cyclic voltammetry in an electrolytic solution of NaCl 3,5% by weight. Through computational simulations, it was predicted that in the chosen parameters, the implantation depth of the ions in the substrate reached up to 40 nm from the surface, with a peak concentration, higher copper content, at the depth of 12 nm. The samples with doses of 1015 ions.cm-2 of copper had the pit potential similar to the austenitic stainless steel AISI 304 without implantation of Cu. In EDS analysis, it was observed that the areas that did not undergo corrosion had better distribution of the alloying elements and of the implanted copper compared to the regions with a higher incidence of pitting. The highest pitting intensity was found for the sample with the highest dose of implanted copper (1016 ions.cm-2 copper).

No description available.

CopA, a thermophilic membrane ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu+ across cellular membranes. CopA contains at least two metal binding domains, a regulatory N-terminal Metal Binding Domain (N-MBD) and an occlusion/coordinating metal binding site in the 6th, 7th and 8th transmembrane segments. Previous studies showed that the presence of millimolar concentration of Cys is essential for CopA activity. The high affinity of CopA for metal in the presence of millimolar concentration of Cys suggests a multifaceted interaction of the enzyme with Cys. To elucidate the role of Cys, we studied its effect on the partial reactions of the catalytic cycle of CopA. We observed that 2-50 mM Cys accelerates enzyme turnover with little effect on the Cu+ affinity of CopA. Cys accelerates enzyme phosphorylation, but has no effect on the dephosphorylation rates. Thus, Cys increases steady state phosphoenzyme levels. Besides, Cys has no significant effect on E1¡ÃƒÂªE2 equilibrium. Similar results were observed in truncated CopA lacking the N-MBD suggesting that enzyme activation by Cys is independent of the regulatory metal binding sites. These results and the kinetic analysis of activation curves suggest that while Cu+ is delivered to the transport site as a Cu-Cys complex, Cys in the mM range stimulates the ATPase acting as a non-essential activator.

This diploma thesis deals with humic acid gels and their ability to bind metallic ions on their surface. In the thesis, there was studied the adsorption of copper ions on the provided gels. The gels were prepared by dissolving the humic acid in sodium hydroxide or sodium tripolyphosphate and then precipitated with hydrochloric acid or metal chlorides. Adsorption was studied at different concentrations of the copper (II) chloride and measured on UV-VIS spectrophotometer. It was proved from measuring that gels made from sodium tripolyphosphate are adsorbing less copper ions than the ones prepared by sodium hydroxide. Also, the copper ions are bound with smaller force on gels prepared with polyphosphate. Gels precipitated with magnesium chloride adsorb much more than gels precipitated with acid, thus in the case of gels sequestered metal ions extracted higher.

Στην εργασία αυτή προχωρήσαμε στη μελέτη της αλληλεπίδρασης των ιόντων δισθενούς χαλκού, Cu2+ με στατιστικά συμπολυμερή P(ANa-co-SSNa) του ακρυλικού οξέος, (ANa) με το στυρενοσουλφονικό οξύ (SSNa). Συντέθηκαν τα συμπολυμερή P(ANa50-co-SSNa) και P(AΝa80-co-SSNa) που περιέχουν 50 mol% και 80 mol% ακρυλικού οξέος αντίστοιχα. Τα συμπολυμερή αυτά χαρακτηρίστηκαν με φασματοσκοπία πυρηνικού μαγνητικού συντονισμού 1H NMR. Προχωρήσαμε στην φυσικοχημική μελέτη της αλληλεπίδρασης ιόντων δισθενούς χαλκού, Cu2+, με τα συμπολυμερή P(ANa50-co-SSNa) και P(AΝa80-co-SSNa) χρησιμοποιώντας διάφορες τεχνικές όπως η θολομετρία, η ιξωδομετρία και η φασματοσκοπία UV-vis. Τα πειράματα πραγματοποιήθηκαν σε υδατικά διαλύματα, στα οποία χρησιμοποιήθηκε ως διαλύτης τρισαπιονισμένο νερό, H2O-3D. Για λόγους σύγκρισης μελετήθηκαν επίσης τα αντίστοιχα ομοπολυμερή των μονομερών που αποτελούν τα συμπολυμερή P(ANa-co-SSNa). Έτσι, πραγματοποιήθηκε φυσικοχημική μελέτη της αλληλεπίδρασης των ιόντων Cu2+ με το πολυ(ακρυλικό νάτριο), PANa, και το πολυ(στυρενοσουλφονικό νάτριο), PSSNa, αντίστοιχα. Από την ιξωδομετρική μελέτη προέκυψε πως η εισαγωγή ιόντων χαλκού Cu2+ σε υδατικό διάλυμα πολυμερούς οδηγεί στην μείωση του ανηγμένου ιξώδους. Μάλιστα η μείωση αυτή είναι πιο δραστική καθώς αυξανεται η συγκέντρωση ιόντων Cu2+ στο διάλυμα. Αυτή η συμπεριφορά αποτελεί ένδειξη της συμπλοκοποίησης των ιόντων χαλκού, Cu2+, με τις ομάδες καρβοξυλίων του ακρυλικού οξέος (ΑΑ). Διαπιστώθηκε πως στα συμπολυμερή P(ANa-co-SSNa) τα Cu2+ συμπλοκοποιούνται με τα καρβοξύλια του ακρυλικού οξέος σε αναλογία ιόντων χαλκού προς ομάδες καρβοξυλίων ίση με 1:2. Όσον αφορά στην μελέτη με UV-vis, ενώ στην περίπτωση του ομοπολυμερούς PSSNa δεν υπάρχει κάποια ιδιαίτερη αλληλεπίδραση τύπου συμπλόκου ένταξης μεταξύ του πολυμερούς και των ιόντων του Cu2+, στην περίπτωση του συμπολυμερούς Ρ(ΑNa50-co-SSNa), παρατηρήθηκε μετατόπιση του σήματος των ιόντων Cu2+ (από τα 800 στα 745 nm) παρουσία του συμπολυμερούς. Αυτό είναι χαρακτηριστικό του σχηματισμού συμπλόκου ένταξης μεταξύ των ιόντων του Cu2+ και των ομάδων COO- του συμπολυμερούς. / In the present work we studied the interaction of bivalent copper ions Cu2+ with random copolymers P(ANa-co-SSNa) of acrylic acid (ANa) and styrenesulfonic acid (SSNa). Two copolymers, P(ANa50-co-SSNa) and P(AΝa80-co-SSNa) with 50 mol% and 80 mol% in acrylic acid, respectively, were synthesized and characterized by neutron magnetic resonance spectroscopy, 1H NMR. We proceeded in the physicochemical study of the interaction of bivalent copper ions, Cu2+, with P(ANa50-co-SSNa) and P(AΝa80-co-SSNa) copolymers using techniques like turbidimetry, viscometry and UV-vis spectroscopy. The experiments were performed in aqueous media using 3D water, H2O-3D. For reasons of comparison the respective homopolymers of sodium polyacrylate, PANa, and poly(styrene sulfonate), PSSNa, were also studied. From viscometry it was found that the introduction of copper ions in the polymer aqueous solutions leads to the decrease of the reduced viscosity. Moreover, this decrease is more pronounced as the concentration of Cu2+ increases in the solution. This behavior is indicative of the complexation between Cu2+ with the carboxylate groups of acrylic acid (ΑΑ). In the P(ANa-co-SSNa)/Cu2+ system, it was found that complexation occurs when the ratio of copper ions to carboxylates is equal to 1:2. UV-vis spectroscopy showed that, while in the case of PSSNa homopolymer there is not strong interaction with Cu2+, in the case of the Ρ(ΑNa50-co-SSNa) copolymer, the introduction of the polymer lead to a shift of the maximum absorbance of Cu2+ (from 800 to 745 nm). This was due to the complexation of Cu2+ with the COO- groups of the copolymer.

Yeast Saccharomyces cerevisiae as an unicellular organism is one of the best-studied experimental organisms. It is an important model organism for the study of intracellular processes of eukaryotic cells. Yeasts are also social organisms with cell-to-cell communication able to form organized multicellular structures (colonies and biofilms). Yeast and other microorganisms in nature prefer to form colonies on solid substrates rather than to grow as "planktonic" single cells (Palková, 2004; Wimpenny, 2009). The yeast S. cerevisiae typically forms colonies, biofilms were described only rarely. Yeast colonies exhibit an organized morphological pattern characteristic of each particular yeast strain (Kocková-Kratochvílová, 1982). This work is focusing on morphology and differentiation of the S. cerevisiae colonies of common laboratory strains forming less structured colonies, and strains of the Σ1278b genetic background forming highly structured "fluffy" colonies. It shows that polarized budding pattern and especially cell ability to form aggregates enable development of structured morphology. During development of "fluffy" colonies two differently regulated events of dimorphic switch from yeast form to filamentous growth occur. One of these events is dependent on the surface glycoprotein, Flo11p flocculin. This...

Объектом исследования были растения табака (Nicotiana tabacum L.), выращенные в трех разных условиях: контроль (р-р Кнопа), р-р Кнопа с добавлением 100 µM CuSO4 и 300 µM CuSO4. Цель работы – изучить анатомо-морфологическое строение, активность антиоксидантных ферментов растений табака в период последействия ионов меди, оценить способность табака к восстановлению после стресса. Исследовали всхожесть семян, биомассу и анатомо-морфологические характеристики корня, стебля и листа. Определяли биохимические параметры (содержание пероксида водорода, продуктов ПОЛ, фенольных соединений, активность ферментов – КАТ, АПО, СОД, цитозольных и апопластных ГПО и БПО, изоформы пероксидаз). Проведенное исследование дает представление о том, как изменяются прорастание, анатомо-морфологические и биохимические параметры растений N. tabacum при последействии ионов меди. Выявлено, что воздействие 100 µM привело к увеличению биомассы корня, при этом длина корней, высота стеблей, толщина корней и стеблей, а также площадь листьев снизились. Действие 300 µM привело к более сильному снижению данных параметров, а также уменьшению сухой массы стеблей и листьев. Содержание пероксида водорода и уровень ПОЛ увеличился при действии 100 и 300 µM. Содержание фенольных соединений снизилось в корнях при 100 µM и увеличилось в корнях и листьях и 300 µM и 100 µM соответственно. Влияние ионов меди активировало работу ферментов антиоксидантной защиты – АПО и КАТ во всем растении, цитозольной ГПО и апопластных ГПО и БПО в корнях, в то же время активность СОД снизилась в обоих вариантах опыта. Белковый электрофоре показал, снижение активности общих для тканей корня, стебля и листьев изоформ пероксидаз. Отмечено снижение ферментативной активности изоформ, найденных в стеблях, в случае предобработки растений 300 µM, при этом активность специфичных для корня изоформ оставалась высокой во всех вариантах опыта. / The object of the study were tobacco plants (Nicotiana tabacum L.) grown under three different conditions: control (Knop's solution), Knop's solution with 100 µM CuSO4 and 300 µM CuSO4. The purpose of the work was to study the anatomical and morphological structure, the activity of antioxidant enzymes in tobacco plants after prolonged exposure to copper ions and their after-action, to assess the ability of tobacco to recover from stress. Seed germination, biomass and anatomical and morphological characteristics of the root, stem and leaf were studied. Biochemical parameters were determined (the content of hydrogen peroxide and phenolic compounds, the activity of enzymes - CAT, APX, SOD, cytosolic and apoplastic GPX and BPX). The study gives an idea of how the germination, anatomical, morphological and biochemical parameters of N. tabacum plants change under the influence of copper ions. It was revealed that exposure to 100 μm led to an increase in root biomass, whereas the length of roots, height of stems, thickness of roots and stems, as well as leaf area were decreased. The effect of 300 μM led to even lower decrease in these parameters, as well as a decrease in dry weight of stems and leaves. The content of hydrogen peroxide and the level of lipid peroxidation increased upon exposure to 100 and 300 µM. The content of phenolic compounds decreased in roots at 100 µM and increased in roots and leaves at 300 µM and 100 µM, respectively. The influence of copper ions activated the work of antioxidant defense enzymes - APO and CAT in the whole plant, cytosolic GPO and apoplastic GPO and BPO in the roots, at the same time, SOD activity decreased in both variants of the experiment. Protein electrophoresis showed a decrease in the activity of peroxidase isoforms common for the root, stem and leaves. A decrease in the enzymatic activity of the peroxidase isoforms found in the stems was noted in plants pretreated with 300 μM, while activity of the root specific isoforms remained high in all variants of the experiment.