Structural and Biochemical Characterization of Peroxiredoxin Q beta from Xylella fastidiosa CATALYTIC MECHANISM AND HIGH REACTIVITY

The phytopathogenic bacterium Xylella fastidiosa is the etiological agent of various plant diseases. To survive under oxidative stress imposed by the host, microorganisms express antioxidant proteins, including cysteine-based peroxidases named peroxiredoxins. This work is a comprehensive analysis of the catalysis performed by PrxQ from X. fastidiosa (XfPrxQ) that belongs to a peroxiredoxin class still poorly characterized and previously considered as moderately reactive toward hydroperoxides. Contrary to these assumptions, our competitive kinetics studies have shown that the second-order rate constants of the peroxidase reactions of XfPrxQ with hydrogen peroxide and peroxynitrite are in the order of 107 and 106 M(-1) s(-1), respectively, which are as fast as the most efficient peroxidases. The XfPrxQ disulfides were only slightly reducible by dithiothreitol; therefore, the identification of a thioredoxin system as the probable biological reductant of XfPrxQ was a relevant finding. We also showed by site-specific mutagenesis and mass spectrometry that an intramolecular disulfide bond between Cys-47 and Cys-83 is generated during the catalytic cycle. Furthermore, we elucidated the crystal structure of XfPrxQ C47S in which Ser-47 and Cys-83 lie similar to 12.3 angstrom apart. Therefore, significant conformational changes are required for disulfide bond formation. In fact, circular dichroism data indicated that there was a significant redox-dependent unfolding of alpha-helices, which is probably triggered by the peroxidatic cysteine oxidation. Finally, we proposed a model that takes data from this work as well data as from the literature into account.

Structural and Biochemical Characterization of Peroxiredoxin Q beta from Xylella fastidiosa CATALYTIC MECHANISM and HIGH REACTIVITY

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structural and Biochemical Characterization of Peroxiredoxin Q beta from Xylella fastidiosa CATALYTIC MECHANISM and HIGH REACTIVITY

The phytopathogenic bacterium Xylella fastidiosa is the etiological agent of various plant diseases. To survive under oxidative stress imposed by the host, microorganisms express antioxidant proteins, including cysteine-based peroxidases named peroxiredoxins. This work is a comprehensive analysis of the catalysis performed by PrxQ from X. fastidiosa (XfPrxQ) that belongs to a peroxiredoxin class still poorly characterized and previously considered as moderately reactive toward hydroperoxides. Contrary to these assumptions, our competitive kinetics studies have shown that the second-order rate constants of the peroxidase reactions of XfPrxQ with hydrogen peroxide and peroxynitrite are in the order of 107 and 106 M(-1) s(-1), respectively, which are as fast as the most efficient peroxidases. The XfPrxQ disulfides were only slightly reducible by dithiothreitol; therefore, the identification of a thioredoxin system as the probable biological reductant of XfPrxQ was a relevant finding. We also showed by site-specific mutagenesis and mass spectrometry that an intramolecular disulfide bond between Cys-47 and Cys-83 is generated during the catalytic cycle. Furthermore, we elucidated the crystal structure of XfPrxQ C47S in which Ser-47 and Cys-83 lie similar to 12.3 angstrom apart. Therefore, significant conformational changes are required for disulfide bond formation. In fact, circular dichroism data indicated that there was a significant redox-dependent unfolding of alpha-helices, which is probably triggered by the peroxidatic cysteine oxidation. Finally, we proposed a model that takes data from this work as well data as from the literature into account.

Synthesis and Characterization of Magnetic Nanoparticles with High Magnetization and Good Oxidation Resistibility

Magnetic nanoparticles attract increasing attention because of their current and potential biomedical applications, such as, magnetically targeted and controlled drug delivery, magnetic hyperthermia and magnetic extraction. Increased magnetization can lead to improved performance in targeting and retention in drug delivery and a higher efficiency in biomaterials extraction. We reported an approach to synthesize iron contained magnetic nanoparticles with high magnetization and good oxidation resistibility by pyrolysis of iron pentacarbonyl (Fe(CO)[subscript 5]) in methane (CH[subscript 4]). Using the high reactivity of Fe nanoparticles, decomposition of CH[subscript 4] on the Fe nanoparticles leads to the formation of nanocrystalline iron carbides at a temperature below 260°C. Structural investigation indicated that the as-synthesized nanoparticles contained crystalline bcc Fe, iron carbides and spinel iron oxide. The Mössbauer and DSC results testified that the as-synthesized nanoparticle contained three crystalline iron carbide phases, which converted to Fe[subscript 3]C after a heat treatment. Surface analysis suggested that the as-synthesized and subsequently heated iron-iron carbide particles were coated by iron oxide, which originated from oxidization of surface Fe atoms. The heat-treated nanoparticles exhibited a magnetization of 160 emu/g, which is two times of that of currently used spinel iron oxide nanoparticles. After heating in an acidic solution with a pH value of 5 at 60°C for 20 h, the nanoparticles retained 90 percentage of the magnetization.

Reactivity of activated electrophiles and nucleophiles: labile intermediates and properties of the reaction products

The main topic of my Ph.D. thesis is the study of nucleophilic and electrophilic aromatic substitution reaction, in particular from a mechanistic point of view. The research was mainly focused on the reactivity of superactivated aromatic systems. In spite of their high reactivity (hence the high reaction’s rate), we were able to identify and in some case to isolate -complexes until now only hypothesized. For example, interesting results comes from the study of the protonation of the supernucleophiles tris(dialkylamino)benzenes. However, the best result obtained in this field was the isolation and structural characterization of the first stables zwitterionic Wheland-Meisenheimer complexes by using 2,4-dipyrrolidine-1,3-thiazole as supernucleophile and 4,6-dinitrobenzofuroxan or 4,6-dinitrotetrazolepyridine as superelectrophile. These reactions were also studied by means of computational chemistry, which allowed us to better investigate on the energetic and properties of the reactions and reactants studied. We also discovered, in some case fortuitously, some relevant properties and application of the compounds we synthesized, such as fluorescence in solid state and nanoparticles, or textile dyeing. We decided to investigate all these findings also by collaborating with other research groups. During a period in the “Laboratoire de Structure et Réactivité des Systèmes Moléculaires Complexes-SRSMC, Université de Lorraine et CNRS, France, I carried out computational studies on new iron complexes for the use as dyes in Dye Sensitized Solar Cells (DSSC). Furthermore, thanks to this new expertise, I was involved in a collaboration for the study of the ligands’ interaction in biological systems. A collaboration with University of Urbino allowed us to investigate on the reactivity of 1,2-diaza-1,3-dienes toward nucleophiles such as amino and phosphine derivatives, which led to the synthesis of new products some of which are 6 or 7 member heterocycles containing both phosphorus and nitrogen atoms.

Mitigating Risk For Anxiety Among Preschool-Age Children Living In Poverty: Evaluating The Impact Of Adult-Provided Social Support On Autonomic Stress Reactivity

Poverty increases children's exposure to stress, elevating their risk for developing patterns of heightened sympathetic and parasympathetic stress reactivity. Repeated patterns of high sympathetic activation and parasympathetic withdrawal place children at risk for anxiety disorders. This study evaluated whether providing social support to preschool-age children during mildly stressful situations helps reduce reactivity, and whether this effect partly depends on children's previously assessed baseline reactivity patterns. The Biological Sensitivity to Context (BSC) theory proposes that highly reactive children may be more sensitive than less reactive children to all environmental influences, including social support. In contrast, conventional physiological reactivity (CPR) theory contends that highly reactive children are more vulnerable to the impact of stress but are less receptive to the potential benefits present within their social environments. In this study, baseline autonomic reactivity patterns were measured. Children were then randomly assigned to a high-support or neutral control condition, and the effect of social support on autonomic response patterns was assessed. Results revealed an interaction between baseline reactivity profiles and experimental condition. Children with patterns of high-reactivity reaped more benefits from the social support in the experimental condition than did their less reactive peers. Highly reactive children experienced relatively less reactivity reduction in the neutral condition while experiencing relatively greater reactivity reduction in the support condition. Despite their demonstrated stability over time, reactivity patterns are also quite susceptible to change at this age; therefore understanding how social support ameliorates reactivity will further efforts to avert stable patterns of high-reactivity among children with high levels of stress, ultimately reducing risk for anxiety disorders.

Late Transition Metal-Oxo complexes: Synthesis, and Biorelevant Reactivity

High-valent terminal metal-oxygen adducts are supposed to be potent oxidising intermediates in enzymatic catalyses. In contrast to those from groups 6-8, oxidants that contain late transition metals (Co, Ni, Cu) are poorly understood. Because of their high reactivity, only a few examples of these compounds have been observed. The aim of this project was to investigate the reactivity of high-valent Ni(III) complexes, containing a monodentate oxygen-donor ligands, in hydrogen atom abstraction (HAA) and oxygen atom transfer (OAT) reactions which are typical of biological high-valent metal-oxygen species. Particularly, the Ni(III) complexes were generated in situ, at low temperature, from the oxidation of the Ni(II) species.The nickel complexes studied during this work were supported by tridentate ligands, with a strong σ-donating ability and exceedingly resistant to several common degradation pathways. These complexes vary based on the monodentate group in the fourth coordination position site, which can be neutral or anionic. In particular, we prepared four different Ni(III) complexes [NiIII(pyN2Me2)(OCO2H)] (12), [NiIII(pyN2Me2)(ONO2)] (14), [NiIII(pyN2Me2)(OC(O)CH3)] (18) and [NiIII(pyN2Me2)(OC(O)H)] (25). They feature a bicarbonate (-OCO2H), nitrate (-ONO2), acetate (-OC(O)CH3) and formate (-OC(O)H) group, respectively.HAA and OAT reactions were performed by adding 2,6-di-tert-butylphenol (2,6-DTBP) at -40°C, and triphenylphosphine (PPh3) at -80°C, to the in situ generated Ni(III) complexes, respectively. These reactions were carried out by adding 7 to 500 equivalents of substrate, in order to ensure pseudo-first order conditions. Since, the reactivity of the Ni(III) complex featured by the bicarbonate group has been studied in a previous work, we only investigated that of the species bearing the nitrate, acetate and formate ligand. Finally we compared the value of the reaction rate of all the four species in the HAA and OAT reactions.

Intrinsic gas-phase electrophilic reactivity of Ccclic N-alkyl- and N-acyliminium ions

he intrinsic gas-phase reactivity of cyclic N-alkyl- and N-acyliminium ions toward addition of allyltrimethylsilane (ATMS) has been compared using MS2 and MS3 pentaquadrupole mass spectrometric experiments. An order of electrophilic reactivity has been derived and found to agree with orders of overall reactivity in solution. The prototype five-membered ring N-alkyliminium ion 1a and its N-CH3 analogue 1b, as well as their six-membered ring analogues 1c and 1d, lack N-acyl activation and they are, accordingly, inert toward ATMS addition. The five- and six-membered ring N-acyliminium ions with N-COCH3 exocycclic groups, 3a and 3b, respectively, are also not very reactive. The N-acyliminium ions 2a and 2c, with s-trans locked endocyclic N-carbonyl groups, are the most reactive followed closely by 3c and 3d with exocyclic (and unlocked) N-CO2CH3 groups. The five-membered ring N-acyliminium ions are more reactive than their six-membered ring analogues, that is:  2a > 2c and 3c > 3d. In contrast with the high reactivity of 2a, its N-CH3 analogue 2b is inert toward ATMS addition. For the first time, the transient intermediates of a Mannich-type condensation reaction were isolatedthe β-silyl cations formed by ATMS addition to N-acyliminium ionsand their intrinsic gas-phase behavior toward dissociation and reaction with a nucleophile investigated. When collisionally activated, the β-silyl cations dissociate preferentially by Grob fragmentation, that is, by retro-addition. With pyridine, they react competitively and to variable extents by proton transfer and by trimethylsilylium ion abstractionthe final and key step postulated for α-amidoalkylation. Becke3LYP/6-311G(d,p) reaction energetics, charge densities on the electrophilic C-2 site, and AM1 LUMO energies have been used to rationalize the order of intrinsic gas-phase electrophilic reactivity of cyclic iminium and N-acyliminium ions.

Pt/TiO(2)/poly(vinyl sulfonic acid) Layer-by-Layer Films for Methanol Electrocatalytic Oxidation

One major challenge for the widespread application of direct methanol fuel cells (DMFCs) is to decrease the amount of platinum used in the electrodes, which has motivated a search for novel electrodes containing platinum nanoparticles. In this study, platinum nanoparticles were electrodeposited on layer-by-layer (LbL) films from TiO(2) and poly(vinyl sulfonic) (PVS), by immersing the films into a H(2)PtCl(6) solution and applying a 100 mu A current during different electrode position times. Scanning tunnel microscopy (STM) and atomic force microscopy (AFM) images showed increased platinum particle size and electrode roughness for increasing electrodeposition times. The potentiodynamic profile of the electrodes indicated that oxygen-like species in 0.5 mol L(-1) H(2)SO(4) were formed at less positive potentials for the smallest platinum particles. Electrochemical impedance spectroscopy measurements confirmed the high reactivity for the water dissociation and the large amount of oxygen-like species adsorbed on the smallest platinum nanoparticles. This high oxophilicity of the smallest nanoparticles was responsible for the electrocatalytic activity of Pt-TiO(2)/PVS systems for methanol electrooxidation, according to the Langmuir-Hinshelwood bifunctional mechanism. Significantly, the approach used here combining platinum electrodeposition and LbL matrices allows one to both control the particle size and optimize methanol electrooxidation, being therefore promising for producing membrane-electrode assemblies of DMFCs.

XRCC3241 Polymorphism influence on genotoxicity biomarkers frequency in workers occupationaly exposed to formaldehyde

Formaldehyde (FA) is ubiquitous in the environment and is a chemical agent that possesses high reactivity. Occupational exposure to FA has been shown to induce nasopharyngeal cancer and has been classified as carcinogenic to humans (group 1) on the basis of sufficient evidence in humans and sufficient evidence in experimental animals. The exposure to this substance is epidemiologically linked to cancer and nuclear changes detected by the cytokinesis-block micronucleus test (CBMN). This method is extensively used in molecular epidemiology, since it determines several biomarkers of genotoxicity, such as micronucleus (biomarkers of chromosomes breakage or loss), nucleoplasmic bridges (biomarker of chromosome rearrangement, poor repair and / or telomeres fusion) and nuclear buds (biomarker of elimination of amplified DNA). The gene X-ray repair cross-complementing group 3 (XRCC3) is involved in homologous recombination repair of cross-links and chromosomal double-strand breaks and at least one polymorphism has been reported in codon 241, a substitution of a methionine for a threonine.

Application of green zero-valent iron nanoparticles to the remediation of soils contaminated with ibuprofen

Zero-valent iron nanoparticles (nZVIs) are often used in environmental remediation. Their high surface area that is associated with their high reactivity makes them an excellent agent capable of transforming/degrading contaminants in soils and waters. Due to the recent development of green methods for the production of nZVIs, the use of this material became even more attractive. However, the knowledge of its capacity to degrade distinct types of contaminants is still scarce. The present work describes the study of the application of green nZVIs to the remediation of soils contaminated with a common anti-inflammatory drug, ibuprofen. The main objectives of this work were to produce nZVIs using extracts of grape marc, black tea and vine leaves, to verify the degradation of ibuprofen in aqueous solutions by the nZVIs, to study the remediation process of a sandy soil contaminated with ibuprofen using the nZVIs, and to compare the experiments with other common chemical oxidants. The produced nZVIs had nanometric sizes and were able to degrade ibuprofen (54 to 66% of the initial amount) in aqueous solutions. Similar remediation efficiencies were obtained in sandy soils. In this case the remediation could be enhanced (achieving degradation efficiencies above 95%) through the complementation of the process with a catalyzed nZVI Fenton-like reaction. These results indicate that this remediation technology represents a good alternative to traditional and more aggressive technologies.

An SEM investigation of the pozzolanic activity of a waste catalyst oil refinery

The most active phase of the fluid catalytic cracking (FCC) catalyst, used in oil refinery, is zeolite-Y which is an aluminosilicate with a high internal and external surface area responsible for its high reactivity. Waste FCC catalyst is potentially able to be reused in cement-based materials - as an additive - undergoing a pozzolanic reaction with calcium hydroxide (Ca(OH)2) formed during cement hydration [1-3]. This reaction produces additional strength-providing reaction products i.e., calcium silicate hydrate (C-S-H) and hydrous calcium aluminates (C-A-H) which exact chemical formula and structure are still unknown. Partial replacement of cement by waste FCC catalyst has two key advantages: (1) lowering of cement production with the associated pollution reduction as this industry represents one of the largest sources of man-made CO2 emissions, and (2) improving the mechanical properties and durability of cement-based materials. Despite these advantages, there is a lack of fundamental knowledge on pozzolanic reaction mechanisms as well as spatial distribution of porosity and solid phases interactions at the microstructural level and consequently their relationship with macroscopical engineering properties of catalyst/cement blends. Within this scope, backscattered electron (BSE) images acquired in a scanning electron microscope (SEM) equipped with Energy-Dispersive Spectroscopy (EDS) and by X-ray diffraction were used to investigate chemical composition of hydration products and to analyse spatial information of the microstructure of waste FCC catalyst blended cement mortars. For this purpose mortars with different levels of cement substitution by waste catalyst as well as with different hydration ages, were prepared. The waste FCC catalyst used is produced by the Portuguese refinery company Petrogal S.A.

Engineering of metal oxide nanoparticles for application in electrochemical devices

The growing demand for materials and devices with new functionalities led to the increased inter-est in the field of nanomaterials and nanotechnologies. Nanoparticles, not only present a reduced size as well as high reactivity, which allows the development of electronic and electrochemical devices with exclusive properties, when compared with thin films. This dissertation aims to explore the development of several nanostructured metal oxides by sol-vothermal synthesis and its application in different electrochemical devices. Within this broad theme, this study has a specific number of objectives: a) research of the influence of the synthesis parameters to the structure and morphology of the nanoparticles; b) improvement of the perfor-mance of the electrochromic devices with the application of the nanoparticles as electrode; c) application of the nanoparticles as probes to sensing devices; and d) production of solution-pro-cessed transistors with a nanostructured metal oxide semiconductor. Regarding the results, several conclusions can be exposed. Solvothermal synthesis shows to be a very versatile method to control the growth and morphology of the nanoparticles. The electrochromic device performance is influenced by the different structures and morphologies of WO3 nanoparticles, mainly due to the surface area and conductivity of the materials. The dep-osition of the electrochromic layer by inkjet printing allows the patterning of the electrodes without wasting material and without any additional steps. Nanostructured WO3 probes were produced by electrodeposition and drop casting and applied as pH sensor and biosensor, respectively. The good performance and sensitivity of the devices is explained by the high number of electrochemical reactions occurring at the surface of the na-noparticles. GIZO nanoparticles were deposited by spin coating and used in electrolyte-gated transistors, which promotes a good interface between the semiconductor and the dielectric. The produced transistors work at low potential and with improved ON-OFF current ratio, up to 6 orders of mag-nitude. To summarize, the low temperatures used in the production of the devices are compatible with flexible substrates and additionally, the low cost of the techniques involved can be adapted for disposable devices.

Analysis of output properties of the Peroxiredoxin/Thioredoxin/Thioredoxin reductase system

Dissertação de mestrado em Bioinformática

Evaluation of Metakaolin for HPC Deck Overlays, MLR-11-01, 2011

The use of High Performance Concrete (HPC) in Iowa has consisted of achieving slightly higher compressive strengths with an emphasis on reduced permeability. Concrete with reduced permeability has increased durability by slowing moisture and chloride ingress. Achieving reduced permeability has typically been accomplished with combinations of slag and Class C fly ash, or the use of blended cements such as locally available Type IS(20), IS(25) and Type IP(25) in conjunction with Class C fly ash. Fly ash has been used in the majority of concrete placed in Iowa since 1984 and slag has been available in Iowa since 1995. During the economic downturn in 2008, one of the cement plants that produced a Type IS(25) cement was forced to shut down, which reduced the availability of blended cements, typically used on HPC deck overlays. Recently, a source of high reactivity metakaolin has been made available. Metakaolin is produced by heating a pure kaolinite clay to 650 to 700 °C in a rotary kiln (calcining). Metakaolin is a white pozzolan that is used to produce concrete with increased strengths, reduced permeability, reduced efflorescence, and resistance to alkali silica reactivity. The W.R. Grace MK-100 metakaolin will likely be available in dissolvable bags between 25 and 50 pounds. Thus, the mix designs were based on the anticipated bag size range for field use. This research evaluated metakaolin mixes with and without Class C fly ash. Results indicated a seven percent replacement with metakaolin produced concrete with increased strengths and low permeability. When used with Class C fly ash, permeability is reduced to very low rating. Metakaolin may be used to enhance hardened concrete properties for use in high performance concrete (HPC).