Estudos têm mostrado a incidência de mastite bovina associada à alga Prototheca zopfii. O objetivo deste trabalho foi estudar a interação da P. zopfii com neutrófilos recuperados de leite bovino e avaliar o efeito do sistema ácido indol-3-acético/peroxidase de raiz forte (AIA/HRP) sobre a viabilidade deste microrganismo em experimentos in vitro. A P. zopfii foi recuperada de vacas com mastite clínica e, no laboratório, foram realizadas a caracterização molecular, morfológica e crescimento exponencial do microrganismo. Em seguida, neutrófilos recuperados de leite bovino foram incubados na ausência e na presença de P. zopfii opsonizada e foram avaliadas a produção de peróxido de hidrogênio, enzimas antioxidantes dos neutrófilos e microrganismo, e a capacidade fagocitária. Em outro estudo, a P. zopfii foi incubada com o sistema AIA/HRP e foram avaliadas a viabilidade por unidades formadoras de colônias (UFC), atividade de enzimas antioxidantes, integridade de membrana por exclusão com azul de Trypan e integridade do DNA. Os resultados foram analisados pela análise de variância com significância de 5% usando o teste Tukey. Foram observados diversos tamanhos celulares da P. zopfii, presença de autofluorescência, crescimento exponencial ao longo do tempo de incubação em que não foi possível determinar o início da fase de morte. Ainda, foram encontrados os genótipos 1, 2 e 3 nos isolados em estudo. A produção de peróxido de hidrogênio pelos neutrófilos na presença da alga foi estimulada 5 vezes em relação ao controle, estimulou a atividade das enzimas catalase (CAT) em 21% e glutationa redutase (GR) em 27% e não houve diferença significativa quanto à atividade de CAT, GR e superóxido dismutase (SOD) produzido pela P. zopfii. Também foi verificado que a P. zopfii não foi englobada pelo neutrófilo. O sistema AIA/HRP inibiu o crescimento do microrganismo em 45, 82 e 88% nos tempos de 4, 6 e 9 horas de incubação; a atividade da SOD, CAT, Glutationa Peroxidase (GPx) e GR aumentou respectivamente em 90, 120, 150% e 3,4 vezes; houve redução da viabilidade da P. zopfii em 10, 15, 20, 25 e 32% após os tempos de 4, 6, 8, 10 e 12 horas de incubação; e não afetou a integridade do DNA após 6 horas de incubação. Conclui-se que a P. zopfii é altamente resistente frente aos neutrófilos e demonstrou ser susceptível quanto ao efeito microbicida do sistema AIA/HRP. / Studies have shown the incidence of bovine mastitis associated with the algae Prototheca zopfii. The objective of this work was to study the interaction of P. zopfii with neutrophils recovered from bovine milk and to evaluate the effect of system indole-3-acetic acid/horseradish peroxidase (IAA/HRP) on the viability of this microorganism in vitro experiment. P. zopfii was recovered from cows with clinical mastitis and both the molecular and morphological characterization were performed besides the evaluation of exponential growth of the microorganism in the laboratory. Next, neutrophils recovered from bovine milk were incubated in the absence and presence of opsonized P. zopfii and were evaluated the production of hydrogen peroxide, antioxidant enzymes on neutrophils and microorganism, and phagocytic capacity. In another study, P. zopfii was incubated with the system IAA/HRP and the viability assessed by colony forming units (CFU), antioxidant enzymes activity, membrane integrity by exclusion with Trypan blue and DNA integrity. The results were analyzed by analysis of variance with a 5% significance using the Tukey test. Results from P. zopfii characterization showed various cellular sizes, presence of autofluorescence, exponential microorganism growth throughout the incubation time and was not possible to determine the beginning of the death. Moreover it was found genotypes 1, 2 and 3 in the isolates in study. The production of hydrogen peroxide by neutrophils in the presence of algae was stimulated 5 times compared to the control, increase the activity of catalase (CAT) in 21% and glutathione reductase (GR) in 27% was seen in neutrophils; and there was no significant difference in CAT, GR and superoxide dismutase (SOD) activity produced by P. zopfii. P. zopfii was not engulfment by neutrophils. The system IAA/HRP inhibited the growth of the microorganism in 45, 82 and 88% in the times of 4, 6 and 9 hours of incubation, the activity of SOD, CAT, glutathione peroxidase (GPx) and GR increased respectively by 90, 120, 150%, and 3.4 times, decreased the viability of P. zopfii 10, 15, 20, 25 and 32% after the times of 4, 6, 8, 10 and 12 hours of incubation, and did not affect the integrity of DNA after 6 hours of incubation. As a conclusion, P. zopfii is highly resistant to the neutrophils and demonstrated to be susceptible to the effect microbicidal of system IAA/HRP.

<p>The four-electron reduction of dioxygen to water is the most exothermic non-photochemical reaction available to biology. A detailed molecular description of this reaction is needed to understand oxygen-based redox processes. Horseradish peroxidase (HRP) is a haem-containing redox enzyme capable of catalysing the reduction of dioxygen to water. We developed instrumentation and experimental methodology to capture and characterise by X-ray crystallography transient reaction intermediates in this reaction. </p><p>An instrument was designed (“the vapour stream system”) to facilitate reaction initiation, monitoring and intermediate trapping. In combination with single crystal microspectrophotometry, it was used to obtain conditions for capturing a reactive dioxygen complex in HRP. X-ray studies on oxidised intermediates can be difficult for various reasons. Electrons re-distributed in the sample through the photoelectric effect during X-ray exposure can react with high-valency intermediates. In order to control such side reactions during data collection, we developed a new method based on an angle-resolved spreading of the X-ray dose over many identical crystals. Composite data sets built up from small chunks of data represent crystal structures which received different X-ray doses. As the number of electrons liberated in the crystal is dose dependent, this method allows us to observe and drive redox reactions electron-by-electron in the crystal, using X-rays.</p><p>The methods developed here were used to obtain a three-dimensional movie on the X-ray-driven reduction of dioxygen to water in HRP. Separate experiments established high resolution crystal structures for all intermediates, showing such structures with confirmed redox states for the first time. </p><p>Activity of HRP is influenced by small molecule ligands, and we also determined the structures of HRP in complex with formate, acetate and carbon monoxide.</p><p>Other studies established conditions for successfully trapping the M-intermediate in crystals of mutant bacteriorhodopsin, but the poor diffraction quality of these crystals prevented high-resolution structural studies.</p>

<p>The main hypothesis in this study is the development of a nanocomposite mediated amperometric biosensor for detection of hydrogen peroxide. The aim is to combine the electrochemical properties of both polyaniline and ferrocenium hexafluorophosphate into highly conductive nano composites capable of exhibiting electrochemistry in non acidic media / shuttling electrons between HRP and GCE for biosensor applications.</p>

A novel cyclopentadienylruthenium(II) thiolato Schiff base, [Ru(SC6H4NC(H)C6H4OCH2CH2SMe)(&eta / 5-C2H5]2 was synthesized and deposited as a selfassembled monolayer (SAM) on a gold electrode. Effective electronic communication between the Ru(II) centers and the gold electrode was established by electrostatically cycling the Shiff base-doped gold electrode in 0.1 M NaOH from -200 mV to +600 mV. The SAMmodified gold electrode (Au/SAM) exhibited quasi-reversible electrochemistry. The integrity of this electro-catalytic SAM, with respect to its ability to block and electro-catalyze certain Faradaic processes, was interrogated using Cyclic and Osteryoung Square Wave voltammetric experiments. The formal potential, E0', varied with pH to give a slope of about - 34 mV pH-1. The surface concentration, &Gamma / , of the ruthenium redox centers was found to be 1.591 x 10-11 mol cm-2. By electrostatically doping the Au/SAM/Horseradish peroxidase at an applied potential of +700 mV vs Ag/AgCl, a biosensor was produced for the amperometric analysis of hydrogen peroxide, cumene hydroperoxide and tert-butylhydroperoxide. The electrocatalytic-type biosensors displayed typical Michaelis-Menten kinetics with their limits of detection of 6.45 &mu / M, 6.92 &mu / M and 7.01 &mu / M for hydrogen peroxide, cumene hydroperoxide and tert-butylhydroperoxide respectively.

The four-electron reduction of dioxygen to water is the most exothermic non-photochemical reaction available to biology. A detailed molecular description of this reaction is needed to understand oxygen-based redox processes. Horseradish peroxidase (HRP) is a haem-containing redox enzyme capable of catalysing the reduction of dioxygen to water. We developed instrumentation and experimental methodology to capture and characterise by X-ray crystallography transient reaction intermediates in this reaction. An instrument was designed (“the vapour stream system”) to facilitate reaction initiation, monitoring and intermediate trapping. In combination with single crystal microspectrophotometry, it was used to obtain conditions for capturing a reactive dioxygen complex in HRP. X-ray studies on oxidised intermediates can be difficult for various reasons. Electrons re-distributed in the sample through the photoelectric effect during X-ray exposure can react with high-valency intermediates. In order to control such side reactions during data collection, we developed a new method based on an angle-resolved spreading of the X-ray dose over many identical crystals. Composite data sets built up from small chunks of data represent crystal structures which received different X-ray doses. As the number of electrons liberated in the crystal is dose dependent, this method allows us to observe and drive redox reactions electron-by-electron in the crystal, using X-rays. The methods developed here were used to obtain a three-dimensional movie on the X-ray-driven reduction of dioxygen to water in HRP. Separate experiments established high resolution crystal structures for all intermediates, showing such structures with confirmed redox states for the first time. Activity of HRP is influenced by small molecule ligands, and we also determined the structures of HRP in complex with formate, acetate and carbon monoxide. Other studies established conditions for successfully trapping the M-intermediate in crystals of mutant bacteriorhodopsin, but the poor diffraction quality of these crystals prevented high-resolution structural studies.

<p>The main hypothesis in this study is the development of a nanocomposite mediated amperometric biosensor for detection of hydrogen peroxide. The aim is to combine the electrochemical properties of both polyaniline and ferrocenium hexafluorophosphate into highly conductive nano composites capable of exhibiting electrochemistry in non acidic media / shuttling electrons between HRP and GCE for biosensor applications.</p>

A novel cyclopentadienylruthenium(II) thiolato Schiff base, [Ru(SC6H4NC(H)C6H4OCH2CH2SMe)(&eta / 5-C2H5]2 was synthesized and deposited as a selfassembled monolayer (SAM) on a gold electrode. Effective electronic communication between the Ru(II) centers and the gold electrode was established by electrostatically cycling the Shiff base-doped gold electrode in 0.1 M NaOH from -200 mV to +600 mV. The SAMmodified gold electrode (Au/SAM) exhibited quasi-reversible electrochemistry. The integrity of this electro-catalytic SAM, with respect to its ability to block and electro-catalyze certain Faradaic processes, was interrogated using Cyclic and Osteryoung Square Wave voltammetric experiments. The formal potential, E0', varied with pH to give a slope of about - 34 mV pH-1. The surface concentration, &Gamma / , of the ruthenium redox centers was found to be 1.591 x 10-11 mol cm-2. By electrostatically doping the Au/SAM/Horseradish peroxidase at an applied potential of +700 mV vs Ag/AgCl, a biosensor was produced for the amperometric analysis of hydrogen peroxide, cumene hydroperoxide and tert-butylhydroperoxide. The electrocatalytic-type biosensors displayed typical Michaelis-Menten kinetics with their limits of detection of 6.45 &mu / M, 6.92 &mu / M and 7.01 &mu / M for hydrogen peroxide, cumene hydroperoxide and tert-butylhydroperoxide respectively.

A novel cyclopentadienylruthenium(II) thiolato Schiff base,[Ru(SC6H4NC(H)C6H4OCH2CH2SMe)(η5-C2H5]2 was synthesized and deposited as a selfassembled monolayer (SAM) on a gold electrode. Effective electronic communication between the Ru(II) centers and the gold electrode was established by electrostatically cycling the Shiff base-doped gold electrode in 0.1 M NaOH from -200 mV to +600 mV. The SAMmodified gold electrode (Au/SAM) exhibited quasi-reversible electrochemistry. The integrity of this electro-catalytic SAM, with respect to its ability to block and electro-catalyze certain Faradaic processes, was interrogated using Cyclic and Osteryoung Square Wave voltammetric experiments. The formal potential, E0', varied with pH to give a slope of about - 34 mV pH-1. The surface concentration, Γ, of the ruthenium redox centers was found to be 1.591 x 10-11 mol cm-2. By electrostatically doping the Au/SAM/Horseradish peroxidase at an applied potential of +700 mV vs Ag/AgCl, a biosensor was produced for the amperometric analysis of hydrogen peroxide, cumene hydroperoxide and tert-butylhydroperoxide. The electrocatalytic-type biosensors displayed typical Michaelis-Menten kinetics with their limits of detection of 6.45 μM, 6.92 μM and 7.01 μM for hydrogen peroxide, cumene hydroperoxide and tert-butylhydroperoxide respectively / Magister Scientiae - MSc

Magister Scientiae - MSc / A novel cyclopentadienylruthenium(II) thiolato Schiff base, [Ru(SC6H4NC(H)C6H4OCH2CH2SMe)(η5-C2H5]2 was synthesized and deposited as a selfassembled monolayer (SAM) on a gold electrode. Effective electronic communication between the Ru(II) centers and the gold electrode was established by electrostatically cycling the Shiff base-doped gold electrode in 0.1 M NaOH from -200 mV to +600 mV. The SAMmodified gold electrode (Au/SAM) exhibited quasi-reversible electrochemistry. The integrity of this electro-catalytic SAM, with respect to its ability to block and electro-catalyze certain Faradaic processes, was interrogated using Cyclic and Osteryoung Square Wave voltammetric experiments. The formal potential, E0', varied with pH to give a slope of about - 34 mV pH-1. The surface concentration, &Gamma;, of the ruthenium redox centers was found to be 1.591 x 10-11 mol cm-2. By electrostatically doping the Au/SAM/Horseradish peroxidase at an applied potential of +700 mV vs Ag/AgCl, a biosensor was produced for the amperometric analysis of hydrogen peroxide, cumene hydroperoxide and tert-butylhydroperoxide. The electrocatalytic-type biosensors displayed typical Michaelis-Menten kinetics with their limits of detection of 6.45 M, 6.92 M and 7.01 M for hydrogen peroxide, cumene hydroperoxide and tert-butylhydroperoxide respectively. / South Africa

Heavy metal contamination significantly reduces crop yields, causing serious problems in agriculture and having a major impact on human health if these contaminants enter the food chain. Understanding the mechanisms of plant responses could help to increase their resistance to heavy metals as well as their potential use in phytoremediation. Carbohydrates play an important role in plant growth and development as well as in defense reactions. This work summarizes the results of four publications focused on the effects of arsenic and thorium on antioxidant mechanisms in tobacco plants and horseradish root crops. Attention is paid, among other things, to the dynamics of sugar contents, which are potentially important molecules involved in the fight against oxidative stress. The first publication summarizes arsenic effects on plant physiological parameters, focusing on arsenic tolerance-enhancing mechanisms as well as summarizing the ability of plants to cope with arsenic-induced oxidative and nitrosative stress. Emphasis was placed on, among other things, a topic that was unjustly neglected in previous publications - i.e. carbohydrate metabolism. Further work was already experimental and dealt with the study of arsenic as a trigger of oxidative stress in the root culture of horseradish and...