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

Thesis (PhD) -- Macquarie University, Division of Environmental and Life Sciences, Dept. of Chemistry and Biomolecular Sciences, 2007. / Bibliography: leaves 295-322. / Generation of peroxide groups in proteins exposed to a wide variety of reactive oxygen species (ROS) requires an initial formation of protein carbon-centred or peroxyl free radicals, which can be reduced to hydroperoxides. Both protein radicals and protein hydroperoxides are capable of oxidizing important biomolecules and thus initiate biological damage. In this study, we investigated the inhibition of protein hydroperoxide formation by ascorbate and GSH in gamma-irradiated HL-60 cells.--We used HL-60 cells as a model for general protection of living organisms by ascorbate (Asc) and glutathione (GSH) from the deleterious effects of protein hydroperoxides generated by radicals produced by gamma radiation. Measurement by HPLC indicated that incubation of HL-60 cells with Asc in the presence of ascorbate oxidase resulted in the accumulation of intracellular Asc. The intracellular Asc levels were lowered by irradiation, demonstrating intracellular consumption of Asc by the radiation-generated radicals. Exposure of HL-60 cells to increasing gamma irradiation doses resulted in increasing accumulation of protein peroxides in the cells. This was measured by the FOX assay. A significant decrease in intracellular protein hydroperoxides was noted when the cells were treated with ascorbic acid before irradiation. A dose-dependent protective effect of Asc was observed. Asc loading also provided strong protection from radiation-generated protein hydroperoxides independently of the composition of the external medium, showing that only the radicals formed within the cells were effective in oxidizing the cell proteins. Similarly, protein peroxidation was inhibited in cells with enhanced levels of GSH and increased when the intracellular GSH concentration was reduced. These findings indicate that ascorbate and GSH are important antioxidants in protecting cells from oxidative stress associated with the generation of protein hydroperoxide. / Mode of access: World Wide Web. / xxix, 322 leaves ill

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, 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 M, 6.92 M and 7.01 M for hydrogen peroxide, cumene hydroperoxide and tert-butylhydroperoxide respectively. / South Africa

Este estudo investigou propriedades mecânicas do compósito silorano em dois capítulos. Capítulo A Objetivo: avaliou a resistência de união entre a estrutura dental e um composto à base de silorano, após o clareamento dental. Método: Sessenta incisivos bovinos foram distribuídos aleatoriamente em seis grupos (n=10), de acordo com o período de armazenamento entre o tratamento clareador e a restauração com compósito à base de silorano. O tratamento clareador com peróxido de carbamida à 10% seguiu um protocolo de quatro horas diárias durante duas semanas. Após o período de armazenamento, as cavidades foram preparadas e restauradas com uma resina composta à base de silorano. Resultado: Após o tratamento clareador e os devidos períodos de armazenagem, nenhuma diferença significativa foi observada nos resultados de resistência adesiva (p>0,05). Conclusão: a resistência adesiva entre o compósito à base de silorano e o substrato dental não foi afetada pelo tratamento clareador com 10% de peróxido de carbamida. Capítulo B Objetivo: avaliou a resistência coesiva entre as camadas de resinas compostas à base metacrilato e silorano e a microdureza Knoop (KHN) após ciclos de erosão e abrasão. Método: Para a resistência coesiva, vinte amostras foram feitas com cada compósito, metacrilato (MC) e silorano (SC) (n=10). As amostras foram divididas em quatro grupos, de acordo com tipo e quantidade de incrementos de resina composta. O teste de microtração foi realizado 24h após a confecção das amostras. Para a análise da KHN, após os ciclos de erosão/abrasão, foram produzidas quarenta amostras de cada resina composta testada (MC e SC). As amostras foram distribuídas em oito grupos (n=10) de acordo com tipo de compósito e tipo de bebida envolvida no processo de erosão (controle, suco de laranja, Smirnoff Ice e Gatorade) e abrasão com dentifrício de alta abrasividade. Resultado - não foram observadas diferenças na resistência coesiva entre as camadas incrementais dos compósitos estudados (p> 0,05). Os resultados de KHN dos grupos MC apresentaram menor valor para o Smirnoff Ice (p<0,05), no entanto, para os grupos SC nenhuma diferença foi observada (p>0,05). Conclusão: a resistência coesiva entre as camadas incrementais de ambos compósitos testados não foi afetada pelo oxigênio; as bebidas, em conjunto com a abrasão, não tiveram capacidade de interferir na KHN dos compósitos testados, exceto o Smirnoff Ice para o MC. / This study investigated the mechanical properties of the silorane based composite in two chapter. Chapter A Aim: evaluated the bond strength between the dental structure and a silorane-based composite (SC), after bleaching treatment. Method: Sixty bovine incisors were randomly distributed into six groups (n=10) according to the storage period between the bleaching treatment and the SC restoration. The bleaching treatment followed a protocol of 10% carbamide peroxide with one application during four hours per day during two weeks. After the period of storage, cavities were made and filled with SC. Results: After the periods of bleaching treatment, no significant difference was observed in the results of push out bond strength between the SC and the bleached substrate (p>0.05). Conclusion: the bond strength between the SC and the dental substrate was not affected by the bleaching treatment with 10% carbamide peroxide. Chapter B Aim: evaluated the bond strength between incremental layers of methacrylate and silorane composites and the micro hardness Knoop (KHN) after erosion and abrasion cycles. Method: for the bond strength, twenty samples were made with each composite, methacrylate (MC) and silorane (SC) (n=10). The samples were divided into four groups according composite type and quantity of increments. The microtensile test was performed 24 h after the samples were made. For the KHN test, after erosion/abrasion process, forty samples of each composite tested (MC and SC) were made. The samples were distributed into eight groups (n=10) according composite type and beverage type involved in the erosion process (control, orange juice, Smirnoff Ice and Gatorade) and abrasion with high abrasivity detifrice. Results: no differences were observed in the bond strength between increment layers when the same composite was analyzed (p>0.05). The results of KHN in the MC groups showed smaller value for the Smirnoff Ice (p<0.05), however for the SC groups no differences were found (p>0.05). Conclusion: the bond strength between incremental layers of SC was not affected by the oxygen; the others beverages, jointly with the abrasion, have no capacity to interfere in the KHN of the composites tested.

The cyclic peroxides: diphenoyl peroxide (1), spiro-adamantyl-1,2-dioxetanone (2) and 4-(3-tert-butyldimethylsilyloxyphenyl)-4-methoxyspiro[1.2-dioxetane-3.2\'-adamantane] (3) synthesized, purified and characterized, also their kinetic and chemiluminescence (CL) properties were determined. The influence of solvent polarity on the chemiexcitation parameters of the catalyzed decomposition of 1 and 2, as well as the induced decomposition of 3 were examined in several binary solvent mixtures with different polarity and polarizability parameters, but similar viscosities. For solvents with low polarity, singlet quantum yields for the intermolecular catalyzed decomposition of diphenoyl peroxide (1,) and 1,2-dioxetanone (2) increase in function of medium polarity, showing maximum values in mediums of intermediate polarity, and decreasing in highly polar mixtures. For the induced decomposition of 3, the quantum yield showed to increase with increasing solvent polarity, including high polar solvent systems. In the binary solvent systems studied, the polarizability parameter showed to be contrary to the polarity values, therefore, it might appear that an increase of polarizability leads to a decrease in the singlet quantum yields for catalyzed decomposition of diphenoyl peroxide (1) and the induced decomposition of 3, however an increase in the singlet quantum yields for 1,2-dioxetanone 2. The three CL systems were also studied in binary solvent mixtures with different viscosities but similar polarity and polarizability parameters and singlet quantum yields showed an increase with increasing medium viscosity. Data were analyzed by using the collisional and the free volume models. The highly efficient intramolecular system induced 1,2-dioxetane 3 decomposition showed to be much more sensible to viscosity effects than the inefficient intermolecular systems. This surprising fact indicates that, even showing a significant solvent-cage effect, the induced 1,2-dioxetane should occur by an intramolecular electron back transfer. Moreover, chemiluminescence parameters of these systems were studied in several pure solvents. The obtained singlet quantum yields were correlated with viscosity, polarizability and polarity parameters by using multiple linear regression analysis. / Os peróxidos cíclicos: peróxido de difenoila (1), spiro-adamantil-1,2-dioxetanona (2) e 4-(3-terc-butildimetilsililoxifenil)-4-metoxispiro[1,2-dioxetano-3.2\'-adamantano] (3) foram sintetizados, purificados e caracterizados e as suas propriedades cinéticas e de quimiluminescência (CL) determinadas. A influência da polaridade do solvente sobre os parâmetros de quimi-excitação da decomposição catalisada de 1 e 2, e a decomposição induzida de 3 foi examinada em diversas misturas binárias de solventes com parâmetros de polaridade e polarizabilidade diferente, mas viscosidades semelhantes. Para solventes com baixa polaridade, os rendimentos quânticos singlete para a decomposição catalisada intermolecular do peróxido de difenoila (1) e 1,2-dioxetanona (2) aumentam em função da polaridade do meio, mostrando valores máximos em meios com polaridade intermediária, e diminuim para misturas altamente polares. Para a decomposição induzida de 3, o rendimento quântico mostrou aumentar com o aumento da polaridade do solvente, inclusive para sistemas com alta polaridade. Nos sistemas binários de solventes estudados, os parâmetros de polarizabilidade mostraram-se contrários aos valores de polaridade, portanto, parece que um aumento da polarizability leva a uma diminuição nos rendimentos quânticos singlete para a decomposição catalisada do peróxido de difenoila (1) e a decomposição induzida de 3, no entanto, um aumento nos rendimentos quânticos singlete para a 1,2-dioxetanona 2. Os três sistemas de CL também foram estudados em misturas binárias de solventes com diferentes viscosidades, mas com parâmetros de polaridade e polarizabilidade semelhantes e os rendimentos quânticos singlete mostraram aumentar com o aumento da viscosidade do meio. Os dados foram analisados usando tanto o modelos colisional quando o modelo de volume livre. Surpreendentemente, o sistema altamente eficiente decomposição induzida intramolecular do 1,2-dioxetano 3 mostrou-se muito mais sensível aos efeitos da viscosidade do que os sistemas intermoleculares ineficientes, o que indica claramente que a decomposição induzida do 1,2-dioxetano deve ocorrer por um processo de retro-transferência de elétron intramolecular. Além disso, os parâmetros de quimiluminescência destes sistemas foram estudados em vários solventes puros. Os rendimentos quânticos singlete obtidos foram correlacionados com parâmetros de viscosidade, polarizabilidade e polaridade usando análise de regressão linear múltipla.

The cyclic peroxides: diphenoyl peroxide (1), spiro-adamantyl-1,2-dioxetanone (2) and 4-(3-tert-butyldimethylsilyloxyphenyl)-4-methoxyspiro[1.2-dioxetane-3.2\'-adamantane] (3) synthesized, purified and characterized, also their kinetic and chemiluminescence (CL) properties were determined. The influence of solvent polarity on the chemiexcitation parameters of the catalyzed decomposition of 1 and 2, as well as the induced decomposition of 3 were examined in several binary solvent mixtures with different polarity and polarizability parameters, but similar viscosities. For solvents with low polarity, singlet quantum yields for the intermolecular catalyzed decomposition of diphenoyl peroxide (1,) and 1,2-dioxetanone (2) increase in function of medium polarity, showing maximum values in mediums of intermediate polarity, and decreasing in highly polar mixtures. For the induced decomposition of 3, the quantum yield showed to increase with increasing solvent polarity, including high polar solvent systems. In the binary solvent systems studied, the polarizability parameter showed to be contrary to the polarity values, therefore, it might appear that an increase of polarizability leads to a decrease in the singlet quantum yields for catalyzed decomposition of diphenoyl peroxide (1) and the induced decomposition of 3, however an increase in the singlet quantum yields for 1,2-dioxetanone 2. The three CL systems were also studied in binary solvent mixtures with different viscosities but similar polarity and polarizability parameters and singlet quantum yields showed an increase with increasing medium viscosity. Data were analyzed by using the collisional and the free volume models. The highly efficient intramolecular system induced 1,2-dioxetane 3 decomposition showed to be much more sensible to viscosity effects than the inefficient intermolecular systems. This surprising fact indicates that, even showing a significant solvent-cage effect, the induced 1,2-dioxetane should occur by an intramolecular electron back transfer. Moreover, chemiluminescence parameters of these systems were studied in several pure solvents. The obtained singlet quantum yields were correlated with viscosity, polarizability and polarity parameters by using multiple linear regression analysis. / Os peróxidos cíclicos: peróxido de difenoila (1), spiro-adamantil-1,2-dioxetanona (2) e 4-(3-terc-butildimetilsililoxifenil)-4-metoxispiro[1,2-dioxetano-3.2\'-adamantano] (3) foram sintetizados, purificados e caracterizados e as suas propriedades cinéticas e de quimiluminescência (CL) determinadas. A influência da polaridade do solvente sobre os parâmetros de quimi-excitação da decomposição catalisada de 1 e 2, e a decomposição induzida de 3 foi examinada em diversas misturas binárias de solventes com parâmetros de polaridade e polarizabilidade diferente, mas viscosidades semelhantes. Para solventes com baixa polaridade, os rendimentos quânticos singlete para a decomposição catalisada intermolecular do peróxido de difenoila (1) e 1,2-dioxetanona (2) aumentam em função da polaridade do meio, mostrando valores máximos em meios com polaridade intermediária, e diminuim para misturas altamente polares. Para a decomposição induzida de 3, o rendimento quântico mostrou aumentar com o aumento da polaridade do solvente, inclusive para sistemas com alta polaridade. Nos sistemas binários de solventes estudados, os parâmetros de polarizabilidade mostraram-se contrários aos valores de polaridade, portanto, parece que um aumento da polarizability leva a uma diminuição nos rendimentos quânticos singlete para a decomposição catalisada do peróxido de difenoila (1) e a decomposição induzida de 3, no entanto, um aumento nos rendimentos quânticos singlete para a 1,2-dioxetanona 2. Os três sistemas de CL também foram estudados em misturas binárias de solventes com diferentes viscosidades, mas com parâmetros de polaridade e polarizabilidade semelhantes e os rendimentos quânticos singlete mostraram aumentar com o aumento da viscosidade do meio. Os dados foram analisados usando tanto o modelos colisional quando o modelo de volume livre. Surpreendentemente, o sistema altamente eficiente decomposição induzida intramolecular do 1,2-dioxetano 3 mostrou-se muito mais sensível aos efeitos da viscosidade do que os sistemas intermoleculares ineficientes, o que indica claramente que a decomposição induzida do 1,2-dioxetano deve ocorrer por um processo de retro-transferência de elétron intramolecular. Além disso, os parâmetros de quimiluminescência destes sistemas foram estudados em vários solventes puros. Os rendimentos quânticos singlete obtidos foram correlacionados com parâmetros de viscosidade, polarizabilidade e polaridade usando análise de regressão linear múltipla.

Problématique : Durant la période fœtale, le métabolisme global du fœtus fonctionne en hypoxie, ce qui limite la phosphorylation oxydative dans la mitochondrie, et par conséquent la production d’adénosine triphosphate (ATP). Ces conditions sont nécessaires pour le développement intra-utérin. Lors de la naissance, l’augmentation des concentrations d’oxygène et un stress oxydatif permettent une transition métabolique. Une charge oxydative supplémentaire en période néonatale pourrait perturber cette transition métabolique et causer des complications. La nutrition parentérale (NP) administrée aux nouveau-nés prématurés apporte un triple fardeau oxydatif : une exposition à des peroxydes oxydants autogénérés par l’interaction des composants de la NP, une carence en vitamine C (instable en solution), et une déficience en glutathion, vu la charge oxydative élevée. Cette charge oxydative excessive affecte l’homéostasie redox au foie et aux poumons, ainsi que le métabolisme énergétique hépatique, et ce, par des effets immédiats et à long-terme. La méthylation de l’ADN est un possible mécanisme qui explique les effets à long terme. Le but de ce travail était de caractériser l’impact à court- et long-terme de la NP néonatale sur l’homéostasie redox, la méthylation de l’ADN, et le métabolisme des glucides et lipides, en isolant chacun des facteurs nutritionnels. Méthodes : Des cochons d’Inde ont été divisés dans les groupes suivants 1) NP : nutrition intraveineuse complète ; 2) NP+ glutathion disulfure (GSSG) (6 ou 12µM- substrat pour la synthèse intra-cellulaire de glutathion); 3) Diète complète : nutrition orale complète 4) Diète déficiente en Vitamine C; 5) Diète déficiente en Cystéine; 6) Diète double déficiente; ou. À 1 semaine de vie, la moitié des animaux était sacrifié et l’autre moitié a commencé à manger une diète complète jusqu’à l’âge adulte. Résultats et discussion : Les animaux ayant reçu une NP néonatale ont un métabolisme énergétique permettant la synthèse de nicotinamide adénine dinucléotide phosphate (NADPH) par l’augmentation de l’activité de la glucokinase (captation de glucose), et diminution de celles de la phosphofructokinase-1 (PFK-1) (glycolyse) et acétyl-CoA-carboxylase-1 (ACC)(lipogenèse). À l’âge adulte, les animaux ont une diminution des niveaux de GSSG, indiquant un débalancement de l’homéostasie redox vers le côté réducteur programmé par la NP néonatale. L’activité augmentée de l’ACC suggère une tendance à accumuler les lipides au foie à la suite d’une diète riche en glucides. L’ajout de glutathion à la NP ne prévient pas ces perturbations, car les déficiences en glutathion et vitamine C jouent un rôle sur la modulation des niveaux protéiques de l’ACC. Les diètes néonatales déficientes en vitamine C et cystéine augmentent l’activité de la PFK-1. Cette augmentation se maintient jusqu’à l’âge adulte chez les mâles, mais pas chez les femelles. Les niveaux protéiques de la glucokinase et ACC sont diminués à 1 semaine, et ceux de l’ACC sont élevés à 3 mois dans les groupes ayant reçu une des diètes déficientes. Ces effets sont similaires à ceux trouvés dans les animaux nourris avec la NP, suggérant que la déficience de la NP en ces nutriments et non les peroxydes cause ces effets. Dans tous les groupes, un stress oxydatif a été démontré à 1 semaine de vie, soit par l’augmentation des niveaux de GSSG, ou la diminution du GSH. Cet effet est vrai pour le foie et le poumon. Une réponse de Nrf2 est observée aussi au foie, ce qui caractérise un niveau bas de stress oxydatif. La baisse de GSH pulmonaire chez les animaux déficients est secondaire à l’inhibition oxydative de la voie de transméthylation au foie. Une diminution des niveaux d’ARNm de glutathion réductase et glutarédoxine sont observées, ce qui favorise encore le stress oxydatif pulmonaire. À long terme, les effets sont les opposés, soit débalancement de l’homéostasie redox vers le côté réducteur au foie et poumon. La méthylation de l’ADN était diminuée au foie des animaux nouveau-nés recevant les diètes déficientes, mais aucun changement n’a été observé aux poumons. Cette diminution est en accord avec les hauts niveaux d’ARNm des gènes de la protéine régulatrice de la glucokinase, et AMPK. À long-terme, l’effet inverse est observé pour la méthylation de l’ADN Conclusion : La NP modifie le flot d’énergie au foie à 1 semaine visant favoriser le métabolisme redox en détriment du métabolisme énergétique. Cet effet semble créer une déficience énergétique fonctionnelle, qui se développe en une lipogenèse accrue en âge adulte. Cela peut représenter un exemple de la plasticité développementale. Bien qu’un stress oxydatif en âge néonatal ne soit pas létal, il affecte le métabolisme énergétique et redox à long-terme, probablement par la méthylation de l’ADN. Les résultats de ce travail démontrent que ces animaux adultes ont une capacité accrue d’entreposer de l’énergie, soit par une lipogenèse plus élevée, soit par une accumulation d’énergie redox (glutathion). Aucune maladie métabolique n’était observée chez les animaux, mais il est attendu à ce que ces animaux développent ces maladies plus facilement suite à l’exposition à des insultes (habitudes de vie malsaines, tabagisme, etc.). / Problematic: During the fetal period, the general metabolism works under hypoxia, limiting oxidative phosphorylation in mitochondria and adenine triphosphate (ATP) synthesis. These conditions are necessary for intrauterine development. After birth, the increasing oxygen concentrations and the associated oxidative stress induce a metabolic transition. An excessive oxidative load during the neonatal period could perturb this transition. Parenteral nutrition (PN) administered to premature newborns comes with a triple oxidative burden: contaminating peroxides generated in solution, vitamin C deficiency (unstable in solution), and glutathione deficiency (caused by the high oxidative load). This oxidative load affects redox homoeostasis in the liver and lungs, as well as energy metabolism in the liver. These effects are not only immediate, but they are also delayed. DNA methylation is a candidate mechanism explaining the long-term effects. The objective of this work was to characterize the short- and long-term impacts of neonatal PN over redox homoeostasis, DNA methylation and carbohydrate and lipid metabolism by isolating each of these factors. Methods: Six groups of three-day-old guinea pigs received for 4 days either: 1) Total PN; 2) PN+glutathione disulfide (GSSG) (6 or 12µM-anti-peroxide);3) Vitamin C deficient; 4) Cysteine deficient; 5) Double deficient; or 6) Complete diets. At 1 week of life, half of the animals were sacrificed, and the other half started eating nutritionally complete diets until adulthood. Results and discussion: NP animals had energy metabolism shifted favouring nicotinamide adenine dinucleotide phosphate (NADPH) synthesis, as evidenced by the increase in glucokinase activity (glucose trapping in hepatocytes) and decrease in phosphfuctokinase-1 (PFK-1) (glycolysis) and acetyl-CoA-carboxyalase-1 (ACC) (lipogenesis) activities. Adding GSSG to parenteral nutrition prevents these changes. During adulthood, ACC activity is increased, suggesting a tendency to accumulate lipids after a diet rich in carbohydrates. Adding GSSG to PN does not prevent these changes as they seem to be caused by the nutritional deficiencies in vitamin C and cysteine. Neonatal diets deficient in vitamin C and cysteine increase PFK-1 activity. This increase is maintained until adulthood in males but not in females. Protein levels of glucokinase and ACC are decreased at 1 week of life and ACC levels are increased at adulthood in deficient groups. These effects are like the ones observed in PN animals. In all groups, oxidative stress is demonstrated in 1-week-old animals, either by an increase in GSSG levels, or a decrease in GSH. This is true for the liver and lungs. An Nrf2 response is also observed in the liver, suggesting a low level of oxidative stress. The decrease in lung GSH is secondary to the oxidative inhibition of the transmethylation pathway in the liver. Decreased levels of glutathione reductase and glutaredoxin mRNA levels are observed in lungs, favouring pulmonary oxidative stress. At adulthood, an imbalance in redox homeostasis towards a reducing state is observed in lungs and liver. DNA methylation was decreased in the liver of deficient animals at 1-week, but no changes were observed in lungs. This decrease is in accordance with the decrease in mRNA levels of glucokinase regulatory protein and AMPK. At adulthood, the opposite effect was observed for DNA methylation. Conclusion: Parenteral nutrition alters the energy flow in the liver of 1-week-old animals, favouring redox metabolism over energy metabolism. This effect seems to create a phenotype of functional energy deficiency which translates into an increased lipogenesis at adult age. This may be an example of developmental plasticity. Although neonatal oxidative stress is not lethal, it affects energy and redox metabolism at adulthood, probably through DNA methylation. The presented results demonstrate these animals have an increased capacity of storing energy, either through increased lipogenesis, or by an increase in redox energy accumulation (glutathione). No metabolic disease was observed. Although it would be expected that these animals would develop these diseases more easily after exposure to insults, such as unhealthy lifestyle habits, smoking, and others.

Die vorliegende Arbeit beschreibt die Herstellung und Charakterisierung der ersten Antikörper gegen Triacetontriperoxid (TATP), einem hoch empfindlichen und unkonventionellen (nicht-kommerziellen) Initialsprengstoff. Entscheidend dafür war die Synthese eines TATP-imitierenden Haptens, welches die typische nonagonale Struktur des TATP mit seinen drei Peroxid- und sechs Methylgruppen nahezu perfekt nachbildet, aber den Vorzug einer zusätzlichen Carboxygruppe zur kovalenten Kopplung an Proteine aufweist. Dadurch konnte das TATP-Hapten an Rinderserumalbumin (BSA) gebunden werden, um ein immunogenes Konjugat zu erzeugen, welches die erfolgreiche Immunisierung zweier Säugetierarten, Maus und Kaninchen, ermöglichte. Der Verlauf der In vivo-Immunisierungen wurde durch die Analyse der Tierseren in regelmäßigen Abständen mittels enzymgekoppeltem Immunoassay (ELISA) verfolgt. Die polyklonalen Antikörper beider Spezies waren ungewöhnlich selektiv gegenüber TATP. Jedoch unterschied sich die Affinität der Antikörper der zwei Spezies um das 5000-fache, wobei die Kaninchenseren den Mausseren überlegen waren. Entsprechend war auch die mit Kaninchenserum erreichbare TATP-Nachweisgrenze von 0.01 µg/L deutlich besser im Vergleich zu 50 µg/L, die mit Mausserum erzielt wurden. Der Messbereich des TATP-ELISA mit Kaninchenserum deckte zudem mehr als vier Zehnerpotenzen ab, wie mittels Präzisionsprofil bestimmt wurde. Die erhaltenen TATP-Antikörper aus Kaninchen stehen damit Anwendungen in Nachweissystemen für die sehr empfindliche Detektion von TATP zur Verfügung, die u. a. in sicherheitsrelevanten Bereichen zum Einsatz kommen könnten. Als erste Anwendung wurde ein TATP-ELISA realisiert, der im Rahmen dieser Arbeit ausführlich optimiert wurde. Außerdem wurden erste Schritte zur Entwicklung eines TATP-Schnelltests (LFA) unternommen. Weitere Biosensoren auf Grundlage der neu entwickelten TATP-Antikörper sind denkbar. / The present work decribes the production and characterization of the first antibodies against triacetone triperoxide (TATP), a highly sensitive and improvised (non-commercial) primary explosive. Crucial to this work was the synthesis of a TATP-related hapten that mimics almost perfectly the typical nonagonal structure of TATP with its three peroxide and six methyl groups. Advantageously, it has an additional carboxylic acid group, which provides a conjugation site for covalent attachment to proteins. Thus, the TATP hapten could be linked to bovine serum albumin (BSA) to produce an immunogenic conjugate, allowing the successful immunization of two different mammalian species, mouse and rabbit. The in vivo immunization progress was followed by periodically analyzing the animals’ sera using enzyme-linked immunosorbent assay (ELISA). The polyclonal antibodies of both species were remarkably selective to TATP. The affinity of these TATP-antibodies was, however, different between the two species, with the rabbit sera showing an affinity about 5000-fold superior than the murine one. Consequently, the TATP detection limit of 0.01 µg/L was considerably better using the sera from rabbit in contrast to 50 µg/L when mouse serum was used. The working range of the TATP-ELISA with rabbit sera covers more than four decades, calculated from a precision profile. The obtained TATP antibodies from rabbit are now available for applications in highly sensitive detection systems for TATP, which could be employed, among others, in security-relevant areas. The first application was the realization of a TATP-ELISA, which was extensively optimized within the course of this work. Furthermore, the first steps towards the development of a lateral flow assay (LFA) targeting TATP were taken, making conceivable further biosensor platforms based on the newly developed TATP antibodies.