Muitas espécies de animais vivenciam situações nas quais há uma profunda depressão metabólica, como na anóxia, na hipóxia e na hibernação. Durante a reoxigenação ou o despertar, ocorre aumento da produção de espécies reativas de oxigênio (ROS), que tendem a causar danos oxidativos. Diferentes enzimas antioxidantes protegem o organismo contra as ROS, porém não se sabe qual a real importância de cada uma delas durante a reoxigenação. A hibernação é uma das formas de hipometabolismo menos estudadas, fazendo com que haja questionamentos sobre como os hibernantes se protegem das ROS durante o despertar. A análise dos dados existentes é complexa devido à existência de variáveis não controladas, como o efeito do jejum associado à hibernação. Nesta tese, foram desenvolvidos dois projetos. Em um, investigou-se a importância da catalase num ciclo de anóxia e reoxigenação em caramujos pulmonados. No segundo, investigou-se o efeito da hibernação e da privação alimentar no intestino de lagartos teiús. Com base nos resultados, foi possível concluir que a catalase exerce um papel complementar contra os danos oxidativos causados pelas ROS e em conjunto com os demais componentes do sistema antioxidante. Porém, sua função não parece ser essencial, sendo em grande parte compensada pela atividade de glutationa peroxidase. Também foi possível concluir que a hibernação, estudada sem a interferência de drásticas quedas da temperatura, causa nítidas alterações no metabolismo de radicais livres no intestino de lagartos, com queda de atividades enzimáticas e de concentração de glutationa. A ausência de grandes danos oxidativos durante o despertar dos animais mostra que eles têm um sistema antioxidante eficiente. A privação alimentar resultou em respostas semelhantes as da hibernação, mas parece ter causado um certo grau de estresse oxidativo. Os resultados apresentados nesta tese respondem dois questionamentos no estudo do metabolismo de radicais livres em situações que envolvem flutuações na disponibilidade e no consumo de oxigênio. / Many species of animals experience situations in which occurs a profound metabolic depression, like anoxia, hypoxia and hibernation. During reoxygenation or arousal, there is an increase of the production of reactive oxygen species (ROS), which tend to cause oxidative damage. Different antioxidant enzymes protect the organims against the ROS, however the real importance of each one of them during reoxygenation is not known. Hibernation is one of the types of hypometabolism less studied, and questions about how the hibernators protect themselves from ROS during the arousal have not yet been answered. The analysis of the existent data is complex due to the existence of uncontrolled variables. In this thesis were carried out two studies in which were investigated: the importance of catalase in a cycle of anoxia and reoxygenation in pulmonate snails, and the effect of hibernation and of food deprivation in the intestine of tegu lizards. Considering the results, it was possible to conclude that catalase plays an complementary role against the damages caused by ROS and in association with the other components of the antioxidant system. However, its function seems to be non-essential, being greatly compensated by the glutathione peroxidase activity. It was also possible to conclude that hibernation, studied without the interference of drastic falls in temperature, causes clear alterations in free radicals metabolism in the lizards intestine, with a reduction in enzymes activities and in glutathione concentration. The absence of big oxidative damage during the arousal of the animals shows that they have an efficient antioxidant system. Food deprivation resulted in similar responses of those from hibernation, but seemed to cause some degree of oxidative stress. The results presented in this thesis answer two questions in the study of the free radical metabolism in situations that involve fluctuations in oxygen availability and consumption.

Este trabalho teve por objetivos: levantar conceitos alternativos sobre radicais livres na mídia impressa e em livros de Bioquímica; examinar os conceitos alternativos prevalentes em pós-graduandos e em professores de Química e Biologia do Ensino Médio; e, planejar e aplicar intervenção para reduzir a influência destes conceitos. Para o levantamento na mídia impressa, foi selecionada a revista Veja, período de 01/01/2000 a 31/07/2014, e foram criadas as seguintes categorias para classificar o material encontrado: Conceito Alternativo (CA), Informação Indutiva (II), Conceito Indutivo (CI) e Generalização Indevida (GI), formando o grupo dos Conceitos Não Científicos (CNC); Conceito Correto (CC); Conceito Errado (CE); e, não preenche os critérios de inclusão (NPCI). Foram encontrados 294 trechos dos quais: 55,4% CA; 21,1% II; 9,2% CI; 5,4% GI; 4,1% CC; 2,7% CE; 2,0% NPCI; e, 91,2% CNC. Para o levantamento em livros de Bioquímica foram selecionados: Campbell - Bioquímica; Lehninger - Princípios de Bioquímica; Marzzoco e Torres Bioquímica Básica; Stryer - Bioquímica; e, Voet e Voet - Bioquímica. A maior parte dos conceitos encontrados nos livros foram conceitos corretos, contudo 77,5% dos conceitos encontrados destacavam aspectos negativos sobre radicais livres. Para o levantamento de conceitos alternativos em pós-graduandos e professores foi desenvolvido o Questionário de Radicais Livres (QRL), versões piloto e final, aplicadas, respectivamente, a 14 pós-graduandos matriculados na disciplina de pós-graduação Planejamento e Aplicação de Sequências Didáticas Envolvendo o Conteúdo Radicais Livres e 31 professores matriculados no curso de extensão Radicais Livres e suas Interfaces com a Vida, planejado com o auxílio dos pós-graduandos da disciplina. O QRL é um questionário de múltipla-escolha em etapas, composto por uma etapa de avaliação do conteúdo e outra sobre a percepção do participante sobre seu nível de certeza. Os pós-graduandos revelaram baixo domínio sobre o tema e baixo nível de certeza. Os professores, testados pré- e pós-intervenção, também apresentaram alta frequência de erros, bastante semelhante no pré- e no pós-teste, e baixo nível de certeza no pré-teste e aumento do nível de certeza no pós-teste. Acredita-se que estes resultados, tanto entre os pós-graduandos e como entre os professores, sejam reflexo da falta de contato acadêmico com o tema. Aos professores, também foram aplicados dois questionários complementares denominados Perfil Profissional e Conhecimentos Sobre Radicais Livres. Entre outras informações, os dados coletados a partir destes questionários, indicaram que os professores lecionam juntos para aproximadamente 12000 estudantes e que o curso de extensão contribuiu para aumentar seus conhecimentos sobre radicais livres. Estes dados oferecem suporte para a escolha do público-alvo e justificam a execução de cursos de extensão, tanto pelo o potencial de alcançar indiretamente um grande número de pessoas, como pela possibilidade de difusão do tema. Tanto o curso de extensão como a disciplina de pós-graduação foram muito bem avaliados pelos professores e pós-graduandos. Os altos percentuais de conceitos não científicos, principalmente conceitos alternativos, encontrados na mídia impressa, bem como os conceitos alternativos apresentados por pós-graduandos e professores, devem preocupar especialistas e cientistas, pois este tema é objeto de muitos anúncios publicitários que frequentemente incentivam o consumo de suplementos de antioxidantes. Em livros de Bioquímica, o aumento de conteúdo destacando o papel fisiológico dos radicais livres deve contribuir para desfazer o conceito disseminado de que radicais livres seriam prejudiciais à saúde. / This work aimed to survey the misconceptions regarding free radicals published in printed media and in biochemistry textbooks; to examine the misconceptions regarding free radicals prevalent among graduate students and chemistry and biology high school teachers; and to design and implement instructional intervention to reduce the influence of these misconceptions. For surveying printed media, publications from Veja magazine, dating from 01/01/2000 to 07/31/2014, were selected. The published material was categorized as follows: Misconception (MC), Inductive Information (II), Inductive Concept (IC), and Inadequate Generalization (IG) - collectively called Non-Scientific Concepts (NSC) - and Right Concept (RC), Wrong Concept (WC), and not fit for the inclusion criteria (NFIC). A total of 294 text excerpts were obtained, comprising 55.4% MC, 21.1% II, 9.2% IC, 5.4% IG, 4.1% RC, 2.7% WC, 2.0% NFIC, and 91.2% NSC. In order to examine the content in biochemistry textbooks, the following were selected: Campbell - Biochemistry; Lehninger - Principles of Biochemistry; Marzzoco & Torres - Basic Biochemistry; Stryer - Biochemistry; and Voet & Voet - Biochemistry. Most of the excerpts selected from the biochemistry textbooks were right concepts. However, 77.5% of the concepts highlighted negative aspects of free radicals. To investigate misconceptions among graduate students and high school teachers, a Free Radicals Questionnaire (FRQ) was developed. Pilot and final versions were given to 14 students enrolled in the graduate course Planning and Application of Didactical Sequences Encompassing Free Radicals Content and to 31 high school teachers enrolled in the extension course Free Radicals and its Interfaces with Life, respectively. The extension course was planned with the assistance of graduate students enrolled in the graduate course. FRQ is a multiple choice tier test with tiers for content evaluation and the participants perception of their level of certainty. Graduate students exhibited low levels of subject expertise and certainty. Teachers were tested pre- and post-intervention, and exhibited a high frequency of mistakes, comparable to graduate students, with similar pre- and post-test outcomes. However, their certainty level increased post-test. It is believed that these results may have occurred due to an academic lack of contact with the subject of free radicals. Additionally, two complementary questionnaires were given to the teachers: Professional Profile and Free Radicals Knowledge. Data collected from these questionnaires indicated that teachers collectively taught approximately 12,000 students and that the extension course contributed to increasing the teachers knowledge on free radicals. These results support the choice of the target audience and justify the implementation of the extension courses, because these courses have the potential to indirectly reach a large audience and can be used for thematic dissemination of knowledge in the subject area. The extension and graduate courses were highly rated by the teachers and graduate students, respectively. The high percentage of non-scientific concepts, especially misconceptions, found in printed media, as well as the misconceptions regarding free radicals prevalent among graduate students and teachers must concern specialists and scientists, as this topic is the subject of many advertisements encouraging consumption of antioxidant supplements. In biochemistry textbooks, increasing the content highlighting the physiological role of free radicals would contribute considerably in dispelling the widespread notion that free radicals are harmful to health.

by Hui Yee Han, Ellen. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 68-71). / acknowledgements --- p.i / abstract --- p.ii / table of contents --- p.iii-vi / list of figures --- p.vii / LIST OF TABLES --- p.viii / chapter / Chapter chapter i : --- introduction --- p.1 / Chapter chapter ii : --- background knowledge --- p.2 -33 / Chapter 2.1 --- Definition of Free Radical --- p.2 / Chapter 2.2 --- Oxygen Derived Radicals and Their Generation In Vivo --- p.2 -9 / Chapter 2.2.1 --- Production of Singlet Oxygen --- p.4 / Chapter 2.2.2 --- Production of Superoxide Radical (O2-) and Hydrogen Peroxide (H2O2) --- p.4 -8 / Chapter I. --- Endogenous Production --- p.4-7 / Chapter II. --- Exogenous Production --- p.7-8 / Chapter 2.2.3 --- Generation of Hydroxy Radical (OH) through H2O2 and O2- --- p.8 -9 / Chapter 2.3 --- Free Radical Damage and Lipid Peroxidation --- p.9 -13 / Chapter 2.4 --- Lipid Peroxidation and Atherosclerosis --- p.13 -14 / Chapter 2.5 --- Antioxidant --- p.15-21 / Chapter 2.5.1 --- Primary Preventive Antioxidants --- p.15-17 / Chapter 2.5.2 --- Secondary Radical Scavenging Antioxidants --- p.18-21 / Chapter I. --- Lipid Soluble Radical-Scavenging Antioxidants --- p.18-19 / Chapter II. --- Water Soluble Radical-Scavenging Antioxidants --- p.20 -21 / Chapter 2.6 --- Measurement of Oxygen-Derived Radical in Vivo --- p.21 -26 / Chapter 2.7 --- Principle of the TRAP assay --- p.27-33 / Chapter 2.7.1 --- Oxygen Consumption Method --- p.29 -30 / Chapter 2.7.2 --- Chemiluminescence Method --- p.31-33 / Chapter chapter III： --- materials and methods --- p.34 -43 / Chapter 3.1 --- Instrumentation and Materials --- p.34 / Chapter 3.2 --- Method --- p.34-43 / Chapter 3.2.1 --- Establishment of Chemiluminescence Method for Determination of TRAP --- p.34 -36 / Chapter I. --- Preparation of Luminometer --- p.35 / Chapter II. --- Preparation of Sample before Analysis --- p.3 5 / Chapter III. --- Manual Operation of the Chemiluminescence Method --- p.35-36 / Chapter IV. --- Calculation of TRAP --- p.36 / Chapter 3.2.2 --- Evaluation of the Chemiluminescence Method --- p.37 -42 / Chapter I. --- Linearity --- p.37 / Chapter II. --- Recovery --- p.37-38 / Chapter III. --- Precision --- p.39 / Chapter IV. --- Interference Experiment --- p.39 -41 / Chapter V. --- Effect of Storage on TRAP --- p.42 / Chapter 3.2.3 --- "Determination of Analytical, Intraindividual and Interindividual Biological Variation of TRAP in A Group of Healthy Chinese" --- p.42-43 / Chapter CHAPTER IV: --- ANALYTICAL RESULTS --- p.44-56 / Chapter 4.1 --- Method Evaluation --- p.44-51 / Chapter 4.1.1 --- Linearity --- p.44 / Chapter 4.1.2 --- Recovery --- p.45 / Chapter 4.1.3 --- Within-Day and Between-Day Precision --- p.46-47 / Chapter 4.1.4 --- Interference --- p.47-48 / Chapter 4.1.5 --- Effect of Storage on TRAP --- p.48-51 / Chapter 4.2 --- "Analytical, Intraindividual and Interindividual Variation of TRAP in A Group of Healthy Chinese Population" --- p.52 -56 / Chapter 4.2.1 --- Difference in TRAP value obtained from the 22 subjects over time --- p.52 -54 / Chapter 4.2.2 --- The Effect of Genders on Trap --- p.55 / Chapter 4.2.3 --- "Determination of Analytical, Intraindividual and Interindividual Variation of TRAP in A Group of Healthy Chinese" --- p.55-56 / Chapter CHAPTER V: --- DISCUSSION --- p.57-67 / Chapter 5.1 --- Validation of the Method Performance --- p.57 / Chapter 5.2 --- Effect of Storage on TRAP --- p.57 / Chapter 5.3 --- Interference of Hemolysis and Lipemia on TRAP assay --- p.58-60 / Chapter 5.3.1 --- Effect of Hemolysis on TRAP --- p.58-59 / Chapter 5.3.2 --- Effect of Lipemia on TRAP --- p.59-60 / Chapter 5.4 --- Possible Sources of Variation in TRAP Over Time --- p.60 -63 / Chapter 5.5 --- Usefulness of the Variation Data of TRAP obtained from a Group of Healthy Chinese --- p.64 -67 / reference --- p.68 -71

o,o???-Dityrosine (dityrosine), an oxidation product of tyrosine produced by reaction between tyrosyl radicals, is becoming established as a biomarker of free radical oxidative protein damage in vivo. Attempts to measure dityrosine concentrations in various physiological and pathological systems have produced varied and often contradictory results. Dityrosine concentrations in urine, plasma, cerebrospinal fluid (CSF) and brain tissue varying over three orders of magnitude have been reported, together with inconsistent claims of significant dityrosine elevation in several ageing-related pathologies. Some of these findings have contributed to the implication of free radical activity in the pathology of several neurodegenerative disorders, vascular and ocular abnormalities and in phagocyte response to infection. The aim of this study was to test the hypothesis that dityrosine levels are elevated in ageing and ageing-related disease. The study also aims to determine the utility of dityrosine measurement as an index of oxidative damage, and elucidate possible explanations for the inconsistent levels reported. An assay for the quantification of dityrosine was developed using capillary HPLC with electrospray tandem quadrupole mass spectrometry (HPLC-MS/MS). The assay was highly specific for dityrosine and has the highest absolute sensitivity for dityrosine of any method reported to date, with a detection limit of 3 femtomoles of dityrosine on-column. Urine samples from volunteers of different age and from hospital patients with various pathologies were analysed. Plasma protein hydrolysates from control, Alzheimer???s and stroke subjects were analysed, together with hydrolysates of post mortem brain tissue from Alzheimer???s and control subjects. Urinary dityrosine level is elevated in states of acute infection and inflammation, but does not correlate with age or chronic disease. Protein dityrosine in four sections of Alzheimer???s brain was not significantly different from control sections. Dityrosine was present in human plasma and tissue proteins at approximately 5-35 residues per million tyrosine residues, and in normal urine at 5-25 micromol/mol creatinine or 20-200 nM. Most of the discrepancies in the literature relate to inadequate specificity of the analytical method. Interpretation of published data with critical appraisal of measurement technology specificity is essential in developing an accurate understanding of the role of free radicals in ageing and disease.

Abstract This work investigates the kinetics of the emulsion polymerization of vinyl acetate. Several aspects of this system have been clarified, including the induced decomposition of persulfate, retardation by oxygen and entry by, and analysis of, the aqueous phase oligomeric radicals. It has been shown that the retardation period observed in the emulsion polymerization of VAc can be explained by the effect of traces of oxygen (< 10-6 M) on the entry efficiency of the initiator-derived aqueous-phase oligomeric radicals. Comparison of rates of polymerization in V and persulfate -initiated polymerizations together with electrospray mass spectrometry of aqueous phase oligomers, has shown that the mechanism for the induced decomposition of persulfate by vinyl acetate is chain transfer to initiator from aqueous-phase oligomeric radicals. A value has been determined for the rate coefficient for transfer to initiator, by fitting literature data to a model based on this mechanism. The reported independence of the rate of polymerization from the monomer concentration in the emulsion polymerization of vinyl acetate has been investigated. Possible explanations for this behaviour have been proposed and tested in this work, by measuring radical-loss rates directly with y-relaxation techniques. Although the Y relaxations were found to be affected by experimental artefacts, it has been demonstrated that rapid exit is not responsible for the high radical-loss rates in this system. The major artefact identified in the y relaxations was the significant effect of relatively small exotherms on relaxation behaviour, Methodologies were developed for correcting affected data and for avoiding exotherms under certain conditions. Arrhenius parameters were determined for the rate coefficient for chain transfer to monomer using the In^M method, which utilises the whole MWD. This section of the work is incomplete, for reasons detailed in chapter 5. However, as a preliminary indication it was found that the frequency factor was 106.38 M-1 s-1 and the activation energy was 38.8 kJ mol-1.

Doctor of Philosophy (PhD) / This thesis primarily details the application of high-level ab initio quantum chemistry techniques in order to understand aspects of free-radical mediated protein oxidation. Traditionally, product analysis and electron paramagnetic resonance (EPR) spectroscopy are the primary means for elucidating the chemistry of protein oxidation. However, in experiments involving relatively small proteins reacting with a controlled radical-flux, a vast array of compounds can be produced, which are often difficult to analyse. Quantum chemical techniques on the other hand, can calculate the properties of any particular species directly, without suffering from the problems associated with experiment, such as side-reactions and chain processes. The results presented in this thesis are aimed at elucidating mechanistic details of protein oxidation, which might otherwise be difficult to probe experimentally. Chapter 1 gives an overview of the free-radical hypothesis of disease and ageing. Protein-derived radicals can undergo a variety of reactions, with the particular reaction that occurs depending on numerous aspects. Many types of reactions have been identified through radiolysis experiments of amino acids, and these are detailed in this chapter. In addition, the key reactive species are characterized and their different chemistries explained. Chapter 2 details the theoretical tools used throughout this thesis. Species with unpaired electrons (radicals) present unique problems for quantum chemistry to handle, thus an appropriate choice of theoretical technique is needed. The approach taken in this thesis is to use high-level compound methods, many of which have been directly formulated to give improved results for radical species, to provide benchmark quality results by which other less demanding techniques can be assessed. During the course of this study, it became apparent there was a void in the armoury of tools that could be used for the theoretical chemistry calculations. Chapter 3 details the formulation of a new tool in an attempt to fill this gap. Historically, the formulation of this new procedure came after much of the work in this thesis had been carried out. Thus, for the study of many of the reactions of this thesis the new method has not been used. However, it is most appropriate to place its formulation after summarizing the current status of techniques in common use today. Chapters 4 and 5 detail computations carried out on models of peptides containing backbone carbon- and nitrogen-centered radicals. A number of different theoretical techniques are used in these chapters, ranging from the highly accurate and computationally intensive to the less reliable and less demanding. The highly accurate techniques are used to gauge the accuracy of the other less demanding theoretical techniques so that the latter can be used with confidence in larger systems. Not only is the choice of theoretical technique important but also the judicious choice of model is essential. With this in mind, models are incrementally built until convergence of the particular property of interest is reached. Chapters 6 and 7 detail the calculations of β-scission reactions of alkoxyl radicals, which are a particular class of reaction known to occur on peptide backbones. Alkoxyl radicals are particularly difficult for theory to describe correctly. Therefore, Chapter 6 extensively assesses and then identifies the theoretical methods needed to portray them. Chapter 7 uses the techniques identified in the previous chapter in order to predict how the preference for a particular type of β-scission reaction changes.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

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Free radicals are thought to be involved in the destructive process of beta cells in Type 1 diabetes mellitus. Studies were performed to test the hypotheses (1) that malondialdehyde (MDA), a by-product of lipid peroxidation, affects $ beta$-cell function and integrity in vitro and (2) that such effects might be prevented in the BB rat (a model of spontaneous autoimmune diabetes) in vivo by administration of $ alpha$-phenyl-N-tert-butylnitrone (PBN), a free radical spin trap. First, islets of Wistar-Furth rats were studied at 12, 24 and 40 hr of culture in either 5.5, 11 or 16.5 mM glucose, and MDA at a range of concentrations ($6 times10 sp{-12}$-10$ sp{-3}$M). High concentrations of MDA inhibited glucose-stimulated insulin release without corresponding decreases in islet insulin content, suggesting that in situations with high predicted islet free radical content (e.g., autoimmune insulitis) beta cell function may be affected even before the cells are destroyed. Second, 28 diabetes-prone (BBdp) and 13 non diabetes-prone (BBn) rats were given PBN (20 mg/kg) s.c. 2x/day and 27 BBdp and 12 BBn rats received an equal volume of saline. PBN was able to decrease MDA in the absence of the autoimmune process and is remarkably non-toxic. However, it did not prevent diabetes for reasons which may include its concentration at the site of the inflammatory process or specificity to types of radicals trapped. Because it did decrease MDA, either a higher dose or a combination of PBN with other agents may hold promise for disease prevention.